Staphylococcus Aureus Biofilms Potentially Present in the Fishery Industry

Assessment of control strategies against Staphylococcus aureus biofilms potentially present in the fishery industry

(Evaluación de estrategias de control frente a biopelículas de Staphylococcus aureus

potencialmente presentes en la industria pesquera)

Daniel Vázquez Sánchez International Ph.D. Thesis

DEPARTAMENTO DE MICROBIOLOGÍA Y TECNOLOGÍA DE PRODUCTOS MARINOS

DEPARTAMENTO DE BIOQUÍMICA, GENÉTICA E INMUNOLOGÍA

Assessment of control strategies against Staphylococcus aureus biofilms potentially present in the fishery industry

(Evaluación de estrategias de control frente a biopelículas de

Staphylococcus aureus potencialmente presentes en la industria pesquera)

Dissertation presented by Daniel Vázquez Sánchez to aim for the International Ph.D. by the University of Vigo

Memoria presentada por Daniel Vázquez Sánchez para optar al título de Doctor Internacional por la Universidad de Vigo

Vigo, Febrero de 2014

DR. PALOMA MORÁN MARTÍNEZ, CATEDRÁTICA DEL DEPARTAMENTO DE BIOQUÍMICA, GENÉTICA E INMUNOLOGÍA DE LA UNIVERSIDAD DE VIGO,

INFORMA:

Que la presente Tesis Doctoral titulada “Assessment of control strategies against Staphylococcus aureus biofilms potentially present in the fishery industry (Evaluación de estrategias de control frente a biopelículas de Staphylococcus aureus potencialmente presentes en la industria pesquera)” presentada por el Licenciado en Biología D. Daniel Vázquez Sánchez para optar al Grado de Doctor por la Universidad de Vigo, fue realizada bajo la dirección del Dr. Juan José Rodríguez Herrera y de la Dra. Marta López Cabo en el Departamento de Microbiología y Tecnología de Productos Marinos perteneciente al Instituto de Investigaciones Marinas (IIM) del Consejo Superior de Investigaciones Científicas (CSIC).

Que el trabajo realizado tiene una base experimental suficiente y que supone una contribución relevante en el ámbito de la microbiología y seguridad alimentaria.

Que la presente Tesis Doctoral reúne los requisitos necesarios para la obtención de la mención de Tesis Internacional.

Que la Tesis Doctoral cumple los requisitos necesarios para presentarla en la modalidad de “compendio de artículos”, debido a la coherencia argumental de los cinco artículos incluidos en ella y que tres de los artículos científicos presentados han sido publicados en revistas indexadas en el Journal Citations Report, publicado por Thomson Reuters. En todos ellos, el doctorando ha contribuido de manera esencial junto con los coautores.

Por todo lo anterior autorizo su presentación ante el Tribunal correspondiente.

Y para que así conste a los efectos oportunos, se expide la presente en Vigo a 4 de Febrero de 2014,

Asdo.: Dra. Paloma Morán Martínez

DR. JUAN JOSÉ RODRÍGUEZ HERRERA Y DRA. MARTA LÓPEZ CABO, CIENTÍFICOS TITULARES DEL DEPARTAMENTO DE MICROBIOLOGÍA Y TECNOLOGÍA DE PRODUCTOS MARINOS DEL INSTITUTO DE INVESTIGACIONES MARINAS (IIM-CSIC),

INFORMAN:

Que el trabajo realizado tiene una base experimental suficiente y que supone una contribución relevante en el ámbito de la microbiología y seguridad alimentaria.

Que la presente Tesis Doctoral reúne los requisitos necesarios para la obtención de la mención de Tesis Internacional.

Por todo lo anterior autorizamos su presentación ante el Tribunal correspondiente.

Y para que así conste a los efectos oportunos, se expide la presente en Vigo a 31 de Enero de 2014,

Asdo.: Dr. Juan José Rodríguez Herrera Asdo.: Dra. Marta López Cabo

Este trabajo ha sido financiado por:

El proyecto PGIDIT 07 TAL 014402PR concedido por la Xunta de Galicia.

La beca predoctoral JAE-predoc concedida por el Consejo Superior de Investigaciones Científicas (CSIC) a Daniel Vázquez Sánchez.

La beca para estancias en centros de investigación concedida por el CSIC a Daniel Vázquez Sánchez para su estancia en el centro Nofima en Ås (Noruega).

"La vida es una unión simbiótica y cooperativa que permite triunfar a los que se asocian”

Lynn Margulis (1938-2011)

Bióloga estadounidense y Doctora Honoris Causa por la Universidad de Vigo

A Vero

Índice / Contents

Resumen ...... 7

Abstract ...... 15

General Introduction ...... 23 1. Staphylococcus aureus ...... 25 1.1. History ...... 25 1.2. Phylogeny ...... 25 1.3. General characteristics ...... 27 1.4. Pathogenicity ...... 28 1.5. Pathogenic determinants ...... 29 2. Biofilm formation by Staphylococcus aureus ...... 32 2.1. Definition of biofilm ...... 32 2.2. Significance of biofilm formation ...... 32 2.3. The process of biofilm development ...... 33 2.4. Ecological advantages of biofilm formation...... 34 2.4.1. Cell-cell communication ...... 34 2.4.2. Increased resistance to external stimulus ...... 35 2.4.3. Increased dispersal capacity ...... 36 2.5. Staphylococcus aureus biofilms: development, composition and regulation ...... 36 3. Incidence of Staphylococcus aureus in the food industry ...... 38 3.1. Outbreaks ...... 38 3.2. Foods implicated in staphylococcal poisonings ...... 43 3.3. Food contamination by Staphylococcus aureus ...... 44 4. Control of Staphylococcus aureus in the food industry ...... 45 4.1. Hazard Analysis and Critical Control Points (HACCP) ...... 45 4.2. Prevention strategies against biofilm formation ...... 45 4.3. Cleaning procedures ...... 46 4.4. Disinfection treatments ...... 47

Justificación y Objetivos ...... 55

Justification and Objectives ...... 61

Índice / Contents

Chapter 1. Incidence and characterization of Staphylococcus aureus in fishery products marketed in Galicia (Northwest Spain) ...... 67 Abstract ...... 69 1.1 Introduction ...... 70 1.2 Materials and Methods ...... 71 1.2.1 Sampling ...... 71 1.2.2 Isolation and identification of S. aureus ...... 72 1.2.3 RAPD ...... 73 1.2.4 Detection of sea-see and seg-sei genes ...... 74 1.2.5 Antibiotic susceptibility test ...... 76 1.2.6 Detection of blaZ and mecA genes ...... 77 1.3 Results ...... 78 1.3.1 Incidence in fishery products ...... 78 1.3.2 RAPD-PCR ...... 80 1.3.3 Presence of enterotoxin genes ...... 83 1.3.4 Antibiotic sensitivity ...... 84 1.4 Discussion ...... 87 1.5 Conclusions ...... 93

Chapter 2. Impact of food-related environmental factors on the adherence and biofilm formation of natural Staphylococcus aureus isolated from fishery products ...... 95 Abstract ...... 97 2.1 Introduction ...... 98 2.2 Materials and Methods ...... 100 2.2.1 Bacterial strains and growth conditions ...... 100 2.2.2 Evaluation of bacterial cell surface physicochemical properties ...... 100 2.2.3 Measurement of the adherence ability to polystyrene at different ionic strength conditions ...... 101 2.2.4 Quantification of biofilm formation on polystyrene under different environmental conditions...... 101 2.2.5 Transcriptional analysis ...... 102 2.2.6 Statistical analysis ...... 103 2.3 Results ...... 105 2.3.1 Surface hydrophobicity and electron donor/acceptor character ...... 105 2.3.2 Adherence ability of S. aureus to polystyrene surfaces ...... 105

Índice / Contents

2.3.3 Biofilm formation on polystyrene surfaces under different environmental conditions...... 108 2.3.3.1 Effect of incubation temperature ...... 108 2.3.3.2 Effect of glucose and NaCl addition ...... 108

2.3.3.3 Effect of MgCl2 addition ...... 111 2.3.4 Multivariate analysis of the physicochemical, adhesion and biofilm- forming properties of the 28 S. aureus strains ...... 112 2.3.5 Gene expression in relation to biofilm formation ...... 114 2.4 Discussion ...... 116 2.5 Conclusions ...... 119 Appendix 1: PCR-detection of icaA and icaD genes ...... 121 Appendix 2: PCR-detection of bap gene ...... 122

Chapter 3. Biofilm-forming ability and resistance to industrial disinfectants of Staphylococcus aureus isolated from fishery products ...... 125 Abstract ...... 127 3.1. Introduction ...... 128 3.2. Material and Methods ...... 129 3.2.1. Bacterial strains and culture conditions ...... 129 3.2.2. Conditions for biofilm formation...... 129 3.2.3. Biofilm formation assays ...... 131 3.2.3.1. Slime production on Congo red agar (CRA) ...... 131 3.2.3.2. Initial adherence ...... 131 3.2.3.3. Quantification of biofilm formation ...... 131 3.2.3.4. Determination of growth kinetics ...... 132 3.2.4. Biocide resistance assays ...... 132 3.2.4.1. Minimal biofilm eradication concentration (MBEC) ...... 133 3.2.4.2. Minimal bactericidal concentration (MBC) ...... 133 3.2.5. Statistical analysis ...... 134 3.3. Results ...... 134 3.3.1. Biofilm-forming ability of S. aureus ...... 134 3.3.1.1. Detection of biofilm-forming ability on Congo red agar (CRA) ...... 134 3.3.1.2. Initial adhesion studies ...... 135 3.3.1.3. Quantification of biofilm biomass ...... 135 3.3.2. Resistance to industrial disinfectants of S. aureus...... 137 3.3.2.1. Effectiveness of benzalkonium chloride ...... 137

Índice / Contents

3.3.2.2. Effectiveness of peracetic acid and sodium hypochlorite ...... 141 3.4. Discussion ...... 143 3.5. Conclusions ...... 147 Appendix: Growth kinetics ...... 149

Chapter 4. Single and sequential application of electrolyzed water with benzalkonium chloride or peracetic acid for removal of Staphylococcus aureus biofilms ...... 153 Abstract ...... 155 4.1. Introduction ...... 156 4.2. Material and Methods ...... 157 4.2.1. Bacterial strains ...... 157 4.2.2. Antibacterial agents ...... 157 4.2.3. Conditions for biofilm formation...... 158 4.2.4. Single application of electrolyzed water (EW) ...... 159 4.2.5. Sequential application treatments ...... 160 4.2.6. Statistical analysis ...... 161 4.3. Results ...... 162 4.3.1. Single application of electrolyzed water (EW) ...... 162 4.3.1.1. Effects of pH ...... 162 4.3.1.2. Effects of active chlorine concentration ...... 163 4.3.1.3. Effects of exposure time ...... 163 4.3.2. Double sequential application of NEW ...... 164 4.3.3. Sequential application of NEW and BAC ...... 166 4.3.4. Sequential application of NEW and PAA ...... 168 4.4. Discussion ...... 170 4.5. Conclusions ...... 173 Appendix ...... 175

Chapter 5. Antimicrobial activity of essential oils against Staphylococcus aureus biofilms ...... 181 Abstract ...... 183 5.1. Introduction ...... 184 5.2. Material and Methods ...... 185 5.2.1. Bacterial strain ...... 185 5.2.2. Antibacterial agents ...... 185 5.2.3. Conditions for biofilm formation...... 186

Índice / Contents

5.2.4. Biocide resistance assays ...... 187 5.2.4.1. Resistance of planktonic cells ...... 187 5.2.4.2. Resistance of biofilms ...... 187 5.2.4.3. Determination of growth kinetics ...... 189 5.2.5. Statistical analysis ...... 189 5.3. Results ...... 190 5.3.1. Effectiveness of essential oils (EOs) against planktonic cells ...... 190 5.3.2. Effectiveness of EOs against 48-h-old biofilms ...... 191 5.3.3. Effects of sub-lethal doses of thyme oil on bacterial growth ...... 192 5.3.4. Effectiveness of thyme oil against biofilms formed under sub-lethal doses of thyme oil ...... 194 5.3.5. Effectiveness of benzalkonium chloride (BAC) against biofilms formed under sub-lethal doses of thyme oil ...... 195 5.4. Discussion ...... 196 5.5. Conclusions ...... 199

General Discussion ...... 203

Conclusiones Generales ...... 211

General Conclusions ...... 217

Bibliografía / References ...... 223

List of original publications ...... 251

Agradecimientos / Acknowledgements ...... 253

Resumen

Staphylococcus aureus es uno de los principales agentes etiológicos de intoxicaciones alimentarias en el mundo, debido a la ingestión de alimentos con enterotoxinas. España es uno de los principales productores y consumidores de productos pesqueros en la Unión Europea. Sin embargo, S. aureus se detecta reiteradamente en estos alimentos como consecuencia de la contaminación cruzada con manipuladores y superficies de contacto con el alimento. La capacidad de formar biopelículas de S. aureus le proporciona una alta tolerancia a los antimicrobianos, permitiéndole una persistencia en ambientes alimentarios a largo plazo. Las nuevas tendencias en la producción de los alimentos (procesado mínimo, producción masiva, globalización) han introducido además nuevos escenarios que pueden favorecer la presencia y proliferación de S. aureus. Un gran incremento de cepas resistentes a antibióticos ha sido detectado también en ámbitos extra-hospitalarios, incluido el alimentario, lo que puede favorecer la transmisión de resistencias a la microbiota humana a través de los alimentos ingeridos y causar infecciones difíciles de tratar. Por tanto, la finalidad del presente trabajo consistió en mejorar el control de S. aureus en la industria alimentaria (en particular, la pesquera) a través de la identificación de los escenarios de contaminación de mayor riesgo y de la evaluación de prometedoras estrategias de desinfección frente a este patógeno.

El primer objetivo fue pues la estimación de la incidencia de S. aureus en diferentes productos pesqueros comercializados. Un total de 298 productos pesqueros de distinto origen y tipo de procesado fueron muestreados. Los aislados fueron identificados como S. aureus mediante específicas pruebas bioquímicas (producción de coagulasa y ADNasa, fermentación del manitol) y genéticas (secuenciación del 23s ADNr), y caracterizadas por RAPD-PCR con tres cebadores (AP-7, ERIC-2 y S). Asimismo, se evaluó la capacidad de todos los aislados de producir enterotoxinas (es decir, presencia de genes se) y su resistencia a varios antibióticos.

S. aureus fue detectado en una proporción significativa de productos (~ 25%), siendo destacable en productos frescos (43%) y congelados (30%). Además, una proporción significativa de ahumados, surimis, huevas y otros productos listos para consumir no cumplieron con los límites legales vigentes. Los aislados mostraron 33 patrones característicos, y cada uno fue atribuido a un clon bacteriano particular. No se encontró

Resumen relación entre los patrones de RAPD y las categorías de productos. La mayoría de los aislados (88%) son portadores del gen sea. Los recuentos de cepas con capacidad enterotoxigénica mostraron que en 17 productos se alcanzaron niveles de riesgo. No se encontró relación entre la presencia de genes se y los patrones RAPD. Todos los aislados fueron resistentes a la penicilina, cloranfenicol y ciprofloxacina, y muchos a la tetraciclina (82.4%), pero no se detectaron MRSA. De hecho, ninguna cepa portaba el gen mecA, relacionado con la resistencia a antibióticos beta-lactámicos como la meticilina.

Posteriormente, se examinó la prevalencia en instalaciones de procesado de pescado de las cepas de S. aureus con capacidad enterotoxigénica mediante el estudio de su capacidad para formar biopelículas y su resistencia a desinfectantes.

Todas las cepas fueron descritas como S. aureus capaces de producir exopolisacáridos (fenotipo positivo en agar rojo Congo y portadores de los genes icaA e icaD), pero ninguna portaba los genes bap (expresan la proteína accesoria de la biopelícula). La mayoría mostraron una capacidad para formar biopelículas mayor que S. aureus ATCC 6538 -cepa de referencia en los métodos bactericidas estandarizados- sobre materiales habituales de las superficies de contacto (acero inoxidable, poliestireno) y bajo diferentes condiciones ambientales (temperatura, contenido de nutrientes, osmolaridad) potencialmente presentes en las plantas de procesado. En general, la adherencia inicial de S. aureus incrementa bajo condiciones de alta fuerza iónica, mientras que la formación de biopelículas es significativamente favorecida por la presencia de glucosa (por ejemplo, como aditivo en surimis y ahumados), pero moderadamente con cloruro sódico o magnesio (por ejemplo, residuos de agua de mar y productos pesqueros). No obstante, el análisis transcriptacional de genes relacionados con la formación de biopelículas (icaA, sarA, rbf y σB) mostró una alta variabilidad entre las cepas en respuesta a estas condiciones ambientales. Por otra parte, parece que el procesado del alimento pudo haber generado una presión selectiva ya que cepas con una alta capacidad para formar biopelículas fueron aisladas de productos altamente procesados y manipulados.

Las biopelículas formadas por todas las cepas mostraron una marcada resistencia a desinfectantes aplicados habitualmente en la industria alimentaria (cloruro de benzalconio o BAC, ácido peracético o PAA, hipoclorito sódico o NaClO), siendo

Resumen mayor que la de ATCC 6538 en muchos casos. Como era de esperar, la resistencia de las biopelículas de S. aureus fue significativamente mayor que la de las células planctónicas en todos los casos. Pero no se encontró correlación entre la resistencia de las biopelículas y las células planctónicas al BAC, PAA y NaClO, por lo que ninguna extrapolación parece posible. Sin embargo, la mayoría de los métodos bactericidas estándar usados en la Unión Europea están basados en cultivos en suspensión, y sólo EN 13697 se basa en biopelículas, pero no parece simular realmente las condiciones ambientales que se encuentran en la industria alimentaria. La resistencia antimicrobiana aumentó con el desarrollo de la biopelícula. Además, la formación de biopelículas parece atenuar el efecto de las bajas temperaturas sobre la resistencia al BAC. El PAA fue el más efectivo frente a biopelículas y células planctónicas, seguido del NaClO y BAC. Pero la resistencia de las cepas no siguió el mismo orden para cada biocida, lo cual muestra la actual limitación de usar un reducido número de cepas de colección (y sólo un S. aureus) en los métodos estándar para asegurar una apropiada aplicación de los desinfectantes. En consecuencia, las dosis recomendadas por los fabricantes para desinfectar superficies de contacto con el alimento mediante la aplicación de BAC, PAA y NaClO fueron menores que las obtenidas en este estudio, por lo que no garantizan la eliminación de las biopelículas. Por tanto, los microorganismos podrían estar expuestos a dosis sub-letales de desinfectante, lo cual puede generar la emergencia de resistencias antimicrobianas.

Por esta razón, este trabajo se centró a continuación en el estudio de la eficacia y aplicabilidad de innovadoras estrategias de desinfección más respetuosas con el medio ambiente para el control de biopelículas de S. aureus en plantas de procesado de pescado. En particular, se investigó la actividad bactericida del agua electrolizada (EW) y de diversos aceites esenciales (EOs), así como tratamientos combinados con BAC o PAA. Las cuatro cepas de S. aureus con mayor potencial de prevalencia en plantas de procesado del alimento y con una alta incidencia en productos pesqueros (St.1.01, St.1.04, St.1.07 y St.1.08) fueron evaluados.

En el caso del EW, su eficacia frente a biopelículas apenas se ve afectada por variaciones en el pH de producción. El EW neutra (NEW) fue por tanto usada posteriormente porque tiene un mayor potencial de aplicación a largo plazo que el EW ácida (debido a una menor corrosividad y toxicidad) y al mayor rendimiento de la

Resumen unidad de producción a pH neutro. La aplicación de NEW causó una alta reducción en el número de células viables en la biopelícula inicialmente. Sin embargo, se necesitó una alta concentración de cloro activo (800 mg/L ACC) para alcanzar la reducción logarítmica (LR) exigida en el método estándar EN 13697 (≥ 4 log CFU/cm2 tras 5 min). Una aplicación secuencial doble de NEW a más bajas concentraciones durante 5 min cada una permitió alcanzar una LR ≥ 4 log CFU/cm2 en la mayoría del rango experimental. Aplicaciones secuenciales de NEW con BAC o PAA mostraron un efecto similar, con PAA-NEW siendo la secuencia más efectiva. La combinación de NEW con otros tratamientos antimicrobianos puede ser por tanto una alternativa eficaz a los protocolos de desinfección tradicionalmente usados en la industria alimentaria.

Por otra parte, se determinó la efectividad de 19 EOs (anis, zanahoria, citronela, cilantro, comino, Eucalyptus globulus, Eucalyptus radiata, hinojo, geranio, jengibre, hisopo, limoncillo, mejorana, palmarosa, pachuli, salvia, árbol del té, tomillo y vetiver) frente a células planctónicas de S. aureus St.1.01. Las células planctónicas mostraron una gran variabilidad en la resistencia a EOs, siendo el aceite de tomillo el más eficaz, seguido de los aceites de limoncillo y luego vetiver. Los 8 aceites más efectivos frente a células planctónicas fueron a continuación testados frente a biopelículas formadas en acero inoxidable durante 48 h. Todos los EOs redujeron significativamente el número de células viables en la biopelícula, aunque ninguno consiguió eliminar totalmente la biopelícula. Aceites de tomillo y pachuli fueron los más efectivos, pero se necesitaron altas concentraciones para alcanzar LR por encima de 4 log CFU/cm2 tras 30 min de exposición. El uso de dosis sub-letales de aceite de tomillo previno la formación de la biopelícula y mejoró la eficacia del tomillo y BAC frente a biopelículas. Sin embargo, cierta adaptación celular al tomillo fue detectada. Tratamientos basados en EOs deben por tanto basarse en la rotación y combinación de distintos EOs o con otros biocidas para prevenir la emergencia de cepas resistentes a los antimicrobianos. Tratamientos combinados con tomillo pueden ser una alternativa eficaz, respetuosa con el medioambiente y relativamente barata para controlar la formación de biopelículas de S. aureus en instalaciones de procesado de alimentos.

Abstract

Staphylococcus aureus is one of the major bacterial agents causing foodborne diseases in humans worldwide, due to the ingestion of food containing staphylococcal enterotoxins. Spain is one of the largest producers and consumers of fishery products in the European Union. However, S. aureus is repeatedly detected in these products as a consequence of cross-contamination from food handlers and food contact surfaces. Biofilm formation also provides S. aureus a high tolerance to biocides allowing a long- term persistence of this pathogen in food-related environments. Novel trends in food production (e.g. minimal processing, mass production, globalization) have additionally introduced new scenarios that can enhance the presence and subsequent growth of S. aureus. Moreover, an increased number of antibiotic-resistant S. aureus has been detected in non-clinical ambits, including the food industry, which can lead to the transmission of resistances to the human microbiome through the ingested food and causing infections hard to be treated. Therefore, this work was aimed to improve the control of S. aureus in the food industry (particularly, in fisheries) through the identification of the most risky scenarios of contamination and the evaluation of promising disinfection strategies against this pathogen.

The first objective was thus the assessment of the incidence of S. aureus in different commercialized fishery products. A total of 298 fishery products of different origin and type of processing were sampled. Isolates were identified as S. aureus by specific biochemical (coagulase, DNAse and mannitol fermentation) and genetic tests (23s rDNA sequencing), and characterized by RAPD-PCR with three primers (AP-7, ERIC-2 and S). In addition, the enterotoxin-producing ability (i.e. presence of se genes) and the resistance to several antibiotics were also evaluated for all isolates.

S. aureus was detected in a significant proportion of products (~ 25%), being the highest incidence in fresh (43%) and frozen products (30%). In addition, a significant proportion of smoked fish, surimis, fish roes and other ready-to-eat products did not comply with legal limits in force. Isolates displayed 33 fingerprint patterns, and each one was attributed to a single bacterial clone. Cluster analysis based on similarity values between RAPD fingerprints did not find relationship between any RAPD pattern and any product category. Most isolates (88%) were found to be sea positive. Putative

Abstract enterotoxigenic strains counts reached high risk levels in 17 products. No relationship was found between the presence of se genes and RAPD patterns. All isolates were resistant to penicillin, chloramphenicol and ciprofloxacin, and most to tetracycline (82.4%), but MRSA were not detected. In fact, none strain carried mecA gen, which is related to the resistance to beta-lactam antibiotics such as methicillin.

Subsequently, the prevalence in fishery-processing facilities of putative enterotoxigenic S. aureus strains was examined by studying their biofilm-forming ability and disinfectant resistance.

All strains were described as S. aureus able to produce exopolysaccharides (positive phenotype in red Congo agar and icaA- and icaD-carriers), but none carried bap gene (expression of biofilm accessory protein). Most strains showed a biofilm-forming ability higher than S. aureus ATCC 6538 -reference strain in bactericidal standard tests- on common food-contact surface materials (stainless steel, polystyrene) and under different environmental conditions (temperature, nutrient content, osmolarity) potentially present in processing plants. In general, initial adhesion of S. aureus was increased by the presence of high ionic strength conditions, whereas biofilm formation was significantly promoted by the presence of glucose (e.g., additive in surimis and smoked fish), but moderately by sodium chloride or magnesium (e.g., wastes of seawater and seafood). Nevertheless, transcriptional analysis of genes related with biofilm formation (icaA, sarA, rbf and σB) showed a high variability between strains in the response to these environmental conditions. Moreover, it seems that food-processing could have produced a selective pressure and strains with a high biofilm-forming ability were more likely to be found in highly handled and processed products.

Biofilms formed by all strains showed a marked resistance to disinfectants applied commonly in the food industry (benzalkonium chloride or BAC, peracetic acid or PAA, sodium hypochlorite or NaClO), being higher than ATCC 6538 in most cases. As expected, the resistance of S. aureus biofilms was significantly higher than that of planktonic cells in all cases. But no correlation was found between the resistance of biofilms to BAC, PAA and NaClO and that of planktonic cells, so no extrapolation seems thus feasible. However, most standard bactericidal tests used in the European Union are based in suspension cultures, and only EN 13697 is biofilm-based, but it does not seem to truly simulate environmental conditions found in the food industry. The

Abstract antimicrobial resistance increased as biofilm aged. Biofilm formation also seemed to attenuate the effect of low temperatures on BAC resistance. PAA was found to be most effective against both biofilms and planktonic cells, followed by NaClO and BAC. But the resistance of strains did not follow the same order for each biocide, which shows the present limitation of using a few type strains (and only one S. aureus) in standard tests in order to ensure a proper application of disinfectants. Consequently, doses recommended by manufacturers for BAC, PAA and NaClO to disinfect food-contact surfaces were lower than data obtained in this study, so they are not able to guarantee biofilm removal. Microorganisms could therefore be exposed to sub-lethal doses of disinfectants and this could generate the emergence of antimicrobial resistance.

For this reason, this work was then focused in the study of the efficacy and applicability of innovative and more environmentally-friendly disinfection strategies to control S. aureus biofilms on fishery-processing plants. Particularly, it was investigated the bactericidal activity of electrolyzed water (EW) and a range of essential oils (EOs), as well as combined treatments with BAC or PAA. The four S. aureus strains with the highest potential prevalence in food-processing plants and with a high incidence in fishery products (St.1.01, St.1.04, St.1.07 and St.1.08) were evaluated.

In the case of EW, its efficacy against biofilms was hardly any affected by variations in the pH of production. Neutral EW (NEW) was therefore used in subsequent studies as it has a higher potential for long-term application than acidic EW (due to a lower corrosiveness and toxicity) and due to the higher yield rate of the production unit at neutral pH. The application of NEW caused a high reduction in the number of viable biofilm cells initially. However, a high available chlorine concentration (800 mg/L ACC) was needed to achieve logarithmic reductions (LR) demanded by the European quantitative surface test of bactericidal activity (≥ 4 log CFU/cm2 after 5 min). A double sequential application of NEW at much lower concentrations for 5 min each allowed LR ≥ 4 log CFU/cm2 to be reached in most of the experimental range. Sequential applications of NEW and either BAC or PAA showed a similar effect, with PAA-NEW being most effective. The combination of NEW with other antimicrobial treatments can thus be an effective alternative to disinfection protocols traditionally used in the food industry.

Abstract

Otherwise, the effectiveness of nineteen EOs (anise, carrot, citronella, coriander, cumin, Eucalyptus globulus, Eucalyptus radiata, fennel, geranium, ginger, hyssop, lemongrass, marjoram, palmarosa, patchouli, sage, tea-tree, thyme and vetiver) was assessed against planktonic cells of S. aureus St.1.01. Planktonic cells showed a wide variability in resistance to EOs, with thyme oil as the most effective, followed by lemongrass oil and then vetiver oil. The eight EOs most effective against planktonic cells were subsequently tested against 48-h-old biofilms formed on stainless steel. All EOs reduced significantly the number of viable biofilm cells, but none of them could remove biofilms completely. Thyme and patchouli oils were the most effective, but high concentrations were needed to achieve LR over 4 log CFU/cm2 after 30 min exposure. The use of sub-lethal doses of thyme oil prevented biofilm formation and enhanced the efficiency of thyme oil and BAC against biofilms. However, some cellular adaptation to thyme oil was detected. Therefore, EO-based treatments should be based on the rotation and combination of different EOs or with other biocides to prevent the emergence of antimicrobial-resistant strains. Combined thyme oil-based treatments can be an effective, environmentally-friendly, safe-to-use and relatively inexpensive alternative to control the formation of S. aureus biofilms on food-processing facilities.

General Introduction

1. Staphylococcus aureus

1.1. History

Staphylococcus aureus was discovered in Aberdeen (Scotland) in 1880 by A. Ogston, who described grape-like clusters of bacteria in stained slide preparations of pus from patients with post-operative wound infections and abscesses (Ogston, 1882). Four years later, A.J. Rosenbach achieved pure cultures of this pathogen on solid media and named it as Staphylococcus aureus for the golden appearance of the colonies (Rosenbach, 1884).

In 1884, V.C. Vaughan and J.M. Sternberg were the first in associate a food poisoning outbreak in Michigan (USA) with the ingestion of cheese contaminated by staphylococci. But this link was not confirmed until 1914 by M.A. Barber, who showed that consuming milk from a cow with staphylococcal mastitis caused illness (Barber, 1914). Later, Dack et al. (1930) demonstrated that staphylococcal food poisonings were caused by a toxin and not by the microorganism itself. Despite the great advances in food safety, Staphylococcus aureus is still a major human pathogen capable of causing food poisoning outbreaks worldwide.

1.2. Phylogeny

Staphylococcus aureus is a member of the monophyletic genus Staphylococcus, which comprises Gram-positive bacteria of low DNA G + C content (32-36%) belonging to the family Staphylococcaceae, order Bacillales, class Bacilli, phylum Firmicutes (Schleifer and Bell, 2009). As shown in Figure 1, this genus is closely related to bacilli and other Gram-positive bacteria with low DNA G + C content such as enterococci, streptococci, lactobacilli and listeria (Götz et al., 2006). Although S. aureus is the responsible to cause most of foodborne intoxications reported, other species and subspecies of the genus Staphylococcus have also shown a real and potential risk for the human health as they are able to produce coagulase, nuclease and/or enterotoxins (Figure 2).

General Introduction

Figure 1. Maximum likelihood phylogram of the genus Staphylococcus into the order Bacillales based on 16S rRNA analysis performed by Götz et al. (2006). The bar length indicates 5% estimated sequence divergence.

Figure 2. Inference of the staphylococcal phylogeny using Bayesian estimation of species trees (BEST) methodology on 16S rRNA and dnaJ gene fragments (modified from Lamers et al., 2012). The bar length indicates a sequence divergence of 0.1 substitutions per site. Staphylococcal species and subspecies of real and potential risk in foodborne intoxications are marked with an asterisk (data from Jay et al., 2005).

General Introduction

1.3. General characteristics

Staphylococcus aureus is a Gram-positive facultative anaerobe of 0.5-1.0 μm in diameter, which grows individually, in pairs, short chains or grape-like clusters (Figure 3A) (Schleifer and Bell, 2009). Macroscopically, S. aureus form colonies in agar medium of 6-8 mm in diameter, rounded and smooth, glistening, translucent, with entire margins, with pigmentations varying from grey to golden yellow to orange (Figure 3B).

Figure 3A-B. Morphology of S. aureus under scan electron microscopy (A) (from the Centers for Disease Control and Prevention's Public Health Image Library, ID#:6486) and S. aureus colonies on tryptic soy agar (B).

S. aureus is a ubiquitous bacteria detected in numerous environmental sources (e.g. soil, air, water, dust, sand, organic wastes, paper, clothing, furniture, blankets, carpets, linens, utensils, vegetal surfaces), though warn-blooded animals are the main reservoirs. As shown in Figure 4, S. aureus form part of the normal human microbiome associated with an asymptomatic commensal colonization of skin, throat, nose, hair, nails, axillae and perineum (Wertheim et al., 2005).

S. aureus can grow at a wide range of temperatures (6-48ºC, optimal growth at 35-

41ºC), pHs (4-10, optimum at 6-7), water activities (aw = 0.83 ≥ 0.99, optimum at 0.99) and salt content (0-20%, optimum at 0%), and it is also tolerant to desiccation (Hennekinne et al., 2012; Schelin et al., 2011). Nutrients required for bacterial growth are achieved by the secretion of diverse enzymes (e.g. thermonucleases, proteases, lipases, hyaluronidases, catalases, collagenases) and cytolytic exotoxins such as haemolysins (DeLeo et al., 2009; Dinges et al., 2000). Thus, S. aureus can metabolize aerobically fructose, galactose, glucose, glycerol, lactose, maltose, mannitol, mannose, ribose, trehalose, turanose and sucrose generating acid, whereas anaerobically produces D- and L-lactate from glucose (Schleifer and Bell, 2009).

General Introduction

Figure 4. Colonization sites of S. aureus in the human body (Wertheim et al., 2005).

1.4. Pathogenicity

S. aureus is the most human-pathogenic species in the genus Staphylococcus. It acts as an opportunistic bacterial pathogen in human carriers, who might also serve as spread vectors (Chambers and DeLeo, 2009). S. aureus can infect individuals through skin injuries (e.g. acne, styes, burns, wounds) and foreign bodies (e.g. sutures, intravenous lines, prosthetic devices), or associated to the infection with other pathogenic agents (e.g. viruses), chronic underlying diseases (e.g. cancer, alcoholism) or heart diseases (Lowy, 1998; Moreillon and Que, 2004; Van-Belkum and Melles, 2005). As shown in Figure 5, S. aureus can cause from relatively minor skin infections to life-threatening systemic illnesses (Wertheim et al., 2005). Although staphylococcal infections do not necessary impedes the infected person from working, it implies a serious risk of cross- contamination in the case of food-handlers.

General Introduction

Figure 5. Major infections caused by S. aureus in humans (Wertheim et al., 2005).

Besides in humans, S. aureus is also capable of producing diverse infections in animals (e.g. mastitis, synovitis, arthritis, endometritis, furuncles, suppurative dermatitis, pyaemia, and septicaemia), which may have considerable economic losses in the food industry (Schleifer and Bell, 2009).

1.5. Pathogenic determinants

S. aureus carries a wealth of extracellular and cell-wall-associated virulence factors (specified in Figure 6), which are encoded in phages, plasmids, pathogenicity islands and in the staphylococcus cassette chromosome. They are coordinately expressed during the different stages of infection, comprising the colonization and invasion of host

General Introduction tissues, protection against host defences, bacterial proliferation and spread (Bien et al., 2011). The production of pathogenic determinants during infection is mediated by global regulators such as the accessory gene regulator (agr), the staphylococcal accessory regulator (sarA) and the sigma factor B (σB), which activity is influenced by different environmental signals (e.g. changes in nutrient availability, temperature, pH, osmolarity, oxygen tension) (Bien et al., 2011; Cheung et al., 2004).

Figure 6. Major pathogenic determinants of S. aureus.

The colonization and invasion of host tissues by S. aureus is mainly mediated by the surface-associated adhesins MSCRAMMs (microbial surface component recognizing adhesive matrix molecules), including fibronectin-binding proteins (Fnbp), collagen- binding proteins (e.g. CnA) and fibrinogen-binding proteins (e.g. Clf) (Bien et al., 2011; Foster and Höök, 1998; Garzoni and Kelley, 2009). S. aureus also produces different exotoxins and enzymes that enhance the invasion of host tissues, such as exfoliative toxins (e.g. ETA, ETB), proteases, lipases, hyaluronidases and thermonucleases (TNase) (Sandel and McKillip, 2004; Bukowski et al., 2010). Thus, S. aureus may internalize into host cells and persist indefinitely, or even multiply and further disseminate or produce a rapid cellular apoptosis or necrosis (Garzoni and Kelley, 2009).

General Introduction

In addition, S. aureus shows different evasive responses that protects bacteria from the immune system, such as the synthesis of surface-associated staphylococcal protein A (SpA), extracellular capsular polysaccharides, extracellular adherence proteins (Eap), chemotaxis inhibitory proteins (CHIPS), formyl peptide receptor-like-1 inhibitory proteins (FLIPr), extracellular complement-binding proteins (Ecb), staphylococcal complement inhibitors (SCIN), extracellular fibrinogen-binding proteins (Efb), staphylokinases (SAK), haemolysins, leukocidins, phenol-soluble modulins (PSMs), catalases and coagulases (Bokarewa et al., 2006; DeLeo et al., 2009; Dinges et al., 2000; Haas et al., 2004; Haggar et al., 2004; Jongerius et al., 2007, 2010; O´Riordan and Lee, 2004; Prat et al., 2006; Sandel and McKillip, 2004).

Most S. aureus strains also generate pyrogenic toxin superantigens (PTSAgs), which cause the deregulation of the immune response. Apart from the TSST-1 responsible of most toxic shock syndrome cases (Dinges et al., 2000; Lappin and Ferguson, 2009), most S. aureus are able to produce staphylococcal enterotoxins (SEs) that cause most food poisonings in humans (Hennekinne et al., 2012; Le-Loir et al., 2003). SEs can be produced at a wide range of temperatures (10-46ºC, optimal production at 34-45ºC), pH

(4.0-9.6, optimum at 7-8), water activity (aw = 0.85 ≥ 0.99, optimum at aw ≥ 0.98) and salt content (< 12%) without affecting the sensory characteristics of the contaminated food (Hennekinne et al., 2012; Schelin et al., 2011). Moreover, the heat-stability and the resistance to proteolytic degradation allow SEs to retain their emetic activity after food consumption (Bergdoll and Wong, 2006; Jablonski and Bohach, 2001; Omoe et al., 2005). Food poisoning symptoms appear approximately 1-6 h after ingestion of SEs, depending on the amount of toxin consumed and the sensitivity of the individuals involved (Pinchuk et al., 2010).

General Introduction

2. Biofilm formation by Staphylococcus aureus

2.1. Definition of biofilm

Although a number of definitions have been proposed over the years with the increased understanding of biofilms (Characklis and Marshall, 1990; Costerton et al., 1978, 1987, 1995; Costerton and Lappin-Scott, 1995; Davies and Geesey, 1995; Marshall, 1976; Prigent-Combaret and Lejeune, 1999), the next definition is the most widely accepted:

“A biofilm is a microbially derived sessile community characterized by cells that are irreversibly attached to a substratum or interface or to each other, embedded in a matrix of extracellular polymeric substances that they have produced, and with an altered phenotype with respect to growth rate and gene transcription”

Donlan and Costerton (2002)

2.2. Significance of biofilm formation

Biofilms are the prevailing microbial lifestyle in natural habitats due to their protector ability during the growth, allowing the survival under extreme environmental conditions of temperature (e.g. thermal waters, glaciers), acidity (e.g. sulphuric pools and geysers) and humidity (e.g. deserts, rainforests) (Dufour et al., 2012). Microorganisms also grow predominantly in form of biofilms on practically any kind of industrial surface, causing food and water contamination, metal surface corrosion and the obstruction of equipments (Beech et al., 2005; Srey et al., 2013). Particularly in the food industry, biofilm formation may contribute to the persistence of spoilage and pathogenic bacteria in food-processing environments, consequently increasing cross- contamination possibilities, which may involve a serious risk for the consumer health as well as subsequent economic losses due to recalls of contaminated food products. Concerning the medical ambit, the US National Institute of Health estimates that biofilms are involved in up to 75% of microbial infections in humans (Table 1 compiled some examples). However, most laboratory studies and many standard methods are still performed using only planktonic cells, though this state is considered a transitory phase in which aims to translocate bacteria to other surfaces.

General Introduction

Table 1. Diversity of human infections involving biofilms (based on Fux et al., 2005) Infection or disease Common bacterial species involved Dental caries Acidogenic Gram-positive cocci Periodontitis Gram-negative anaerobic oral bacteria Otitis media Non-typeable Haemophilus influenzae Chronic tonsillitis Various species Cystic fibrosis pneumonia P. aeruginosa, Burkholderia cepacia Endocarditis Viridans group streptococci, staphylococci Necrotizing fasciitis Group A streptococci Musculoskeletal infections Gram-positive cocci Osteomyelitis Various species Biliary tract infection Enteric bacteria Infectious kidney stones Gram-negative rods Bacterial prostatitis E. coli and other Gram-negative bacteria Infections related to medical devices Contact lens P. aeruginosa, Gram-positive cocci Sutures Staphylococci Ventilation-associated pneumonia Gram-negative rods Mechanical heart valves Staphylococci Vascular grafts Gram-positive cocci Arteriovenous shunts Staphylococci Endovascular catheter infections Staphylococci Cerebral spinal fluid-shunts Staphylococci Peritoneal dialysis (CAPD) peritonitis Various species Urinary catheter infections E. coli, Gram-negative rods IUDs Actinomyces israelii and others Penile prostheses Staphylococci Orthopaedic prosthesis Staphylococci

2.3. The process of biofilm development

The development of bacterial biofilms is a dynamic process affected by the substratum (i.e. texture or roughness, hydrophobicity, surface chemistry, charge, pre- conditioning film), the medium (i.e. nutrient levels, ionic strength, temperature, pH, flow rate, presence of antimicrobial agents) and intrinsic properties of cells (i.e. cell surface hydrophobicity, extracellular appendages, extracellular polymeric substances (EPS), signalling molecules) (Donlan, 2002; Renner and Weibel, 2011).

General Introduction

As shown in Figure 7, the process of biofilm formation comprises 1) a reversible attachment of bacterial cells by weak interactions (i.e., Van der Waals forces) to a pre- conditioning film formed on the abiotic or biotic surface (Bos et al., 1999; Donlan, 2002); 2) an irreversible adsorption to the surface by hydrophilic/hydrophobic interactions, electrostatic forces and Lewis acid-base interactions mediated by several attachment structures (e.g., flagella, fimbriae, lipopolysaccharides, adhesive proteins) (Bos et al., 1999; Donlan, 2002); 3) the proliferation of adsorbed cells and production of a self-produced EPS matrix mainly composed by polysaccharides, proteins and extracellular DNA (Branda et al., 2005; Flemming et al., 2007); 4) the formation of a mature biofilm, whose structure can be flat or mushroom-shaped and that contains water channels that effectively distribute nutrients and signalling molecules within the biofilm (Dufour et al., 2012; Hall-Stoodley et al., 2004); 5) the detachment of biofilm cells individually or in clumps as a response to external or internal factors and, finally, 6) the spread and colonization of other niches (Srey et al., 2013).

Figure 7. Processes involved in the development of a bacterial biofilm.

2.4. Ecological advantages of biofilm formation

2.4.1. Cell-cell communication

Biofilms provide microorganisms a great adaptability to the different environmental conditions (Davey and O´Toole, 2000). The establishment of bacteria on surfaces generates a higher degree of stability in the cell growth and enables beneficial cell-cell interactions, such as quorum sensing and genetic exchange (Daniels et al., 2004; Davey and O´Toole, 2000; Elias and Banin, 2012; Hall-Stoodley et al., 2004; Watnick and

General Introduction

Kolter, 2000). In the case of quorum sensing, low-molecular mass signalling molecules called auto-inducers (AI) regulate gene expression, metabolic cooperativity and competition, physical contact and bacteriocin production, providing thus a mechanism for self-organization and regulation of biofilm cells (Daniels et al., 2004; Donlan, 2002; Elias and Banin, 2012; Parsek and Greenberg, 2005; Van-Houdt and Michiels, 2010). The quorum sensing effects depend on the concentration of AI, which increases in a cell-density-dependent manner (Parsek and Greenberg, 2005). Meanwhile, the transmission of mobile genetic elements between nearby biofilm cells allows the acquisition of new antimicrobial resistance, virulence factors and environmental survival capabilities (Madsen et al., 2012).

2.4.2. Increased resistance to external stimulus

Bacterial cells in biofilms are protected against a wide range of environmental stresses, leading to a higher survival and persistence when compared with planktonic state, which is particularly problematic in clinical and food environments. Several physiological characteristics and mechanisms are responsible of this increase of resistance:

Presence of extracellular matrix. It acts as a barrier that slows down the infiltration, neutralizes, binds and effectively diffuses to sub-lethal concentrations antimicrobial and antibiotic agents (e.g. chlorine species, oxacillin, vancomycin) before they can reach cell targets (Bridier et al., 2011a; Singh et al., 2010), and also reduce other adverse external effects such as UV light, toxic metals, acidity, desiccation, salinity and host defences (Hall-Stoodley et al., 2004).

Specific physiology of biofilm cells. Changes in the expression of specific genes associated with sessile growth and physiological differentiation between biofilm cells actively growing located in the outer surface and dormant or even in the oxygen- and nutrient-deprived inner interfaces (Bridier et al., 2011a) represents the basis of the biofilm-specific adaptive response and it can explain their reduced susceptibility to antimicrobials (Bridier et al., 2011a; Gilbert et al., 2002; Høiby et al., 2010). As an example, persistent cells are able to produce particular cellular toxins that block cellular processes like translation, thus rendering protection against biocides that act only against active cells (Lewis, 2010).

General Introduction

Expression of specific mechanisms of defence. In between others, biofilm cells can respond to antimicrobials by the activation of chromosomal β-lactamases and efflux pumps (e.g. QAC efflux system of S. aureus), the induction of mutations in antimicrobial target molecules or, indirectly, by the release of extracellular DNA that promotes the synthesis of biofilm matrix (Anderson and O’Toole, 2008; Bridier et al., 2011a; Høiby et al., 2010; Kaplan et al., 2012).

2.4.3. Increased dispersal capacity

The dispersal of biofilm cells in clumps may provide a sufficient number of cells for an infective dose that is not typically found in bulk fluid, enabling an enhanced transmission and infection of bacterial pathogens (Hall-Stoodley and Stoodley, 2005). Detachment can be promoted by environmental changes such as nutrient starvation or by internal biofilm processes such as endogenous enzymatic degradation, or the release of EPS or surface-binding proteins (Srey et al., 2013).

2.5. Staphylococcus aureus biofilms: development, composition and regulation

S. aureus biofilms have been detected on diverse biotic and abiotic surfaces, including human tissues, indwelling medical devices (e.g., implanted catheters, artificial heart valves, bone and joint prostheses), food products and food-processing facilities (Devita et al., 2007; Herrera et al., 2006; Sattar et al., 2001; Simon and Sanjeev, 2007; Trampuz and Widmer, 2006).

The initial attachment of S. aureus to abiotic surfaces is mostly mediated by hydrophobic or electrostatic interactions, but bacterial surface molecules such as autolysins or teichoic acids can be also participate in this process (Gross et al., 2001; Houston et al., 2011). In contrast, S. aureus adhere to biotic surfaces through much more specific interactions governed by the surface-anchored proteins MSCRAMMs (Foster and Höök, 1998).

General Introduction

Once adhered, S. aureus develop a multi-layered biofilm embedded within a matrix composed by secreted polysaccharides (e.g. PIA-PNAG), lipids and proteins, extracellular DNA originating from lysed cells, and some molecules trapped from the environment (Branda et al., 2005; Cramton et al., 1999; Fitzpatrick et al., 2005; Flemming et al., 2007; Høiby et al., 2010; Maira-Litrán et al., 2002), though the composition can vary between strains and at different environmental conditions.

The extracellular polysaccharide intercellular adhesins (PIA) participates in the quorum-sensing contacts that coordinate the biofilm formation process, as well as in the cell detachment induced by changes in environmental conditions (Fitzpatrick et al., 2005). PIA is formed by poly-β(1,6)-N-acetyl-d-glucosamine glycans (PNAG), which are synthetized by the products of the chromosomal intercellular adhesion (ica) operon carried by most S. aureus strains (Cramton et al., 1999; Fitzpatrick et al., 2005; Maira- Litrán et al., 2002). IcaA and IcaD form a transmembrane protein N-acetyl-glucosamine transferase that produce N-acetyl-glucosamine oligomers, which are elongated and translocated to the cell surface by the membrane protein IcaC (Gerke et al., 1998), where the surface-attached protein IcaB finally de-acetylates the polymers, introducing positive charges that enhance the adhesion of PIA to the bacterial surface (Vuong et al., 2004). The ica operon is fundamentally repressed by the icaR gene products (Jefferson et al., 2003), which are regulated by the stress-induced sigma factor B (σB), the staphylococcus accessory regulator A (SarA), the LuxS/AI-2 quorum-sensing system and, indirectly, by the rbf gene (Cerca et al., 2008; Cue et al., 2009; Yu et al., 2012). The role of the ica operon in the biofilm formation of S. aureus is complex and likely to be both strain- and environment-dependent (O’Gara, 2007). In fact, the expression of the ica operon is influenced by different environmental factors such as anaerobic conditions, glucose, ethanol, osmolarity, temperature and antibiotics such as tetracycline (Cramton et al., 2001; Fitzpatrick et al., 2005).

The extracellular DNA, meanwhile, has been found to play a structure-stabilizing role in the extracellular matrix of S. aureus biofilms. Thus, the negative charge of extracellular DNA seems to aid in interacting with other surface structures and lead to the formation of an adherent staphylococcal biofilm (Izano et al., 2008). The extracellular DNA is released from lysed adhered cells since the early stages of biofilm

General Introduction formation under the control of cid/lgr operons (Mann et al., 2009; Rice et al., 2007). Furthermore, balance of the extracellular DNA content by the degrading activity of secreted thermonucleases (e.g. nuc1, nuc2) seems to be important in the maintenance of mature biofilms (Mann et al., 2009).

The presence of extracellular carbohydrate-binding proteins and surface adhesins also contribute to the formation and stabilization of the polysaccharide matrix as well as to enhance the intercellular adhesion. Several biofilm adhesive proteins are implicated in biofilm accumulation by S. aureus, including the multifactorial virulence factor SpA (Merino et al., 2009), FnbpA and FnbpB (O’Neill et al., 2008), the biofilm-associated protein (Bap) expressed by bovine strains of S. aureus (Lasa and Penadés, 2006) and the surface proteins SasG (Corrigan et al., 2007; Geoghegan et al., 2010) and SasC (Schroeder et al., 2009). The expansion and dispersal of S. aureus biofilms is also controlled by secreted proteins, particularly the phenol-soluble modulins (PSMs), which originate the characteristic channels of mature biofilms (Periasamy et al., 2012). The proteases aureolysins seem to be also implicated in these processes, though there is no clear evidence currently (Otto, 2013). The production of both effectors is strictly regulated by the quorum-sensing system Agr (accessory gene regulator), which is stimulated by auto-inducing peptides (Wang et al., 2007).

3. Incidence of Staphylococcus aureus in the food industry

3.1. Outbreaks

A selection of the most notable foodborne outbreaks worldwide caused by the ingestion of S. aureus enterotoxins is shown in Table 2.

General Introduction

(2007)

1985) 1986)

1987)

1995)

1975) ( (

(

1993)

1988)

972)

(

1996)

(

1981) 1989)

( (

Reference

1976) 1983)

1992)

(

Buyser et al. et Buyser

Johnson et al. (1990) al. et Johnson (1968) CDC al. et Morris ( al. et Eisenberg CDC al. et Todd ( CDC al. et Waterman De al. et Bone al. et Woolaway ( al. et Evenson (2007) al. et Kérouanton al. et Levine al. et Thaikruea etal. Richards al. et Kérouanton FDA (2007) al. et Kérouanton (2007) al. et Kérouanton

SEE

SED SEA SEA SEA SEA SEA SEA SEA SEA

SEA, SEC SEA, SEC SEA,

SEA, SED SEA,

Unspecified Unspecified Unspecified Unspecified Unspecified Unspecified Unspecified

SE involved SE

80 62 20 27 50 70 32 87 47

200 100 197 121 215 860 102 485 100

1300 1364

Cases

enterotoxins

S. aureus S.

sandwich, stuffed chicken stuffed sandwich,

Incriminated food Incriminated

Raw milk cheese milk Raw salad Chicken sandwiches ham rolls, Sausages Ham éclairs Chocolate curd Cheese Ham/cheese pastry cream Dessert cheese eve Farm milkcheese Sheep’s lasagne Dried milk Chocolate Spaghettis mushrooms Canned Éclairs corn and beans Ham, salads rice and Potato salad Chicken cheese milk Raw cheese milk Raw

USA

Denmark

USA USA USA USA USA USA USA

France France France France France

Canada

Scotland

Thailand

Localization

Flight Brazil Flight

Caribbean cruise ship cruise Caribbean

Flight Japan Flight

France, UK, Italy, Luxembourg Italy, UK, France,

Excerpt of reported foodborne intoxications caused by by caused intoxications foodborne reported of Excerpt

1958 1968 1971 1975 1976 1980 1982 1983 1983 1984 1985 1985 1988 1989 1990 1990 1990 1992 1997 1997

Year Table 2. Table

General Introduction

Reference

al. (2003) al.

uanton et al. (2007) al. et uanton

James et al. (2008) al. et James

Carmo et al. (2002) al. et Carmo (2007) al. et Colombari (2002) al. et Carmo (2007) al. et Kérouanton et Asao (2005) al. et Ikeda (2007) al. et Kérouanton (2007) al. et Kérouanton Kéro (2007) al. et Kérouanton (2007) etal. Nema (2009) al. et Hennekinne Fitz (2009) al. et Schmid (2009) al. et Kitamoto (2010) al. Ostynet (2013) al. et Solano

SEE

SEC

SEA SEA SEA SED SEA

SEB, SED SEB,

SEA, SEH SEA, SED SEA, SED SEA, SED SEA, SED SEA,

SE involved SE

SEA, SEB, SEC SEB, SEA, SEC SEB, SEA,

SEA, SEB, SED SEB, SEA,

enterotoxins

100

50 21 85 17 45 17 15 75 23 42

180 160 166

4000

> >

Cases

13420

S. aureus S.

Incriminated food Incriminated

hard cheese hard

sauce

peas

fat milkpowder fat

Chicken Chicken pancake, rice, beans, chick mashed tomato sauce and Vegetable salad with mayonnaise, broiled chicken, tomato in pasta cheese white milk Raw salad Mixed Low Pancakes Cream semi milk Raw cheese milk sheep´s Raw balls potato Fried dessert) (Chinese pearls nut Coco Hamburger milk vanilla milk, cacao Milk, Crepes cheese milk Raw Macaroni

Excerpt of reported foodborne intoxications caused by by caused intoxications foodborne reported of Excerpt

India

Japan Japan Spain

Brazil Brazil Brazil

France France France France France France France

Austria

Belgium

Localization

1998 1998 1999 2000 2000 2001 2001 2001 2002 2005 2006 2007 2007 2009 2009 2011

Year Table 2 (continuation). (continuation). 2 Table

General Introduction

The European Food Safety Authority (EFSA) informed that staphylococcal toxins comprise 4.6% of food poisoning outbreaks reported since 2005 (Table 3), being the fourth main causative agent after Salmonella spp. (42.3%), foodborne viruses (12.5%) and Campylobacter spp. (8.1%) (EFSA, 2006, 2007, 2009a, 2010, 2011, 2012). Moreover, the hospitalization rate average due to staphylococcal intoxications (14.1%) was higher than that of foodborne viruses (5.5%) and Campylobacter spp. (7.1%), causing also a higher proportion of deaths (7% out of total deaths against 2.2% for foodborne viruses and 1.1% for Campylobacter spp.). Particularly in Spain, staphylococcal toxins had higher impact in foodborne outbreaks (5.9%) than in the EU between 2008 and 2010 (previous data were not included in EFSA reports), but a lower hospitalization rate average (3.4%) and none death was reported.

Similar results were reported in Japan, where staphylococcal intoxications involve 4.3% of foodborne outbreaks with identified causative agent reported in 2009, producing 690 cases but none deaths (MHLW, 2011). S. aureus enterotoxins were associated with approximately 7.8% of foodborne outbreaks reported in China between 1994 and 2005, causing 3055 cases but none deaths (Wang et al., 2007). In contrast, the Food and Drug Administration (FDA) estimated that staphylococcal toxins are responsible for causing only 0.5% of food poisoning outbreaks reported in the USA, with a hospitalization rate of 0.4% and 6 deaths each year (FDA, 2012).

However, it is known that the number of reported cases is underestimated by the healthcare services due to misdiagnosis of the illness (symptomatically similar than other types of food poisoning, e.g., Bacillus cereus emetic toxin), the self-limiting nature of the illness and the rapid recovery of people intoxicated by staphylococcal toxins (within 24 to 48 h after onset). In fact, only 40% out of the foodborne outbreaks caused by staphylococcal toxins in the EU were verified. Furthermore, the notification of staphylococcal intoxications is not mandatory in a number of member states of the EU. Therefore, the actual incidence of staphylococcal food poisonings is assumed to be much higher than reported (Lawrynowicz-Paciorek et al., 2007; Smyth et al., 2004).

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1 2 4 2 0 3 0 0 0 0

no no no 12

Deaths

(EFSA, (EFSA,

no no no 10 13 23

365 378 272 362 303 357

2037

Hospitalised

no no no

132 477 166 775

1692 2373 2728 2749 2671 2796

Cases

15009

no no no no no

90.70 27.84 46.88 30.03 36.67 13.87 37.50 40.61 40.35

% verified %

Outbreaks caused by staphylococcal toxins staphylococcal by caused Outbreaks

no no no 32 30 24 86

164 240 258 291 293 274

1520

, , including those caused by staphylococcal toxins

7 1 3 1 2 7

24 50 19 32 46 25 21

196

Deaths

23 no

362 181 297 378

5330 5525 3291 6230 4356 4695 1241

29427

Hospitalised

between between 2005 and 2010

7682 3491 6705 2372 5584 4025

Cases

47251 53568 39727 45622 48964 43473 29859

278605

Totaloutbreaks

92.00 83.70 31.12 41.03 16.69 38.84 17.60 33.89 13.26 40.66 42. 59.07

100.00 100.00

% verified %

460 351 619 551 416 482

5311 5710 5733 5332 5550 5262 2879

32898

EFSA reports EFSA

EU EU EU EU EU EU EU

Spain Spain Spain Spain Spain Spain Spain

included

Geographic area Geographic

Foodborne Foodborne outbreaks reported in the European Union

2005 2006 2007 2008 2009 2010

Year Total

no, data not not data no,

Table Table 2012) 2011, 2010, 2009a, 2007, 2006,

General Introduction

Diseases caused by food intoxications additionally generate considerable economic costs and a wide impact in public health worldwide. For example, the Centres for Disease Control and Prevention (CDC) estimate that foodborne diseases generate costs of over 1.4 trillion of dollars in the USA each year (Roberts, 2007). Moreover, the recall of contaminated products, factory closing, extraordinary cleanings and compensation to infected people can generate additional expenses to the food industry.

3.2. Foods implicated in staphylococcal poisonings

Different food vehicles have been incriminated in staphylococcal intoxications. As shown in Figure 8, the largest proportion of verified outbreaks with known food vehicle caused by staphylococcal toxins in the EU since 2006 (no data included in the EFSA report of 2005) was attributed to meat products (32.9%), milk and dairy products (23.5%) and mixed or buffet meals (10.9%), followed by fishery products (3.8%).

Figure 8. Identified food vehicles incriminated in verified foodborne outbreaks (n = 340) caused by staphylococcal toxins in the European Union between 2006 and 2010 (data from EFSA, 2007, 2009a, 2010, 2011, 2012).

However, foods incriminated in staphylococcal intoxications varying widely among countries due to differences in the food consumption and habits (Bhatia and Zahoor, 2007; Le-Loir et al., 2003). For instance, in France are frequently reported staphylococcal poisonings associated with the consumption of milk and dairy products (Cretenet et al., 2011), whereas meat products are mostly involved in outbreaks reported

General Introduction in Anglo-Saxon countries (Gormley et al., 2011), and cereals (above all rice) and fishery products in Asiatic countries such as Japan (MHLW, 2011). In Spain, staphylococcal intoxications possibly have a higher incidence in fishery products than the European mean, as the second largest consumer in the European Union (FAO, 2012). However, from our knowledge, only two studies have evaluated the presence of S. aureus in fishery products in the last decade, particularly in vacuum-packed cold‐smoked salmon (González-Rodríguez et al., 2002) and fresh marine fish (Herrera et al., 2006).

3.3. Food contamination by Staphylococcus aureus

Human handlers are considered the major source of transference of S. aureus to food and food-contact surfaces during the preparation and processing of food products (Devita et al., 2007; Sattar et al., 2001; Simon and Sanjeev, 2007), as S. aureus form part of the normal human microbiome (see Figure 4). Nevertheless, foods of animal origin (e.g. milk from dairy animals with mastitis) (Kérouanton et al., 2007; Morandi et al., 2010) as well as endemic strains present in the food-processing environment (Le- Loir et al., 2003) can be also a potential source of primary contamination.

S. aureus is a poor competitor in complex microbial populations, being often inhibited or displaced by food-spoilage microorganisms of faster growing such as Acinetobacter, Aeromonas, Bacillus, Pseudomonas, the Enterobacteriaceae or the Lactobacillaceae, among others. Thus, the greatest risk of staphylococcal food poisoning is often associated with food contaminated with S. aureus after the normal microflora has been destroyed (e.g. cooked products) or inhibited (e.g. frozen/salted foods) (Bore et al., 2007).

Food-related environmental conditions such as the physicochemical characteristics of food and food-contact surfaces, as well as inadequate temperatures and hygiene practices during processing, storage or distribution of products can favour the growth of S. aureus and stimulate the production of toxins (Hennekinne et al., 2012). Finally, novel trends in food production such as minimal processing, mass production and globalization, among others, have additionally introduced new factors and conditions that can enhance the staphylococcal proliferation on food environments (Abee and Wouters, 1999; Cebrián et al., 2007; Rendueles et al., 2011).

General Introduction

4. Control of Staphylococcus aureus in the food industry

4.1. Hazard Analysis and Critical Control Points (HACCP)

HACCP is an effective management system that assures food safety from growing to consumption, by anticipating and preventing health hazards before they occur. Prerequisite programs provide the basic environmental and operating conditions needed for the production of safe, wholesome food. It combines the Good Manufacturing Practice (GMPs), Good Hygienic Practices (GHPs) and Good Agricultural Practices (GAPs) to determine the strategy ensuring hazard control. Some of these basic conditions are the good quality of raw material, the effective training of employees in food hygiene, and the appropriate design of the factory and equipment to assure a correct cleaning and disinfection of the surfaces. All prerequisite programs should be documented and regularly audited, and are established and managed separately from the HACCP plan.

Once prerequisites are controlled, critical points in the process where contamination can occur should be identified and controlled. In the case of S. aureus, the lack of hygiene in food handlers and food-contact surfaces have been found to be the major factors involved in the contamination of food products (DeVita et al., 2007; Sattar et al., 2001; Simon and Sanjeev, 2007), so their control is essential to provide an appropriate food safety conditions.

4.2. Prevention strategies against biofilm formation

Several approaches were proposed to prevent or limit bacterial attachment in food contact surfaces, including the incorporation of antimicrobial products in the surface materials themselves (Weng et al., 1999; Park et al., 2004; Knetsch and Koole, 2011), the coating of surfaces with antimicrobials (Gottenbos et al., 2002; Knetsch and Koole, 2011; Thouvenin et al., 2003), the modification of the physicochemical properties of surfaces (Chandra et al., 2005; Mauermann et al., 2009; Rosmaninho et al., 2007) or the pre-conditioning of surfaces with surfactants (Chen, 2003; Choi et al., 2011), among others.

In another line of research, different biofilm detectors were developed to monitor the bacterial colonization of surfaces, allowing thus the control of biofilms in the early

General Introduction stages of development when cells are more susceptible to antimicrobials (Pereira et al., 2008; Philip-Chandy et al., 2000). However, even though the efforts to solve the problem, there is currently no known technique that is able to successfully prevent or control the formation of unwanted biofilms without causing adverse side effects (Simões et al., 2010).

4.3. Cleaning procedures

Cleaning procedures aim to remove any food residues and other compounds that may promote bacterial proliferation and biofilm formation (Simões et al., 2010). Cleaning agents are frequently combined with disinfectants to synergistically enhance disinfection efficiency due to the low efficacy of most disinfectants in the presence of organic materials (Forsythe and Hayes, 1998; Simões et al., 2010).

Mechanical cleaning procedures (e.g., water turbulence, brushing and scrubbing) and high temperature treatments have been traditionally used as efficient cleaning methods in the food industry (Chmielewski and Frank, 2006; Gibson et al., 1999; Maukonen et al., 2003), but the former usually are an arduous task while the latter may favour the growth of opportunistic pathogens such as S. aureus.

Food residues can also be removed through the application of surfactants and alkaline products, which decrease surface tension, emulsify fats and denature proteins (Forsythe and Hayes, 1998; Maukonen et al., 2003), or acid cleaners, in the case of food residues with high mineral content (Marriott and Gravani, 2006). However, the use of these cleaning agents usually produce corrosiveness to metal surfaces, harmful effects in workers and a high environmental impact (Marriott and Gravani, 2006).

Enzyme-based detergents and quorum-sensing inhibitors have been proposed as a promising alternative (Høiby et al., 2010; Thallinger et al., 2013), but the complexity to achieve a wide-spectrum enzymatic formulation and the high production costs have reduced their potential application in the food industry (Lequette et al., 2010; Simões et al., 2010).

Dry-ice cleaning was recently proposed as an effective procedure to remove impurities adhered to surfaces by local undercooling (Otto et al., 2011), although it must

General Introduction be still fully automated, and sound levels and physical stresses generated by compression units should be greatly reduced.

4.4. Disinfection treatments

Disinfection aims to kill the remaining surface population left after cleaning and thus prevent the microbial regrowth before production restart (Simões et al., 2010).

Physical treatments such as the application of heated solutions (e.g. hot water) and ultraviolet (UV) radiation have been frequently used in the food industry (Marriott and Gravani, 2006), but the limited effectiveness against spores and biofilms as well as the possibility to form films or scale on equipment has questioned their validity. Pulsed UV light (Gómez-López et al., 2007), ultrasonication (Chemat et al., 2011; Oulahal-Lagsir et al., 2000), ionizing radiations (Byun et al., 2007; Niemira and Solomon, 2005; Niemira, 2008) and low temperature atmospheric pressure plasmas (Ehlbeck et al., 2011) have shown promising results for the eradication of bacteria attached to surfaces, but they require high initial investments and have low consumer acceptance.

Many chemical disinfectants have been also used in the food industry. They generally interact with multiple cellular targets (Figure 9), leading to lethal bactericidal effects. Thus, the rotation and combination of biocides is widely accepted to prevent the emergence of antimicrobial-resistant strains.

The choice of a chemical disinfectant depends on efficacy, safety, toxicity, corrosive effects and ease of removal, among other factors (Marriott and Gravani, 2006). However, the efficacy of chemical disinfectants are often affected by the presence of organic materials, pH, temperature, water hardness, chemical inhibitors, concentration, exposure time and bacterial resistance (Bessems, 1998; Marriott and Gravani, 2006). Advantages and disadvantages of some disinfectants widely used in the food industry are summarized in Table 4.

General Introduction

potentially explosive and and explosive potentially

Low efficacy against yeasts

Disadvantages

(Wirtanen (Wirtanen and Salo, 2003; Marriott and

place place (CIP). Reduced efficacy at high pH and at

aterials. aterials. Highly foaming, unsuitable for cleaning -

esistance development. expensive. development. Relatively esistance

Biostatics. Ineffective against spores. Biostatics.Ineffective unstable, irritating, Toxic, corrosive. Inactivated in the presence material. of pH organic sensitive. Discoloration of products. development. Resistance Biostatic. Not biodegradable. Low penetration in biofilms. Flavour and odour. m Stain plastics in and porous 50°C. above Expensive. temperatures Loss of effectiveness in the material presence and some metals of contained in water. organic May corrode some metals. moulds. expensive. Relatively and Limited effectiveness, which is affected water, by low temperatures and low hard pH. Incompatible with most detergents. Highly foaming, unsuitable for CIP. Residual R antimicrobial film forming.

ood ood industry

- -

use.

non

use. Low

toxic, toxic, non

use, colourless,

ss, ss, flavourless and

oxic oxic and easy

use use and unaffected by hard

Advantages

corrosive to stainless steel and

film film forming without residual

toxic, easy

active agents, non

irritating and easy

corrosive, corrosive, odourle

acting acting oxidizers of broad microbial

acting biocides of broad microbial

acting acting oxidizers of broad microbial

Cheap, Cheap, fast spectrum, non volatile. and skin, on soluble water in harmless Cheap, fast spectrum. Easy Effective water. against planktonic cells and spores, even at low detachment. temperatures. Non Supports activity. microbial Cheap biocide of broad microbial spectrum, corrosive. Sanitizers of corrosive, broad non activity toxicity and stable at a very low pH. Little affected spectrum, materials. organic by non Strong, fast spectrum, relatively non Low foaming, suitable for CIP. Effective bacterial against biofilms temperatures. Non and spores, aluminium. even Stable, at surface irritating, low non colourless. Little affected detachment. microbial Supports by organic materials.

acid,

based compounds based

Alcohols

Iodophors

Advantages Advantages and disadvantages of some disinfectants widely used in the f

Peroxygens

Disinfectant

(e.g., ethanol) (e.g.,

Glutaraldehyde

hydrogen peroxide) hydrogen

compounds (QACs) compounds

(e.g., peracetic peracetic (e.g.,

Quaternary ammonium ammonium Quaternary

(e.g., sodium hypochlorite) (e.g.,

Chlorine

(e.g., benzalkonium chloride) benzalkonium (e.g.,

Table 4. 2006) Gravani,

General Introduction

Figure 9. Cellular targets and effects of disinfectants commonly used in the food industry (based on Denyer and Stewart (1998) and Maillard (2002)).

Interestingly, numerous working-safe, environmentally-friendly and cost-effective disinfection options have been introduced ‒or at least proposed to be introduced‒ in the food industry to be in concordance with present and future regulatory landscapes, highlighting:

Ozone: it is considered a suitable and safety sanitizer for decontaminating food products, food-contact surfaces, equipments and the food-processing environment (Graham et al., 1997; Khadre et al., 2001). Likewise EW, ozone is generated on-site and it poses low environmental impact because it decomposes rapidly without leaving toxic residues, but it is more expensive, unstable and corrosive than EW (O’Donnell et al., 2012). Ozone acts as a powerful and non-selective oxidant and disinfectant against a wide range of microorganisms including food-related bacteria (Restaino et al., 1995). Ozone kills cells by oxidizing polyunsaturated fatty acids and the sulfhydryl groups of certain enzymes, which leads to the disruption or disintegration of the cell envelope and subsequent leakage of cellular contents (Victorin, 1992); or by the destruction and damage of nucleic acids (Guzel-Seydim et

General Introduction

al., 2004). As a consequence, bacteria seems to cannot develop resistance to ozone disinfection (Pascual et al., 2007). However, the stability of ozone is affected by temperature and pH conditions and its efficacy is decreased by the presence of organic matter and depends on the type of microorganism targeted (Khadre et al., 2001; O’Donnell et al., 2012).

Electrolyzed water (EW): this disinfectant is considered safety, environmentally- friendly and non-corrosive to stainless steel surfaces (Huang et al., 2008). Moreover, EW is more cost effective than traditional disinfectants because, once the initial capital investment is made to purchase an EW generator unit, the only operating expenses are water, salts and electricity to run the unit (Walker et al., 2005). The bactericidal activity of EW derives from the combined action of pH, oxidation- reduction potential (ORP) and available chlorine concentration (ACC). Thus, EW damages the bacterial protective barriers, increases membrane permeability leading to the leakage of intracellular DNA, K+ and proteins, and causes an activity decrease on critical enzymatic pathways (Liao et al., 2007; Marriott and Gravani, 2006; Zeng et al., 2010). The efficacy of EW against foodborne pathogens has been widely demonstrated in suspension cultures and foods, including S. aureus (Deza et al., 2005; Fenner et al., 2006; Guentzel et al., 2008; Horiba et al., 1999; Issa-Zacharia et al., 2010; Rahman et al., 2010; Vorobjeva et al., 2004). Nonetheless, a reduced number of studies were performed against bacterial biofilms (Ayebah and Hung, 2005; Huang et al., 2008; Liu et al., 2006; Monnin et al., 2012; Park et al., 2002; Phuvasate and Su, 2010; Venkitanarayanan et al., 1999; Walker et al., 2005), which may question the antimicrobial potential of EW in contaminated food-processing facilities.

Bacteriophages: the application of viruses infecting bacteria along the food chain (phage therapy, biosanitation, biopreservation) is considered as a versatile biofilm control tool, highly active and specific (without adverse effects on the intestinal microbiota and innocuous to mammalian cells), relatively low cost, and genetically amenable (García et al., 2008; Loc-Carrillo and Abedon, 2011). Bacteriophages are often engineered to express specific proteins such as endolysins and other hydrolases that directly degrade or interfere in the biosynthesis of bacterial peptidoglycans (Sutherland et al., 2004; Lu and Collins, 2007; García et al., 2010). Several studies

General Introduction reported the capability of bacteriophages to remove S. aureus biofilms (García et al., 2007; Obeso et al., 2008; Sass and Bierbaum, 2007). However, the limited spectrum of infectivity of each bacteriophage necessitates the identification of causative bacterial pathogens and their susceptibility to phages (Lu and Koeris, 2011). In fact, some bacteria can evolve resistance to phages by blocking phage adsorption, inhibiting the injection of phage genomes, restriction-modification systems and abortive infection systems (Labrie et al., 2010). Moreover, bacteriophages should be genetically well-characterized to avoid the dissemination of undesirable traits (virulence and antibiotic-resistance genes) that could involve safety concerns (Loc- Carrillo and Abedon, 2011). In food systems, different factors such as the concentration and diffusion rate of phages, the physiological status and proportion of cells immersed into the biofilm, as well as environmental conditions (e.g., temperature, pH, presence of inhibitory compounds) can additionally affect the infection of biofilm cells by phages (García et al., 2008).

Essential oils (EOs): they comprise a wide variety of aromatic oily liquids (approximately 3000 EOs are known nowadays) extracted from different plant materials such as flowers, fruits, herbs, leaves, roots and seeds (Bakkali et al., 2008; Burt, 2004). EOs show a versatile composition, which may vary depending on geographical source, climate, harvesting season, soil composition, plant organ, age and vegetative cycle stage, as well as the extraction method used (Angioni et al., 2006; Ennajar et al., 2010; Guan et al., 2007; Hussain et al., 2010; Paolini et al., 2010). EOs acts as antioxidants (Brenes and Roura, 2010) and possess broad-range antibacterial (Oussalah et al., 2007), antiparasitic (George et al., 2009), insecticidal (Nerio et al., 2010), antiviral (Astani et al., 2011), antifungal (Tserennadmid et al., 2011) properties. In fact, several studies have used EOs against S. aureus biofilms (Table 5). EOs cause changes in cell morphology, in the physicochemical properties of membranes, as well as in the transcriptome, proteome and toxin production; disruptions in the membrane potential, intracellular pH and Ca+2 homeostasis, and cellular respiration; alterations in the thiol groups; and the inhibition of essential enzymatic pathways and cell division (Hyldgaard et al., 2012). Nonetheless, the practical application of EOs has been limited due to their strong flavour, poor solubility and partial volatility (Delaquis et al., 2002; Kalemba and Kunicka, 2003).

General Introduction

(2012)

Reference

uave et al. (2008) al. uaveet

Quave et al. (2008) al. et Quave (2012) Ribbeck and Kavanaugh (2012) al. et Millezi (2012) Ribbeck and Kavanaugh (2008) al. et Quave (2008) al. et Quave (2008) al. et Schillaci Q (2008) al. et Quave (2011) al. et Budzynska (2012) al. et Millezi (2011) al. et Budzynska (2011) al. et Aiemsaard (2008) al. et Quave (2007) al. et Nostro Ribbeck and Kavanaugh (2008) al. et Quave (2011) al. et Budzynska (2008) al. et Quave (2008) al. et Quave

2 2

biomass

biofilms

S. aureus S.

against against

biofilms. biofilms.

Effectiveness

1000 mg/L 1000

500 mg/L 500

500

S. aureus S.

= =

= 8 mg/L 8 = mg/L 8 = mg/L 32 = mg/L 128 = mg/L 64 = mg/L 32 = mg/L 16 = mg/L 16 = mg/L 8 =

50 50 50 50 50 50 50 50 50

against against

biofilm biomass. biofilm

IC MBEC CFU/cm log 2.2 in cells biofilm reduce mg/L 1000 of Application mg/L 1700 = MBEC IC IC biofilm of 75% reduces mg/L 1500 of Application IC IC mg/L 1560 = MBEC CFU/cm log 2.2 in cells biofilm reduce mg/L 1000 of Application mg/L 380 = MBEC completely biofilms eradicates h 1 for mg/L 4000 of Application IC MBEC mg/L 1700 = MBEC IC mg/L 780 = MBEC IC IC

applied applied

)

) )

)

) )

)

essential oils oils essential

vulgare

Ballota nigra Ballota

Rubus ulmifolius Rubus

Essential oil Essential

Melissa officinalis Melissa

(Boswellia papyrifera) (Boswellia

Cymbopogon citrates Cymbopogon

Vitis vinifera Vitis

Thymus vulgaris Thymus

Cyclamen hederifolium Cyclamen officinalis Rosmarinus

Cymbopogon nardus Cymbopogon

Lavandula angustifolia Lavandula

Rosa canina Rosa

Origanum

Melaleuca alternifolia Melaleuca

Examples of of Examples

Malva sylvestris Malva

Juglans regia Juglans

Citrus limonia Citrus

Cinnamomum aromaticum Cinnamomum

Syzygium aromaticum Syzygium

, concentration that reduces the 50% of the50% reduces that concentration ,

Table 5. Table ( Blackhorehound ( Cassia ( Citronella ( Clove ( Cyclamen ( rose Dog Frankincense donax) (Arundo cane Giant ( vine Grape ( Lavender ( Lemon ( balm Lemon ( Lemongrass ( Mallow ( Oregano ( thyme Red ( Rosemary ( tree Tea ( Walnut ( blackberry Wild Concentration. Eradication Biofilm Minimum MBEC, IC

General Introduction

Nanoparticles: several engineered nanomaterials have also shown strong antimicrobial properties against S. aureus, including those made of silver (Li et al., 2011), gold (Bresee et al., 2011) and chitosan (Xing et al., 2009) nanoparticles. This high efficacy of nanoparticles is accounted by their high reactivity, unique interactions with biological systems, small size and large surface to volume ratio (Li et al. 2008; Weir et al. 2008; Taylor and Webster 2009). Moreover, these nanoparticles can additionally load other antimicrobials by physical encapsulation, adsorption or chemical conjugation, controlling their release and improving their bactericidal activity against microbial pathogens (Zhang et al., 2010).

Justificación y Objetivos

Teniendo en cuenta que Staphylococcus aureus es uno de los principales agentes etiológicos de intoxicaciones alimentarias en el mundo y que España es uno de los mayores productores y consumidores de productos pesqueros en la Unión Europea, hay dos motivos concretos que justifican la consecución de los objetivos científicos propuestos en esta tesis:

. Que S. aureus es repetidamente detectado en productos pesqueros como consecuencia de la contaminación cruzada de los manipuladores y superficies de contacto con el alimento.

. Que la capacidad de formación de biopelículas le proporciona a S. aureus una alta tolerancia a biocidas, permitiéndole persistir a largo plazo en ambientes alimentarios.

En este contexto, y con el objetivo final de mejorar el control de S. aureus en la industria alimentaria mediante la identificación de los escenarios de mayor riesgo de contaminación y la evaluación de estrategias de desinfección prometedoras frente a este patógeno, los siguientes objetivos específicos fueron realizados:

1. Evaluar la incidencia de S. aureus en diferentes productos pesqueros comercializados. Este objetivo comprende el aislamiento, la caracterización química y genética de los S. aureus encontrados (incluyendo la tipificación y diferenciación de las cepas bacterianas mediante RAPD-PCR) y estudios de la capacidad para producir enterotoxinas y resistir antibióticos.

2. Examinar la prevalencia en plantas de procesado de productos pesqueros de S. aureus con capacidad para producir enterotoxinas, distinguiendo dos objetivos más específicos:

2.1. Determinar su capacidad para formar biopelículas sobre materiales habituales de superficies de contacto con el alimento (acero inoxidable, poliestireno) y bajo diferentes condiciones ambientales (temperatura, presencia de nutrientes, osmolaridad) potencialmente presentes en las plantas de procesado.

Justificación y Objetivos

2.2. Evaluar la resistencia de las biopelículas de S. aureus a desinfectantes aplicados comúnmente en la industria alimentaria (cloruro de benzalconio, ácido peracético, hipoclorito sódico).

3. Estudiar la eficacia y aplicabilidad de dos estrategias de desinfección innovadoras y más respetuosas con el medioambiente para controlar las biopelículas de S. aureus en las plantas de procesado de productos pesqueros:

3.1. Efectividad de la aplicación individual y secuencial de agua electrolizada con cloruro de benzalconio o ácido peracético.

3.2. Eficacia de la aplicación de aceites esenciales frente a biopelículas de S. aureus y de la aplicación combinada del más efectivo con cloruro de benzalconio.

Justification and Objectives

Bearing in mind that Staphylococcus aureus is one of the major bacterial agents causing foodborne diseases in human worldwide and that Spain is one of the largest producers and consumers of fishery products in the European Union, two specific reasons justify the accomplishment of the scientific objectives proposed in this PhD thesis:

. That S. aureus is repeatedly detected in fishery products as a consequence of cross- contamination from food handlers and food contact surfaces.

. That biofilm formation provides S. aureus a high tolerance to biocides allowing a long-term persistence of this pathogen in food-related environments.

In this context, and with the final aim of improving the control of S. aureus in the food industry through the identification of the most risky scenarios of contamination and the evaluation of promising disinfection strategies against this pathogen, the following specific objectives were carried out:

1. Assessment of the incidence of S. aureus in different commercialized fishery products. This aim comprises isolation, chemical and genetic characterization of the S. aureus found (including the typing and differentiation of bacterial strains by RAPD-PCR) and studies of enterotoxin-producing ability and antibiotic resistance.

2. Examine the prevalence in fishery-processing facilities of putative enterotoxigenic S. aureus strains, with two more specific objectives:

2.1. Determine their biofilm-forming ability on common food-contact surface materials (stainless steel, polystyrene) and under different environmental conditions (temperature, nutrient content, osmolarity) potentially present in processing plants.

2.2. Assess the resistance of S. aureus biofilms to disinfectants applied commonly in the food industry (benzalkonium chloride, peracetic acid, sodium hypochlorite).

3. Study the efficacy and applicability of two innovative and more environmentally- friendly disinfection strategies to control S. aureus biofilms on fishery-processing plants:

Justification and Objectives

3.1. Effectiveness of single and sequential application of electrolyzed water with benzalkonium chloride or peracetic acid.

3.2. Effectiveness of the application of essential oils against S. aureus biofilms and combined application of the most effective with benzalkonium chloride.

Chapter 1. Incidence and characterization of Staphylococcus aureus in fishery products marketed in Galicia (Northwest Spain)

Incidence and characterization of S. aureus in fishery products

Abstract A total of 298 fishery products purchased from retail outlets in Galicia (NW Spain) between January 2008 and May 2009 were analysed for the presence of Staphylococcus aureus. S. aureus was detected in a significant proportion of products (~ 25%). Incidence was highest in fresh (43%) and frozen products (30%), but it was high in all other categories: salted fish (27%), smoked fish (26%), ready-to-cook products (25%), non-frozen surimis (20%), fish roes (17%) and other ready-to-eat products (10%). A significant proportion of smoked fish, surimis, fish roes and other ready-to-eat products did not comply with legal limits in force. RAPD-PCR of 125 S. aureus isolated from fishery products was carried out using three primers (AP-7, ERIC-2 and S). Isolates displayed 33 fingerprint patterns. Each pattern was attributed to a single bacterial clone. Cluster analysis based on similarity values between RAPD fingerprints did not find relationship between any RAPD pattern and any product category. Isolates were also tested for se genes and susceptibility to a range of antibiotics (cephalothin, clindamycin, chloramphenicol, erythromycin, gentamicin, oxacillin, penicillin G, tetracycline, vancomycin, methicillin, ciprofloxacin and trimethoprim- sulfamethoxazole). Most isolates (88%) were found to be sea positive. Putative enterotoxigenic strains counts reached high risk levels in 17 products. No relationship was found between the presence of se genes and RAPD patterns. All isolates were resistant to penicillin, chloramphenicol and ciprofloxacin, and most to tetracycline (82.4%), but none was methicillin-resistant.

Chapter 1

A revision of pre-requisite programs leading to improve hygienic practices in handling and processing operations from fishing or farming to retail is recommended to ensure fishery products safety.

Keywords: Staphylococcus aureus; fishery products; retail level; enterotoxin genes; antibiotic resistance.

1.1 Introduction

Although it is necessary to ensure food safety for the health of consumers and industry, Salmonella spp., Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and pathogenic vibrio species have been repeatedly detected in a diverse variety of fishery products (EFSA, 2010; Garrido et al., 2009; Herrera et al., 2006; Kumar et al., 2009; Novotny et al., 2004; Papadopoulou et al., 2006; Yang et al., 2008). Novel trends in food production such as minimal processing, mass production and globalization, among others, have additionally introduced new factors and conditions that can enhance the presence and subsequent growth of bacterial pathogens (Abee and Wouters, 1999; Cebrián et al., 2007; Rendueles et al., 2011). S. aureus is one of the major bacterial agents causing foodborne diseases in humans worldwide (EFSA, 2010; Le-Loir et al., 2003). Staphylococcal food poisoning is usually self-limiting and resolves within 24 to 48 h after onset. Most cases are therefore not reported to healthcare services. As a result, the actual incidence of staphylococcal food poisoning is known to be much higher than reported (Lawrynowicz-Paciorek et al., 2007; Smyth et al., 2004). In addition, the notification of staphylococcal intoxications is not mandatory in a number of member states of the European Union. Staphylococcal food poisonings result from the ingestion of food containing staphylococcal enterotoxins (SEs) preformed by enterotoxigenic strains (Kérouanton et al., 2007; Le- Loir et al., 2003). SEs are resistant to proteolysis and heat-stable, so the presence of SEs involves a significant food safety risk (Omoe et al., 2005). The widespread use of antibiotics has evolved the emergence of multidrug resistant strains, and it makes eradication more difficult and incidence to increase. Multi-resistant S. aureus is rather common in hospital settings and farms (Livermore, 2000; Sakoulas and Moellering, 2008). Community-associated multi-resistant S. aureus is becoming an emerging problem too (Popovich et al., 2007; Ribeiro et al., 2007; Stankovic et al.,

Incidence and characterization of S. aureus in fishery products

2007). Antibiotic-resistant strains of S. aureus have been detected in food animals (Lee, 2003) and food like meat (Normanno et al., 2007; Pesavento et al., 2007), milk and dairy products (Gündoğan et al., 2006; Peles et al., 2007; Pereira et al., 2009) and also fishery products (Beleneva, 2011), and it may be very hazardous for human health. The identification of bacterial clones with enhanced virulence or increased ability to spread is important. Nowadays, PCR-based techniques are commonly used for typing, as they are easy, fast and cost-effective. Among such techniques, random amplified polymorphic DNA (RAPD-PCR) has been considered a very useful tool for rapid differentiation of clones with no prior information of the gene sequence (Van-Belkum et al., 1995; Fueyo et al., 2001; Nema et al., 2007; Nikbakht et al., 2008; Shehata, 2008). Nowadays, Spain is the largest fishery producer, particularly in Galicia (NW Spain), and the second largest consumer in the European Union (Eurostat, 2007). However, no results have been found on the incidence of bacterial pathogens in fishery products made or sold in Galicia, apart from one study on molluscan shellfish farmed in Galician waters (Martínez et al., 2009). The situation is not different for fishery products marketed in other parts of Spain, and only two studies on smoked fish (Garrido et al., 2009; Herrera et al., 2006) and another study on freshwater fish (González-Rodríguez et al., 2002) have been carried out in the last decade. Therefore, the present study was aimed to determine the incidence of S. aureus in fishery products marketed in Galicia and subsequently identify most common clones by RAPD-PCR, as well as cases of increased risk according to the presence of enterotoxin genes and antibiotic-resistance of isolates.

1.2 Materials and Methods

1.2.1 Sampling

A total of 298 fishery products marketed at different retail outlets in Vigo (Galicia, Northwest Spain) were purchased and analysed between January 2008 and April 2009. Fourteen samplings (approximately one each month) were carried out. Products were classified into eight different categories: fresh products, frozen products, salted fish, ready-to-cook products, smoked fish, fish roes, non-frozen surimis and other ready-to- eat products (seafood salads, pâtés and anchovies in oil). Between 24 and 43 products of each category were analysed.

Chapter 1

1.2.2 Isolation and identification of S. aureus

About 50 g of product mixed with 200 mL of peptone water was homogenized in a stomacher masticator (IUL instruments, Spain). Subsequently, homogenates were serially diluted in peptone water (1:50 and 1:500). Aliquots (0.5 mL) of each dilution spread onto Baird Parker agar supplemented with egg yolk tellurite emulsion (Biolife, Italy) (BP-EY). Plates were incubated at 37ºC for 48 h. Typical colonies of S. aureus as well as non-typical colonies (showing no white margin and smaller than 2 mm) were counted. Between 1 and 9 colonies from each product were selected and sub-cultured twice on BP-EY agar for isolation of single colonies (isolates). Isolates cultured in Brain Heart Infusion broth (Biolife) (BHI) for 24 h at 37ºC were subjected to three different biochemical tests: coagulase, DNAse and mannitol fermentation. Coagulase production was tested by adding 100 µL of bacterial culture into 300 µL of reconstituted rabbit plasma with EDTA (Bactident® Coagulase rabbit, Merck, Germany) followed by incubation of tubes at 37ºC. Clotting of plasma was assessed at 1-h intervals during 6 h and after 24 h of incubation. Isolates were streaked onto DNAse agar (Cultimed, Panreac Quimica, Spain) supplemented with D-mannitol and bromothymol blue and plates were incubated at 37ºC for 24 h. The surface of the plates was then flooded with 0.1 N HCl during 15-20 min for DNA precipitation. DNAse activity was observed by the presence of a transparent halo around the colonies on the agar. Mannitol fermentation was observed as a colour change of the pH indicator -from blue to yellow- due to acid production. Colonies found to be coagulase positive, DNAse positive and able to ferment mannitol (suspected S. aureus) were confirmed to be S. aureus by species-specific 23S rDNA PCR. Genomic DNA was extracted from 24 h cultures in BHI using an InstaGene™ Matrix kit (Bio-Rad Laboratories, S.A., Spain) following manufacturer’s instructions. DNA was quantified by assuming that an absorbance value at 260 nm of 0.100 corresponds to 5 µg/mL of DNA. Primers staur4 (5´-ACGGAGTTACAAAGGA CGAC-3´) and staur6 (5´-AGCTCAGCCTTAACGAGTAC -3´) (Straub et al., 1999) were used for each strain. Expected size of amplified PCR products was 1250 bp. Each PCR mixture contained 100 ng DNA, 1x Taq Buffer Advanced, 2.5 U Taq DNA

Incidence and characterization of S. aureus in fishery products polymerase (5 Prime, Germany), 40 nmol of each dNTP (Bioline, UK), 0.25 nmol of forward and reverse primers (Thermo Fisher Scientific, Germany) and sterile Milli-Q water up to a final volume of 50 µL. PCR was performed with a MyCycler™ Thermocycler (Bio-Rad). The conditions proposed by Vautor et al. (2008) were used to target the 23S rDNA gen. An initial step of 5 min at 94ºC was followed by 30 cycles of 30 s at 94ºC, 30 s at 58ºC and 75 s at 72ºC, and a final step at 72ºC for 5 min. PCR products were subjected to electrophoresis on 1.5% agarose gel containing ethidium bromide for 90 min at 75 V and 100 mAmp. Gels were photographed in a Gel Doc XR system (Bio-Rad) using the Quantity One® software (Bio-Rad). A DNA ladder of 50- 2000 bp (Hyperladder II, Bioline) was included as a molecular size marker. Stock cultures of S. aureus isolates were maintained in 50% glycerol (w/w) at ─80ºC. When needed, stock cultures were thawed and subcultured twice in tryptic soy broth (Cultimed) for 24 h at 37ºC prior to being used.

1.2.3 RAPD Genotypic characterization of isolates was performed by RAPD-PCR. DNA was extracted and quantified as previously described from two different cultures of each isolate to check reproducibility of banding profiles. Primers S (5´-TCACGATGCA-3´) (Martín et al., 2004), AP-7 (5´-GTGGATGCGA-3´) and ERIC-2 (5´-AAGTAAGTGAC TGGGGTGAGCG-3´) (Van-Belkum et al., 1995) were individually used in separate reactions with each isolate. Each PCR mixture consisted of 200 ng DNA; 1x Taq Buffer Advanced and 2.5 U Taq DNA polymerase (5 Prime); 40 nmol of each dNTP (Bioline); 0.25 nmol primer (Thermo Fisher Scientific) and sterile Milli-Q water up to a final volume of 50 µL. PCR mixtures with primers AP-7 and ERIC-2 were further supplemented with 1mM MgCl2 (5 Prime). RAPD-PCR was performed with a MyCycler™ Thermocycler (Bio-Rad). PCRs containing primer S consisted of an initial cycle at 95ºC for 5 min, followed by 35 cycles of 95ºC for 1 min, 37ºC for 1 min and 72ºC for 2 min, with a final extension of 5 min at 72ºC. Amplification conditions for AP-7 and ERIC-2 included a denaturation cycle at 94ºC for 4 min, 35 cycles of 94ºC for 1 min, 25ºC for 1 min and 72ºC for 2 min, and a last extension at 72ºC for 7 min. PCR products were subjected to electrophoresis on 1.5% agarose gel containing ethidium bromide as aforementioned. A DNA ladder of 50-2000 bp was included in all gels.

Chapter 1

A second-order polynomial relationship between molecular size and mobility was obtained for each gel (r > 0.99) and used to determine the molecular size of DNA bands. A RAPD pattern was described as different when at least one band difference was found. Reproducibility of patterns was checked twice using independent DNA samples. Variations in band intensity were not considered. Bands too faint to be reproduced were not considered. A binary value (0 or 1) denoting absence or presence of each band was assigned to each pattern. Similarity analysis determining the Dice coefficients (Struelens et al., 1996) was performed by IBM SPSS 19.0. Cluster analysis by UPGMA (Sneath and Sokal, 1973) and dendrograms were performed with StatistiXL 1.8.

1.2.4 Detection of sea-see and seg-sei genes

A slight modification of the method described by Omoe et al. (2002) was followed to analyse the presence of staphylococcal enterotoxin (se) genes. Two multiplex PCR detecting sea-see and seg-sei genes were performed for each strain. DNA was extracted and quantified as previously described. Primer nucleotide sequences and expected sizes of amplicons are shown in Table 1.1. Each PCR mixture contained 100 ng DNA; 10x KCl reaction buffer and 40 nmol of each dNTP (Bioline); 40 pmol SEC-3/SEC-4 primers, 80 pmol SEB-1/SEB-4 primers and 20 pmol for other primers (Thermo Fisher Scientific); 2.5 U Taq DNA polymerase (5 Prime) and sterile Milli-Q water up to a final volume of 50 µL. S. aureus ATCC 12600 was used as a negative control in all PCRs, whereas S. aureus ATCC 13565 was used as a positive control for sea and sed, S. aureus ATCC 19095 for sec, seg, seh and sei, S. aureus ATCC 14458 for seb, and S. aureus ATCC 27664 for see. All these strains were obtained from the Spanish Type Culture Collection (CECT, Valencia, Spain). All PCRs were performed with a MyCycler™ Thermocycler (Bio-Rad) as follows: an initial cycle of 95ºC for 2 min, 55ºC for 1 min and 68ºC for 2 min, followed by 28 cycles of 95ºC for 1 min, 55ºC for 1 min and 68ºC for 2 min, and a final cycle of 95ºC at 1 min, 55ºC for 1 min and 68ºC for 5 min. PCR products were subjected to electrophoresis on 2.5% agarose gel containing ethidium bromide. Run conditions and gel display were as aforementioned. A DNA ladder of 50-2000 bp was also included in all gels.

Incidence and characterization of S. aureus in fishery products

Reference

Omoe et al. (2002) al. et Omoe

Becker et al. (1998) al. et Becker

127 477 271 319 178 287 213 454

Amplicons size (bp) size Amplicons

3´)

→

ACT GAC AAA CG GAC AAA ACT

Nucleotide sequences (5´ sequences Nucleotide

TAG ACA TTT TTG GCG TTC C TTC GCG TTG TTT ACA TAG

CCT TTG GAA ACG GTT AAA ACG AAA GTT ACG GAA TTG CCT CAA CAT TCG

TCT GAA CCT TCC CAT CAA AAA C AAA CAA CAT TCC CCT GAA TCT GC CCT TAA GTG CTA ACT GGT GCA GG CTA AAG TAA ACA TAG AAC AAG CTC CC TAT TAA CAT GAT TCG AAA TCA ACG TAA CTT TAT CTC TAA TGG GTT CTA TC ACA TAA GGG ATA CTT TAT CTA ATG TTA GC CAA AAA TTA AAG ATA TAG CTA TAC CAG TC GAC CCT GTG ACC CTT TAA AAG C TAG GTA CTC AAA ATC ACC AGA T CAC CAA GTA GAG ATG TAT GTC C TGT TTT CGC TTA TAC CTT GAC AAC GGT GTA GGT ATT GAT GGT CAG TAC CTT TGC CTT ATT CAT ATC

3 4 3 4 1 2 1 2

1 4 3 4

3 2

1 2

------

- - - -

- -

SEI SEI

SEE SEE

SEB SEB SEC SEC

SEA SEA SED SED SEG SEG SEH SEH

Primer

Nucleotide sequences of primer pairs and predicted sizes of resulting PCR products. PCR resulting of sizes predicted and pairs of primer sequences Nucleotide

sei

sec see

sea seb sed seg seh

Gene Table 1.1. Table

Chapter 1

1.2.5 Antibiotic susceptibility test Minimal inhibitory concentration (MIC) of twelve antibiotics was determined against S. aureus isolated. The EUCAST guidelines (2003) for broth microdilution and disk diffusion testing were followed. Broth microdilution test was performed with nine antibiotics: cephalothin, oxacillin, penicillin G and vancomycin (Sigma-Aldrich Química, Spain); clindamycin and erythromycin (Acofarma, Spain); and chloramphenicol, gentamicin and tetracycline (Fagron Iberica, Spain). Adjusted cultures of each isolate to 5·105 CFU/mL in Muller

Hinton broth (Cultimed) supplemented with CaCl2·2H2O (25 mg/mL) and MgCl2·6H2O (12.5 mg/mL) were exposed to each antibiotic into a microtiter plate (Falcon®, Becton Dickinson Labware, USA) for 18-20 h (or 24h for oxacillin and vancomycin) at 35ºC.

The OD655nm was measured in an iMark Microplate Reader through Microplate Manager 6® software (Bio-Rad). MIC was defined as the minimum antibiotic concentration at which no growth was observed. Disk diffusion test was performed with methicillin (Oxoid, UK), ciprofloxacin and trimethoprim-sulfamethoxazole (bioMerieux España, Spain). Adjusted cultures were evenly spread on Muller Hinton agar (Cultimed) and commercially prepared antibiotic disks were placed on the agar surface. The length of the inhibition halo was measured after 16-18 h (24 h for methicillin) at 35ºC. S. aureus ATCC 29213 and S. aureus ATCC 43300 (CECT) was used as negative control and positive control, respectively. Antibiotic susceptibility was classified as sensitive, intermediate or resistant on the basis of the breakpoints reported in Table 1.2.

Table 1.2. Antibiotic breakpoints used for interpretation of susceptibility tests. Breakpoints (µg/mL) Antibiotic Sensitive ≤ Resistant > a Cephalothin 8 32 b Chloramphenicol 8 8 b Ciprofloxacin, b Gentamicin 1 1 b Clindamycin 0.250 0.500 b Erythromycin, b Tetracycline 1 2 a Methicillin 8 16 a Oxacillin, b Trimethoprim-Sulfamethoxazole 2 4 b Penicillin G 0.125 0.125 b Vancomycin 2 2 a b Breakpoints from the CLSI (2011); breakpoints from the EUCAST (2011).

Incidence and characterization of S. aureus in fishery products

1.2.6 Detection of blaZ and mecA genes

A slight modification of the methods described by Baddour et al. (2007) and Olsen et al. (2006) was followed for detecting genes encoding penicillin (blaZ) and methicillin resistance (mecA), respectively.

DNA was extracted with DNeasy® kit (Qiagen, Germany) according to the manufacturer. Extraction was tested by using λ HindIII DNA Ladder as a reference (564-23130 bp) (New England BioLabs™, USA).

Primers blaZF487 (5´-TAAGAGATTTGCCTATGCTT-3´) and blaZR373 (5´- TTAAAGTCTTACCGAA AGCAG-3´) for blaZ gen, and mecA1-F (5´-TGGCTAT CGTGTCACAATCG-3´) and mecA2-R (5´-CTGGAACTTGTTGAGCAGAG-3´) for mecA gen, were used. Expected sizes of amplified PCR products were 377 bp for blaZ gen and 309 bp for mecA gen.

PCR mixtures were composed of 20 ng of DNA; 5 nmol of each dNTP (Invitrogen Corporation, USA); 2.5 µL of Dynazym buffer 10x and 1.2 U of Dynazym Hot Start (Bio-Rad); 10 pmol of forward and reverse primer and sterile Milli-Q water up to a final volume of 25 µL.

PCRs were performed with an 80 Gene Amp PCR System 9700 (Applied Biosystems, USA). For mecA gen, PCR consisted of an initial denaturation at 94ºC for 5 min, followed by 30 cycles at 94ºC for 1 min, 54ºC for 1 min and 72ºC for 1 min, and a final cycle at 72ºC for 7 min. Conditions for blaZ gen detection consisted of denaturation at 94ºC for 5 min, 35 cycles of 94ºC for 1 min, 54ºC for 1 min and 72ºC for 1 min, and a last extension at 72ºC for 10 min. S. aureus ATCC 29213 was used as negative control, whereas S. aureus ATCC 43300 was used as positive control.

Amplicons were subjected to electrophoresis on 1.2% agarose gel containing ethidium bromide for 30 min at 100 V and 200 mAmp. Gels were visualized and saved in a Typhoon Scanner 8600 (Molecular Dynamics, GE Healthcare, UK). A DNA ladder of 154-2176 bp (DNA Molecular Weight Marker VI, Roche Applied Science, USA) was included in all gels.

Chapter 1

1.3 Results

1.3.1 Incidence in fishery products

Colonies were observed on BP-EY for 167 out of 298 fishery products. A total of 728 colonies were picked up, isolated and subjected to phenotypic (coagulase, DNAse and mannitol fermentation) confirmation tests. Out of them, 125 were positive by all tests and thus identified as S. aureus.

Additionally, one representative isolate of each RAPD global pattern (see below) was analysed by species-specific 23S rDNA PCR. All of them were confirmed as S. aureus (Figure 1.1). These isolates were obtained from 75 fishery products, which represented an incidence of 25.16%.

Figure 1.1. Agarose gels showing the identification of isolates as S. aureus by species-specific 23S rDNA PCR. Lane 1 and 19, DNA molecular size marker (HyperLadder II, 50-2000 bp; Bioline); lanes 2-18 and 20-35, PCR products for different isolates.

Incidence and characterization of S. aureus in fishery products

As shown in Figure 1.2, incidence was different in each product category, and it decreased in the following order: fresh products (43%), frozen products (30%), salted fish (27%), smoked fish (26%), ready-to-cook products (25%), non-frozen surimis (20%), fish roes (17%), and lastly other ready-to-eat products (10%).

Figure 1.2. Incidence (%) of S. aureus in fishery products marketed at retail level in Galicia. The number of products surveyed in each category is shown (n).

A significant proportion of surimis (9%), fish roes (4%) and other ready-to-eat products (5%) as well as ready-to-cook products (13%) exceeded 102 CFU/g of food, which was the maximum number of S. aureus allowed by legislation in force when the study was conducted (O. 2/8/1991, RD 3484/2000 and Commission Regulation (EC) No 2073/2005). Additionally, counts were higher than 103 CFU/g in 9 out of 35 surimis, 5 out 41 other ready-to-eat products and 8 out of 40 ready-to-cook products. In the same way, S. aureus must have been absent in anchovies in oil, but it was detected in 2 out of 4 products.

The present results have also shown that a significant proportion (18.6%) of smoked products exceeded the limit set for smoked fish products in O. 2/8/1991, that is, 2·101 CFU/g of food. Later, Commission Recommendation 2001/337/EC proposed that counts higher than 102 CFU/g (M value) should not be permitted in smoked fish. The value set for M value was exceeded in 16.3% of the smoked products tested in this study. Additionally, counts were higher than exceeded 103 CFU/g of food in 3 out of 43 smoked products (7%).

Chapter 1

Although it is not subject to legal regulations, it is also worthy to mention that the number of S. aureus was higher than 102 CFU/g of food in 19.4% of fresh products, 14% of frozen products and 13.3% of salted fish, and 103 CFU/g of food in 7% of frozen products, 4.8% of fresh products and 6.7% of salted fish.

1.3.2 RAPD-PCR

Genotypic characterization of isolates was performed by RAPD-PCR with three different primers (S, AP-7 and ERIC-2). As shown in Figure 1.3., RAPD analysis with primer S yielded 13 visually different banding profiles, whereas 12 profiles were obtained with each of the two other primers. A total of 31 different bands with a size between 115 and 2292 bp were amplified by primer S, whereas primer AP-7 and ERIC- 2 amplified 19 and 18 different bands ranging from 127 to 2023 bp and 125 to 1420 bp, respectively. A good reproducibility of patters was achieved when DNA from different cultures of each isolate was used as a template.

The combination of RAPD fingerprints obtained in separate reactions with different primers has been employed as a strategy to increase the discriminatory power of the analysis (Byun et al., 1997; Nema et al., 2007). The combination of the patterns obtained with the three primers generated a higher discriminatory power (D = 0.926) than those of single primers (0.818 for primer S, 0.698 for ERIC-2 and 0.622 for AP-7) as well as of pairwise-combinations of primers (0.883 for S and ERIC-2, 0.878 for S and AP-7 and 0.850 for AP-7 and ERIC-2). As a result, 33 combined or global fingerprints were distinguished. A three-digit code number were assigned to combined patterns, each one corresponding to the number assigned to patterns obtained with primers S, AP-7 and ERIC-2, respectively. The most common combined pattern was 4.4.1., which was characteristic of 23 isolates. Also, two other patterns (1.1.1 and 13.1.11) were shared by 12 and 16 isolates, respectively. Thus, 40.8% of isolates were included in one of these three patterns. In contrast, 18 patterns were specific to one isolate. Isolates showing identical combined RAPD-PCR fingerprints were considered to be a single genetic type, that is, a single bacterial clone or strain (Fueyo et al., 2001). Thus, strains St.1.10 and St.1.31 were the most prevalent in fishery products, being found in 15 and 16 products, respectively.

Incidence and characterization of S. aureus in fishery products

Figure 1.3A-C. Agarose gels showing RAPD-PCR fingerprints obtained for S. aureus isolates using primers S (A), AP-7 (B) and ERIC-2 (C). Lane 1: DNA Molecular Weight Marker (HyperLadder II, 50-2000 bp; Bioline). Pattern number is shown on the top of each fingerprint.

Chapter 1

Cluster analyses based on similarity measurements from RAPD fingerprints were carried out with the aim of finding possible relationships between RAPD patterns and product categories. Cluster analysis of combined RAPD patterns classified isolates into 17 groups at a relative genetic similarity of 0.84 (Figure 1.4). Clusters 1 and 2 were the largest ones and contained 4 and 11 patterns, respectively, which included most isolates (31 and 57, respectively) from all product categories. In contrast, there were 10 single clusters which were formed by only one isolate. The other two single clusters (13.5.11 and 9.4.7) were composed of 3 and 4 isolates.

Figure 1.4. Dendrogram from cluster analysis based on the global combination of RAPD-PCR patterns obtained with all three primers. Combined patterns were assigned a three-digit code number, with digits corresponding to numbers assigned to patterns obtained with primers S, AP- 7 and ERIC-2, respectively.

The validity assessment of cluster analyses rendered low values of hierarchical F- measures for banding patterns generated with primers S (0.314), ERIC-2 (0.311) and AP-7 (0.289). Similarly, low values were also obtained for F-measure, precision and

Incidence and characterization of S. aureus in fishery products recall in all cases. Hierarchical F-measure did not increase when banding patterns generated with each primer were combined (0.329). Values of F-measure, precision and recall did not increase in this case either. This assessment showed that no cluster was relatively pure and included most isolates of only one product category. No relationship was therefore found between any RAPD pattern and any product category.

1.3.3 Presence of enterotoxin genes

Over 91% of S. aureus isolates (n = 114) carried enterotoxin genes, from which 110 were sea positive. However, only four isolates carried several enterotoxin genes. In two isolates, the seg and sei genes were detected, whereas two others carried the sea, sec and seh genes. Each of these isolates shared a different combined RAPD pattern, so they were different bacterial strains. Nevertheless, no further relationship was found between RAPD patterns of se-carrying isolates.

All isolates sharing an identical global RAPD pattern carried the same se gen pattern, and this supports the thesis that they are bacterial clones. A total of 26 out of the 33 strains identified by RAPD-PCR analysis were se gene carriers. S. aureus St.1.10 and St.1.31, which were most prevalent, were se positive. No se-carrying strain was characteristic of a single product category.

Non-se-carrying strains showed distinct RAPD fingerprints, which were not shared by any se-carrying strain. However, no further relationship was found between the presence of se genes and any RAPD pattern. Cluster analysis did not discriminate any cluster comprising either all non-se-carrying strains or all multi-se-carrying strains only.

A significant proportion of the fishery products tested (23.5%) were contaminated with se-positive S. aureus. Furthermore, counts of se-carrying S. aureus exceeded 102 and 103 CFU/g of food in 34 and 18 products, respectively, and were even higher than 105 CFU/g of food in two dried-salted tuna loin products (i.e. mojama) and one surimi product. The incidence of se-positive S. aureus was different in each product category (Figure 1.5). Isolates carrying se genes were found in all salted fish (n = 8), surimis (n = 7) and fish roes (n = 4) which were contaminated with S. aureus. The presence of se- positive isolates was also very high in fresh products (94%), smoked fish (91%), ready- to-cook products (90%), frozen products (85%) and other ready-to-eat products (75%) in which S. aureus was detected.

Chapter 1

Figure 1.5. Presence of se genes (%) in S. aureus isolated from fishery products marketed at retail level in Galicia. Presence of se genes was defined as the number of se-carrying S. aureus- containing products respect to the number of S. aureus-containing products. The number of fishery products carrying se positive S. aureus in each category is shown (n).

The presence of se-positive and se-negative isolates was not detected in a same fishery product, except in one frozen hake nuggets product and one fresh perch fillet, in which both types were found. Moreover, one cod pâté with pepper carried sea positive and sea, sec and seh positive isolates.

1.3.4 Antibiotic sensitivity

No differences were found among isolates with regard to sensitivity profiles, except for tetracycline. All strains were thus found to be resistant to penicillin G, chloramphenicol and ciprofloxacin. However, differences were found with regard to MIC values. Thus, strains St.1.11 and St.1.18 were the most resistant to penicillin G, St.1.01 and St.1.31 were the most resistant to chloramphenicol and St.1.26 and St.1.33 showed the highest MIC value (1.58 µg/mL) for ciprofloxacin. On the contrary, none of the strains was found to be resistant to beta-lactam antibiotics such as oxacillin and methicillin, but they all had intermediate resistance to methicillin (MIC from 9.5 to 14.5 µg/mL). Strains St.1.11 and St.1.13 showed the highest levels of resistance to methicillin (MIC = 14.5 µg/mL). No strain was resistant to vancomycin, cephalothin, clindamycin, erythromycin, gentamicin or trimethoprim-sulfamethoxazole.

Incidence and characterization of S. aureus in fishery products

Tetracycline was the only antibiotic on which both resistant- and sensitive-strains were found. Thus, 17 out of the 33 strains (51.5%) identified by RAPD-PCR analysis were tetracycline-resistant, being S. aureus St.1.20 and St.1.31 the most resistant. However, over 82% of isolates were tetracycline-resistant. Tetracycline-resistant isolates were detected in 65 out of 75 fishery products contaminated with S. aureus (86.7%). St.1.10 and St.1.31, which were most prevalent, were also tetracycline- resistant. No differences in tetracycline susceptibility of isolates sharing identical global RAPD patterns with all three primers were found. However, no relationship was found between any RAPD pattern and susceptibility to tetracycline.

The incidence of tetracycline-resistant S. aureus was different in each product category (Figure 1.6). Tetracycline-resistant isolates were detected in all salted fish and fish roes which were found to be contaminated with S. aureus. Tetracycline-resistant isolates were also found in most contaminated fishery products of other categories, with an incidence decreasing in the following order: surimis (93.7%), ready-to-cook products (87.5%), smoked fish (82.4%), other ready-to-eat products (80.0%), fresh products (76.0%) and frozen products (64.3%).

Figure 1.6. Tetracycline resistance (%) of S. aureus isolated from fishery products marketed at retail level in Galicia. Resistance was defined as the number of tetracycline-resistant isolates with respect to the number of isolates. The number of isolates from each category is shown (n).

Additionally, one representative isolate of each se-carrying strain was tested for the presence of blaZ and mecA genes, which are major determinants of the resistance of staphylococci to penicillin (Olsen et al., 2006; Vesterholm-Nielsen et al., 1999) and methicillin and all other beta-lactam antibiotics (Baddour et al., 2007; Strommenger et

Chapter 1 al., 2003), respectively. No PCR product was detected for mecA in any of the 26 isolates tested (Figure 1.7A), whereas blaZ was detected in all of them (Figure 1.7B). These results are in agreement with those of antibiotic sensitivity testing.

Figure 1.7A-B. Agarose gels showing PCR products obtained for the putative enterotoxigenic S. aureus during detection of genes mecA (A) and blaZ (B). Lane 1 and 17, DNA molecular size marker (DNA Molecular Weight Marker VI, Roche Applied Science, USA); lanes 2-3, reference strains S. aureus ATCC 29213 and S. aureus ATCC 43300, respectively; lanes 4-16 and 18-30, amplicons of each putative enterotoxigenic strain; lane 31, blank.

Incidence and characterization of S. aureus in fishery products

1.4 Discussion A high incidence of S. aureus was found in fishery products marketed in Galicia (25.16%) in the present study. Although data on the incidence of S. aureus in fishery products was scarce, a high incidence had also been reported over the last ten years in some studies (Abrahim et al., 2010; Herrera et al., 2006; Oh et al., 2007; Papadopoulou et al., 2006; Simon and Sanjeev, 2007), and only one work on fishery products collected at retail outlets in Italy found a low incidence, i.e. < 3% (Normanno et al., 2005). Only a few studies on microbial safety of fish products marketed in Spain have been carried out in the last decade (Garrido et al., 2009; González-Rodríguez et al., 2002; Herrera et al., 2006; Martínez et al., 2009). Considering the importance of fishery products at national level, the lack of information available on microbial safety in fishery products made or sold in Spain, and particularly in Galicia, was found surprising. Only Martínez et al. (2009) tested for the presence of bacterial pathogens in fishery products made in Galicia, specifically in farmed molluscan shellfish.

A wide range of product categories has been examined in the present work, comprising between 24 and 43 products of each one. In contrast, previous studies on the incidence of S. aureus in fish products have focused in only one or two product categories. Thus, Da-Silva et al. (2010), Herrera et al. (2006), Oh et al. (2007) and Papadopoulou et al. (2007) only tested fresh fishery products obtained from retail stores in Brazil, Spain, Korea and Greece, respectively, Simon and Sanjeev (2007) focused on frozen products and dried fish products sold in India, and Basti et al. (2006) examined smoked and salted Iranian fish products. Similarly, González-Rodríguez et al. (2002) only surveyed vacuum-packed cold-smoked freshwater fish.

It was also found surprising that most previous studies dealt with low risk fishery products, such as fresh fish, frozen products and salted or dried fish products, and hardly a work on the incidence of S. aureus in fishery products having to comply with legal regulations in Spain or other European Union member states was found to be published in the last ten years. As an exception, a study on vacuum-packed cold-smoked freshwater fish by González-Rodríguez et al. (2002) reported that 3 packages out of 54 were contaminated with S. aureus. In addition, Normanno et al. (2003) reported an incidence of 10% in a particular raw-eaten Italian fishery product, i.e. strips of cuttlefish, and Alarcón et al. (2006) detected the presence of S. aureus in 1 out of 10

Chapter 1 ready-to-eat products within a study aimed to establish a RTQ-PCR procedure suitable for detection and quantification of S. aureus in food.

Incidence was found to be high in all categories (10-43%), but notable differences were found among them. Interestingly, the incidence was higher in those categories not covered by legislation, that is, fresh products, frozen products and salted fish, than in those having to comply with legal regulations in force at the time the study was conducted, i.e. ready-to-eat products and ready meals. This result seems to underline the effectiveness of regulations on the efforts of the industry to ensure food hygiene.

Although incidence was lower in fishery products subject to legal regulations, the presence of S. aureus was also detected in a high proportion of ready-to-cook products, smoked fish, non-frozen surimis, fish roes and other ready-to-eat products. Fishery products of these categories do not need to be cooked prior to being consumed. Therefore, the risk for the consumer can become significant if S. aureus is above regulatory limits, and food exceeding legal limits cannot be placed on the market or must be recalled. However, this study has revealed that a significant proportion of fishery products marketed in Galicia (11.3%) did not comply with regulatory limits in force.

A previous work on cold-smoked fish obtained at retail level in a nearby location showed that 3 out of 54 packages (5.5%) were contaminated with S. aureus at levels lower than 4 log CFU/g (González-Rodríguez et al., 2002), but authors did not report if levels were higher than regulatory limits (O. 2/8/1991) or laid down in Commission Recommendation 2001/337/EC). In the present study, a higher proportion of smoked fish (18.6%) was found not to comply with legal regulations.

Incidence was highest in fresh products, followed by frozen products. Incidence values reported for fresh products in previous studies were also high, ranging between 10 and 30%, with the highest value for those marketed in Northwest Spain (Herrera et al., 2006). These results were slightly lower to those presented in this work. Incidence was also reported to be slightly lower in frozen products sold in India, i.e. 17% (Simon and Sanjeev, 2007). In the present work, samples were homogenized in a lower volume of diluent and the volume of bacterial suspension spread on agar plates was higher than or equal to those used in all aforementioned studies. These slight differences in the

Incidence and characterization of S. aureus in fishery products methodology resulted in a higher limit of detection, and it could account for at least part of the differences.

Conditions such as low-temperature storage, particularly in frozen fish, a low water activity typical of frozen and salted products or the activity of specific spoilage microorganisms of fresh fish prevent S. aureus to grow and, as a result, enterotoxin production. Also, fresh, frozen and salted products are commonly cooked prior to being consumed and it should destroy all or most S. aureus. The risk for the consumer is thus lower and no regulatory limits have been laid down for these fishery products neither in Spanish nor European legislation. However, an improper storage (temperature abuse) or processing (e.g. long desalting), can enable SEs to be formed. For instance, desalting at 20ºC was found to result in unsafe levels of S. aureus (≥ 106 CFU/g) in cod, and possible toxin formation (Pedro et al., 2004). The risk can increase if the number of microorganisms is low (such as in thawed products), shelf-life is long (such as in salted products) or the product is consumed raw, undercooked or, in general, lightly processed. Additionally, staphylococcal enterotoxins (SEs) are highly heat-resistant and therefore, in many cases, thermal processes cannot be used as a measure to prevent staphylococcal food poisoning (Balaban and Rasooly, 2000; Cremonesi et al., 2005).

Most of the S. aureus isolates found in this work carried se genes so their incidence in fishery products marketed in Galicia was high (23.5%). A lower proportion of S. aureus se positive had been previously found among isolates from fishery products in other geographical regions (Normanno et al., 2005; Oh et al., 2007; Simon and Sanjeev, 2007).

At present, nine different serological types of SEs (SEA-SEE and SEG-SEJ) have been proven to have emetic activity (Ortega et al., 2010). Except for two, all se-carrying isolates found in this study carried the sea gene, and therefore could produce SEA, but none was found to be seb-see positive. Classical staphylococcal enterotoxins (SEA- SEE) have been reported to cause 95% of staphylococcal food poisoning. Among them, SEA is the most common in staphylococcus-related food poisoning (Pinchuk et al., 2010), probably due to a very high resistance to proteolytic enzymes (Le-Loir et al., 2003). Several studies have reported that a high proportion of isolates from outbreaks of staphylococcal food poisoning occurring in South Korea, France, Japan and UK could produce SEA, either alone or with another toxin (Cha et al., 2006; Kérouanton et al.,

Chapter 1

2007; Shimizu et al., 2000; Wieneke et al., 1993). In contrast, sea had been found not to be the most predominant se gene in fishery products in some previous studies (Normanno et al., 2005; Simon and Sanjeev, 2007).

Only four of the isolates were multi-se-carriers, two harboured the seg and sei genes and two others, the sea, sec and seh genes. In contrast, Cha et al. (2006) found that most isolates (ca. 85%) from staphylococcal food poisoning incidents in South Korea were multi-se-carriers and detected seg-sei genes in a significant proportion of isolates, either alone (ca. 4%) or along with other se genes (17.5%). Nonetheless, SEG and SEI have been considered to play minor role in food poisoning (Chen et al., 2004).

A dose lower than 1 µg of SE has been reported to make symptoms of staphylococcal food poisoning to appear within 1-6 h after consumption of contaminated food in an adult healthy individual (FDA, 1992; Pinchuk et al., 2010). This toxin level can be reached when cell number exceeds 105 CFU/g of food (Bhatia and Zahoor, 2007). As a preventive measure, legal limits of 102-103 CFU/g had been set for S. aureus in different fishery products. However, in this study, counts of se-carrying S. aureus exceeded such limits in a significant number of products and in some of them even by one or two orders of magnitude.

S. aureus has been reported as the third major causative agent of foodborne illness by fish and fishery products in the European Union (EFSA, 2009a). Moreover, the actual incidence of staphylococcal food poisoning is known to be much higher than reported (Lawrynowicz-Paciorek et al., 2007; Smyth et al., 2004). Thus, the notification of staphylococcal intoxications is not mandatory in many member states of the European Union and most cases are not reported to healthcare services as they resolve within 24 to 48 h after onset -hospitalization rate was 19.5% for verified outbreaks caused by S. aureus in 2008 (EFSA, 2010)-. However, microbiological criteria laid down in national regulations (O. 2/8/1991 and RD 3484/2000) have been recently repealed (RD 135/2010) following Commission Regulation (EC) No 2073/2005 on microbiological criteria for foodstuff. Coagulase positive staphylococci (mainly S. aureus) are thus no longer a microbiological criterion for ready-to-cook products and most ready-to-eat products. At present, S. aureus is set as a process hygiene criterion only for shelled and shucked products of cooked crustaceans and molluscan shellfish, with a value for M of 103 CFU/g. In the present study, 3 out of 12 products (25%) exceeded this value.

Incidence and characterization of S. aureus in fishery products

The emergence of multidrug resistant pathogens is recognized as an environmental hazard to the food supply and human health, as it makes eradication more difficult and incidence to increase (Livermore, 2000; Popovich et al., 2007; Ribeiro et al., 2007). S. aureus has developed multidrug resistance worldwide, but wide variations in incidence exist regionally (Gündoğan et al., 2006; Normanno et al., 2007; Peles et al., 2007; Pesavento et al., 2007). All isolates found in fishery products marketed in Galicia were resistant to penicillin, chloramphenicol and ciprofloxacin and most of them were resistant to tetracycline too. Beleneva (2011) also found a high incidence of ciprofloxacin-resistant S. aureus (84.7%) in fishery products from the Sea of Japan and South China Sea, but the percentage of penicillin- and tetracycline-resistant strains was lower (47.2% and 27.5%, respectively). Variations in antibiotic resistance are also the result of other different factors. For instance, Pereira et al. (2009) isolated a high number of penicillin-resistant S. aureus (73%) from meat and dairy products in a nearby geographical area (North of Portugal), but the number of chloramphenicol-, ciprofloxacin- and tetracycline-resistant isolates was extremely low (0-2%). Tetracycline was the only antibiotic on which both resistant- and sensitive-strains were found. Tetracycline-resistance seemed to enhance the presence of S. aureus in fishery products obtained at retail level in Galicia.

Methicillin-resistant S. aureus (MRSA) are being increasingly found outside clinical settings (Popovich et al., 2007; Ribeiro et al., 2007; Stankovic et al., 2007). MRSA have thus been found in food animals (Lee, 2003) and different foods (Gündoğan et al., 2006; Peles et al., 2007; Pesavento et al., 2007; Pereira et al., 2009) and also in fishery products recently (Beleneva, 2011). In the present study, however, no MRSA was isolated from fishery products and no isolate carried the mecA gene, though intermediate resistance to methicillin was detected in all isolates. Although EFSA (2009b) reported that there is no current evidence that eating food contaminated with MRSA may lead to an increased risk of humans becoming healthy carriers or infected with MRSA strains, the risk of multidrug-resistant MSSA, which are present in food more frequently than MRSA, has not been examined yet. In fact, it has been recently underlined the need of incidence studies of multidrug-resistant strains in food to clarify their public health relevance (Waters et al., 2011). Meanwhile, it is important to take some preventive control measures.

Chapter 1

The identification of bacterial clones with enhanced virulence or increased ability to spread is important. RAPD is a fast and cost-effective PCR method for typing and differentiation of bacterial strains with no prior information of the gene sequence. However, a lack of standardization and a low reproducibility have been claimed as major drawbacks of RAPD (Deplano et al., 2000; Van-Belkum et al., 1995). Nevertheless, RAPD-PCR binding patterns were found to be reproducible in this study when DNA from different cultures of a same isolate was used as a template. A good reproducibility has been also observed in epidemiological studies using RAPD for typing S. aureus isolates (Aras et al., 2012; Fueyo et al., 2001; Nikbakht et al., 2008; Shehata, 2008). Accordingly, RAPD could be used for initial screening of isolates in public health epidemiological studies (outbreak and endemic strains), prior to other complementary typing methods such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST), which must be used for global epidemiology and population genetic studies of S. aureus (Al-Thawadi et al., 2003; Byun et al., 1997; Deplano et al., 2006).

The use of RAPD to discriminate strains has been also questioned. Morandi et al. (2010) has recently reported that multilocus variable number tandem repeat analysis (MLVA) is more powerful than RAPD-PCR for typing of S. aureus (discriminatory power of 0.99 and 0.94, respectively). Nonetheless, the discriminatory power of RAPD was determined by using only one primer (AP-4). In the present study, the combination of RAPD fingerprints obtained in separate reactions with three different primers allowed the discriminatory power of the analysis to be increased (increases of 11.7%, 24.6% and 32.8% for primers S, ERIC-2 and AP-7, respectively). Other authors have used even a much higher number of primers for strain differentiation (Byun et al., 1997; Nema et al., 2007). This allows strains to be differentiated when RAPD patterns are rather similar, i.e. low-yield patterns. However, the number of polymorphic bands generated by primers S, AP-7 and ERIC-2 was considered to be high enough to distinguish S. aureus strains among isolates found in this study. Nonetheless, it is not unlikely that the use of a higher number of primers had increased the number of different strains, but this had been time-consuming.

Isolates sharing a same global RAPD fingerprint also showed identical enterotoxin gene and antibiotic susceptibility patterns, and this supports the thesis that RAPD

Incidence and characterization of S. aureus in fishery products analysis allowed bacterial clones to be distinguished. Nema et al. (2007) also found that S. aureus isolates with a same RAPD fingerprint had identical se gen patterns and suggested a clonal origin of isolates.

Cluster analyses based on similarity measurements between RAPD patterns found no relationship between any RAPD pattern and any product category and, though se- negative and se-positive strains did not shared RAPD fingerprints, no further relationship was found between the presence of se genes and any RAPD pattern either.

The use of RAPD-PCR fingerprinting with several primers to assess the genetic relationship between S. aureus isolated from fishery products marketed in Galicia has therefore exclude a clonal origin.

1.5 Conclusions A significant proportion (~ 25%) of fishery products surveyed from retail sector in Galicia in 2008 and 2009 was found to be contaminated with S. aureus, mostly with se- carrying strains. About 12% of products did not comply with regulatory limits, and a higher proportion of products not subject to regulations were contaminated too. These results suggest some effect of regulations on the efforts of the industry to ensure food hygiene. However, a number of microbiological criteria laid down in national regulations have been recently repealed and coagulase positive staphylococci (mainly S. aureus) are thus no longer a microbiological criterion for ready-to-cook products and most ready-to-eat products. However, S. aureus has been reported as the third major causative agent of foodborne illness by fish and fish products in the European Union (EFSA, 2009a). In addition, the actual incidence of staphylococcal food poisoning is known to be much higher than reported -the notification of is not mandatory in many member states of the European Union and most cases are not reported to healthcare services as they resolve within 24 to 48 h after onset-. A revision of pre-requisite programs and an improvement of hygienic practices in handling and processing operations from fishing or farming to retail outlet is therefore recommended in order to ensure the safety of fishery products marketed in Galicia. Nonetheless, at present, S. aureus is still under surveillance by a significant part of the industrial sector.

Chapter 2. Impact of food-related environmental factors on the adherence and biofilm formation of natural Staphylococcus aureus isolated from fishery products

Impact of food-related environmental factors on the biofilm formation of S. aureus

Abstract Staphylococcus aureus is a pathogenic bacterium capable of developing biofilms on food-processing surfaces, a pathway leading to cross-contamination of foods. The purpose of this study was to investigate the influence of environmental stress factors found during seafood production on the adhesion and biofilm-forming properties of S. aureus. Adhesion and biofilm assays were performed on 26 S. aureus isolated from seafoods and two S. aureus reference strains (ATCC 6538 and ATCC 43300). Cell surface properties were evaluated by affinity measurements to solvents in a partitioning test, while adhesion and biofilm assays were performed in polystyrene microplates under different stress conditions of temperature, osmolarity and nutrients content. The expression of genes implicated in the regulation of biofilm formation (icaA, sarA, rbf and σB) was analysed by reverse transcription and quantitative real time PCR.

In general, S. aureus isolates showed moderate hydrophobic properties and a marked Lewis-base character. Initial adhesion to polystyrene was positively correlated with the ionic strength of the growth medium. Most of the strains had a higher biofilm production at 37ºC than at 25ºC, promoted by the addition of glucose, whereas NaCl and MgCl2 had a lower impact markedly affected by incubation temperatures. Principal Component Analysis revealed a considerable variability in adhesion and biofilm- forming properties between S. aureus isolates. Transcriptional analysis also indicated variations in gene expression between three characteristic isolates under different environmental conditions. These results suggested that the prevalence of S. aureus strains on food-processing surfaces is above all conditioned by the ability to adapt to the environmental stress conditions present during food production.

Chapter 2

These findings are relevant for food safety and may be of importance when choosing the safest environmental conditions and material during processing, packaging and storage of seafood products.

Keywords: Staphylococcus aureus; Polystyrene; Hydrophobicity; Adhesion; Biofilm; icaA.

2.1 Introduction

Staphylococcus aureus is a common human pathogen responsible for food-borne intoxications worldwide, caused by the ingestion of food containing staphylococcal heat-stable enterotoxins (Lowy, 1998; Le-Loir et al., 2003). The greatest risk of staphylococcal food poisoning is associated with food contaminated with S. aureus after the normal microflora has been destroyed or inhibited (Bore et al., 2007). In 2009, the European Union witnessed staphylococcal outbreaks which led to a hospitalization rate of 16.9% (EFSA, 2011). Both food products and food-contact surfaces are often contaminated through handling during processing and packaging (Sattar et al., 2001; DeVita et al., 2007; Simon and Sanjeev, 2007), as S. aureus is part of the normal microbiota associated with human skin, throat and nose. Consequently, S. aureus has been repeatedly detected in a diverse variety of food, including seafood (Novotny et al., 2004; Herrera et al., 2006; Papadopoulou et al., 2007). One recent study (Vázquez- Sánchez et al., 2012) reported a high incidence of S. aureus (~ 25%) in seafood marketed in Spain, which is the largest seafood producer and the second largest consumer in the European Union (Eurostat, 2007).

Biofilm is considered as part of the normal life cycle of S. aureus in the environment (Otto, 2008), in which planktonic cells present attach to solid surfaces, proliferating and accumulating in multilayer cell clusters embedded in an organic polymer matrix. This structure protects the bacterial community from environmental stresses, from the host immune system and from antibiotic attacks, as opposed to the situation for vulnerable and exposed planktonic cells (Costerton et al., 1987). This may contribute to the persistence of S. aureus in food-processing environments, consequently increasing cross-contamination risks as well as subsequent economic losses due to recalls of contaminated food products.

Impact of food-related environmental factors on the biofilm formation of S. aureus

Several studies have shown the attachment of S. aureus on work surfaces such as polystyrene, polypropylene, stainless steel and glass, and also in food products (Costerton et al., 1978; Sattar et al., 2001; Herrera et al., 2006; DeVita et al., 2007; Simon and Sanjeev, 2007). However, changes in surface physicochemical properties and substratum topography, as well as in environmental factors such as osmolarity, nutrient content and temperature may lead to staphylococcal biofilm development and, consequently, influence their persistence on food-contact environments (Kumar and Anand, 1998; Ammendolia et al., 1999; Bos et al., 1999; Poulsen, 1999; Akpolat et al., 2003; Møretrø et al., 2003; Planchon et al., 2006; Rode et al., 2007; Pagedar et al., 2010; Xu et al., 2010).

The extracellular matrix of S. aureus is mainly composed by polysaccharide intercellular adhesins (PIA), i.e., poly-β(1,6)-N-acetyl-d-glucosamines (PNAG), which are synthetized by N-acetyl-glucosamine transferase (Cramton et al., 1999; Maira-Litrán et al., 2002; Fitzpatrick et al., 2005; O’Gara, 2007). This enzyme is induced by the co- expression of icaA with icaD, products of the chromosomal intercellular adhesion (ica) operon carried by most S. aureus strains (Cramton et al., 1999; Maira-Litrán et al., 2002; Jefferson et al., 2003; Fitzpatrick et al., 2005). The expression of the ica operon is controlled by the repressor icaR, which is regulated by the staphylococcus accessory regulator (sarA), the stress-induced sigma factor B (σB) (Cerca et al., 2008) and, indirectly, by the rbf gene (Cue et al., 2009), among others. These genes are also involved in the resistance of S. aureus to various environmental stresses (Gertz et al., 2000; Rachid et al., 2000; Lim et al., 2004).

The present study aimed at investigating the persistence of 26 natural S. aureus isolates on polystyrene surfaces, a material frequently used in the food industry, through the evaluation of their physicochemical, adhesion and biofilm-forming properties under different environmental stress conditions found during processing, packaging and storage of food products. Moreover, the variability of the expression of genes implicated in the regulation of biofilm formation between three strains selected during the screening was also investigated under different stress conditions.

Chapter 2

2.2 Materials and Methods

2.2.1 Bacterial strains and growth conditions

Twenty six S. aureus isolates from seafood marketed in Galicia (Northwest Spain) were investigated. They were previously identified as S. aureus by specific biochemical (coagulase, DNAse and mannitol fermentation) and genetic tests (23s rDNA) and characterized by RAPD-PCR (Vázquez-Sánchez et al., 2012). These isolates carried sea (n = 22), sea-c-h (n = 2) or seg-i (n = 2) genes, whose expression produce enterotoxins. S. aureus ATCC 6538 (a known biofilm former) and ATCC 43300 (MRSA strain), provided from the Spanish Type Culture Collection (CECT, Valencia, Spain), were used as reference strains. Stock cultures were maintained in 20% glycerol at ─80ºC. All strains were thawed and subcultured in tryptic soy broth (TSB, Oxoid, UK) for 24 h at 37ºC, 200 rpm prior to use.

2.2.2 Evaluation of bacterial cell surface physicochemical properties

Microbial Adhesion to Solvents (MATS) was used as a method to determine the hydrophobic character of the cell surface of S. aureus strains and their Lewis acid-base properties (Bellon-Fontaine et al., 1996). This method is based on the comparison between microbial cell surface affinity to a monopolar solvent and an apolar solvent, which both exhibit similar Lifshitz-van der Waals surface tension components.

Chloroform (an electron-acceptor solvent), hexadecane (nonpolar solvent), ethyl acetate (an electron-donor solvent) and decane (nonpolar solvent) were used of the highest purity grade (Sigma-Aldrich, USA). Experimentally, overnight bacterial cultures were washed twice in phosphate buffer (7.6 g/L NaCl, 0.2 g/L KCl, 0.245 g/L

Na2HPO4 and 0.71 g/L K2HPO4; Merck, Inc.) and resuspended to a final OD400nm of 0.8 (~ 108 CFU/mL). Individual bacterial suspensions (2.4 mL) were first mixed with 0.4 mL of the respective solvent and then manually shaken for 10 s prior to vortexing for 50 s. The mixture was allowed to stand for 15 min to ensure complete separation of phases.

1 mL from the aqueous phase was removed and the final OD400nm measured. The percentage of cells residing in the solvent was calculated by:

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Impact of food-related environmental factors on the biofilm formation of S. aureus

where (ODi) was the optical density of the bacterial suspension before mixing with the solvent and (ODf) the absorbance after mixing and phase separation. Each measurement was performed in triplicate and the experiment was performed twice using independent bacterial cultures.

2.2.3 Measurement of the adherence ability to polystyrene at different ionic strength conditions

The ability of S. aureus strains to adhere to polystyrene was evaluated in terms of biomass using the crystal violet method described by Giaouris et al. (2009), but with some modifications.

Overnight cultures were washed twice and resuspended to a final OD600nm of 0.8 in 150 mM NaCl or 1.5 mM NaCl. 200 µL of each sample was added in a flat-bottomed 96-well microtiter plate with Nunclon Surface (Nunc, Denmark) and then incubated for

4 h at 25ºC. After measuring the OD600nm, the microplates were washed three times with peptone water (Oxoid, UK), using an automatic microplate washer (Wellwash AC, Thermo Electron Corporation, Inc.), and air-dried for 2 h. Wells were then stained for 15 min using 150 µL of 0.5% (w/v) Crystal Violet (CV) (Merck, Inc.) followed by three rinsing steps with distilled water. The microplates were air-dried for 15 min and the bound CV was extracted with 150 µL of 33% (v/v) Glacial Acetic Acid (Merck, Inc.) for 30 min at room temperature. 100 µL of the mixture was diluted in a new microplate with 100 µL of 33% Glacial Acetic Acid prior to read the OD562nm. Each measurement was performed in triplicate and the experiment was repeated twice using independent bacterial cultures.

2.2.4 Quantification of biofilm formation on polystyrene under different environmental conditions

The biofilm-forming ability of S. aureus strains on polystyrene microtiter plates was also investigated in terms of biomass using an optimized protocol based on previously described methods (Song and Leff, 2006; Rode et al., 2007; Peeters et al., 2008). Each well was added with 100 μL of growth medium and 100 μL of an overnight bacterial culture diluted 1:100 in TSB. Negative control wells contained TSB only. Biofilm formation was evaluated after 24 and 48 h in TSB with or without 5% glucose,

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5% NaCl, 5% glucose + 5% NaCl, 0.1 mM MgCl2 or 1 mM MgCl2 (Merck, Inc.) at 25 and 37ºC. After measuring the OD600nm, the microplates were washed three times with peptone water using the automatic microplate washer and air-dried for 2 h. The microplates were then stained with 150 µL of 0.5% (w/v) CV for 15 min followed by three rinsing steps with distilled water. After air-dried for 15 min, the bound CV was extracted with 150 µL of 33% (v/v) Glacial Acetic Acid for 30 min. The mixture added to a new microplate was then diluted 1:1 in 33% Glacial Acetic Acid prior to read the

OD562nm. Each measurement was performed in triplicate and the experiment was repeated twice using independent bacterial cultures.

2.2.5 Transcriptional analysis

To assess the expression levels of the genes reported in Table 2.1, RNA was extracted from St.1.07, St.1.14 and St.1.29 grown in TSB with or without 5% glucose, 5% NaCl or 5% glucose + 5% NaCl. An overnight culture was diluted 1:100 in each medium and cultivated at 37ºC with 200 rpm of agitation until an OD600 ~ 0.5. After incubation, two volumes of bacterial culture were diluted in four volumes of RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany). The mixture was vortexed for 15 s, incubated for 5 min at room temperature and centrifuged (5000 × g) for 10 min at room temperature. The supernatant was discarded and 200 μL of a mixture containing TE buffer, 40 mg/mL lysozyme and 1 mg/mL lysostaphin (Sigma, USA) was added for enzymatic lysis of bacteria. RNA was isolated using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions and including a DNase treatment. The concentration and purity of total RNA were analysed using a

NanoDrop, ND-1000 spectrophotometer (NanoDrop Technologies, Inc.). Reverse transcription of the RNA isolated was carried out using random primers, as previously described (Rode et al., 2007) with slight modifications. A reaction mixture (13 μL) with 300 ng RNA, 100 ng Random Primers and 10 mM of each dNTP (Invitrogen) was denatured at 65ºC for 5 min, incubated on ice immediately for at least 1 min and centrifuged briefly. A mixture (6 μL) of 5x first strand buffer, 0.1 M DTT and 200 U Superscript III reverse transcriptase (Invitrogen) was then added to the reaction. The samples were incubated at 25ºC for 5 min, heated at 50ºC for 45 min and immediately incubated at 70ºC for 15 min to inactivate the reaction. A brief centrifugation between each step was done. Six reverse transcriptase reactions were

102

Impact of food-related environmental factors on the biofilm formation of S. aureus made for each biological replicate of RNA, of which three were without enzyme as negative controls. Quantitative real-time PCR (qRT-PCR) was performed in an Abi Prism 7900 HT Sequence Detection System (Applied Biosystems, Inc.). The PCR mixture contained 1×

TaqMan Buffer A, 5 mM MgCl2, 0.2 mM of dATP, dCTP and dGTP, 0.4 mM dUTP, 0.2 μM primer, 0.1 μM probe, 0.1 U AmpErase uracil N-glycosylase, 1.25 U Ampli-Taq

Gold DNA Polymerase (Applied Biosystems, Roche, Inc.), 10 ng of cDNA and dH2O ultrapure DNAse and RNAse free (Gibco, Invitrogen Corporation) up to a final volume of 25 μL. Primers and Taqman® probes were designed previously by Rode et al. (2007). Reaction mixtures were subjected to an initial cycle of 50ºC for 2 min and 95ºC for 10 min, followed by 40 cycles of 95ºC for 30 s and 60ºC for 1 min.

CT values were estimated on SDS 2.2 software (Applied Biosystems, Inc.). The difference between CT of the reference gene 16S and CT of other gene analysed (ΔCT) were calculated to see possible changes in gene expression. One unit change represents a log of 2-fold change.

2.2.6 Statistical analysis

Results from the analytical determinations were statistically treated with the software package IBM SPSS 19.0. They were averaged and the standard error of the mean was calculated. Data of the adhesion and biofilm formation assays were normalized and expressed as OD562nm/OD600nm, due to the variation in total growth at 25ºC and 37ºC and to have a clearer view of biofilm formation for the conditions where growth was limited, as Rode et al. (2007) proposed. Significance of the data was determined using a one way ANOVA and the homogeneity of variances was examined by a post-hoc least significant difference (LSD) test. Otherwise, a Dunnett´s T3 test was performed. An independent-samples T test was also done to compare strains in pairs. Bivariate correlations were analysed using the Pearson correlation coefficient. Significance was expressed at the 95% confidence level (P < 0.05) or greater. Principal Components Analysis (PCA) was performed to group the 28 S. aureus strains by their similar physicochemical, adhesion and biofilm formation properties showed on polystyrene. Varimax normalization method with Kaiser was used to build the rotated component matrix.

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Reference

Lim et al. (2004) al. et Lim

Bore et al. (2007) al. et Bore

Valle et al. (2003) al. et Valle

Weinrick et al. (2004) al. et Weinrick (2004) al. et Weinrick

′

′ → → ′

TT G TT

Nucleotide sequence 5 sequence Nucleotide

TTG CTT CCA AAG A CCA AAG CTT TTG

CGT AGG TGG CAA GCG TTA TCC GGA TCC TTA GCG CAA TGG AGG CGT AT GTA GCG GCC GCA CCA CCA ATA CGC TTA GCT CGC G GGA TTG ACA GAA CCA GTT TTG ATG TGG CAT GTG TGC GCT ACC TGA GCG CAC G GGA TTG ACA GAA CCA GTT TTG ATG TGG AAT AA TTG TTA ACC AAA TTT ATC GGA GAA TTA ACA CGG CTT CTT TTT ATT TGA TTG T ATC GTA GAA ATC TTT TTT ACG ATT CTC ATG ATC TTC TCT CA TAA CAT GTG GTT GTT TAC TTT TTT G CAA CCA AAA CAA TAC AAC TTA CAT T TAA TTA AAG ACA GGG AAT GGA AAT AGC AGA GTT AGT AGA G GAA TCA GTC GAA TTA CGA GAT GCT ATA G AAC CTT ACA TCA TGA GAA ATG TCA

F F

R R

Pr Pr

Pr - -

F -

- -

R -

- -

Pr -

rbf

16S

Name

16S

sigB

16S icaA sarA

sigB

icaA sarA

sigB

icaA sarA

Nucleotide sequences of primer pairs (F: forward; R: reward) and Taqman probes (Pr) used in the transcriptional analysis. transcriptional the in used (Pr) probes Taqman and reward) R: forward; (F: pairs of primer sequences Nucleotide

rbf

16S

icaA

sarA

Gene Table 2.1. 2.1. Table

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Impact of food-related environmental factors on the biofilm formation of S. aureus

2.3 Results

2.3.1 Surface hydrophobicity and electron donor/acceptor character

The physicochemical surface properties of the 28 S. aureus strains were studied to estimate their potential for adhesion and subsequent biofilm formation on surfaces. Affinities of the strains to different polar and apolar solvents are shown in Figure 2.1. Considerable variations in the percentage of adhesion to decane between S. aureus tested strains reveal the degree of diversity in their hydrophobic character. Affinity to decane ranged from 22.32% to 74.82%. However, affinity to hexadecane were less variable ranging from 56.40% to 84.14%, revealing a moderate hydrophobic character for the majority of S. aureus tested strains. High percentage of adhesion to chloroform was observed for all tested strains (ranging between 74.37% and 95.75%), which in all cases were higher than that to hexadecane. This also reveals the diversity in electron donor (Lewis base) properties among tested S. aureus, highlighting the strain St.1.19 with the highest electron donor character. S. aureus tested strains generally expressed non electron acceptor (Lewis acid) properties, as seen by the higher affinity to decane compared to ethyl acetate with values below 19.75%.

2.3.2 Adherence ability of S. aureus to polystyrene surfaces

Initial adhesion to polystyrene surfaces of the 28 S. aureus strains to polystyrene was quantified in terms of biomass at two different ionic strengths (1.5 mM and 150 mM NaCl) to evaluate their electrostatic interactions.

The results showed that initial adhesion to polystyrene was positively correlated (r = 0.577, P < 0.01) with ionic strengths presented in the suspension. Thus, initial adhesion to polystyrene was reduced at lower ionic strength conditions compared to high ionic conditions, except for strains St.1.08 and St.1.21 (Figure 2.2). Moreover, the variability of adhesive properties to polystyrene among S. aureus strains at low ionic strength medium may also be an indication of the diversity in cell wall electronegativity among the tested S. aureus strains, as previously described (Giaouris et al., 2009). The strains St.1.08 and St.1.09 showed the most remarkable adherence ability under low and high ionic strength conditions, respectively.

105

Chapter 2

0.05) 0.05) in affinity to each solvent between the

decane decane and ethyl acetate. Mean and SD values: three

28) 28) to the solvents chloroform, hexadecane,

strains strains (n

S. S. aureus

Affinity Affinity of

Figure 2.1. replicates of each sample. Different letters on the top of each column show significant differences ( tested. strains

106

Impact of food-related environmental factors on the biofilm formation of S. aureus

ee replicates of each sample.

28) under ionic different strength conditions (NaCl 1.5 mM and 150 mM).

strains strains (n

S. S. aureus

0.05) 0.05) between the adherence ability of strains at each condition were indicated by different letters on the top of each

Initial adhesion Initial toadhesion polystyrene surfaces of

Adhesion Adhesion ability of each strain was expressed in terms of biofilm biomass after Significant 4 differences h ( at 25ºC. Mean and SD values: thr column. Figure 2.2.

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2.3.3 Biofilm formation on polystyrene surfaces under different environmental conditions

The ability of the 28 S. aureus strains to develop biofilms on polystyrene surfaces under different conditions of temperature (25ºC and 37ºC), osmolarity and nutrient content (TSB with or without 5% glucose, 5% NaCl, 5% glucose + 5% NaCl, 0.1 mM

MgCl2 and 1 mM MgCl2) was investigated after 24 and 48 h to understand the direct effects of environmental factors in staphylococcal biofilm formation. These two temperatures were selected by their relevance to the food industry and hospitals (25ºC) and in infectious disease (37ºC). To compensate for variations in cell mass at stationary phase at the two different temperatures, the biofilm formation values were expressed as

OD562nm/OD600nm. Significant differences (P < 0.05) between strains for each treatment and viceversa were observed, as indicated by the different letters showed in Figure 2.3.

2.3.3.1 Effect of incubation temperature

Biofilm formation in a medium without nutrient addition (TSB only) was positively correlated (r = 0.386, P < 0.01) with the temperature of incubation. Thus, incubation at 37ºC increased biofilm-forming ability for the majority of tested isolates (84%), compared to incubation at 25ºC. S. aureus St.1.22 and St.1.11 showed the highest biofilm formation at 37ºC, while St.1.31 was able to form biofilms with high cell densities at 25ºC (Figure 2.3A).

Biofilm formation was also positively correlated (P < 0.01) with incubation temperature when TSB was added with 5% glucose (r = 0.522), 5% glucose + 5% NaCl

(r = 0.637), 0.1 mM MgCl2 (r = 0.487) or 1 mM MgCl2 (r = 0.405), but addition of 5% NaCl generated a negative correlation (r = ‒0.418, P < 0.01). In fact, 78.5% of the strains showed a higher biofilm formation in TSB with 5% NaCl when they were incubated at 25ºC than at 37ºC.

2.3.3.2 Effect of glucose and NaCl addition

Addition of 5% glucose to TSB generally led to enhanced staphylococcal biofilm formation (Figure 2.3B), as shown its positive correlation (P < 0.01) with biofilm formation under all tested conditions (Table 2.2). However, these increases on biofilm development with the addition of glucose were affected by incubation temperatures. The

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Impact of food-related environmental factors on the biofilm formation of S. aureus highest increases in biofilm formation with the addition of 5% glucose were produced in the first 24 h at 37ºC and after 48 h at 25ºC, with 3-fold and 2-fold increases respectively. In the presence of 5% glucose, isolates St.1.01, St.1.02, St.1.04 and St.1.08 expressed a 4-fold biofilm increase after 24 h at 37ºC, while isolates St.1.05 and St.1.06 showed 5-fold increases after 48 h at 25ºC.

The effect of NaCl on biofilm formation was markedly affected by incubation temperatures. Thus, a negative correlation (P < 0.01) at 37ºC between biofilm formation and the addition of 5% NaCl was observed (Table 2.2). In fact, 75% of tested isolates expressed lower biofilm formation in environments with supplemented salt than those grown in the absence of salt (Figure 2.3C). Nevertheless, a positive correlation (P < 0.01) was reported at 25ºC for the first 24 h between NaCl addition and biofilm formation, slightly improving the production of biofilm by most isolates (75%). Under similar conditions, isolates St.1.02, St.1.21 and St.1.29 grown in the presence of 5% NaCl showed a remarkable 2-fold increase in biofilm formation compared to those grown in the absence of salt. These isolates were isolated from a Paella (containing mussels and squids), frozen shelled prawns and a Panga fillet respectively, three seafood products with high amounts of NaCl (> 100 mg per 100 g of product) (FEM and FROM, 2012). No significant correlation was observed after 48 h at 25ºC between biofilm formation and the addition of NaCl.

Comparing with individual effects, no synergy was observed between the addition of glucose and NaCl (Figure 2.3D). Moreover, no significant correlations were observed between biofilm formation and the addition of both nutrients, except a negative correlation (P < 0.01) reported when the strains were incubated for 24 h at 37ºC. The addition of 5% glucose + 5% NaCl therefore slightly increased the biofilm formation compared to growth in the absence of glucose and NaCl in 64.3% of all tested isolates after 48 h growth for both 25ºC and 37ºC. Two-fold biofilm increases were observed in non-supplemented TSB for isolates St.1.07, St.1.12 and St.1.28 grown at 25ºC, and isolates St.1.03, St.1.05, St.1.06, St.1.08 and St.1.14 grown at 37ºC.

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), ),

), 5% NaCl (

) ) or added with 5% glucose (

). Mean and SD values: nine replicates of each sample. Different letters on on letters Different each sample. of replicates values: nine SD and ).Mean

(

28) 28) on in polystyrene TSB only (

0.05) in biofilm formation between strains for each condition. each for strains between formation in biofilm 0.05)

strains (n strains

)or 1 MgCl mM

(

S. S. aureus

MgCl

), 0.1 mM mM 0.1 ),

Biofilm formation Biofilm of

Figure 2.3A ( NaCl 5% + 5%glucose ( differences significant indicate eachcolumn

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Impact of food-related environmental factors on the biofilm formation of S. aureus

2.3.3.3 Effect of MgCl2 addition

Generally, addition of 0.1 mM MgCl2 did not significantly affect biofilm formation compared to growth in the absence of MgCl2 (Figure 2.3E). No correlation was observed between the addition of 0.1 mM MgCl2 and biofilm formation, except a positive correlation (P < 0.05) for growth at 37ºC after 48 h (Table 2.2). In fact, 57.1% of the strains increased significantly their biofilm formation with the addition of MgCl2 under these conditions, highlighting St.1.05, St.1.07, St.1.14, St.1.20 and St.1.31 with a 3-fold biofilm increase.

Otherwise, the increment of the MgCl2 concentration from 0.1 to 1 mM did not induce an increase on the biofilm formation (Figure 2.3F). Consequently, only 21.4% isolates showed on average a 2-fold increase with the addition of 1 mM MgCl2 after 48 h at 37ºC, highlighting St.1.01 and St.1.03 isolated from smoked swordfish, a seafood with high magnesium levels (57 mg per 100 g of product) (FEM and FROM, 2012). Biofilm formation was positively correlated (P < 0.05) with the addition of 1 mM

MgCl2 after 48 h at 37ºC, but no significant correlations were observed under the other conditions tested (Table 2.2).

Table 2.2. Correlations between the biofilm formation and nutrient content expressed as r values. An r value of zero indicates no correlation, whereas a value of 1 or ‒1 indicates a perfect positive or negative correlation.

Incubation condition Nutrient added 25ºC 24 h 25ºC 48 h 37ºC 24 h 37ºC 48 h

5% glucose 0.478b 0.588b 0.733b 0.470b 5% NaCl 0.499b 0.082 ‒0.521b ‒0.439b 5% glucose + 5% NaCl ‒0.031 ‒0.040 ‒0.356b 0.133 a 0.1 mM MgCl2 0.148 0.033 0.149 0.176 a 1 mM MgCl2 0.139 0.049 0.068 0.161 a P < 0.05; b P < 0.01

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2.3.4 Multivariate analysis of the physicochemical, adhesion and biofilm-forming properties of the 28 S. aureus strains

The variables (n = 30) defined during adhesion and biofilm formation assays as well as two additional variables (the type of seafoods from which isolates were sampled and the type of processing used during their production) were used to perform a Principal Components Analysis (PCA) for the 28 S. aureus strains. However, the two principal components (PC) obtained only accounted for 33% of total variance. The selection of the most significant parameters (n = 8) indicated in the rotated component matrix for each PC allowed increase up to 79.3% the total variance accounted (Table 2.3). PC1 and PC2 accounted individually a variance of 55.7% and 23.6%, respectively. PC1 was positively correlated (P < 0.01) with biofilm formation in TSB with 5% glucose at 25ºC and in TSB added with or without 5% glucose, 5% glucose + 5% NaCl or 1 mM MgCl2 at 37ºC. PC1 was also correlated with type of product (r = 0.444, P < 0.05) and processing (r = 0.625, P < 0.01). Meanwhile, PC2 was positively correlated (P < 0.01) with biofilm formation in TSB with 5% NaCl or 5% glucose + 5% NaCl at 25ºC.

Table 2.3. Component score coefficients matrix obtained from the PCA for the eight relevant parameters selected, which account for 79.3% of the total variance.

Indicator Condition PC 1 PC 2

TSB 37ºC 24h 0.843 ‒0.254 TSB + 5% glucose 25ºC 48h 0.754 0.122 TSB + 5% glucose 37ºC 24h 0.921 ‒0.055 TSB + 5% glucose 37ºC 48h 0.928 ‒0.003 TSB + 5% NaCl 25ºC 48h 0.030 0.938 TSB + 5% glucose + 5% NaCl 25ºC 48h ‒0.072 0.949 TSB + 5% glucose + 5% NaCl 37ºC 48h 0.885 ‒0.127

TSB + 1 mM MgCl2 37ºC 24h 0.825 0.108

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S. aureus isolates were located in a scatter plot based on the results from both PC obtained (Figure 2.4). They were distributed in four groups, each one corresponding to a defined quadrant. Considerable variations in the ability to develop biofilms on polystyrene were showed by the isolates under environmental conditions selected.

Figure 2.4. PCA score plot of the S. aureus strains (n = 28) for first two components. PC1: impact on biofilm formation of glucose at 25ºC and glucose, glucose + NaCl and MgCl2 at 37ºC. PC2: impact of NaCl and glucose + NaCl on biofilm formation at 25ºC. First quadrant delimited by a solid line; second quadrant delimited by dots; third quadrant delimited by broken lines; fourth quadrant delimited by dots inserted between broken lines.

Five isolates were distributed in the first quadrant (delimited by a solid line), which showed a biofilm formation ability significantly influenced by the addition of 5% NaCl alone or together with 5% glucose at 25ºC. Both the biofilm former reference strain ATCC 6538 as well as the two isolates carrying sea, sec and seh genes (St.1.07 and St.1.24) tested in this study were located in this quadrant. However, the five strains had a different origin: ATCC 6538 were isolated from a human lesion, St.1.07 and St.1.28 from fresh fish and St.1.20 and St.1.24 from precooked products.

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The second quadrant (delimited by dots) included six isolates which biofilm development on polystyrene was highly influenced by the environmental conditions selected, highlighting St.1.04 and St.1.12. However, they were isolated from seafood with a different processing: St.1.01, St.1.06 and St.1.12 were isolated from smoked fish, St.1.02 and St.1.05 from precooked products and St.1.04 from a salted product.

The third quadrant (delimited by broken lines) clustered the highest number of strains (n = 10), including the antibiotic resistant strain ATCC 43300 and the two strains carriers of seg and sei genes (St.1.16 and St.1.19) of this study. Biofilm formation of these isolates was not significantly affected by the environmental conditions selected. Given that most of them were isolated from frozen (5) and fresh (2) products, other conditions such as cold temperatures could be the environmental limiting factor during biofilm formation of these isolates. Moreover, two strains (St.1.13 and St.1.15) of this group were isolated from shellfish growth by aquaculture, where the application of antibiotics is widely used.

Finally, the fourth quadrant (delimited by dots inserted between broken lines) grouped seven strains which biofilm formation was mainly influenced by the addition to TSB of 5% glucose at 37ºC. They were isolated from precooked (St.1.10, St.1.11 and St.1.14), smoked (St.1.22) and salted (St.1.23) products, and two from products made with squids (St.1.09 and St.1.30).

From these results, S. aureus St.1.07, St.1.14 and St.1.29 strains were selected for their characteristic biofilm-forming ability under food-related environmental stresses tested to investigate the expression of different genes involved in biofilm formation.

2.3.5 Gene expression in relation to biofilm formation

The genes icaA, rbf, sarA and σB are reported to be involved in the regulation of biofilm formation. Their statistical significant (P < 0.05) changes in expression were investigated under different biofilm promoting growth conditions (TSB with 5% glucose, 5% NaCl or 5% glucose + 5% NaCl) and compared with expression in TSB by reverse transcriptase real-time PCR for the three selected strains. All the genes were highly expressed in TSB (CT ≤ 30), with significant (P < 0.05) differences between the strains. Thus, St.1.14 showed the highest expression of icaA (CT = 26.9) and rbf (CT =

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Impact of food-related environmental factors on the biofilm formation of S. aureus

B 28.4) genes, whereas St.1.07 expressed remarkably genes sarA (CT = 22.7) and σ (CT = 24.6).

Each strain showed a different expression pattern of the analysed genes under the different growth conditions tested (Figure 2.5). The most variable expression was observed in icaA gene. An additive effect on icaA expression was seen in St.1.07 when both NaCl and glucose were added, whereas icaA expression in St.1.29 was down- regulated in high NaCl conditions (without glucose additions) and up-regulated by the presence of glucose in the medium. In contrast, icaA expression in St.1.14 was highly affected by the presence of NaCl, while an up-regulation was observed upon glucose addition.

Figure 2.5. ΔCt for the expression of genes icaA, rbf, sarA and σB in three S. aureus strains (St.1.07, St.1.14 and St.1.29) under different conditions compared to expression in TSB. Different letters on the top of each column show significant differences (P < 0.05) in the expression of these genes at each condition tested between the selected strains.

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Otherwise, the genes rbf, sarA and σB were also highly expressed by the three strains selected. In St.1.07, the expression of these genes was up-regulated by NaCl with a dominant down-regulating effect of glucose. For strain St.1.14, σB expression was increased when glucose, NaCl or both were added, whereas the presence of glucose in the medium had a dominant effect in the expression of the other genes, leading to up- regulation of rbf and down-regulation of sarA. Finally, an additive effect on rbf expression was seen in St.1.29 when both NaCl and glucose were added, whereas expression of sarA and σB was up-regulated by glucose addition.

2.4 Discussion

The present study showed considerable variations between the adhesion and biofilm formation properties of 26 natural S. aureus isolates from seafoods on polystyrene surfaces under different food-related environmental stress conditions. This surface is frequently used in the food industry, above all in the packaging of products, and its bacterial colonization may cause food-spoilage, consequently increasing risk for the consumer health as well as subsequent economic losses due to recalls of contaminated food products.

Bacterial adhesion to surfaces is directly correlated with cell surface hydrophobicity (Rosenberg, 1981; Pagedar et al., 2010). According to our results, all S. aureus strains expressed moderate hydrophobicity, suggesting a lower initial adhesion to hydrophobic polystyrene compared to hydrophilic surfaces such as glass. Mafu et al. (2011) also reported a moderate hydrophobicity and a low tendency to attach to polystyrene in S. aureus, but a single strain was used.

The electrostatic interactions between the tested S. aureus strains and polystyrene surface showed a significantly (P < 0.01) higher adhesion when the ionic strength conditions were increased from 1.5 mM NaCl to 150 mM NaCl, except for strains St.1.08 and St.1.21. As previously reported (Habimana et al., 2007; Giaouris et al., 2009), adhesion at high ionic conditions was probably caused by the attenuation of repulsive electrostatic interactions between the highly negatively charged bacteria and the negatively charged polystyrene surface. The initial adhesion of S. aureus to polystyrene could therefore be enhanced in situations involving the use of seawater during seafood-processing, consequently increasing the risk of biofilm formation and

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Impact of food-related environmental factors on the biofilm formation of S. aureus cross-contamination. Therefore, the use of fresh water as a mean to reduce the attachment of negatively charged bacteria to polystyrene should be considered. Moreover, obtained results showed that adhesion was dependent on both tested strain and ionic strength conditions. A high variability in adhesion to polystyrene among S. aureus strains was observed for both ionic conditions, hence suggesting possible differences in cell wall electronegativity, as described by Giaouris et al. (2009) in Lactococcus lactis. To our knowledge, this is the first time that such variability of surface physicochemical properties is described for natural S. aureus strains from fisheries. Therefore, these findings provide important information for the development of novel surfaces and control strategies against the adhesion of natural S. aureus during processing, packaging and storage of food products, especially in fisheries.

Principal Components Analysis also showed a considerable variability in biofilm formation between the 26 S. aureus strains tested under relevant environmental conditions of temperature, osmolarity and nutrient content found during seafood production. Thus, isolates had generally higher biofilm production at 37ºC as expected, although four strains (St.1.14, St.1.16, St.1.24 and St.1.31) showed a significantly higher biofilm development during the first 48 h at 25ºC. Pagedar et al. (2010) also reported a higher cell count of S. aureus growth in TSB at 25ºC than at 37ºC after 48 h, but these biofilms were formed on stainless steel surfaces.

Meanwhile, the presence of glucose increased biofilm formation of all tested S. aureus, although significant differences between isolates were observed. This nutrient is considered a limiting factor of biofilm formation due to its requirement during the production of the extracellular matrix components (Ammendolia et al., 1999). Therefore, our results are in totally agreement with those obtained by Rode et al. (2007), considering that the presence of glucose promotes biofilm formation in S. aureus. In fisheries, glucose is an additive frequently used to reduce the water activity of products, above all in surimis and smoked fish. Data obtained in this study showed that the presence of glucose significantly influenced biofilm formation of most isolates (70%) from surimis and smoked fish, as shown their distribution in the PCA score plot. Thus, the presence of glucose in these products could potentially increase the contamination by S. aureus, involving a serious risk for the health of consumers and probable economic losses.

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Another important environmental factor is the amount of NaCl present on food- processing surfaces, which could be increased by the presence of seawater and seafood wastes generated during seafood production. Different authors showed that NaCl could promote bacterial aggregation and enhanced the stability of biofilms in polystyrene (Møretrø et al., 2003; Rode et al., 2007). However, the addition of NaCl generally decreased the biofilm formation of tested S. aureus strains at 37ºC, whereas it was improved at 25ºC. Xu et al. (2010) also reported that the number of adhered cells of S. aureus ATCC 12600 in polystyrene was higher in a medium without NaCl for the first 48 h at 37ºC. A possibility proposed by Lim et al. (2004) could be the repression of biofilm formation either directly or through overexpression of rbf gen with concentrations of 5% NaCl approximately. However, a rather average expression of rbf gen was observed in this study during transcriptional analysis by qRT-PCR of S. aureus St.1.07, St.1.14 and St.1.29 ‒isolates selected by their characteristic biofilm-forming properties for PCA‒ when they were growth in TSB added with 5% NaCl. Rachid et al. (2000) described an osmotic stress resistance and biofilm formation induced by σB, but a lower expression of σB was reported in S. aureus St.1.29, which had a remarkable biofilm formation in the presence of 5% NaCl compared to those grown in the absence of salt. Similarly, the presence of 5% NaCl also caused the down-regulation of the biofilm-forming promoter sarA in St.1.29, whereas a higher expression was detected in the other two strains tested under these osmolarity conditions. Therefore, these results indicate a great variability of regulatory responses against osmolarity stress conditions during the development of staphylococcal biofilms. Further investigations (e.g. using knock-out mutants) should be done in the future to deepen this study. Results of such studies could lead to new biofilm control strategies on food-contact surfaces.

Several authors also indicated the influence of MgCl2 in the adhesion to food-contact surfaces of Staphylococcus spp. (Barnes et al., 1999; Dunne, 2002; Akpolat et al., 2003; Planchon et al., 2006). In fisheries, both seawater and seafood wastes are an important source of magnesium. However, biofilm formation of S. aureus isolates tested in this study generally was not affected by the presence of MgCl2, although rather favoured after 48 h at 37ºC. These results are in accordance as those previously reported, suggesting that MgCl2 are implicated in biofilm stabilization at optimal growth conditions.

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The results obtained in this study hence supported that environmental conditions found in the food industry affected the adhesion and biofilm formation in S. aureus. Different regulatory pathways are involved in biofilm development of S. aureus highlighting the ica operon, which is associated in the regulation of extracellular matrix synthesis (Cramton et al., 1999). Several authors reported that the addition of glucose, NaCl or both together promote biofilm formation by inducing the ica operon in S. aureus (Møretrø et al., 2003; Rode et al., 2007). In this study, all the tested strains carried icaA and icaD (see Appendix 1). Moreover, an increase in icaA expression with the addition of glucose was also observed during transcriptional analysis by qRT-PCR of the selected S. aureus isolates St.1.07, St.1.14 and St.1.29. However, although icaA expression remained high, biofilm formation was lowered when both glucose and NaCl were added, suggesting that other ica-independent pathways are implicated as proposed previously different authors (Fitzpatrick et al., 2005; Kogan et al., 2006). Other internal factors supposedly involved in the initial adhesion to surfaces and host molecules and in the intercellular adhesion are the biofilm-associated proteins or Bap (Cucarella et al., 2002). However, none of the natural S. aureus isolates from seafoods carried bap gen (see Appendix 2). These results are in accordance with Vautor et al. (2008), which concluded that the prevalence of this gene among S. aureus isolates should be very low. In fact, the bap gene has only been identified in a small proportion of S. aureus strains originating from bovine mastitis (Cucarella et al., 2001).

2.5 Conclusions

According to results obtained in the present study, natural S. aureus seems to show a high ability to adhere and form biofilms on polystyrene surfaces. Food-contact surfaces made of this material can thus be a hazardous reservoir for S. aureus in the food industry and, therefore, an important source of food contamination unless appropriate food safety procedures are applied.

Our results also support that staphylococcal biofilm formation is influenced by environmental conditions relevant for the food industry such as temperature, osmolarity, nutrients content and cell surface properties. In fact, considerable variations in biofilm- forming ability were observed between the different strains tested under these environmental conditions. Therefore, the prevalence of S. aureus isolates on food-

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These findings are relevant for food safety and may be of importance when choosing the safest environmental conditions and material during processing, packaging and storage of seafood products. The maintenance of thermal conditions that avoid or reduce the bacterial growth in food products, the use of low-adherent materials in food- processing facilities as well as the application of proper cleaning and disinfection procedures to food-contact surfaces are essentials to ensure food safety.

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Appendix 1: PCR-detection of icaA and icaD genes

Two genes encoded in ica operon (icaA, icaD) were studied for all S. aureus strains. Genomic DNA was extracted from 24 h cultures in BHI using an InstaGene™ Matrix kit (Bio-Rad Laboratories, S.A., Spain) following manufacturer’s instructions. DNA was quantified by assuming that an absorbance value at 260 nm of 0.100 corresponds to 5 µg/mL of DNA. Primers ICAAF (5´-CCTAACTAACGAAAGGTAG-3´) and ICAAR (5´-AAGATATAGCGATAAGTGC-3´) for icaA gen, ICADF (5´-AAACGTAAGAGA GGTGG-3´) and ICADR (5´-GGCAATATGATCAAGATAC-3´) for icaD gen were used for each strain (Cramton et al., 1999). Expected size of amplified PCR products were 1315 bp for icaA gen, 381 bp for icaD. Each PCR mixture contained 100 ng DNA, 1x Taq Buffer Advanced, 2.5 U Taq DNA polymerase (5 Prime, Germany), 40 nmol of each dNTP (Bioline, UK), 0.25 nmol of forward and reverse primers (Thermo Fisher Scientific, Germany) and sterile Milli-Q water up to a final volume of 50 µL. PCR was performed with a MyCycler™ Thermocycler (Bio-Rad). PCR conditions consisted of denaturation at 92ºC for 5 min, 30 cycles at 92ºC for 45 s, 49ºC for 45 s and 72ºC for 1 min, and a final cycle at 72ºC for 7 min (Vasudevan et al., 2003). PCR products were subjected to electrophoresis on 1.5% agarose gel containing ethidium bromide for 90 min at 75 V and 100 mAmp. Gels were photographed in a Gel Doc XR system (Bio- Rad) using the Quantity One® software (Bio-Rad). A DNA ladder of 50-2000 bp (Hyperladder II, Bioline) was included as a molecular size marker.

Figure Ap.1A-B. Agarose gels showing PCR products obtained for all S. aureus strains during detection of genes icaA (A) and icaD (B). Lane 1 and 17, DNA molecular size marker (HyperLadder II, 50-2000 bp; Bioline); lanes 2-3, reference strains S. aureus ATCC 29213 and S. aureus ATCC 43300, respectively; lanes 4-16 and 18-30, amplicons of each strain; lane 31, blank.

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Appendix 2: PCR-detection of bap gene The presence of gene involved in the production of biofilm-associated proteins (bap) was investigated for all S. aureus strains. DNA was extracted with DNeasy® kit (Qiagen, Germany) according to the manufacturer. Extraction was tested by using λ HindIII DNA Ladder as a reference (564-23130 bp) (New England BioLabs™, USA). Primers sasp-6m (5´-CCCTATATCGAAGGTGTAGAATTGCAC-3´) and sasp-7c (5´- GCTGTTGAAGTTAATACTGTACCTGC-3´) were used (Cucarella et al., 2004). The expected size of amplified PCR products was 971 bp. PCR mixtures were composed of 20 ng of DNA; 5 nmol of each dNTP (Invitrogen Corporation, USA); 2.5 µL of Dynazym buffer 10x and 1.2 U of Dynazym Hot Start (Bio-Rad); 10 pmol of forward and reverse primer and sterile Milli-Q water up to a final volume of 25 µL. PCRs were performed with an 80 Gene Amp PCR System 9700 (Applied Biosystems, USA). PCR conditions consisted of denaturation at 94ºC for 2 min, 40 cycles of 94ºC for 30 s, 55ºC for 30 s and 72ºC for 75 s, and a last extension at 72ºC for 5 min (Vautor et al., 2008). S. aureus ATCC 29213 was used as negative control, whereas S. aureus ATCC 43300 was used as positive control. Amplicons were subjected to electrophoresis on 1.2% agarose gel containing ethidium bromide for 30 min at 100 V and 200 mAmp. Gels were visualized and saved in a Typhoon Scanner 8600 (Molecular Dynamics, GE Healthcare, UK). A DNA ladder of 154-2176 bp (DNA Molecular Weight Marker VI, Roche Applied Science, USA) was included in all gels.

Figure Ap.2. Agarose gel showing PCR products obtained for all S. aureus strains during detection of bap gene. Lane 1 and 17, DNA molecular size marker (DNA Molecular Weight Marker VI, Roche Applied Science); lanes 2-3, reference strains S. aureus ATCC 29213 and ATCC 43300; lanes 4-16 and 18-30, amplicons of each strain; lane 31, blank; lane 32, bap- positive strain S. aureus V329 (Cucarella et al., 2001).

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Chapter 3. Biofilm-forming ability and resistance to industrial disinfectants of Staphylococcus aureus isolated from fishery products

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Biofilm-forming ability and resistance to industrial disinfectants of S. aureus

* Corresponding author at: Instituto de Investigaciones Marinas (CSIC), Eduardo Cabello 6, 36208, Vigo Spain. Tel.: +34 986 231 930; fax: +34 986 292 762. E-mail address: [email protected] (J.J. Rodríguez-Herrera). Abstract Adhesion and biofilm-forming ability of twenty six S. aureus strains previously isolated from fishery products on stainless steel was assessed. All strains reached counts higher than 104 CFU/cm2 after 5 h at 25°C. Most strains also showed a biofilm-forming ability higher than S. aureus ATCC 6538 ‒reference strain in bactericidal standard tests‒ by crystal violet staining. In addition, it seems that food-processing could have produced a selective pressure and strains with a high biofilm-forming ability were more likely found in highly handled and processed products.

The efficacy of the industrial disinfectants benzalkonium chloride (BAC), sodium hypochlorite (NaClO) and peracetic acid (PAA) against biofilms and planktonic counterparts was also examined in terms of minimum biofilm eradication concentration (MBEC) and minimum bactericidal concentration (MBC), respectively. Biofilms showed an antimicrobial resistance higher than planktonic cells in all cases. However, no correlation was found between MBEC and MBC, likely due to differences in biofilm extracellular matrix (composition, content and architecture) between strains. BAC resistance increased as biofilms aged. Generally, biofilm formation seemed to attenuate the effect of low temperatures on BAC resistance. PAA was found to be most effective against both biofilms and planktonic cells, followed by NaClO and BAC. Resistance did not follow the same order for each biocide, which remarks the need of using a wide collection of strains in standard tests of bactericidal activity to ensure a proper application of disinfectants. Doses recommended by manufacturers for BAC, PAA and

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NaClO to disinfect food-contact surfaces were lower than doses for complete biofilm removal (i.e. MBEC) under some environmental conditions common in the food industry, which questions bactericidal standard tests and promotes the search for new strategies for biofilm removal.

Keywords: Staphylococcus aureus; biofilm; benzalkonium chloride; peracetic acid; sodium hypochlorite.

3.1. Introduction

Many disinfectants are used to kill microorganisms in the food industry, being quaternary ammonium compounds, chlorine-based biocides and peroxides among most used. However, bacteria form biofilms on any kind of surface, which allows them to tolerate the presence of biocides much better than free-living counterparts (Bridier et al., 2011a; Donlan and Costerton, 2002; Van-Houdt and Michiels, 2010). The resistance of biofilms to biocides can increase the persistence of bacterial pathogens in the food environment (Srey et al., 2013; Van-Houdt and Michiels, 2010). In fact, an improper use of biocides (due to short exposure times, sub-lethal doses or unsuitable equipment designs) can provoke an increase in incidence or even the emergence of antimicrobial- resistance (Gibson et al., 1999; Langsrud et al., 2003; Sharma and Anand, 2002). Controversy has therefore arisen over the validity of official standardised tests EN 1040 (CEN, 2005), EN 1276 (CEN, 2009) and EN 13697 (CEN, 2002) applied in the European Union to determine the bactericidal efficacy of disinfectants, particularly if they truly simulate conditions found in the food industry as well as if they cover the spectrum of biological variation that can be found in nature (Briñez et al., 2006; Langsrud et al., 2003; Meyer et al., 2010). Staphylococcus aureus is a major foodborne bacterial pathogen causing food poisoning due to the ingestion of food containing staphylococcal enterotoxins (Bhatia and Zahoor, 2007; EFSA, 2010; Le-Loir et al., 2003). S. aureus can spread from food handlers and food-contact surfaces to food products throughout the food chain (DeVita et al., 2007; Sattar et al., 2001; Simon and Sanjeev, 2007; Sospedra et al., 2012). Nonetheless, recent changes in Spanish national regulations (RD 135/2010) following Commission Regulation (EC) No 2073/2005 have revoked the use of S. aureus as a microbiological criterion for a number of foods, including several fishery products.

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However, a high incidence of S. aureus (~ 25%) has been recently found in fishery products marketed in Spain (Vázquez-Sánchez et al., 2012). Being Spain the largest producer and the second largest consumer of seafood in the European Union (Eurostat, 2010; FAO, 2012), the safety of seafood produced or consumed in Spain is of outmost importance. The present study was therefore aimed to examine if the formation of biofilms could be a potential source of contamination with S. aureus in industries processing fishery products. With this aim, the biofilm-forming ability of the strains previously isolated from fishery products and the resistance of such biofilms to three traditional biocides (benzalkonium chloride, peracetic acid and sodium hypochlorite) was determined.

3.2. Material and Methods

3.2.1. Bacterial strains and culture conditions Twenty six S. aureus strains isolated from fishery products from different countries of origin were investigated (Table 3.1). They have been previously identified as S. aureus by specific biochemical (coagulase, DNAse and mannitol fermentation) and genetic tests (23s rDNA) (Vázquez-Sánchez et al., 2012). Strains were also formerly characterized by RAPD-PCR and considered to be putative enterotoxigenic strains because they carried se genes (Vázquez-Sánchez et al., 2012). S. aureus ATCC 6538, which is used as a reference gram-positive strain in United States and European bactericidal standard tests, was provided by the Spanish Type Culture Collection. Bacterial stocks of each strain were maintained at ‒80°C in tryptic soy broth (TSB) (Panreac Química, Spain) containing 20% glycerol (v/v). All strains were thawed and subcultured twice in TSB at 37°C for 24 h under static conditions prior to each experiment.

3.2.2. Conditions for biofilm formation Stainless steel coupons (AISI 304, 2B finish) (Markim Galicia S.L., Spain) of approximately 10 mm × 10 mm (and 0.8 mm thickness) were used as experimental surfaces. Coupons were soaked in 2 M NaOH to remove residues, rinsed several times with distilled water, air-dried and autoclaved before use. One sterile coupon was placed into each well of a sterile 24-well flat-bottom microtiter plate (Falcon®, Becton Dickinson Labware, USA).

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Table 3.1. Origin and putative enterotoxigenicity (as se gene carried) of S. aureus strains studied. Strain Fishery product Type of processing Producing country se gene St.1.01 Smoked swordfish Smoking Spain (Andalucía) a St.1.02 Shellfish Paella Precooking Spain (Valencia) a St.1.03 Smoked swordfish Smoking Spain (Andalucía) a St.1.04 Mojama (salted dry tuna) Salting Spain (Valencia) a St.1.05 Herring in Madeira sauce Precooking Denmark a St.1.06 Smoked angel fish Smoking Spain (Andalucía) a St.1.07 Fresh cuttlefish Fresh France a, c, h St.1.08 Surimi elver Precooking Spain (Basque Country) a St.1.09 Fresh squid Fresh France a St.1.10 Surimi elver Precooking Portugal a St.1.11 Elver with shrimps Precooking Denmark a St.1.12 Smoked tuna Smoking Spain (Andalucía) a St.1.13 Frozen shellfish paella Freezing Spain (Galicia) a St.1.14 Surimi elver Precooking Spain (Galicia) a St.1.15 Frozen shellfish paella Freezing Spain (Galicia) a St.1.16 Perch fillet Fresh France g, i St.1.19 Frozen cod Freezing Spain (Galicia) g, i St.1.20 Elver with shrimps Precooking Portugal a St.1.21 Frozen Shelled prawn Freezing Spain (Valencia) a St.1.22 Smoked trout Smoking Spain (Catalonia) a St.1.23 Salted cod Salting Spain (Galicia) a St.1.24 Cod pâté with peppers Precooking Spain (Valencia) a, c, h St.1.28 Panga fillet Fresh Vietnam a St.1.29 Panga fillet Fresh Vietnam a St.1.30 Frozen Squid ring Freezing Spain (Galicia) a St.1.31 Frozen hake nuggets Freezing Spain (Galicia) a

Overnight bacterial cultures were adjusted to an absorbance value at 700 nm of 0.100 ± 0.01 with phosphate buffer saline (PBS, composed by 7.6 g/L NaCl, 0.2 g/L KCl and

0.245 g/L Na2HPO4 (BDH Prolabo, VWR International Eurolab, Spain); and 0.71 g/L

K2HPO4 (Panreac Química, Spain)). This value corresponds to a cell concentration of approximately 108 CFU/mL for all strains. PBS-suspended cells were then serially diluted in TSB, and a 700 μL aliquot (containing approximately 7×105 CFU) was added into each well with a coupon. Inoculum size was checked in all cases by plating on tryptic soy agar (TSA) (Cultimed, Panreac Química, Spain). A negative control with no inoculum was included in all assays. Microplates were incubated at 25°C under static conditions until analysis.

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3.2.3. Biofilm formation assays

3.2.3.1. Slime production on Congo red agar (CRA)

A slight modification of the method proposed by Freeman et al. (1989) was used. CRA was composed of 37 g/L brain heart infusion broth (Biolife, Italy), 36 g/L sucrose (Probus, Spain), 10 g/L agar-agar (Scharlau, Spain) and 0.8 g/L Congo red stain (Panreac Química, Spain). Congo red solution was autoclaved separately and then added to sterile agar medium at 55°C. A 0.1 mL inoculum of each S. aureus strain was spread on CRA (by duplicate), incubated at 37°C for 72 h. Two plates were visually inspected for slime production each 24 h. The assays were repeated twice using independent bacterial cultures.

3.2.3.2. Initial adherence

After 5 h of incubation at 25°C, coupons were removed from microplates and washed with 1 mL of PBS for 10 s to eliminate non-adhered cells. Adhered cells were collected by thoroughly rubbing with two sterile swabs (Deltalab, Spain). Cells were resuspended by vortexing swabs vigorously for 1 min in 9 mL of peptone water (10 g/L triptone (Cultimed, Panreac Química, Spain) and 5 g/L sodium chloride (BDH Prolabo, VWR International Eurolab, Spain)). Ten-fold serial dilutions of resuspended cells were made in peptone water and aliquots of 0.1 mL of appropriate dilutions were spread on TSA plates. Plates were incubated at 37°C for 24 h. Four coupons were analysed for each strain and each assay was repeated twice using independent bacterial cultures.

3.2.3.3. Quantification of biofilm formation

Biofilm formation was quantified in terms of biomass after 5, 24 and 48 h of incubation at 25°C. At each sampling time, coupons were placed into a new microplate and washed with 1 mL of PBS for 10 s to remove non-adhered cells. Coupons were then air-dried and biofilms were fixed in Bouin´s solution (Sigma-Aldrich Química, Spain) for 45 min. Subsequently, coupons were further washed with 1 mL of PBS for 10 s to discard excess fixative. Once fixed, biofilms were stained with 0.5 mL of 0.5% (w/v) crystal violet solution (Panreac Química, Spain) for 15 min. Excess stain was rinsed off by placing microplates under running tap water. Coupons were air-dried and biofilm- bound crystal violet was solubilized in 1 mL of 33% (v/v) glacial acetic acid (BDH

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Prolabo, VWR International Eurolab, Spain) by shaking at 100 rpm for 30 min. Crystal violet acid solution was then transferred to a 96-well round-bottom microplate (Falcon®, Becton Dickinson Labware, USA) and absorbance was read out at 595 nm using a iMark™ Microplate Absorbance Reader equipped with Microplate Manager software 6.0 (Bio-Rad Laboratories, Spain). Coupons without biofilms were used in each assay as a negative control to subtract background staining. Six coupons were analysed for each strain at each sampling time and all assays were repeated twice using independent bacterial cultures.

3.2.3.4. Determination of growth kinetics

A set of test tubes containing 5 ml TSB each was inoculated with a 100 μL aliquot (containing approximately 105 CFU/mL) from bacterial cultures prepared as aforementioned. Cultures were incubated at 12°C and 25°C for 14 and 8 days, respectively, under static conditions. Absorbance was read out at 700 nm after each 24 h of incubation. Three replicates were used each day for each strain. A negative control without inoculum was included in all assays. Growth kinetics was determined using two independent bacterial cultures. Once obtained, experimental data of growth were fitted to the Gompertz equation proposed by Zwietering et al. (1990):

( ) { [( ) ]}

where ODt is the optical density at 700 nm at time t (days), OD0 is the optical density at the time of inoculation, A is a dimensionless asymptotic value, µmax is the maximum specific growth rate (1/days) and λ is the detection time (days), i.e., the time at which OD at 700 nm is firstly noted to increase.

3.2.4. Biocide resistance assays

Three traditional chemical disinfectants were tested: benzalkonium chloride (50% (v/v) BAC solution, Guinama Absoluta Calidad, Spain), peracetic acid (40% (v/v) PAA solution in acetic acid:water, Fluka, Sigma-Aldrich Química, Spain) and sodium hypochlorite (NaClO, Scharlau, Spain). BAC was studied in a greater detail than PAA and NaClO as BAC is more widely used for disinfection of stainless steel surfaces in the food industry. Disinfectants were diluted in ultrapure water to working concentrations

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Biofilm-forming ability and resistance to industrial disinfectants of S. aureus just before each assay. Each concentration was evaluated in triplicate in two independent experiments.

3.2.4.1. Minimal biofilm eradication concentration (MBEC)

The resistance of biofilms was assessed in terms of the minimal biofilm eradication concentration (MBEC), which was defined as the lowest disinfectant concentration that kills all biofilm cells under the experimental conditions tested. Once non-adhered cells were removed, three coupons were exposed to 1.5 mL of each disinfectant concentration for 30 min. Thirteen different concentrations were tested for BAC (5000, 7500, 10000, 12000, 14000, 16000, 18000, 20000, 22000, 24000, 26000, 28000 and 30000 mg/L), twelve for PAA (1000, 1500, 2000, 2500, 3000, 3500, 4000, 5000, 7000, 9000, 11000 and 13000 mg/L) and eleven for NaClO (5000, 6000, 7000, 8000, 9000, 10000, 12000, 14000, 16000, 18000, 20000 mg/L). Afterwards, coupons were placed into sterile glass vials and 9 mL of neutralizing broth (0.34 g/L KH2PO4 and 5 g/L Na2S2O3 (Probus, Spain); and 3 g/L soy lecithin, 1 g/L L-histidine and 3% (v/v) polysorbate 80 (Fagron Iberica, Spain)) was added and left to stand for 10 min at room temperature, as proposed by Luppens et al. (2002). Finally, each coupon was incubated in TSB at 37°C for 24 h and bacterial growth was monitored visually. In addition, visually undetectable growth was checked by plating an aliquot of 0.1 mL of culture medium on TSA and searching for the presence of colonies after 24 h at 37°C. In any case, bacterial growth indicated the presence of viable cells in disinfectant-treated biofilms.

3.2.4.2. Minimal bactericidal concentration (MBC)

The resistance of planktonic cells was assessed in terms of the minimum bactericidal concentration (MBC), which was considered to be the lowest disinfectant concentration necessary to kill all free-living bacterial cells under the experimental conditions tested. Planktonic counterparts (i.e. non-adhered cells) were diluted in TSB to achieve a similar cell concentration to that in biofilms. Planktonic cells (0.1 mL) were exposed to each disinfectant concentration (0.1 mL) for 30 min (by triplicate) and then immediately neutralized (2.0 mL). Eleven different concentrations were tested for BAC (750, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 6000, 7000 and 8000 mg/L), PAA (100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 750 mg/L) and NaClO (500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1000 mg/L). An aliquot of 0.3 mL of each neutralized

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Chapter 3 disinfectant-treated bacterial culture was added to 1.7 mL of TSB and incubated at 37°C for 24 h. Bacterial growth was monitored as for MBEC.

3.2.5. Statistical analysis

Experimental results were statistically analysed with the software packages Microsoft Excel 2010 and IBM SPSS 19.0. Statistical significance analysis was carried out using a one-way ANOVA. Homogeneity of variances was examined by a post-hoc least significant difference (LSD) test. Otherwise, a Dunnett´s T3 test was performed. An independent-samples Student´s t-test was also done to determine if there were statistical differences between pairs of strains. A bivariate correlation analysis was conducted using Pearson’s correlation coefficient to measure the strength of the linear relationship between two variables. Statistical significance was accepted at a confidence level greater than 95% (P < 0.05). Occasionally, a level greater than 99% (P < 0.01) is considered to remark differences between variables. The variability among strains was calculated by means of the coefficient of variation (CV), which is defined as the ratio of the standard deviation to the mean.

3.3. Results

3.3.1. Biofilm-forming ability of S. aureus

The biofilm-forming ability of a number of S. aureus strains isolated from food products was compared with that of S. aureus ATCC 6538. This strain is used as a reference in several antimicrobial tests, including the quantitative surface test (EN 13697) of bactericidal activity of disinfectants used in food, industrial, domestic and institutional areas, on the basis that it is a biofilm former.

3.3.1.1. Detection of biofilm-forming ability on Congo red agar (CRA)

Biofilm-forming ability of each strain was also visually investigated by the CRA plate test following the colorimetric scale proposed by Arciola et al. (2002). Colonies coloured from very black to almost black have been described as typical of a biofilm- positive phenotype, whereas negative slime producers form colonies coloured from very red to burgundy. All strains formed black colonies after 24 h, with colour changing

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Biofilm-forming ability and resistance to industrial disinfectants of S. aureus from red to black in the surrounding agar too. Thus, all strains showed slime production ability after 24 h. All of them maintained this biofilm-positive phenotype during the following 48 h. No strain formed red colonies onto CRA.

3.3.1.2. Initial adhesion studies

The adhesion of strains to stainless steel was determined after 5 h at 25°C. The number of cells adhered was higher than 104 CFU/cm2 for all strains. A small variability was found among strains (CV of 4.0%), but significant differences (P < 0.05) were observed between strains (Figure 3.1). S. aureus St.1.01, St.1.09 and St.1.30 showed the highest adhesion ability (6-7×104 CFU/cm2), which was significantly higher than that of S. aureus ATCC 6538. On the contrary, S. aureus St.1.03, St.1.06 and St.1.13 showed the lowest number of adhered cells (< 2×104 CFU/cm2).

Figure 3.1. Adherence of S. aureus strains to stainless steel. Adherence was assessed after 5 h at 25°C. Statistically significant differences (P < 0.05) are indicated by different letters.

3.3.1.3. Quantification of biofilm biomass

Biofilm formation was quantified in terms of biomass by using the crystal violet staining method. Biofilm biomass of all strains increased continuously (and significantly, P < 0.01) during incubation for 48 h at 25°C, except for the case of St.1.28 between 24 and 48 h (Figure 3.2A-C). The increase was higher for most of the strains under study than for S. aureus ATCC 6538. Thus, only three strains (St.1.09, St.1.30 and St.1.31) showed a biofilm-forming ability significantly higher (P < 0.05) than S. aureus ATCC 6538 after 5 h, but the majority showed a biofilm biomass significantly higher (P < 0.05) after 24 h (76.9%) and 48 h (69.2%). A high inter-strain variability in

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Chapter 3 biofilm production ‒involving statistically significant (P < 0.05) differences‒ was found at each time of study (CV of 36.4%, 30% and 29.7% after 5, 24 and 48 h, respectively). A positive correlation (r = 0.890, P < 0.01) between the number of adhered cells and biofilm density was also found after 5 h at 25°C.

Interestingly, 48-h-old biofilm biomass was found to be positively correlated (r = 0.401, P < 0.01) with the type of processing applied to products that strains were isolated from (Table 3.1). In fact, eight strains out of the ten with highest biofilm biomass were found in highly-processed products (including smoked products, salted fish and precooked products), whereas seven strains out of the ten with lowest biofilm- forming ability were isolated from low-processed products (i.e., fresh and some frozen products).

Figure 3.2A-C. Biofilm-forming ability of S. aureus on stainless steel. Biofilm-forming ability was measured after 5 h (A), 24 h (B) and 48 h (C) at 25°C in terms of biofilm biomass by crystal violet staining. Different letters denote statistically significant differences (P < 0.05).

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3.3.2. Resistance to industrial disinfectants of S. aureus

3.3.2.1. Effectiveness of benzalkonium chloride

3.3.2.1.1. Variability in BAC resistance

The resistance to benzalkonium chloride (BAC) was firstly assessed in 48-h-old biofilms and planktonic counterpart cells. As shown in Figure 3.3, the resistance of both biofilms and planktonic cells showed a high variability between strains (CVs of 27.1% and 34.7%, respectively). Thus, whereas nearly half of strains (44.4%) formed biofilms that only resisted up to 14000 mg/L (included ATCC 6538), seven others showed a much higher resistance (MBEC = 20000-26000 mg/L). In the case of planktonic cells, most strains (65.4%) resisted up to 2000-3000 mg/L, but St.1.09, St.1.11, St.1.28 and St.1.30 achieved MBC values of 4000 mg/L, 4-fold higher than that of ATCC 6538 (MBC = 1000 mg/L).

Figure 3.3. Effectiveness of BAC against S. aureus biofilms and planktonic cells after 48 h at 25°C. MBEC: Minimal Biofilm Eradication Concentration; MBC: Minimal Bactericidal Concentration.

As expected, biofilms formed by S. aureus on stainless steel showed a higher resistance to BAC than their planktonic counterpart cells, but this increase was highly strain-dependent. Thus, S. aureus St.1.12 biofilms showed a resistance 16-fold higher than planktonic cells, whereas St.1.11 biofilms, which had shown a high biofilm- forming ability, were only about 3-fold more resistant than planktonic counterparts. As an overall result, no correlation was found between MBEC and MBC, neither between MBEC and 48 h biofilm biomass.

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From these results, those strains highly-BAC resistant (St.1.01, St.1.03, St.1.07, St.1.14, St.1.23, St.1.24, St.1.28, St.1.30 and St.1.31) were chosen for the subsequent studies. S. aureus St.1.04, St.1.08 and St.1.10 were also selected for these studies on the basis of a higher incidence in fishery products (Vázquez-Sánchez et al., 2012) and a higher risk to food safety associated to the type of product that they were isolated from (Table 3.1).

3.3.2.1.2. Effects of growth kinetics on BAC resistance

Foreseeing a different response during the time course of biofilm formation, the resistance to BAC of biofilms formed by the strains selected was determined after 5, 24, 48 and 168 h at 25°C. The resistance of planktonic counterparts after 24, 48 and 168 h was also determined for the interest in studying post-exponential and stationary-phase cells able to express antimicrobial resistance responses.

The development of biofilms induced a progressive increase in BAC resistance (Figure 3.4A), particularly during the first 24 h, when most of strains (83.3%) showed MBEC values at least 2-fold higher than after 5 h. MBEC was thus only found to be positively correlated with biofilm biomass after 24 h (r = 0.558, P < 0.01), but no after 5 or even 48 h. In fact, the increase slowed down subsequently, with most strains (75%) displaying only slight increases (2000 mg/L) or remaining similar otherwise. Nevertheless, 168-h-old biofilms of the majority of the strains (83.3%) were able to resist up to 20000-28000 mg/L, with St.1.01 forming the most resistant biofilms.

Figure 3.4A-B. Effectiveness of BAC against biofilms (A) and planktonic counterparts (B) after 5, 24, 48 and 168 h at 25°C.

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The resistance of planktonic cells also increased progressively as bacterial cultures aged, particularly during the first 48 h (Figure 3.4B). However, likewise in biofilms, increases slowed down or simply did not occur in most strains (75%) between 48-168 h. Nevertheless, 168-h-old planktonic cells of five strains were able to resist up to 4000- 5000 mg/L.

These results have shown that the formation of biofilms decreased markedly the efficacy of BAC from the first stages of biofilm development. In fact, the resistance of 5-h-old-biofilms was higher than that of 168-h-old planktonic cells in all cases. However, MBEC and MBC were only found to be positively correlated after 168 h of incubation (r = 0.603, P < 0.01). Interestingly, a positive correlation was found between initial adhesion and MBEC after 24 h (r = 0.637, P < 0.01), 48 h (r = 0.683, P < 0.01) and 168 h (r = 0.652, P < 0.01).

3.3.2.1.3. Effects of growth temperature on BAC resistance

The effects of the growth temperature on the BAC resistance of biofilms were analysed at 12°C and 25°C after 168 h of incubation. The use of 168-h-old biofilms is accounted for the interest in studying highly mature biofilms as examples of worst-case scenarios as well as for the slow development of biofilms at 12°C.

As shown in Figure 3.5A, the temperature affected the resistance of biofilms, with lower MBEC values at 12°C (10000-24000 mg/L) than at 25°C (12000-28000 mg/L). These differences can be at least partially accounted for the growth kinetics of these strains (see Appendix), particularly in terms of detection times (λ) much longer and maximum specific growth rates (µmax) much lower at 12°C than at 25°C (Table 3.2). For instance, λ at 12°C was positively correlated (r = 0.711, P < 0.01) with the decrease in resistance of biofilms. That is, the longer the λ, the higher the decrease in resistance between temperatures. Besides, differences in these parameters between strains were much higher at 12°C than at 25°C. Also, MBEC and cell density were found to be positively correlated at 12°C (r = 0.604, P < 0.01), but not at 25°C. Nevertheless, a positive correlation (r = 0.755, P < 0.01) was found between the resistance of biofilms at both temperatures.

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Figure 3.5A-B. Effectiveness of BAC against 168-h-old biofilms (A) and planktonic counterparts (B) grown at 12°C and 25°C.

Table 3.2. Detection time (λ) and maximum specific growth rate (µmax) of S. aureus strains grown at 12ºC and 25ºC. Letters denote statistically significant differences (P < 0.05).

λ (days) µmax (1/days) Strain 12ºC 25ºC 12ºC 25ºC St.1.01 2.09 ± 0.21c 0.00 1.25 ± 0.05cd 3.52 ± 0.05a St.1.03 8.15 ± 0.08a 0.00 1.44 ± 0.10b 2.81 ± 0.06bc St.1.04 0.47 ± 0.12f 0.00 0.77 ± 0.01g 2.58 ± 0.10de St.1.07 0.24 ± 0.06g 0.00 0.67 ± 0.12g 2.50 ± 0.16de St.1.08 1.77 ± 0.06c 0.00 0.99 ± 0.06ef 3.07 ± 0.19b St.1.10 1.83 ± 0.20c 0.00 1.59 ± 0.02a 3.89 ± 0.38a St.1.14 1.42 ± 0.18d 0.00 1.11 ± 0.08de 2.78 ± 0.05c St.1.23 1.36 ± 0.03d 0.00 0.91 ± 0.07f 2.39 ± 0.16e St.1.24 0.00h 0.00 0.97 ± 0.09ef 2.39 ± 0.17e St.1.28 0.88 ± 0.05e 0.00 1.19 ± 0.06d 3.51 ± 0.15a St.1.30 6.47 ± 0.51b 0.00 1.58 ± 0.30abc 2.73 ± 0.13bcd St.1.31 0.71 ± 0.17ef 0.00 0.91 ± 0.12f 2.19 ± 0.29e

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The effect of the temperature was particularly noticeable in S. aureus St.1.03, St.1.07 and St.1.30, with decreases in MBEC of 8000-10000 mg/L from 25°C to 12°C. This seems to be a consequence of the slow adaptation to low temperatures of these strains

(St.1.03 and St. 1.30 showed the longest λ and St.1.07 the lowest µmax in planktonic state of all strains at 12°C). In contrast, St.1.01 and St.1.14 biofilms seemed to be weakly affected by low temperatures as they also showed a high resistance at 12°C.

This could be partially accounted for the high µmax shown by these strains in the planktonic state. Surprisingly, St.1.24 showed a MBEC slightly lower at 25°C than at 12°C, which was presumably due in part to the absence of λ at 12°C.

The temperature also influenced the resistance of planktonic cells to BAC (Figure 3.5B), which showed a lower MBC at 12°C (1000-3000 mg/L) than at 25°C (2000-5000 mg/L). MBC was found to be positively correlated with cell density at 12°C (r = 0.670, P < 0.01), but not at 25°C. MBEC and MBC were also found to be positively correlated at 12°C (r = 0.736, P < 0.01). Unlike biofilms, however, the resistance of planktonic cells at 12°C and 25°C were not found to be correlated. The variability of BAC resistance was higher in planktonic cells than in biofilms, particularly at 12°C (CV of 38.5% and 26.5%, respectively).

3.3.2.2. Effectiveness of peracetic acid and sodium hypochlorite

The antimicrobial efficacy of peracetic acid (PAA) and sodium hypochlorite (NaClO) against 48-h-old biofilms and planktonic counterparts of the selected strains grown at 25°C was assessed and compared with that of BAC. The type strain ATCC 6538 was also included in this study.

Likewise for BAC, biofilms showed a much higher resistance than planktonic cells to PAA (up to 11-fold for St.1.01 and St.1.14) and NaClO (up to 23-fold for St.1.24) in all cases. However, strains were ranked in a different order according to the resistance of biofilms ‒and planktonic cells‒ to each disinfectant (Table 3.3). As a result, no correlation was found between the resistance of biofilms or planktonic cells to different disinfectants. No correlation was found either between MBEC and MBC or between MBEC for PAA, NaClO or BAC and biofilm biomass after 48 h of incubation at 25°C.

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PAA was found to be the most effective disinfectant against 48-h-old biofilms in all cases. In fact, PAA was up to 11-fold (St.1.31) and 9-fold (St.1.24) more effective against biofilms than BAC and NaClO, respectively. The efficacy of NaClO against biofilms was over 2-fold higher than BAC in many cases (50%) and similar in others (St.1.04 and St.1.10), whereas BAC was more effective than NaClO in one case (St.1.08).

PAA also showed the highest effectiveness against planktonic counterparts, up to 11- fold higher than BAC (St.1.28 and St.1.30) and 2-fold higher than NaClO in all cases. Meanwhile, NaClO was more effective than BAC in all cases, being up to 4-fold higher (St.1.03, St.1.28 and St.1.30).

Lastly, the resistance of biofilms to PAA showed higher variability (coefficient of variation of 33.4%) than to BAC (25.8%) and NaClO (14.4%). However, the resistance of planktonic cells showed the highest variability in the case of BAC (21.6%) followed by PAA (10.0%) and NaClO (6.1%).

Table 3.3. Effectiveness of BAC, PAA and NaClO against S. aureus biofilms and planktonic counterpart cells after 48 h at 25°C. MBEC: Minimal Biofilm Eradication Concentration; MBC: Minimal Bactericidal Concentration.

MBEC (mg/L) MBC (mg/L) Strain BAC PAA NaClO BAC PAA NaClO ATCC 6538 10000 1500 5000 1000 300 600 St.1.01 26000 4000 14000 2500 350 850 St.1.03 18000 3000 12000 3000 350 800 St.1.04 12000 4000 12000 2500 400 800 St.1.07 22000 4000 16000 2500 350 950 St.1.08 10000 2000 12000 2500 350 900 St.1.10 14000 1500 14000 2500 300 800 St.1.14 26000 4000 12000 2500 350 850 St.1.23 20000 4000 16000 2000 400 800 St.1.24 20000 2000 18000 2500 450 800 St.1.28 18000 2000 12000 4000 350 900 St.1.30 24000 2500 14000 4000 350 900 St.1.31 22000 2000 12000 2500 350 900

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3.4. Discussion

The results obtained in the present study have shown that a number of S. aureus strains (n = 26) isolated from fishery products have, in general, a high biofilm-forming ability on stainless steel surfaces and a high resistance to some disinfectants commonly used in the food industry. Accordingly, the entrance of these strains in food-processing facilities does not only involve an immediate risk to food safety but more importantly the risk of long-term presence (even persistence) unless appropriate measurements are applied to kill them.

Biofilm-forming ability was assessed in terms of biomass using the crystal violet staining method. It could thus be observed that all strains had a high biofilm-forming ability on stainless steel under experimental conditions simulating situations normally found in the food industry. Stainless steel, a common material in food-processing facilities, can thus serve as an important reservoir for S. aureus in the food industry. Several studies have also detected the presence of S. aureus biofilms on stainless steel food-processing surfaces (Bagge-Ravn et al., 2003; Sattar et al., 2001; Sospedra et al., 2012).

Biofilm biomass increased proportionally as biofilms aged. A high variability in biofilm biomass was found among strains throughout the time course of biofilm formation, which is in accordance with previous studies (Marino et al., 2011; Melchior et al., 2007; Rode et al., 2007). The risk of the presence of S. aureus would thus be highly dependent on both the strain and the age of the biofilm. As previously observed by the authors (Herrera et al., 2007), the importance of defining the biofilm formation kinetics (or at least some stages, as done here) seems thus clear to draw reliable conclusions.

Interestingly, a statistical trend was found between 48-h-old biofilm biomass and the type of processing applied to the fishery products that strains were isolated from. This trend seems to show a selective pressure of food-processing conditions on S. aureus. Thus, most strains isolated from highly-processed products showed a biofilm-forming ability higher than those from low-processed products. Generally, a high degree of food-processing involves a high degree of contact with surfaces and a high degree of handling of raw materials and intermediate products. As S. aureus is a major component

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Chapter 3 of human microbiome, a high degree of handling can enhance the spread of S. aureus to food and food-contact surfaces (DeVita et al., 2007; Sattar et al., 2001; Simon and Sanjeev, 2007; Sospedra et al., 2012), where it forms biofilms that increase the resistance to external stresses such as antimicrobial compounds, high salt contents, relatively high temperatures, etc. (Bridier et al., 2011a; Donlan and Costerton, 2002; Van-Houdt and Michiels, 2010).

Biofilm formation was also inspected by the CRA plate test, which is easier to perform and less time-consuming than staining methods. This method has been used successfully for the detection of biofilm-forming strains of S. epidermidis (Handke et al., 2004; Oliveira et al., 2006), but the interpretation of results has been controversial in the case of S. aureus, with phenotypic colony changes taking place during incubation and false negative being reported (Milanov et al., 2010; Vasudevan et al., 2003). In this study, only black-coloured colonies after 72 h of incubation were considered to be typical of a biofilm-positive phenotype, as Arciola et al. (2002) recommended. All strains formed black-coloured colonies after 24 h, and this biofilm-positive phenotype was maintained subsequently. These results are in accordance with those of crystal violet staining, and it contrasts with claims that CRA test results do not always correspond to biofilm production determined by staining methods (Jain and Agarwal, 2009; Oliveira et al., 2006). Its qualitative nature, however, limits significantly the scope of this method. According to Arciola et al. (2002), CRA test results were correlated to the presence of icaA and icaD genes (Vázquez-Sánchez et al., 2013), which are usually involved in staphylococcal biofilm formation. However, recent studies have observed a lack of correspondence between slime production and the presence of ica genes (Ciftci et al., 2009; Zmantar et al., 2010).

Biofilms formed by all strains showed a marked resistance to benzalkonium chloride, peracetic acid and sodium hypochlorite, being higher than ATCC 6538 in most cases. However, a high variation in biofilm resistance was observed between strains. Moreover, strains followed a different order according to the resistance of biofilms to each biocide, which reveals a different response between strains. Similarly, Melchior et al. (2007) had previously indicated that each strain requires a specific exposure time to each antimicrobial for complete biofilm removal. Therefore, the use of a wide collection of strains for the assessment of the bactericidal activity of disinfectants seems to be

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Biofilm-forming ability and resistance to industrial disinfectants of S. aureus necessary to ensure that they are correctly applied. However, the current European Union standard bactericidal tests (EN 1040, EN 1276 and EN 13697) use a small number of type strains (and only one S. aureus) to assess the efficacy of disinfectants. Doses considerably lower than the MBECs determined in this study under some conditions common in the food industry are thus often recommended by manufacturers for BAC (200-1000 mg/L), PAA (50-350 mg/L) and NaClO (50-800 mg/L) (Gaulin et al., 2011). Microorganisms could therefore be exposed to sub-lethal doses of disinfectants and this can generate the emergence of antimicrobial resistance (Langsrud et al., 2003; Sheridan et al., 2012). Whether or not standard bactericidal tests truly simulate conditions found in the food industry and clinical settings has been also subject of a long debate (Briñez et al., 2006; Langsrud et al., 2003; Meyer et al., 2010).

Likewise for biomass, the resistance of biofilms was found to increase as biofilms aged. Therefore, ensuring biofilm removal would need to adequate antimicrobial treatments to highly-mature biofilms, or a biofilm representing a worst-case scenario. Accordingly, it would be expected that the higher the biofilm biomass, the higher the MBEC. However, this was not always the case and, in fact, biomass and BAC resistance were found not to be correlated. This lack of correlation is likely accounted by a high variation in the expression of genes involved in biofilm formation (e.g. icaA, rbf and σB), such as it has been recently observed between different strains used in this study (Vázquez-Sánchez et al., 2013). This could in turn produce differences in the composition and architecture of the extracellular matrix and, therefore, in the stability and resistance of biofilms. A thorough knowledge of the composition and architecture of the extracellular matrix could thus be helpful to improve the efficacy of disinfection strategies. However, biofilms are highly complex and the extraction and purification of extracellular matrix components is difficult, so providing a complete biochemical profile still remains an important challenge (Flemming et al., 2007).

Although the resistance of biofilms was much higher than planktonic counterparts, no correlation was found between MBEC and MBC. Therefore, this lack of correlation is presumably not as much due to differences in cell resistance as to the protective role of the extracellular matrix (Bridier et al., 2011a; Donlan and Costerton, 2002; Russell, 2003). In fact, differences between MBEC and MBC increased as biofilms aged and the extracellular matrix developed. However, an unexpected positive correlation between

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MBEC and MBC of BAC was obtained after 168 h of incubation, probably due to cell detachment from mature biofilms caused by nutrient depletion (Hunt et al., 2004). S. aureus biofilms often detach in form of cell clumps which are partially protected from antimicrobials by the extracellular matrix (Fux et al., 2004; Hall-Stoodley and Stoodley, 2005). Although MBCs were much lower than MBECs, 168-h old planktonic cultures would have shown an antimicrobial behaviour more similar (and thereof correlated) to that of biofilms than in earlier stages of culture, when only free-living cells (and no cell clumps) would have been present.

The antimicrobial resistance was also affected by the temperature, as observed in 168-h-old biofilms and planktonic counterpart cells for BAC, with resistance being lower at 12°C than at 25°C. A similar effect had been previously reported (Meira et al., 2012; Taylor et al., 1999). This effect was highly strain dependent and this dependence can be partially accounted for the different adaptive response of each strain to low temperatures (as defined by the growth kinetics). The importance of the growth kinetics explains in turn that both MBEC and MBC were found to be positively correlated with cell density at 12°C, but no at 25°C, temperature at which all cultures were in stationary phase after 168 h of incubation. It also explains that the correlation between MBEC and MBC was higher at 12°C than at 25°C. Nonetheless, the development of biofilms attenuated the adverse effect of low temperatures on BAC resistance in most cases. Thus, the resistance of biofilms developed at 12°C and 25°C were found to be positively correlated, but no correlation was found in the case of planktonic cells.

Significant differences in the efficacy of the industrial disinfectants tested were observed. Peracetic acid was found to be the most effective against both biofilms and planktonic cells, followed by sodium hypochlorite and then benzalkonium chloride. Bridier et al. (2011b) had also reported that PAA was more effective than BAC against planktonic cells of several S. aureus strains. Meanwhile, PAA was found to have a higher effectiveness than NaClO against biofilms of S. aureus on different food-contact surfaces (Meira et al., 2012). The lack of correlation between the efficacies of disinfectants against biofilms is indicative of significant differences in the diffusion and reactivity of each disinfectant in biofilms, which also depends on the composition of the extracellular matrix. The high antimicrobial activity of PAA is considered to be accounted for a high oxidative potential, a non-specificity and a high reactivity against

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Biofilm-forming ability and resistance to industrial disinfectants of S. aureus intracellular enzymes, proteins and DNA as well as against bacterial cell membrane and extracellular matrix components, and also a small size enabling diffusion through the biofilm matrix (Bessems, 1998; Denyer and Stewart, 1998; Kitis, 2004; Saá-Ibusquiza et al., 2011). NaClO is a powerful oxidizing agent which disturbs the synthesis of DNA and reacts with intracellular proteins, cell wall and extracellular matrix components (Russell, 2003), but it has a limited diffusion in the biofilm (De-Beer et al., 1994). Meanwhile, BAC forms mixed micellar aggregates that solubilize membrane phospholipids and proteins and thus causes a decrease of fluidity and the appearance of hydrophilic voids in the membrane leading to a generalized and progressive leakage of cytoplasmic materials to the environment and eventually cell lysis (Gilbert and Moore, 2005). However, the interaction of BAC with extracellular matrix components slows down penetration into the biofilm (Bridier et al., 2011c).

The rotation and combination of biocides is widely accepted to prevent the emergence of antimicrobial-resistant strains. However, in practice, different disinfectants with similar cell targets or similar mechanisms of action are often applied, which increases the risk of cross-resistance, particularly in biofilms (Braoudaki and Hilton, 2004; Chapman, 2003; Langsrud et al., 2004; Saá-Ibusquiza et al., 2011; Schweizer, 2001). The introduction of novel antimicrobials (e.g. electrolyzed water, essential oils, ozone, etc.) as well as the development of new control strategies (e.g. incorporation of antimicrobials on surface materials, modification of the physicochemical properties of surfaces, etc.) could be an effective alternative to avoid, or at least reduce, the risk of biofilm formation and antimicrobial resistance. Moreover, the use of disinfectant compounds less corrosive to metal surfaces, less hazardous for the health of workers and more environmentally-friendly have to be imperative nowadays.

3.5. Conclusions S. aureus can spread from humans to food and food-contact surfaces. All strains tested in this study showed a significant ability to adhere and form biofilms on stainless steel surfaces. In fact, most showed a biofilm-forming ability higher than S. aureus ATCC 6538, a common reference strain in bactericidal standard tests. Stainless steel food-contact surfaces can thus be an important reservoir for S. aureus in the food

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Chapter 3 industry and play an important role as a source of food contamination unless appropriate protocols of cleaning and disinfection are applied.

It is well known that biofilms show a much higher resistance to disinfectants than planktonic counterparts. This has driven to some controversy on the validity of standard bactericidal tests used in the European Union, since most are suspension-based. In this study, no correlation was found between the resistance of biofilms to BAC, PAA and NaClO and that of planktonic cells. No extrapolation seems thus feasible. Nonetheless, a biofilm-based standard bactericidal test (EN 13697) has been developed, but it does not seem to truly simulate environmental conditions found in the food industry. Moreover, the resistance of the strains did not follow the same order for BAC, NaClO and PAA, which shows the present limitation of using a few type strains (and only one S. aureus) in standard tests in order to ensure a proper application of disinfectants. These different reasons could at least partially account for the fact that doses recommended by manufacturers for BAC, PAA and NaClO to disinfect food-contact surfaces are lower than the MBEC values determined in this study under some conditions common in the food industry and therefore are not able to guarantee biofilm removal.

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Biofilm-forming ability and resistance to industrial disinfectants of S. aureus

Appendix: Growth kinetics

Figures showed the growth kinetics of the twelve selected S. aureus strains (St.1.01, St.1.03, St.1.04, St.1.07, St.1.08, St.1.10, St.1.14, St.1.23, St.1.24, St.1.28, St.1.30 and

St.1.31) at 12°C and 25°C. ODt represents the optical density at 700 nm at time t (days), whereas OD0 is the optical density at the time of inoculation. Experimental values are represented by symbols, whereas corresponding expected values are shown as solid lines.

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Chapter 4. Single and sequential application of electrolyzed water with benzalkonium chloride or peracetic acid for removal of Staphylococcus aureus biofilms

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Single and sequential application of EW with BAC or PAA for removal of S. aureus biofilms

Single and sequential application of electrolyzed water with benzalkonium chloride or peracetic acid for removal of Staphylococcus aureus biofilms

Daniel Vázquez-Sánchez, Marta López Cabo, Juan José Rodríguez-Herrera

Seafood Microbiology and Technology Section, Marine Research Institute (IIM), Spanish National Research Council (CSIC), Eduardo Cabello 6, 36208, Vigo (Spain).

Abstract

The effectiveness of electrolyzed water (EW) against biofilms formed on stainless steel by S. aureus strains isolated from fishery products was assessed. The bactericidal activity of EW against biofilms was hardly any affected by variations in the pH of production. Neutral EW (NEW) was therefore used in subsequent studies as it has a higher potential for long-term application than acidic EW (due to a lower corrosiveness and toxicity) and due to the higher yield rate of the production unit at neutral pH.

The application of NEW caused a high reduction in the number of viable biofilm cells initially. However, a high available chlorine concentration (800 mg/L ACC) was needed to achieve logarithmic reductions (LR) demanded by the European quantitative surface test of bactericidal activity (≥ 4 log CFU/cm2 after 5 min of exposure). A double sequential application of NEW at much lower concentrations for 5 min each allowed LR ≥ 4 log CFU/cm2 to be reached in most of the experimental range. Sequential applications of NEW and either benzalkonium chloride (BAC) or peracetic acid (PAA) showed a similar effect, with PAA-NEW being most effective. The combination of NEW with other antimicrobial treatments can thus be an environmentally-friendly alternative to disinfection protocols traditionally used in the food industry.

Keywords: Staphylococcus aureus; biofilm; electrolyzed water; benzalkonium chloride; peracetic acid.

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4.1. Introduction

The ingestion of food containing staphylococcal enterotoxins is a major cause of foodborne intoxications in humans worldwide (EFSA, 2012; Hennekinne et al., 2012). Nonetheless, staphylococcal food poisoning (SFP) usually resolves within 24-48 h after onset, so most cases are not reported to healthcare services. As a result, the actual incidence of SFP is known to be much higher than reported (Argudín et al., 2010; Lawrynowicz-Paciorek et al., 2007).

In a recent study, a high incidence of Staphylococcus aureus (~ 25%) was found in fishery products marketed in Spain (Vázquez-Sánchez et al., 2012). Meanwhile, changes in Spanish national regulations (RD 135/2010), following Commission Regulation (EC) No 2073/2005, revoked the use of S. aureus as a microbiological criterion for a number of foods, including several types of fishery products. Being the largest producer and the second largest consumer of seafood in the European Union (Eurostat, 2010; FAO, 2012), the safety of seafood produced or consumed in Spain are of outmost importance.

Because humans are natural reservoirs of S. aureus, most of the concern has focused on preventing the spread from food handlers to food products. However, S. aureus can also attach and form biofilms on food-contact surfaces (DeVita et al., 2007; Gutiérrez et al., 2012; Herrera et al., 2007; Simon and Sanjeev, 2007; Sospedra et al., 2012). The formation of biofilms increases the likelihood of long-term presence of S. aureus in food-related environments due to an increased tolerance to adverse conditions, including the application of biocides (Bridier et al., 2011a; Van-Houdt and Michiels, 2010; Vázquez-Sánchez et al., 2014).

Many disinfectants are used to kill undesirable microorganisms in the food industry, but most have a high environmental impact and produce harmful effects on workers, and many are also corrosive to metal surfaces (Zabala et al., 2011). Electrolyzed water (EW) is a promising alternative to traditional disinfectants applied in the food industry. It is prepared on-site by electrolysis of a saturated salt solution, so that it is less dangerous for workers, more environmentally-friendly and less expensive (Ayebah and Hung, 2005; Huang et al., 2008; Issa-Zacharia et al., 2010; Ozer and Demirci, 2006). The bactericidal activity of EW derives from the combined action of a relatively high

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Single and sequential application of EW with BAC or PAA for removal of S. aureus biofilms available chlorine concentration (ACC) and a high positive oxidation-reduction potential (ORP). Numerous studies have shown the effectiveness of EW against different foodborne pathogens, including S. aureus, but only a few studies have been conducted to determine the potential of EW to eliminate S. aureus from food contact surfaces (Deza et al., 2005; Guentzel et al., 2008; Park et al., 2002; Sun et al., 2012).

The present study was therefore aimed to examine the potential of EW against biofilms formed on stainless steel surfaces by several strains of S. aureus isolated from fishery products. With this aim, the resistance of such biofilms to a single application of different types of EW was firstly determined. Subsequently, the efficacy of the sequential application of EW, either on its own or combined with classical disinfectants such as benzalkonium chloride and peracetic acid, was assessed.

4.2. Material and Methods

4.2.1. Bacterial strains

Four se-positive strains of S. aureus (St.1.01, St.1.04, St.1.07 and St.1.08) isolated from different commercial fishery products were used (Vázquez-Sánchez et al., 2012). The strains were formerly identified as S. aureus by specific biochemical (coagulase, DNAse and mannitol fermentation) and genetic tests (23s rDNA), and subsequently characterized as different strains by RAPD-PCR (Vázquez-Sánchez et al., 2012). Bacterial stocks of each strain were maintained at ─80°C in tryptic soy broth (TSB) (Panreac Química, Spain) containing 20% glycerol (v/v). A stock culture of each strain was thawed and then subcultured twice in TSB at 37°C for 24 h under static conditions prior to each experiment.

4.2.2. Antibacterial agents

Electrolyzed water (EW) was generated using an Envirolyte® EL-400 Unit (membrane electrolytic cell, model R-40, Envirolyte Industries International Ltd., Estonia) following manufacturer´s recommendations. A saturated sodium chloride solution and tap water were simultaneously pumped into the equipment at room temperature and at a current intensity of 20-25 A. Acidic (AEW), neutral (NEW) and basic electrolyzed water (BEW) were produced by redirecting appropriate amounts of

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Chapter 4 acidic anolyte solution (pH = 2.0-3.0, ORP ~ 1200 mV) to the cathode chamber containing the alkaline catholyte solution (pH = 11.0-12.0, ORP ~ ─900 mV) after electrolysis. The oxidation-reduction potential (ORP) and the pH of each EW were measured using a portable pH & REDOX 26 multimeter (Crison Instruments S.A, Spain), and the available chlorine concentration (ACC) was determined by iodometric titration (APHA-AWWA-WPCF, 1992). The physicochemical properties of each EW are indicated in Table 4.1.

Table 4.1. Physicochemical properties of acidic (AEW), neutral (NEW) and basic electrolyzed water (BEW) generated. ORP: oxidation-reduction potential. ACC: available chlorine concentration. Electrolyzed water pH ORP (mV) ACC (mg/L) AEW 2.99 ± 0.19 1171.75 ± 53.32 565.00 ± 35.22 NEW 6.06 ± 0.12 967.17 ± 36.52 856.33 ± 19.63 BEW 7.95 ± 0.13 843.00 ± 52.53 541.00 ± 39.64

Benzalkonium chloride (50% (v/v) solution, Guinama Absoluta Calidad, Spain) and peracetic acid (40% (v/v) solution in acetic acid:water, Fluka, Sigma-Aldrich, Spain) were also used in sequential applications.

All disinfectants were diluted in ultrapure water to working concentrations just before each assay.

4.2.3. Conditions for biofilm formation

Stainless steel coupons (AISI 304, 2B finish; Markim Galicia S.L., Spain) of approximately 10 mm × 10 mm (and 0.8 mm thickness) were used as experimental surfaces. Coupons were soaked in 2 M NaOH to remove residues, and then rinsed several times with distilled water, air-dried in a biosafety cabinet and autoclaved before use. One sterile coupon was placed into each well of a sterile 24-well flat-bottom microtiter plate (Falcon®, Becton Dickinson Labware, USA).

Overnight cultures of each strain were adjusted to an absorbance value at 700 nm of 0.100 ± 0.01 with phosphate buffer saline (PBS, composed by 7.6 g/L NaCl, 0.2 g/L

KCl and 0.245 g/L Na2HPO4 (BDH Prolabo, VWR International Eurolab, Spain); and

0.71 g/L K2HPO4 (Panreac Química, Spain)). This value corresponds to a cell concentration of 108 CFU/mL for all strains. PBS-suspended cells were then serially

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Single and sequential application of EW with BAC or PAA for removal of S. aureus biofilms diluted in TSB, and a 700 μL aliquot (containing approximately 7×105 CFU) was added into each microtiter well. Inoculum size was checked in all cases by plating on tryptic soy agar (TSA) (Cultimed, Panreac Química, Spain). A negative control with no inoculum was included in all assays. Microplates were incubated at 25°C for 48 h under static conditions.

4.2.4. Single application of electrolyzed water (EW)

The resistance of biofilms to each EW was assessed in terms of the logarithmic reduction (LR) in the number of viable biofilm cells per square centimetre under the experimental conditions tested. After biofilms being formed for 48 h at 25°C, coupons were placed into a new microplate and washed with 1 mL of PBS for 10 s to remove non-adhered cells. Coupons were then exposed to 1.5 mL of EW for 30 min (unless otherwise indicated). Three coupons were used for each treatment. Three positive controls (i.e., coupons exposed to sterile water) were also included in each assay. Subsequently, coupons were placed into sterile glass vials and 9 ml of neutralizing broth

(0.34 g/L KH2PO4 and 5 g/L Na2S2O3 (Probus, Spain); and 3 g/L soy lecithin, 1 g/L L- histidine and 3% (v/v) polysorbate 80 (Fagron Iberica, Spain)) were added and left to stand for 10 min at room temperature, according to Luppens et al. (2002). Surviving viable cells were collected by thoroughly rubbing the surface of coupons with two sterile swabs (Deltalab, Spain). Cells were resuspended by vigorously vortexing swabs for 1 min in 9 ml of peptone water (10 g/L triptone (Cultimed, Panreac Química, Spain) and 5 g/L sodium chloride (BDH Prolabo, VWR International Eurolab, Spain)). Ten- fold serial dilutions of resuspended cells were made in peptone water and aliquots of 0.1 mL of appropriate dilutions were spread on TSA plates. Also, the number of viable biofilm cells washed out during neutralization was quantified by plating 0.1 mL aliquots of appropriate dilutions of neutralizing broth on TSA. The total number of surviving biofilm cells was determined by adding up both counts after incubation at 37°C for 24 h. LR was then calculated as the difference between the logarithm of the total number of viable cells in non-EW-exposed biofilms and the logarithm of the number of surviving viable cells in EW-exposed biofilms. All assays were repeated twice using independent bacterial cultures.

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The resistance of planktonic cells was assessed in terms of the minimal bactericidal concentration (MBC), which was considered to be the lowest disinfectant concentration necessary to kill all free-living bacterial cells under the experimental conditions tested. Planktonic counterparts (i.e. non-adhered cells) were diluted in TSB to achieve a similar cell concentration to that in biofilms. Planktonic cells (0.1 mL) were exposed to 0.1 mL of EW for 30 min and then immediately neutralized (2.0 mL). Eleven different concentrations were tested for each EW (ranging between 50-550 mg/L for AEW and BEW, and 300-800 mg/L for NEW). Each concentration was tested in triplicate in two independent experiments. An aliquot of 0.3 mL of each neutralized disinfectant-treated bacterial culture was added to 1.7 mL of TSB and incubated at 37°C for 24 h. Bacterial growth was monitored visually. In addition, visually undetectable growth was checked by plating a 0.1 mL aliquot of culture medium on TSA and searching for the presence of colonies after 24 h at 37°C. Bacterial growth indicated the presence of viable cells in disinfectant-treated cultures.

4.2.5. Sequential application treatments

Sequential treatments consisted in the application of NEW either twice or in combination with benzalkonium chloride (BAC) or peracetic acid (PAA). A second order rotatable factorial design was carried out for each sequential application assay, according to Box et al. (1989). Each assay consisted of nine combinations of variables with five replicates in the centre of the domain. Biocide concentrations applied during the first (NEW1, BAC1, PAA1) and the second disinfection (NEW2, BAC2, PAA2) were used as independent variables. Ranges and codifications for each variable are shown in Table 4.2.

The resistance of biofilms was also assessed in terms of logarithmic reduction. Once non-adhered cells were removed, coupons were exposed to 1.5 mL of the first biocide for 5 min. To prevent cross-reactions between different disinfectants, each coupon was washed with 1 mL of PBS for 10 s before being exposed to 1.5 mL of the second biocide for another 5 min. The number of viable biofilm cells washed out was determined by plating a 0.1 mL aliquot of the washing PBS on TSA followed by incubation at 37°C for 24 h. Coupons were then neutralized (9 mL) for 10 min and the number of surviving biofilm cells were determined as aforementioned. Counts of cells

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Single and sequential application of EW with BAC or PAA for removal of S. aureus biofilms rubbed from coupons and washed out by PBS or by neutralizing broth were added up and LR was calculated as aforementioned. LR data were fitted to a second-order polynomial model by means of a least-squares method. Each combination of concentrations of biocides was tested in triplicate (i.e. three coupons) in two independent experiments.

Table 4.2. Natural and coded values of independent variables used in central composite rotatable experimental designs. Natural values

Codified values NEW1 BAC1 PAA1 NEW2 BAC2 PAA2 (mg/L ACC) (mg/L) (mg/L) (mg/L ACC) (mg/L) (mg/L) 1 1 500 500 250 500 500 250 1 ‒1 500 500 250 100 100 50 ‒1 1 100 100 50 500 500 250 ‒1 ‒1 100 100 50 100 100 50 1.41 0 583 583 291 300 300 150 ‒1.41 0 17 17 9 300 300 150 0 1.41 300 300 150 583 583 291 0 ‒1.41 300 300 150 17 17 9 0 0 300 300 150 300 300 150

4.2.6. Statistical analysis

In the case of sequential application assays, a Student´s t-test was done to determine if there were statistical differences between the coefficients of the equations obtained in the factorial design. Additionally, the model consistency was tested by the Fisher´s F- test applied to the following mean squares ratios: model / total error; (model + lack of fitting) / model; total error / experimental error; lack of fitting / experimental error.

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4.3. Results

4.3.1. Single application of electrolyzed water (EW)

4.3.1.1. Effects of pH

Biofilms containing between 7.0-7.2×107 CFU/cm2 were exposed to 500 mg/L ACC of acidic (AEW), neutral (NEW) or basic electrolyzed water (BEW) during 30 min to evaluate how the pH of EW affected the antimicrobial activity. Logarithmic reductions in the number of viable biofilm cells (LR) higher than 4 log CFU/cm2 were obtained in all cases (Figure 4.1). However, no significant differences were observed in the resistance of biofilms to the different types of EWs.

Figure 4.1. Logarithmic reduction (LR) in the number of viable cells of 48-h-old biofilms of S. aureus after exposure to 500 mg/L ACC of acidic (AEW), neutral (NEW) and basic electrolyzed water (BEW) for 30 min.

The effects of the pH of EW on the resistance of 48-h-old planktonic counterpart cells were assessed in terms of the minimum bactericidal concentration (MBC). AEW showed the highest effectiveness against planktonic cells, with MBC values ranging from 500 (S. aureus St.1.07) to 550 mg/L ACC (St.1.01, St.1.04 and St.1.08). Meanwhile, the MBC of NEW ranged from 700 (St.1.07) to 750 mg/L ACC (St.1.01, St.1.04 and St.1.08). Unfortunately, the MBC of BEW could not be determined for any of the strains, because the highest ACC concentration generated by the EW production unit at pH 8.0 (~ 540 mg/L ACC) was not high enough to kill all cells.

Although AEW showed the highest efficacy against planktonic cells, no differences against biofilms were observed between the different types of EW. Consequently, NEW was selected for the following studies bearing in mind that the yield rate of the production unit was much higher when conditions to produce NEW were set (Table 4.1).

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4.3.1.2. Effects of active chlorine concentration

The antimicrobial effectiveness of NEW was firstly assessed by exposing 48-h-old biofilms of each strain to a number of ACC during 30 min. As expected, the effect of NEW increased significantly (P < 0.01) with increasing ACC (Figure 4.2). Thus, average LR values of 3.84, 4.44 and 5.51 log CFU/cm2 were reached by applying 200, 500 and 800 mg/L ACC, respectively. However, no significant differences were detected among strains at each dose applied. S. aureus St.1.01 was therefore chosen for the following studies because it forms the most resistant biofilms to BAC and PAA among the strains tested (Vázquez-Sánchez et al., 2014).

Figure 4.2. Logarithmic reduction (LR) in the number of viable cells of 48-h-old biofilms of S. aureus strains after exposure to different doses of neutral electrolyzed water (NEW) for 30 min.

4.3.1.3. Effects of exposure time

The effects of the exposure time on the resistance to NEW of biofilms formed by S. aureus St.1.01 was examined after 5, 10, 15, 20, 25 and 30 min of exposure to 200, 500 and 800 mg/L ACC.

As expected, the number of viable biofilm cells decreased as either ACC or exposure time increased (Figure 4.3). No significant differences were thus found, for instance, between the effects of 200 mg/L ACC for 30 min and 500 mg/L ACC for 5 min, neither between 500 mg/L ACC for 30 min and 800 mg/L ACC for 5 min. The effects of both ACC and exposure time were clearly non-linear within the ranges of study, with the net effect increasing noticeably after 10-15 min of exposure at 800 mg/L ACC.

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Figure 4.3. Logarithmic reduction (LR) in the number of viable cells of 48-h-old biofilms of S. aureus St.1.01 caused by different doses of NEW at several exposure times.

According to the European standard EN 13697 (CEN, 2002), a disinfectant has to be able to kill over 4 log CFU/cm2 after 5 min of exposure. However, this effect could only be achieved when 800 mg/L ACC of NEW were applied. It seemed thus clear that another strategy was needed for NEW to be effective enough against biofilms.

4.3.2. Double sequential application of NEW

As could be inferred from Figure 4.3, the application of NEW produced a high reduction in the number of biofilm viable cells initially. Thus, a reduction of approximately 3 log CFU/cm2 was reached after only 5 min of exposure to 200 mg/L ACC. The resistance of 48-h-old biofilms of S. aureus St.1.01 to a double sequential application of NEW during 5 min each was thus examined next. A central composite rotatable experimental design with two independent variables (the ACC applied firstly

[NEW1] and secondly [NEW2]) was used.

The logarithmic reduction (LR) in the number of viable biofilm cells was adequately described by the following second order polynomial model (statistical analyses are specified in Appendix):

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This model is composed by a positive first order term and a lower negative second order term for each application. These terms reflect that the antimicrobial activity of NEW increases sub-linearly with concentration for both applications. Hardly differences were found in the values of the parameters and therefore in the bactericidal efficacies as a function of the order of application. A low negative interaction term between applications was also found. This term shows that the net effect of NEW2 decreased when high doses of NEW1 had been applied and, on the contrary, it increased when

NEW1 was used at low doses.

As shown in Figure 4.4, a double sequential application of NEW killed over 4 log CFU/cm2 of biofilm cells after only 5 min of exposure in most of the experimental range. Although this strategy was highly effective, the repeated application of a same disinfectant would involve a risk of adaptation. Therefore, it seemed convenient that sequential applications were based on the use of different biocides, preferably with different cellular targets. The sequential application of NEW with traditional industrial disinfectants such as benzalkonium chloride (BAC) or peracetic acid (PAA) was subsequently tested by means of a similar approach, based on a central composite rotatable experimental design for two variables (the concentration of each biocide).

Figure 4.4. Logarithmic reduction (LR) in the number of viable cells of 48-h-old biofilms of S. aureus St.1.01 after a double sequential application of NEW for 5 min each.

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4.3.3. Sequential application of NEW and BAC

When biofilms were exposed to the sequential applications of NEW1-BAC2 and

BAC1-NEW2 during 5 min each biocide, LR was described by the following polynomial models (statistical analyses are showed in Appendix):

Likewise for the double sequential application of NEW, the effects of NEW and BAC on LR were described by a positive first order term and a negative second order term each, independently of the order of application. A negative interaction term was also noted for both combinations. Significant (P < 0.01) differences were observed between the first and second order terms of NEW1 and BAC1. Accordingly, BAC was found to be more effective and less reactive than NEW. In contrast, interaction terms were similar in both sequences.

LR values higher than 4 log CFU/cm2 were reached in most of the experimental range by both NEW1-BAC2 (Figure 4.5A) and BAC1-NEW2 (Figure 4.5B) sequences. Differences in the bactericidal efficacy of both sequences hardly existed, except in two cases. Thus, when high concentrations of BAC were used in the first application, the killing effect was slightly higher than when NEW was applied instead. Similarly, when high concentrations of BAC were applied following low-medium concentrations of NEW, the effect was slightly higher than that of the reverse sequence. Both sequences showed a first order term for the first biocide significantly (P < 0.01) higher than that of

NEW1-NEW2, but no differences were detected for the other terms.

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Figure 4.5A-B. Logarithmic reduction (LR) in the number of viable cells of 48-h-old biofilms of S. aureus st.1.01 after sequential application of NEW and BAC for 5 min each. Sequences were NEW1-BAC2 (A) and BAC1-NEW2 (B).

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4.3.4. Sequential application of NEW and PAA

The effectiveness of the sequential application of NEW and PAA was also assessed in both possible forms: NEW1-PAA2 and PAA1-NEW2. Like previous models, the effects of each NEW and PAA were described by a positive first order term and a negative second order term, and a negative interaction term also appeared, as shown in the following equations (statistical analyses are specified in Appendix):

Both first and second order terms of PAA1 were significantly (P < 0.01) higher than those of NEW1. The effect and the reactivity of PAA were therefore higher than of

NEW. Meanwhile, the interaction term was higher (P < 0.01) in the case of NEW1-

PAA2.

LR values higher than 4 log CFU/cm2 were also achieved in most of the experimental range by both NEW1-PAA2 (Figure 4.6A) and PAA1-NEW2 (Figure 4.6B) sequences. Unlike previously, differences between sequences were found in a large part of the experimental range, so the order of application seemed to have some influence on the bactericidal effect. Thus, the effect of PAA1-NEW2 was generally higher than

NEW1-PAA2 when low-medium concentrations of the second biocide were applied, whereas the opposite was found when low-intermediate concentrations of the first biocide were followed by medium-high concentrations of the second.

In comparison with previous sequence NEW1-NEW2, the single terms of PAA1-

NEW2 and NEW1-PAA2 sequences were significantly (P < 0.01) higher, whereas the interaction term was lower (P < 0.01). In contrast, no significant differences were observed between the first order terms of BAC1 and PAA1, but the second order term of

PAA1 was significantly (P < 0.01) higher than that of BAC1. In addition, the interaction terms of the sequences applying PAA were significantly (P < 0.01) lower than those of BAC-based sequences.

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Figure 4.6A-B. Logarithmic reduction (LR) in the number of viable cells of 48-h-old biofilms of S. aureus St.1.01 after sequential application of NEW and PAA for 5 min each. Sequences were NEW1-PAA2 (A) and PAA1-NEW2 (B).

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4.4. Discussion

In the present study, the effects of several parameters potentially affecting the antimicrobial activity of EW (pH, dose and exposure time) on biofilms of S. aureus were examined in a relatively wide experimental range. In contrast, most studies dealing with the antimicrobial effect of EW have been carried out in liquid cultures (i.e. planktonic cells) and only a few have been made on biofilms, despite they are a common source of microbial contamination in the food industry. Actually, most works have been focused on assessing the efficacy of EW generated at a specific pH (Chen et al., 2013; Deza et al., 2005; Guentzel et al., 2008; Liu et al., 2006; Monnin et al., 2012; Park et al., 2002; Phuvasate and Su, 2010). Often too, only one dose has been applied during a particular exposure time against a small number of strains. In the case of S. aureus, the strain ATCC 6538 is the only one used in these studies as well as in standard bactericidal tests. However, the strains tested in this study had previously shown a higher biofilm-forming ability on stainless steel and a higher resistance to disinfectants commonly used in the food industry than S. aureus ATCC 6538 (Vázquez- Sánchez et al., 2014). Therefore, it could be expected that they would have a higher likelihood of long-term presence in food-related environments (clearly a worse-case scenario than S. aureus ATCC 6538), which was considered to make them more suitable for the study.

The results obtained in this study have shown that the variation in the pH of production affects the effectiveness of EW against S. aureus in the planktonic state. Thus, the effectiveness of EW increased with decreasing pH. These results are in concordance with previous studies in which the effects of several types of EWs against planktonic cells of different foodborne pathogens were assessed (Cao et al., 2009; Rahman et al., 2010a), but the range of pH tested in such studies was lower than in this study.

The higher efficacy of AEW and NEW in comparison with BEW is at least partially accounted for the presence of a much higher proportion of hypochlorous acid (HClO) (Len et al., 2000; Vorobjeva et al., 2004), which has a higher oxidizing ability than hypochlorite ions (ClO─), predominantly present at pH above 7.5. Meanwhile, differences between the bactericidal activities of AEW and NEW are presumably due to

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a much higher amount of dissolved Cl2 gas, hydrogen peroxide (H2O2) and reactive oxygen species (ROS) in the former, and of ClO─ in the latter (Len et al., 2000; Vorobjeva et al., 2004). The low pH and the high oxidation-reduction potential (ORP) of AEW (Table 4.1) could additionally enhance the entrance of HClO into bacterial cells by disruption of membranes (Huang et al., 2008; McPherson, 1993).

However, the variation of pH did not affect the effectiveness of EW against S. aureus biofilms. This lack of effect is considered to be due to the role of the extracellular matrix of biofilms, which acts as a protective barrier that limits molecular diffusion towards cellular targets and also reacts with active chlorine compounds (Chen and Stewart, 1996; Oomori et al., 2000; White, 1999). Therefore, other factors were taken into account to decide which type of EW was most appropriate for biofilm removal. In this sense, AEW would enhance the corrosion of food-processing equipments and could cause health issues in handlers as a consequence of Cl2 off-gassing (Ayebah and Hung, 2005; Guentzel et al., 2008), so NEW seemed to have a higher potential than AEW for long-term application in the food industry. In addition, the yield rate of the production unit used in the study was much higher when conditions to produce NEW were set (with respect to AEW or BEW). Consequently, the study focused on the application of NEW subsequently.

As expected, the effectiveness of NEW against S. aureus biofilms increased with increasing ACC and exposure time. In fact, the high reactivity of NEW caused a high initial reduction of viable biofilm cells, presumably by killing cells located in the outer layers of biofilms, which are more exposed to biocide molecules. Ayebah et al. (2005) had also shown that acidic electrolyzed water (pH ~ 2.4) produced a reduction of 4.3 to 5.2 log CFU/coupon in the number of viable cells of L. monocytogenes biofilms after only 30-120 s. Afterwards, the inactivation rate of viable biofilm cells was much lower. Once easily accessible cells are killed, the chemical species of EW need to diffuse through the extracellular matrix of the biofilm to reach bacterial cells, but this matrix acts as a barrier that physically hinders diffusion and present many reactive sites for biocide molecules (Chen and Stewart, 1996; Oomori et al., 2000; White, 1999). When 800 mg/L ACC were applied, the antimicrobial effect of NEW increased over that linearly expected, particularly after 15 min, which is thought to be due to a higher exposition of bacterial cells as a result of the disruption of the biofilm matrix. Similarly,

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Kim et al. (2001) described that acidic electrolyzed water (pH ~ 2.5) decreased the number of viable cells in Listeria monocytogenes biofilms noticeably for the first 30 s, followed by a considerable deceleration of the antimicrobial activity and a final increase in the inactivation rate of biofilm cells.

According to the European quantitative surface test of bactericidal activity EN 13697, a disinfectant has to be able to kill at least 4 log CFU/cm2 biofilm cells after 5 min of exposure with the aim of achieving an effective disinfection of food-contact surfaces in a short time. High concentrations of NEW (800 mg/L ACC) were needed to achieve this effect on S. aureus biofilms, but this would be costly and not environmentally-friendly. Nevertheless, logarithmic reductions higher than 3 log CFU/cm2 were obtained by applying only 200 mg/L of NEW during 5 min, which suggested that consecutive applications of low concentrations of NEW could increase significantly the bactericidal effect against biofilms, with clear advantages in terms of both dose and exposure time.

As expected, a double sequential application of NEW at low concentrations was enough to achieve logarithmic reductions higher than 4 log CFU/cm2 after 5 min of exposure each. However, this procedure could involve a risk of adaptation to EW, as previously observed after the repeated use of a same chlorinated disinfectant (Braoudaki and Hilton, 2004; Bridier et al., 2011a; Lundén et al., 2003; Maalej et al., 2006; Sanderson and Stewart, 1997). Therefore, it seemed convenient that sequential applications were based on the use of different biocides, preferably with different cellular targets.

The potential of combining NEW with benzalkonium chloride (BAC) or peracetic acid (PAA) was thus examined. Logarithmic reductions demanded by the European standard test EN 13697 were also reached in all cases by applying low concentrations of each biocide. Comparatively, PAA was more effective than NEW and BAC, as the concentrations of PAA used in the study were about 2-fold lower than those of NEW and BAC. A high oxidative potential, non-specificity, a high reactivity (higher than that of BAC, as shown by the magnitude of the second order terms) and a small size enabling diffusion through the biofilm matrix have been indicated to account for a higher antimicrobial activity of PAA against biofilms than BAC and chlorine-based compounds such as NEW (Bessems, 1998; Bridier et al., 2011b; Denyer and Stewart,

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1998; Kitis, 2004; Saá-Ibusquiza et al., 2011). However, when sequences were based on the use of different disinfectants, washing was mandatory to prevent cross-reaction between them. As a result, operation time increased and approximately 10% of cells surviving after the first disinfection were washed out. Therefore, wash water would require to be collected and subjected to additional biocidal processes (e.g., heat, UV radiation, chlorination, ozonation) to prevent dissemination and reattachment of dispersed cells (Casani and Knøchel, 2002; Fähnrich et al., 1998). In contrast, no intermediate washing was needed when NEW was applied twice.

Various combined treatments have also shown an enhanced antimicrobial effect against S. aureus biofilms respect to single disinfection (Caballero-Gómez et al., 2013; Hendry et al., 2009; Xing et al., 2012). However, in these studies, biofilms were formed under conditions rarely found in the food industry such as a growth temperature of 37°C (Hendry et al., 2009; Xing et al., 2012) or exposed to antimicrobials for a longer time (60 min) than that specified in EN 13697 (Caballero-Gómez et al., 2013), so they hardly have any application in the food industry. In the case of EW-based combined treatments, many studies have dealt with food products (Chen et al., 2013; Kim et al., 2003; Koseki et al., 2004; Mahmoud et al., 2006; Rahman et al., 2010b, 2011; Wang et al., 2004, 2006), whereas studies performed on food-contact surfaces have been sporadic (Ayebah et al., 2005; Chen et al., 2013), despite biofilms are a common source of food contamination. However, these two studies were focused on combining acidic EW with alkaline EW or ultrasounds, respectively. Therefore, the NEW-based combined treatments proposed in the present study represent an effective, safe, environmentally-friendly and less expensive alternative to be applied in the food industry.

4.5. Conclusions

Electrolyzed water (EW) is considered a promising alternative for bacterial disinfection in the food industry as it is environmentally-friendly, safe-to-use and relatively inexpensive. In contrast to planktonic cells, the bactericidal activity of EW against biofilms was not affected by variations in the pH of production. Neutral EW (NEW) seems thus to show a higher potential for long-term application in the food industry, as it is less corrosive to metal surfaces and less toxic to handlers than acidic

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EW. However, a high ACC of NEW was required to comply with the specifications set in the European quantitative surface test of bactericidal activity (LR ≥ 4 log CFU/cm2 after 5 min of exposure). Nevertheless, the combination of NEW and other disinfectants resulted in a promising alternative to control the contamination of food-processing facilities by foodborne pathogens in terms of dose and exposure time. This strategy can be also extended to almost any other biocide combination, although working-safe and environmentally-friendly options such as NEW should be preferentially selected to be in concordance with present and future regulatory landscapes. Furthermore, the combination of biocides should be addressed to search intelligent solutions of disinfection in the sense described by the principles of hurdle technology for food products. Thus, biocides should be chosen on a mechanistic basis (with the aim to obtain some synergistic effect), so a much greater knowledge on the mechanisms of action of biocides is required, and disinfection treatments (dose, time of exposure and type of sequence) should be optimized by a predictive microbiology-based approach. This strategy could reduce considerably adverse effects on working, environmental and economic aspects.

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Appendix

Statistical data of factorial design and test of significance for the different second order polynomial models showed in this work.

Table Ap.1. Sequence NEW1-NEW2. C N Y Ŷ Coefficients t Model 1 1 6.056 5.990 5.191 124.510 5.191

1 ‒1 5.254 5.114 0.573 17.373 0.573 NEW1

‒1 1 5.278 5.162 0.597 18.079 0.597 NEW2

‒1 ‒1 3.842 3.652 ‒0.159 3.401 ‒0.159 NEW1 NEW2

1.41 0 5.688 5.781 ‒0.110 3.110 ‒0.110

‒1.41 0 3.998 4.161 ‒0.101 2.854 ‒0.101 0 1.41 5.755 5.831 0 ‒1.41 3.966 4.148 Average value 5.0610 0 0 5.191 5.191 Expected average value 5.1911 0 0 5.278 5.191

0 0 5.278 5.191 Var (Ee) 0.0087 0 0 5.056 5.191 t (α < 0.05; ʋ = 4) 2.7760 0 0 5.153 5.191

SS ʋ MS MSMd/MSE 43.737 (α = 0.05) 3.860

Md 5.701 5 1.1402 MSMdLF/MSM 0.641 (α = 0.05) 9.120

E 0.182 7 0.0261 MSE/MSEe 3.000 (α = 0.05) 6.000

Ee 0.035 4 0.0087 MSLF/MSEe 5.666 (α = 0.05) 6.260 LF 0.148 3 0.0492 r2 0.969 2 Total 5.884 12 r adjusted 0.947 Y, observed response; Ŷ, expected response; SS, sum of squares; ʋ, degrees of freedom; MS, mean squares; Md, Model; E, total error; Ee, experimental error; LF, lack of fitting; MSE, mean squares for total error; MSEe, mean squares for experimental error; MSLF, mean squares for lack of fitting; MSMd, mean squares for model; MSMdLF, mean squares for (model + lack of fitting).

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Table Ap.2. Sequence NEW1-BAC2. C N Y Ŷ Coefficients t Model 1 1 6.040 5.994 5.145 376.660 5.145

1 ‒1 5.040 5.029 0.608 56.313 0.608 NEW1

‒1 1 5.102 5.074 0.630 58.296 0.630 BAC2

‒1 ‒1 3.510 3.517 ‒0.148 9.690 ‒0.148 NEW1 BAC2

1.41 0 5.704 5.736 ‒0.134 11.610 ‒0.134

‒1.41 0 4.009 4.016 ‒0.107 9.197 ‒0.107 0 1.41 5.776 5.821 0 ‒1.41 4.048 4.043 Average value 4.9953 0 0 5.137 5.145 Expected average value 5.1447 0 0 5.137 5.145

0 0 5.174 5.145 Var (Ee) 0.0009 0 0 5.102 5.145 t (α < 0.05; ʋ = 4) 2.7760 0 0 5.174 5.145

SS ʋ MS MSMd/MSE 907.777 (α = 0.05) 3.860

Md 6.398 5 1.2795 MSMdLF/MSM 0.626 (α = 0.05) 9.120

E 0.010 7 0.0014 MSE/MSEe 1.511 (α = 0.05) 6.000

Ee 0.004 4 0.0009 MSLF/MSEe 2.193 (α = 0.05) 6.260 LF 0.006 3 0.0020 r2 0.998 2 Total 6.408 12 r adjusted 0.997 Y, observed response; Ŷ, expected response; SS, sum of squares; ʋ, degrees of freedom; MS, mean squares; Md, Model; E, total error; Ee, experimental error; LF, lack of fitting; MSE, mean squares for total error; MSEe, mean squares for experimental error; MSLF, mean squares for lack of fitting; MSMd, mean squares for model; MSMdLF, mean squares for (model + lack of fitting).

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Table Ap.3. Sequence BAC1-NEW2. C N Y Ŷ Coefficients t Model 1 1 6.037 6.034 5.149 556.271 5.149

1 ‒1 5.116 5.073 0.643 87.888 0.643 BAC1

‒1 1 4.996 5.017 0.615 83.904 0.615 NEW2

‒1 ‒1 3.537 3.518 ‒0.134 12.984 ‒0.134 BAC1 NEW2

1.41 0 5.815 5.843 ‒0.108 13.737 ‒0.108

‒1.41 0 4.030 4.024 ‒0.131 16.576 ‒0.131 0 1.41 5.774 5.756 0 ‒1.41 3.983 4.022 Average value 5.0025 0 0 5.172 5.149 Expected average value 5.1490 0 0 5.134 5.149

0 0 5.134 5.149 Var (Ee) 0.0004 0 0 5.134 5.149 t (α < 0.05; ʋ = 4) 2.7760 0 0 5.172 5.149

SS ʋ MS MSMd/MSE 1302.425 (α = 0.05) 3.860

Md 6.573 5 1.3147 MSMdLF/MSM 0.626 (α = 0.05) 9.120

E 0.007 7 0.0010 MSE/MSEe 2.356 (α = 0.05) 6.000

Ee 0.002 4 0.0004 MSLF/MSEe 4.165 (α = 0.05) 6.260 LF 0.005 3 0.0018 r2 0.999 2 Total 6.580 12 r adjusted 0.998 Y, observed response; Ŷ, expected response; SS, sum of squares; ʋ, degrees of freedom; MS, mean squares; Md, Model; E, total error; Ee, experimental error; LF, lack of fitting; MSE, mean squares for total error; MSEe, mean squares for experimental error; MSLF, mean squares for lack of fitting; MSMd, mean squares for model; MSMdLF, mean squares for (model + lack of fitting).

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Table Ap.4. Sequence NEW1-PAA2. C N Y Ŷ Coefficients t Model 1 1 6.037 6.033 5.149 556.276 5.149

1 ‒1 4.881 4.886 0.606 82.752 0.606 NEW1

‒1 1 5.009 5.023 0.674 92.016 0.674 PAA2

‒1 ‒1 3.450 3.473 ‒0.101 9.734 ‒0.101 NEW1 PAA2

1.41 0 5.701 5.705 ‒0.150 19.175 ‒0.150

‒1.41 0 4.014 3.992 ‒0.145 18.377 ‒0.145 0 1.41 5.815 5.812 0 ‒1.41 3.926 3.910 Average value 4.9676 0 0 5.134 5.149 Expected average value 5.1490 0 0 5.172 5.149 5.149 0 0 5.134 Var (Ee) 0.0004 0 0 5.172 5.149 t (α < 0.05; ʋ = 4) 2.7760 0 0 5.134 5.149

SS ʋ MS MSMd/MSE 2908.016 (α = 0.05) 3.860

Md 6.869 5 1.3738 MSMdLF/MSM 0.625 (α = 0.05) 9.120

E 0.003 7 0.0005 MSE/MSEe 1.103 (α = 0.05) 6.000

Ee 0.002 4 0.0004 MSLF/MSEe 1.240 (α = 0.05) 6.260 LF 0.002 3 0.0005 r2 0.9995 2 Total 6.872 12 r adjusted 0.9992 Y, observed response; Ŷ, expected response; SS, sum of squares; ʋ, degrees of freedom; MS, mean squares; Md, Model; E, total error; Ee, experimental error; LF, lack of fitting; MSE, mean squares for total error; MSEe, mean squares for experimental error; MSLF, mean squares for lack of fitting; MSMd, mean squares for model; MSMdLF, mean squares for (model + lack of fitting).

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Table Ap.5. Sequence PAA1-NEW2. C N Y Ŷ Coefficients t Model 1 1 6.037 6.045 5.206 321.421 5.206

1 ‒1 4.923 4.939 0.634 49.485 0.634 PAA1

‒1 1 4.851 4.889 0.613 47.825 0.613 NEW2

‒1 ‒1 3.495 3.551 ‒0.060 3.341 ‒0.060 PAA1 NEW2

1.41 0 5.736 5.732 ‒0.185 13.469 ‒0.185

‒1.41 0 4.000 3.940 ‒0.162 11.771 ‒0.162 0 1.41 5.774 5.748 0 ‒1.41 4.056 4.018 Average value 4.9924 0 0 5.172 5.206 Expected average value 5.2057 0 0 5.213 5.206

0 0 5.259 5.206 Var (Ee) 0.0013 0 0 5.213 5.206 t (α < 0.05; ʋ = 4) 2.7760 0 0 5.172 5.206

SS ʋ MS MSMd/MSE 556.104 (α = 0.05) 3.860

Md 6.598 5 1.3197 MSMdLF/MSM 0.626 (α = 0.05) 9.120

E 0.017 7 0.0024 MSE/MSEe 1.809 (α = 0.05) 6.000

Ee 0.005 4 0.0013 MSLF/MSEe 2.889 (α = 0.05) 6.260 LF 0.011 3 0.0038 r2 0.997 2 Total 6.615 12 r adjusted 0.996 Y, observed response; Ŷ, expected response; SS, sum of squares; ʋ, degrees of freedom; MS, mean squares; Md, Model; E, total error; Ee, experimental error; LF, lack of fitting; MSE, mean squares for total error; MSEe, mean squares for experimental error; MSLF, mean squares for lack of fitting; MSMd, mean squares for model; MSMdLF, mean squares for (model + lack of fitting).

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Chapter 5. Antimicrobial activity of essential oils against Staphylococcus aureus biofilms

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Antimicrobial activity of essential oils against S. aureus biofilms

Antimicrobial activity of essential oils against Staphylococcus aureus biofilms

Daniel Vázquez-Sánchez, Marta López Cabo, Juan José Rodríguez-Herrera

Seafood Microbiology and Technology Section, Marine Research Institute (IIM), Spanish National Research Council (CSIC), Eduardo Cabello 6, 36208, Vigo (Spain).

Abstract

The effectiveness of nineteen essential oils (EOs) against planktonic cells of S. aureus was firstly assessed by minimal inhibitory concentration (MIC). Planktonic cells showed a wide variability in resistance to EOs, with thyme oil as the most effective, followed by lemongrass oil and then vetiver oil. The eight EOs most effective against planktonic cells were subsequently tested against 48-h-old biofilms formed on stainless steel. All EOs reduced significantly (P < 0.01) the number of viable biofilm cells, but none of them could remove biofilms completely. Thyme and patchouli oils were the most effective, but high concentrations were needed to achieve logarithmic reductions over 4 log CFU/cm2 after 30 min exposure.

The use of sub-lethal doses of thyme oil prevented biofilm formation and enhanced the efficiency of thyme oil and benzalkonium chloride against biofilms. However, some cellular adaptation to thyme oil was detected. Combined thyme oil-based treatments were thus considered as a suitable alternative for the removal of S. aureus biofilms in food-processing facilities. But EO-based treatments should be based on the rotation and combination of different EOs or with other biocides to prevent the emergence of antimicrobial-resistant strains.

Keywords: Staphylococcus aureus; biofilm; essential oil; thyme; benzalkonium chloride.

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5.1. Introduction

One of the major causes of foodborne intoxications in humans worldwide is the ingestion of food containing staphylococcal enterotoxins (Bhatia and Zahoor, 2007; EFSA, 2012; Hennekinne et al., 2012). Nonetheless, it is known that the actual incidence of staphylococcal food poisoning is underestimated, because it usually resolves 24-48 h after onset (Argudín et al., 2010; Lawrynowicz-Paciorek et al., 2007).

Most strategies aimed to prevent the contamination of food with S. aureus are focused on the importance of food handlers as natural reservoirs. However, this pathogen has also a high ability to form biofilms on food-contact surfaces (DeVita et al., 2007; Gutiérrez et al., 2012; Herrera et al., 2007; Simon and Sanjeev, 2007; Sospedra et al., 2012). The formation of biofilms increases the resistance to adverse conditions, such as the application of disinfectants (Bridier et al., 2011a; Van-Houdt and Michiels, 2010; Vázquez-Sánchez et al., 2014). Consequently, forming biofilms increases the likelihood of long-term presence in food-related environments and, therefore, the risk of food contamination as well as the spread of the bacterium.

Many disinfectants are applied to remove undesirable microorganisms from food- contact surfaces, but most are corrosive to metal surfaces, harmful for workers or have a high environmental impact (Zabala et al., 2011). Thus, scientific interest in the antimicrobial properties of plant essential oils (EOs) has emerged. EOs comprise a wide variety of aromatic oily liquids (approximately 3000 EOs are known nowadays) with a versatile composition, which are usually extracted from different plant materials such as flowers, fruits, herbs, leaves, roots and seeds (Bakkali et al., 2008; Burt, 2004) EOs have shown to have antioxidant (Brenes and Roura, 2010), antibacterial (Oussalah et al., 2007), antiparasitic (George et al., 2009), insecticidal (Nerio et al., 2010), antiviral (Astani et al., 2011) and antifungal (Tserennadmid et al., 2011) properties. Nonetheless, few studies have evaluated the potential of EOs to remove S. aureus from food-contact surfaces (Adukwu et al., 2012; Lebert et al., 2007; Millezi et al., 2012). Also, the poor solubility and partial volatility of EOs have limited their practical application (Delaquis et al., 2002; Kalemba and Kunicka, 2003).

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The present study was therefore aimed to evaluate the potential of essential oils against biofilms formed by S. aureus on stainless steel surfaces. With this aim, the resistance of such biofilms to a single application of an array of EOs was firstly determined. Subsequently, it was investigated the potential of applying thyme oil (the EO with the highest effect) on the formation and removal of biofilms (on its own and in combination with benzalkonium chloride), as well as the risk of bacterial adaptation to thyme oil.

5.2. Material and Methods

5.2.1. Bacterial strain

The sea-positive strain S. aureus St.1.01 was used. It was identified as S. aureus by specific biochemical (coagulase, DNAse and mannitol fermentation) and genetic tests (23s rDNA sequencing), and characterized by RAPD-PCR with three primers (Vázquez-Sánchez et al., 2012). The RAPD pattern of St.1.01 was also shown by different S. aureus isolates from several commercial fishery products. Bacterial stocks were maintained at ─80°C in tryptic soy broth (TSB) (Panreac Química, Spain) containing 20% glycerol (v/v). A stock culture of the strain was thawed and subcultured twice in TSB at 37°C for 24 h under static conditions prior to each experiment.

5.2.2. Antibacterial agents

Nineteen pure essential oils (EOs) industrially produced by steam distillation of different plant parts (Table 5.1) were tested in the present study. All EOs were diluted in ultrapure water with 0.15% (w/v) bacteriological agar (Panreac Química, Spain) before each experiment. Benzalkonium chloride was purchased as a 50% (v/v) solution (Guinama Absoluta Calidad, Spain), which was diluted in ultrapure water to working concentrations prior to each assay.

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Table 5.1. Origin of pure essential oils used in this study Common name Plant species Distilled part

Anise1 Pimpinella anisum Seeds Carrot2 Daucus carota Plant and flowers Citronella1 Cymbopogon nardus Herbs Coriander2 Coriandrum sativum Plant and flowers Cumin1 Cuminum cyminum Seeds Eucalyptus globulus2 Eucalyptus globulus Leaves Eucalyptus radiata3 Eucalyptus radiata Leaves Fennel1 Foeniculum vulgare Plants Geranium2 Pelargonium graveolens Plant and flowers Ginger2 Zingiber officinale Plant and flowers Hyssop1 Hyssopus officinalis Plant with flowers Lemongrass2 Cymbopogon citratus Plant and flowers Marjoram2 Origanum majorana Plant and flowers Palmarosa2 Cymbopogon martinii Plant and flowers Patchouli2 Pogostemon patchouli Leaves Sage2 Salvia officinalis Plant and flowers Tea tree2 Melaleuca alternifolia Leaves Thyme1 Thymus vulgaris Plant with flowers Vetiver2 Vetiveria zizanioides Plant and flowers

1Esential Arôms, Dietéticos Intersa S.A. (Spain) 2Mon Deconatur S.L. (Spain) 3Pranaróm International (Belgium)

5.2.3. Conditions for biofilm formation

Stainless steel coupons (AISI 304, 2B finish) (Markim Galicia S.L., Spain) of approximately 10 mm × 10 mm (and 0.8 mm thickness) were used as experimental surfaces. Coupons were soaked in 2 M NaOH to remove residues, rinsed several times with distilled water, air-dried in a biosafety cabinet and autoclaved before use. One sterile coupon was placed into each well of a sterile 24-well flat-bottom microtiter plate (Falcon®, Becton Dickinson Labware, USA).

Overnight cultures of S. aureus St.1.01 were adjusted to an absorbance value at 700 nm of 0.100 ± 0.01 with phosphate buffer saline (PBS, composed by 7.6 g/L NaCl, 0.2 g/L KCl and 0.245 g/L Na2HPO4 (BDH Prolabo, VWR International Eurolab, Spain); and 0.71 g/L K2HPO4 (Panreac Química, Spain)), which corresponds to a cell

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5.2.4. Biocide resistance assays

5.2.4.1. Resistance of planktonic cells

The resistance of planktonic cells to EOs was evaluated in terms of minimal inhibitory concentration (MIC), which was defined as the lowest concentration at which no bacterial growth was detected under experimental conditions. The method proposed by Mann and Markham (1998) was followed, but with some modifications.

Aliquots of 95 μL of bacterial cultures adjusted to an absorbance of 0.100 ± 0.01 as aforementioned (containing thus approximately 105 CFU) were exposed to 95 μL of a specific series of concentrations of each EO in a sterilized 96-well U-bottom microtiter plates (Falcon®, Becton Dickinson Labware, USA). For each EO, sixteen different concentrations (0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.50%, 1%, 1.5%, 2%, 2.5% and 3% (v/v)) were tested in triplicate in two independent experiments. A positive control with no EOs, a negative control with no inoculum and a blank with medium only were included in all assays.

Microplates were incubated for 24 h at 37°C under static conditions. Wells were then stained with 10 µl of 0.01% (w/v) resazurin sodium salt solution (Sigma-Aldrich Química, Spain) and incubation at 37°C continued for further 2 h. Bacterial growth was monitored visually as colour change from blue to pink, which indicates the presence of viable cells in cultures. Visually undetectable growth was also checked by plating 0.1 mL aliquots of cultures on TSA and searching for the presence of colonies after 24 h at 37°C.

5.2.4.2. Resistance of biofilms

The resistance of biofilms to EOs was determined in terms of the logarithmic reduction of viable biofilm cells (LR) caused by treatment with EO.

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The EOs with the highest effectiveness against planktonic cells were assessed against 48-h-old biofilms (i.e., biofilms representing an example of worst-case scenario in the food industry). After incubation time, coupons were placed into a new microplate and washed with 1 mL of PBS to remove non-adhered cells. Coupons were then exposed to 1.5 mL of EO for 30 min. For each EO, twelve concentrations (0.10%, 0.25%, 0.50%, 0.75%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 6% and 8% (v/v)) were tested in triplicate. Three positive controls consisting of biofilms exposed to sterile water were also included in each assay. Subsequently, coupons were placed into sterile glass vials and 9 mL of neutralizing broth (0.34 g/L KH2PO4 and 5 g/L Na2S2O3 (Probus, Spain); and 3 g/L soy lecithin, 1 g/L L-histidine and 3% (v/v) polysorbate 80 (Fagron Iberica, Spain)) were added and left to stand for 10 min at room temperature, following Luppens et al. (2002). Biofilm cells were then collected by thoroughly rubbing the surface of coupons with two sterile swabs (Deltalab, Spain). Swabs were immersed in 9 mL of peptone water (10 g/L triptone (Cultimed, Panreac Química, Spain) with 5 g/L sodium chloride (BDH Prolabo, VWR International Eurolab, Spain)) and vigorously vortexed for 1 min for resuspension of cells. Ten-fold serial dilutions of resuspended cells were made in peptone water and 0.1 mL aliquots of appropriate dilutions were spread on TSA plates. Also, the number of viable biofilm cells washed out during neutralization was quantified by plating 0.1 mL aliquots of appropriate dilutions of neutralizing broth on TSA. Surviving biofilm cells were counted by adding up both counts after incubation at 37°C for 24 h. LR was determined as the difference between the logarithm of the total number of viable cells in non-disinfectant-exposed biofilms and the logarithm of the number of surviving viable cells in disinfectant-exposed biofilms.

Subsequently, the effects of the presence of thyme oil at sub-lethal doses (0.020%, 0.025%, 0.030% and 0.040% (v/v)) during biofilm formation on the efficacy of thyme oil and BAC against such biofilms was evaluated. Because those sub-lethal doses inhibited the growth of S. aureus, biofilms were formed during 7, 10, 14 and 26 days, respectively, to have a concentration of viable cells between 5-9×107 CFU/cm2 in all cases. Biofilms formed during 4 days in the absence of thyme oil were also included as a control. After incubation, biofilms were exposed to thyme oil (the same sub-lethal concentrations) or BAC (50, 200, 1000, 2500, 5000, 10000, 15000 and 20000 mg/L) for

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30 min and then immediately neutralized. Finally, viable biofilm cells were counted and LR was calculated as aforementioned.

All assays were repeated twice using independent bacterial cultures.

5.2.4.3. Determination of growth kinetics

A series of test tubes containing 5 mL TSB with 0.15% agar and different sub-lethal doses of thyme oil (0.020%, 0.025%, 0.030% and 0.040% (v/v)) were inoculated with 100 μL of cultures of S. aureus containing approximately 105 CFU and subsequently incubated at 25°C under static conditions for up to 30 days. Absorbance was read out at 700 nm in triplicate every 24 h of incubation. Each tube was used for only one reading and then discarded. A positive control with no thyme oil, a negative control with no inoculum and a blank with medium only were included in all assays. Growth kinetics was determined using two independent bacterial cultures.

Experimental data were fitted to the Gompertz equation proposed by Zwietering et al. (1990):

( ) { [ ]}

where ODt is the optical density at 700 nm at time t (days), OD0 is the optical density at the time of inoculation, A is a dimensionless asymptotic value, µmax is the maximum specific growth rate (1/days), and λ is the detection time (days), i.e., the time at which OD at 700 nm is firstly noted to increase.

5.2.5. Statistical analysis

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5.3. Results

5.3.1. Effectiveness of essential oils (EOs) against planktonic cells

The resistance of S. aureus planktonic cells to 19 different EOs was determined in terms of the minimum inhibitory concentration (MIC). As shown in Figure 5.1, a very high degree of variability was found in the effectiveness of the different EOs tested (CV = 88.35%). Thus, whereas thyme oil showed the highest efficacy (MIC = 0.04% (v/v)), followed closely by lemongrass (MIC = 0.06%) and vetiver (MIC = 0.07%) oils, anise, carrot and ginger oils were very much less effective, with a MIC over 50-fold higher (MIC = 3%). In between, the application of citronella, cumin, geranium, palmarosa and patchouli oils showed a MIC of 0.5%, whereas coriander, Eucalyptus globulus, E. radiata, fennel, hyssop, marjoram, sage and tea tree oils showed a MIC of 1%. On the basis of these results, the most effective EOs (i.e., thyme, lemongrass, vetiver, citronella, cumin, geranium, palmarosa and patchouli oils) were tested against biofilms subsequently.

Figure 5.1. Minimum inhibitory concentration (MIC) of essential oils against S. aureus St.1.01 planktonic cells.

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5.3.2. Effectiveness of EOs against 48-h-old biofilms

The resistance of 48-h-old biofilms to the selected EOs was determined in terms of the logarithmic reduction of viable biofilm cells (LR) caused by EO-treatments.

None of the EOs was able to eradicate completely biofilms. Likewise for planktonic cells, a high variability was found in the effectiveness of EOs. The variability was higher at high concentrations (CV ~ 24% at 8%) than at low concentrations (CV ~ 5.6% at 0.10%).

Thyme and patchouli oils showed the highest antimicrobial efficacy against biofilms in all cases, followed by citronella oil (Figure 5.2A-L). Although no significant differences were found between these three EOs in the range 0.10-0.25%, citronella oil was significantly (P < 0.05) less effective than thyme and patchouli oils at higher concentrations. Thus, a LR ~ 3.5 log CFU/cm2 was achieved with the application of 8% citronella oil, whereas the same dose of patchouli and thyme oils caused a LR of 4.2 and 4.3 log CFU/cm2, respectively.

The efficacy of the remaining EOs against biofilms was found to be dependent on the dose. In general, palmarosa and vetiver were least effective, with LR significantly (P < 0.05) lower than those of lemongrass (in the range 0.75-8%), geranium (1.5-8%) and cumin (2-8%) oils. In fact, only palmarosa showed a significantly (P < 0.05) higher effect than cumin at 0.25% and 0.75%. Lemongrass was significantly (P < 0.05) more effective than cumin and geranium at low doses (0.25-2.5% and 0.5-1.5% respectively), whereas cumin showed a significantly (P < 0.05) higher efficacy than lemongrass in the range 6-8%. Meanwhile, geranium was significantly (P < 0.05) more effective than cumin between 1-2.5%.

Noteworthy differences were found between the ranking of EOs on the basis of the effectiveness against biofilms and planktonic cells. In particular, lemongrass and vetiver oils were highly efficacy against planktonic cells but poorly against biofilms, whereas patchouli and citronella oils showed a high effect against biofilms but rather moderate against planktonic cells. In contrast, thyme oil showed the highest efficacy against both biofilms and planktonic cells, so this EO was chosen for use in subsequent studies.

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Figure 5.2A-L. Logarithmic reduction of viable cells (LR) in 48-h-old biofilms exposed to different EOs for 30 min. Each figure (A-L) corresponds to each dose tested. Letters denote statistically significant differences (P < 0.05) between EOs at each dose, being defined the most effective EO with the letter a.

5.3.3. Effects of sub-lethal doses of thyme oil on bacterial growth

Cultures of S. aureus St.1.01 were exposed to four different sub-lethal doses of thyme oil (0.020%, 0.025%, 0.030% and 0.040% (v/v)) for 30 days at 25°C to evaluate whether it inhibited growth kinetics.

The application of sub-lethal doses of thyme oil slowed down the growth of St.1.01 (Figure 5.3A). The detection time (λ) increased significantly (P < 0.01) with the dose of thyme oil (Table 5.2). Thus, λ was found to be 25-fold higher when the dose of thyme oil was increased from 0.020 to 0.040%. The addition of thyme oil also produced a

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significant (P < 0.01) reduction in µmax, which was approximately 2-fold lower between

0.025-0.040% than when no EO was added (µmax = 3.375 ± 0.014 1/days). Consequently, whereas bacterial cells reached stationary phase after about 4 days in the absence of thyme oil, it was delayed for approximately 3, 6, 10 and 22 days when 0.020%, 0.025%, 0.030% and 0.040% of thyme oil, respectively, were present.

Bacterial cells of stationary-phase cultures obtained after 26 days at 25°C in the presence of 0.040% of thyme oil were subsequently used to find out if they had adapted to thyme oil. The kinetics of growth of these cells showed slight differences in λ, with values 0.3-0.9 days shorter than non-thyme oil-exposed cells (Table 5.2). It was also observed a slight increase in µmax at 0.020-0.025% of thyme oil (0.14-0.25 1/days faster), but no significant differences were found in µmax between 0.030-0.040%. As a result, cultures of thyme oil-exposed cells reached stationary phase about 1 day earlier (Figure 5.3B). Therefore, thyme oil-exposed cells would seem to show some adaptation to sub-lethal doses of thyme oil.

Figure 5.3A-B. Growth kinetics of S. aureus in the presence of sub-lethal doses of thyme oil. Non-thyme oil-exposed cells (A) and cells previously exposed to thyme oil (0.040% (v/v)) for 26 days (B) were cultured at 25°C. Experimental values are represented by symbols, whereas corresponding expected values are shown as solid lines.

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Table 5.2. Detection time (λ) and maximum specific growth rate (µmax) of non-thyme oil- exposed cells and thyme oil-exposed cells of S. aureus grown at 25°C in the presence of several sub-lethal doses of thyme oil (0.020%, 0.025%, 0.030% and 0.040% (v/v)). Thyme oil-exposed cells were obtained by previously culturing in the presence of 0.040% thyme oil for 26 days at 25°C. Parameter Thyme oil (% v/v) Non-thyme oil-exposed cells Thyme oil-exposed cells 0.020* 0.749 ± 0.012 0.416 ± 0.047

λ 0.025* 3.719 ± 0.022 3.026 ± 0.021 (days) 0.030* 6.898 ± 0.043 6.618 ± 0.080 0.040* 18.550 ± 0.050 17.634 ± 0.089 0.020* 1.851 ± 0.013 2.097 ± 0.032

µmax 0.025* 1.575 ± 0.023 1.719 ± 0.012 (1/days) 0.030 1.500 ± 0.020 1.467 ± 0.043 0.040 1.310 ± 0.044 1.239 ± 0.031

*Statistically significant differences (P < 0.05) in λ or µmax between both types of cells at same sub-lethal dose.

5.3.4. Effectiveness of thyme oil against biofilms formed under sub-lethal doses of thyme oil

A study was carried out next to assess whether the resistance of biofilms to thyme oil changed as a result of the presence of sub-lethal doses of thyme oil during biofilm formation. Because of the inhibitory effect of sub-lethal doses of thyme oil on the growth of S. aureus, biofilms obtained when cultures reached stationary phase were tested. All biofilms contained between 5-9×107 CFU/cm2.

As shown in Figure 5.4, the application of 0.040% of thyme oil produced a slight decrease in the number of viable cells (LR ~ 0.5 log CFU/cm2) of biofilms formed in the absence of this EO. However, the resistance of biofilms to thyme oil decreased significantly (P < 0.01) when sub-lethal doses of thyme oil were present during biofilm formation, with LR > 3 log CFU/cm2 being frequently achieved. Moreover, some combinations of thyme oil caused LR > 4 log CFU/cm2 (0.020% + 0.040%, 0.030% + 0.030%, 0.025% + 0.040% and 0.040% + 0.030%) and even LR > 5 log CFU/cm2 (0.030% + 0.040% and 0.040% + 0.040%), where the former figure is the concentration of thyme oil present during biofilm formation, and the latter is the concentration applied for biofilm removal.

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Figure 5.4. Logarithmic reductions of viable cells (LR) in biofilms formed under different sub- lethal doses of thyme oil after treatment with thyme oil for 30 min.

Although combining the presence of a sub-lethal dose of thyme oil during biofilm formation with the subsequent application of thyme oil for biofilm removal seems to be a highly effective strategy, it involves some risk of adaptation to thyme oil, as reported in the previous section. It was therefore considered convenient to examine the effectiveness of applying a different biocide to remove biofilms, in particular benzalkonium chloride, which is commonly used in the food industry.

5.3.5. Effectiveness of benzalkonium chloride (BAC) against biofilms formed under sub-lethal doses of thyme oil

The effectiveness of BAC against biofilms formed under sub-lethal doses of thyme oil (0.020%, 0.025%, 0.030% and 0.040% (v/v)) was therefore assessed subsequently. As shown in Figure 5.5, the presence of sub-lethal doses of thyme oil during biofilm formation also increased significantly (P < 0.01) the susceptibility of biofilms to BAC, with LR > 3 log CFU/cm2 being achieved in most of the experimental range. Some combinations of thyme oil and BAC also caused LR > 4 log CFU/cm2 (0.020% thyme oil and 10000-20000 mg/L BAC, 0.025% thyme oil and 10000-15000 mg/L BAC, 0.030-0.040% thyme oil and 5000-10000 mg/L BAC) and even LR > 5 log CFU/cm2 (0.020-0.025% thyme oil and 20000 mg/L BAC, and 0.030-0.040% thyme oil and 15000-20000 mg/L BAC).

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Figure 5.5. Logarithmic reductions of viable cells (LR) in biofilms formed under different sub- lethal doses of thyme oil after the application of BAC for 30 min.

5.4. Discussion

The results obtained in this study have shown a high variability in the effectiveness against S. aureus planktonic cells among nineteen EOs. Variations in the nature and concentration of active compounds, as well as synergistic interactions between some of them, likely explain some variability in antimicrobial activity of EOs (Bakkali et al., 2008; Burt, 2004; Hyldgaard et al., 2012). Thus, phenol compounds were found to be most active, followed by aldehydes, ketones, alcohols, ethers and hydrocarbons (Kalemba and Kunicka, 2003). A high variability in the efficacy of EOs against planktonic cells of S. aureus had been previously reported (Ertürk, 2010; Hammer et al., 1999; Mayaud et al., 2008; Prabuseenivasan et al., 2006; Silva et al., 2013). However, the ranking of EOs based on the biocidal effect on S. aureus planktonic cells was considerably different from those previous studies, probably due to the use of different strains and assay conditions. Standard bactericidal tests used as a reference the clinical strain S. aureus ATCC 6538. However, a food-related strain (St.1.01) was tested in the present study because it has both a biofilm-forming ability on stainless steel and a resistance to disinfectants commonly used in the food industry higher than S. aureus ATCC 6538 (Vázquez-Sánchez et al., 2014). Thus, it was considered more suitable (at least clearly a worse-case scenario than S. aureus ATCC 6538) to evaluate the potential of the application of EOs against biofilms in the food industry.

As expected, the resistance of S. aureus to EOs increased notably as a result of biofilm formation with respect to free-living counterparts. However, the majority of

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Antimicrobial activity of essential oils against S. aureus biofilms studies about the antimicrobial effects of EOs have been carried out using suspension cultures (i.e. planktonic cells), whereas only a few studies have been made on biofilms despite they are a common source of microbial contamination in the food industry. In fact, this is the first study that evaluates the activity of cumin, geranium, palmarosa, patchouli and vetiver oils against S. aureus biofilms. It is well-known that the use of suspension cultures as experimental models underestimates considerably the doses needed for removal of bacterial cells. But more importantly, this study clearly shows that the ranking of EOs by antimicrobial effectiveness against biofilms and planktonic cells was dissimilar. It thus seems that factors such as the composition and architecture of the biofilm as well as the reactivity, hydrophobicity and diffusion rate of EOs into the biofilm matrix affect significantly the effectiveness of EOs.

The eight most effective EOs against planktonic cells also reduced the number of biofilm viable cells significantly, but none of them was able to completely eradicate S. aureus biofilms at doses tested (0.1-8% (v/v)). It seems thus clear that preventing biofilm formation should be targeted rather than disruption and removal of biofilms, as suggested by Kelly et al. (2012). In fact, Adukwu et al. (2012) also reported that lemongrass and grapefruit oils did not remove 48-h-old biofilms of three MSSA and two MRSA strains at any concentration tested (i.e., 0.06-4%), despite biofilms were exposed for 24 h to EOs. Neither the exposure to 1% of lemon or citronella oils for 15 min was able to kill 240-h-old biofilms of S. aureus formed on polypropylene (Millezi et al., 2012). Only in the case of the clinical strain S. aureus DMST 4745, 24-h-old biofilms formed on polystyrene at 37ºC were totally eradicated with the application of 4% of lemongrass for 1 h (Aiemsaard et al., 2011).

Likewise for planktonic cells, thyme oil (from Thymus vulgaris) was found to be the most effective EO against biofilms of S. aureus. Kavanaugh and Ribbeck (2012) also observed a high efficacy of thyme oil against biofilms formed by a clinical MRSA strain, with a minimum biofilm eradication concentration (MBEC) lower than tee tree oil (0.15% and > 5%, respectively). The high efficacy of thyme oil has been allocated to a high content in thymol and carvacrol (Hudaib et al., 2002; Rota et al., 2008). These two phenolic compounds cause alterations in fluidity, permeability and composition of membrane, depletion of intracellular ATP and ATPase inhibition, as well as perturbation of different metabolic pathways and gene responses (Di-Pasqua et al.,

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2007; La-Storia et al., 2011; Trombetta et al., 2005; Walsh et al., 2003). In S. aureus, thymol also inhibits the synthesis and secretion of α-hemolysin, SEA and SEB (Qiu et al., 2010). The high hydrophilicity of thymol and carvacrol has been suggested to account for the antimicrobial activity of thyme oil against biofilms, as it could enhance the diffusion of thyme oil into the extracellular matrix (Griffin et al., 1999; Nostro et al., 2007).

The presence of sub-lethal doses of thyme oil inhibited growth kinetics of planktonic cultures of S. aureus St.1.01 (the detection time increased whereas the maximum specific growth rate decreased). It was therefore considered that it could also affect biofilm formation. Thus, when biofilms were formed in the presence of sub-lethal doses of thyme oil, the resistance of biofilms to thyme oil and BAC decreased highly. This decrease was proportional to the sub-lethal dose applied. As a result, the doses of biocide needed to significantly reduce viable biofilm cells (i.e. LR ≥ 4 log CFU/cm2) were much lower than in biofilms formed with no thyme oil present. The treatment of food-contact surfaces with thyme oil would thus be an interesting alternative to inhibit or at least slow down the formation of S. aureus biofilms as well as to reduce the doses of disinfectant required to be eradicated.

Thyme oil also showed a higher antimicrobial potential against S. aureus biofilms than BAC, a chemical widely used for disinfection of stainless steel surfaces in the food industry. This is probably due to the high reactivity of BAC with extracellular matrix components, which would diminished the concentration of active compounds that are effective against biofilm cells (Bridier et al., 2011b). Thyme oil-based treatments can thus be considered as an effective, safe, less corrosive and more environmentally- friendly alternative in the food industry.

Nonetheless, growth kinetics of planktonic cultures of S. aureus St.1.01 has shown that a long-term exposure to thyme oil can produce some adaptation (shown by a decrease in λ and an increase in µmax). Previously, bacterial adaptation to individual active compounds of EOs (e.g., carvacrol, thymol, camphor or cineole) had been detected (Di-Pasqua et al., 2010; Turina et al., 2006; Ultee et al., 2000), but not to crude EOs (including thyme oil). However, it is thought that adaptation was affected by the volatility of some active compounds of thyme oil, so it actually occurred at a concentration lower than 0.040%. Thus, the growth of thyme oil-adapted cells would

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Antimicrobial activity of essential oils against S. aureus biofilms also start at a concentration lower than the initial dose, particularly at high initial sub- lethal doses.

To prevent the emergence of antimicrobial-resistant strains, antimicrobial treatments using EOs should be based on the rotation and combination of different EOs (or combinations of different active compounds that produce a synergistic lethal effect) or with other biocides. Several EO-based combined treatments have been proposed as an antimicrobial strategy against S. aureus. Blends of oregano, basil and bergamot have shown a synergistic bactericidal effect (Lv et al., 2011), whereas an additive bactericidal effect was detected for mixtures of cinnamon and clove (Goñi et al., 2009). Combinations of clove and rosemary (Fu et al., 2007) or thyme and anise (Al-Bayati, 2008) have been also tested, but the efficacy did not significantly increase with respect to the application of these EOs individually. However, none of these studies has dealt with S. aureus biofilms. In fact, only Oliveira et al. (2010) has evaluated the effectiveness of an EO-based combined treatment (blends of citronella and lemongrass) against biofilms of L. monocytogenes, but they were formed at a temperature rather uncommon in the food industry (37°C). Therefore, the combined treatments using thyme oil proposed in this study represent a novel strategy for prevent biofilm formation on food-contact surfaces as well as a highly effective disinfectant procedure.

5.5. Conclusions

Many essential oils (EOs) are well-known to have antimicrobial properties, so they are considered a promising alternative for disinfection in the food industry. However, not all EOs are highly effective antimicrobials. Thus, a high variability in the effectiveness against S. aureus biofilms and planktonic cells was found among EOs tested. But more importantly, the rankings of EOs by antimicrobial effectiveness against biofilms and planktonic cells were dissimilar. Therefore, biofilm-related factors should be also taken into account to select the most effective EO to be applied in the food industry.

Though several EOs showed a high antimicrobial effect against S. aureus biofilms, with thyme oil as most effective, none of them was able to completely eradicate biofilms at doses tested. The use of EOs should thus be targeted to the prevention of

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Chapter 5 biofilm formation rather than disruption and removal of biofilms formed. The presence of thyme oil at sub-lethal doses was found to slow down the development of S. aureus biofilms, as well as to notably enhance their subsequent removal by exposure to antimicrobials such as thyme oil or BAC. The treatment of food-contact surfaces with thyme oil would be an interesting preventive strategy. However, loss of active compounds due to volatility would have to be solved. Also, long-term exposure to thyme oil could cause bacterial adaptation. Therefore, treatments against biofilms should be based on the rotation and combination of different EOs (or active compounds) or with other biocides to prevent the emergence of antimicrobial-resistant strains. And the combination of biocides should be addressed to search intelligent solutions of disinfection in a similar sense to hurdle technology. This approach could reduce adverse effects on working, environmental and economic aspects. The choice of biocides should thus be aimed to obtain synergistic effects so a greater knowledge of the mechanisms of action of biocides would be required. Individual active compounds of EOs would be used preferentially instead of crude EOs.

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Despite the great importance of fishery products in Spain –it is the largest producer and the second largest consumer in the European Union (Eurostat, 2010; FAO, 2012)–, the first chapter of the present thesis represents the first incidence study that analyses different categories of fishery products of different origin and type of processing commercialized in Spain. In fact, previous information available on microbial safety of fishery products made or sold in Spain is scarce, and only González-Rodríguez et al. (2002) and Herrera et al. (2006) examined the presence of S. aureus in cold-smoked salmon and 50 samples of fresh marine fish. A high incidence of S. aureus was detected in commercialized fishery products (~ 25%), being most of them se-carriers (i.e., putative enterotoxigenic strains). In addition, a significant proportion of these products (~ 11%) exceeded the regulatory limits in force at the time the study was conducted, which involves a serious risk of staphylococcal food poisoning for the consumer. These results also questioned the adequacy of recent changes in Spanish national regulations (RD 135/2010), following Commission Regulation (EC) No 2073/2005, which revoked the use of S. aureus as a microbiological criterion for a number of foods, including several fishery products.

Moreover, the emergence of multidrug resistant pathogens is additionally generating an environmental hazard to the food supply and human health, as it makes eradication more difficult and incidence to increase (Livermore, 2000; Popovich et al., 2007; Ribeiro et al., 2007). Particularly, methicillin-resistant S. aureus (MRSA) are being increasingly found outside clinical settings (Popovich et al., 2007; Ribeiro et al., 2007; Stankovic et al., 2007), including in fishery products (Beleneva, 2011). In the present thesis, however, all S. aureus isolated from marketed fishery products were found to be multidrug-resistant MSSA (Chapter 1). Although EFSA (2009b) reported that there is no current evidence that eating food contaminated with MRSA may lead to an increased risk of humans becoming healthy carriers or infected with MRSA strains, the risk of multidrug-resistant MSSA, which are present in food more frequently than MRSA, has not been examined yet. In fact, it has been recently underlined the need of incidence studies of multidrug-resistant strains in food to clarify their public health relevance (Waters et al., 2011). Meanwhile, some preventive control measures should be taken.

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It is also well known that biofilm formation allows bacteria to tolerate adverse environmental conditions (e.g. presence of biocides) much better than free-living counterparts (Srey et al., 2013; Van-Houdt and Michiels, 2010). In this thesis, putative enterotoxigenic S. aureus strains isolated from fishery products exhibited, generally, a high biofilm-forming ability on two common materials of food-contact surfaces, polystyrene and stainless steel (Chapters 2 and 3). As a result, these strains also showed a high resistance to three disinfectants traditionally used in the food industry such as benzalkonium chloride (BAC), peracetic acid (PAA) and sodium hypochlorite (NaClO). Moreover, the minimum biofilm eradication concentrations (MBECs) determined in this work under some conditions usual in the food industry were considerably higher than doses often recommended by manufacturers for BAC (200-1000 mg/L), PAA (50-350 mg/L) and NaClO (50-800 mg/L) (Gaulin et al., 2011). Among these disinfectants, PAA was found to be the most effective against both biofilms and planktonic cells, followed by NaClO and then BAC, but latters are more frequently used in the food industry. Accordingly, the entrance of these strains in food-processing facilities does not only involve an immediate risk to food safety but more importantly the risk of long-term presence (even persistence) unless appropriate measurements are applied to kill them.

The risk of the presence of S. aureus on food-contact surfaces was found to be highly strain dependent. In fact, a high variation in the expression of genes involved in biofilm formation (e.g. icaA, rbf, sarA and σB) was detected between different strains (Chapter 2), which could in turn produce differences in the composition and architecture of the extracellular matrix and, therefore, in the stability and resistance of biofilms. Nonetheless, the current European Union standard bactericidal tests EN 1040 (CEN, 2005), EN 1276 (CEN, 2009) and EN 13697 (CEN, 2002) use a small number of type strains (and only one S. aureus, the strain ATCC 6538) to assess the efficacy of disinfectants. In this regard, most strains isolated in this thesis from fishery products showed a higher biofilm-forming ability and biocide resistance than S. aureus ATCC 6538. Thus, the use of a wide collection of strains for the assessment of the bactericidal activity of disinfectants seems to be necessary to ensure that they are correctly applied on surfaces.

Data obtained in this thesis also showed that different environmental factors present in the food industry (e.g., temperature, ionic strength, availability of nutrients) can

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General Discussion affect the adhesion and biofilm formation of S. aureus on food-contact surfaces (Chapters 2 and 3). Interestingly, a statistical trend was found between biofilm-forming ability and the type of processing applied to the fishery products that strains were isolated from, which seems to show a selective pressure of food-processing conditions on S. aureus. Moreover, it was observed that temperature also affected the antimicrobial resistance of 168-h-old biofilms and planktonic counterpart cells to BAC, being this effect highly dependent to the adaptive response of each strain to low temperatures (as defined by the growth kinetics). Therefore, it is also important that standard bactericidal tests truly simulate conditions found in the food industry and clinical settings, as previously recommended some authors (Briñez et al., 2006; Langsrud et al., 2003; Meyer et al., 2010).

The introduction of innovative disinfection strategies could be an effective alternative to avoid, or at least reduce, the risk of biofilm formation and antimicrobial resistance. In the present thesis, electrolyzed water (EW) and several essential oils (EOs) have been evaluated as alternatives to traditional disinfectants used in the food industry, because they are less hazardous for the health of workers and more environmentally-friendly.

In the case of EW (Chapter 4), data provided in this thesis showed that variations in the pH of production not significantly affect the effectiveness of EW against S. aureus biofilms, in contrast to planktonic cells. This lack of effect is considered to be due to the role of the extracellular matrix of biofilms, which acts as a protective barrier that limits molecular diffusion towards cellular targets and also reacts with active chlorine compounds (Chen and Stewart, 1996; Oomori et al., 2000; White, 1999). Neutral EW (NEW) was therefore used in subsequent studies as it has a higher potential for long- term application than acidic EW (i.e., a lower corrosiveness and toxicity (Ayebah and Hung, 2005; Guentzel et al., 2008)) and due to the higher yield rate of the production unit at neutral pH.

The efficacy of NEW against S. aureus biofilms increased with increasing ACC and exposure time. The application of NEW caused a high initial reduction in the number of viable biofilm cells, presumably by killing cells located in the outer layers of biofilms, which are more exposed to biocide molecules. Afterwards, the inactivation rate of biofilm cells by NEW slowed down considerably, probably as the extracellular matrix

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General Discussion physically hinders diffusion and present many reactive sites for biocide molecules (Chen and Stewart, 1996; White, 1999; Oomori et al., 2000), and it only increase with the exposure to 800 mg/L ACC during at least 15 min as a result of the disruption of the biofilm matrix. Similar effects were reported by Kim et al. (2001) on Listeria monocytogenes biofilms with the application of AEW.

High concentrations of NEW (800 mg/L ACC) were thus needed to achieve the logarithmic reductions (LR) demanded by the European quantitative surface test of bactericidal activity EN 13697 (≥ 4 log CFU/cm2 after 5 min of exposure), but this would be costly and not environmentally-friendly. Nevertheless, a noticeable effect (LR > 3 log CFU/cm2) was obtained by applying only 200 mg/L of NEW during 5 min, which suggested that consecutive applications of low concentrations of NEW could increase significantly the bactericidal effect against biofilms, with clear advantages in terms of both dose and exposure time.

As expected, a double sequential application of NEW at low concentrations was enough to achieve the LR higher than 4 log CFU/cm2 after 5 min of exposure each. However, this procedure could involve a risk of adaptation to EW, as previously observed after the repeated use of a same chlorinated disinfectant (Braoudaki and Hilton, 2004; Bridier et al., 2011a; Lundén et al., 2003; Maalej et al., 2006; Sanderson and Stewart, 1997). Therefore, it seemed convenient that sequential applications were based on the use of different biocides, preferably with different cellular targets.

The potential of combining NEW with benzalkonium chloride (BAC) or peracetic acid (PAA) was thus examined. LR demanded by the European standard test EN 13697 were also reached in all cases by applying low concentrations of each biocide. But washing was mandatory to prevent cross-reaction between them, so operation time increased and wash water would require additional biocidal processes (e.g., heat, UV radiation, chlorination, ozonation) to kill dispersed cells during washing (Casani and Knøchel, 2002; Fähnrich et al., 1998).

Alternatively, the efficacy of commercial essential oils (EOs) against biofilms and planktonic counterparts of S. aureus St.1.01 was also assessed in this thesis (Chapter 5). A high variability in the biocidal effectiveness against planktonic cells was observed between the nineteen commercial EOs tested (anise, carrot, citronella, coriander, cumin,

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Eucalyptus globulus, Eucalyptus radiata, fennel, geranium, ginger, hyssop, lemongrass, marjoram, palmarosa, patchouli, sage, tea-tree, thyme and vetiver). This variability could be explained by variations in the nature and concentration of active compounds, as well as synergistic interactions between some of them (Burt, 2004; Bakkali et al., 2008; Hyldgaard et al., 2012). Thus, phenol compounds were found to be most active, followed by aldehydes, ketones, alcohols, ethers and hydrocarbons (Kalemba and Kunicka, 2003).

The resistance of S. aureus to EOs increased notably as a result of biofilm formation with respect to free-living counterparts. However, the ranking of EOs by antimicrobial effectiveness against biofilms and planktonic cells was dissimilar. It thus seems that factors such as the composition and architecture of the biofilm as well as the reactivity, hydrophobicity and diffusion rate of EOs into the biofilm matrix affect significantly the effectiveness of EOs. The eight most effective EOs against planktonic cells (thyme, lemongrass, vetiver, citronella, cumin, geranium, palmarosa and patchouli) also reduced the number of biofilm viable cells significantly, but none of them was able to completely eradicate S. aureus biofilms at doses tested (0.1-8% (v/v)). It seems thus clear that preventing biofilm formation should be targeted rather than disruption and removal of biofilms, as suggested by Kelly et al. (2012).

Thyme oil (from Thymus vulgaris) was found to be the most effective EO against both biofilms and planktonic cells of S. aureus, presumably by its high content in the phenolic compounds thymol and carvacrol (Hudaib et al., 2002; Rota et al., 2008). The use of sub-lethal doses of thyme oil prevented biofilm formation and enhanced the efficiency of thyme oil and BAC against biofilms. The treatment of food-contact surfaces with thyme oil would be an interesting preventive strategy. Nonetheless, a long-term exposure to thyme oil can produce some adaptation and problems of volatility of active compounds. Therefore, antimicrobial treatments using EOs should be based on the rotation and combination of different EOs (or active compounds) or with other biocides to prevent the emergence of antimicrobial-resistant strains.

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Los resultados obtenidos en esta tesis han permitido obtener las siguientes conclusiones:

1. S. aureus fueron detectados en una significativa proporción (~ 25%) de productos pesqueros comercializados en España en 2008 y 2009, sobre todo cepas con capacidad enterotoxigénica. Una cantidad significativa de los productos regulados no cumplían los límites vigentes, y una alta proporción de los no regulados también estaban contaminados, lo cual revela una cierta eficacia de las políticas legales para asegurar la higiene en la industria alimentaria. Por tanto, se recomienda revisar los programas de prerrequisitos y mejorar las prácticas de higiene en las operaciones de manipulación y procesado, desde la captura o cultivo del pescado hasta su punto de venta, para certificar la seguridad de los productos pesqueros consumidos en España. Además, estos resultados cuestionan que se haya eliminado a los estafilococos coagulasa positivos (mayormente S. aureus) como criterio microbiológico en productos listos para consumir y listos para cocinar. 2. Una considerable variabilidad en la capacidad de adhesión y formación de biopelículas fue encontrada entre las cepas con capacidad enterotoxigénica de S. aureus aisladas de productos pesqueros. Está variabilidad estaba claramente influenciada por diferentes condiciones ambientales relevantes en la industria alimentaria como temperatura, osmolaridad y disponibilidad de nutrientes, así como propiedades intrínsecas de cada cepa. Así, se detectó que la adhesión inicial incrementa al aumentar la fuerza iónica del medio, mientras que la formación de biopelículas aumenta en presencia de glucosa y, en menor medida, de cloruro sódico y magnesio. Además, se encontraron diferencias significativas en la expresión de ciertos genes relacionados con la formación de biopelículas entre cepas y a distintas condiciones ambientales, lo cual sugiere que la presencia de biopelículas en plantas de procesado de alimentos esté condicionada por la adaptación a los estreses ambientales. En este sentido, parece que el procesado del alimento genera una presión selectiva, por lo que cepas con una alta capacidad de formación de biopelículas se detectan más frecuentemente en productos altamente manipulados y procesados.

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3. La mayoría de las cepas con capacidad enterotoxigénica mostraron una capacidad de formación de biopelículas sobre superficies de poliestireno y acero inoxidable, así como una resistencia a cloruro de benzalconio (BAC), ácido peracético (PAA) e hipoclorito sódico (NaClO) mayor que la de S. aureus ATCC 6538, la cepa de referencia usada en los métodos bactericidas estándar. Como resultado, fueron necesarias mayores dosis de estos desinfectantes que las recomendadas por los fabricantes para eliminar biopelículas formadas por todas las cepas sobre superficies de contacto con el alimento. No se encontró correlación entre la concentración mínima necesaria para erradicar biopelículas y la concentración bactericida mínima, y las cepas mostraron un orden de clasificación distinto para cada desinfectante. Todos estos resultados cuestionan y sugieren una revisión de los actuales métodos bactericidas estándar (la mayoría basados en cultivos líquidos usando unas pocas cepas, y sólo un S. aureus). Por tanto, se recomienda tomar decisiones sobre la aplicación de desinfectantes basándose en el uso de biopelículas como sistema experimental, analizando un amplio número de cepas, y en la simulación de las condiciones ambientales presentes en la industria alimentaria.

4. La evaluación de estrategias de desinfección más respetuosas con el medioambiente, basadas en la aplicación de agua electrolizada (EW) y ciertos aceites esenciales (EOs), ha permitido obtener las siguientes conclusiones:

4.1 Apenas hubo diferencias en la actividad bactericida del EW frente a biopelículas debido a variaciones en el pH de producción. Esto reveló que el EW neutra (NEW) puede tener un mayor potencial como desinfectante a largo plazo que el EW ácida debido a su menor corrosión en superficies metálicas y a su menor toxicidad sobre los manipuladores. Sin embargo, se necesitó una alta concentración de cloro activo para cumplir con las especificaciones demandadas por el método cuantitativo europeo de actividad bactericida en superficie. La aplicación secuencial doble de NEW o la aplicación secuencial de NEW con desinfectantes clásicos (BAC o PAA) resultó en una prometedora alternativa para eliminar S. aureus de instalaciones de procesado de alimento en términos de dosis y tiempo de exposición.

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4.2 Se encontró una alta variabilidad en la eficacia de los EOs evaluados frente a células planctónicas y biopelículas formadas en acero inoxidable, pero ninguno fue capaz de erradicar completamente las biopelículas. El aceite de tomillo fue el más efectivo en ambos casos, pero altas concentraciones fueron necesarias para reducir significativamente el número de células viables en la biopelícula. Alternativamente, la presencia de dosis sub-letales de aceite de tomillo ralentizó el desarrollo de biopelículas, y mejoró la eficacia del aceite de tomillo y del BAC frente a estas. Por tanto, el uso de EOs debe enfocarse a estrategias de prevención de la formación de biopelículas más que a la erradicación de biopelículas establecidas. Sin embargo, cierta adaptación al aceite de tomillo fue detectada, lo cual podría ser un inconveniente este uso. Por tanto, estas estrategias se deben basar en la rotación y combinación de diferentes EOs o con otros biocidas para prevenir la emergencia de cepas resistentes a los antimicrobianos.

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The results obtained in this thesis have permitted the following conclusions to be drawn:

1. A remarkable number (~ 25%) of fishery products collected from the retail sector in Spain in 2008 and 2009 was found to be contaminated with S. aureus, mostly with strains able to produce enterotoxins. A significant proportion of regulated products did not comply with legal limits in force, but most contaminated products was not subject to any regulation, which seems to reveal some effects of legal policies on the efforts of the industry to ensure food hygiene. A revision of pre-requisite programs leading to improve hygienic practices in handling and processing operations from fishing or farming to retail is therefore recommended to ensure the safety of seafood consumed in Spain. Also, these results call into question the following repeal of coagulase positive staphylococci (mainly S. aureus) as a microbiological criterion for ready-to-cook products and most ready-to-eat products.

2. A significant variability in adhesion and biofilm-forming properties was found among putative enterotoxigenic S. aureus strains isolated from fishery products. This variability was clearly influenced by environmental conditions relevant for the food industry such as temperature, osmolarity and nutrient availability, as well as by intrinsic properties of strains. Thus, initial adhesion was increased by high ionic strength conditions, whereas biofilm formation was enhanced by glucose and, to a lower extent, by sodium or magnesium chloride. Significant differences in the expression of some biofilm-related genes were also found among strains and environmental conditions, which suggest that the presence of biofilms in food- processing facilities would be conditioned by the adaptation to environmental stresses. In this sense, it seems that food processing could have applied some selective pressure, so strains with a high biofilm-forming ability were more likely to be found in highly handled and processed products.

3. Most putative enterotoxigenic strains showed a biofilm-forming ability on polystyrene and stainless steel and a resistance to benzalkonium chloride (BAC), peracetic acid (PAA) and sodium hypochlorite (NaClO) higher than that of S. aureus ATCC 6538, the reference strain used in bactericidal standard tests. As a result,

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doses of BAC, PAA and NaClO higher than those recommended by manufacturers were needed to remove biofilms of all strains from food-contact surfaces. No correlation was found between minimum biofilm eradication concentration and minimum bactericidal concentration either, and strains were ranked differently according to biofilm resistance to each disinfectant. All these results question and call for a revision of current bactericidal standard tests (mostly suspension-based using a few strains, and only one S. aureus). Therefore, decision-making on the application of disinfectants must be based on a widespread use of biofilms as experimental systems, testing a relatively wide number of strains, and on the simulation of environmental conditions found in the food industry.

4. The assessment of disinfection strategies more environmentally-friendly, based on the application of electrolyzed water (EW) and some essential oils (EOs), have allowed to obtain the following conclusions:

4.1. Hardly differences were noticed in the bactericidal activity of EW against biofilms as a result of variations in the pH of production. This revealed that neutral EW (NEW) would have a higher potential than acidic EW for long-term use as an antimicrobial in the food industry due to a lower corrosiveness to metal surfaces and a lower toxicity to handlers. However, a high available chlorine concentration was needed to comply with specifications demanded by the European quantitative surface test of bactericidal activity. A double sequential application of NEW or a sequential application of NEW and a classical disinfectant (either BAC or PAA) was found to be a promising alternative to remove S. aureus from food-processing facilities in terms of dose and exposure time.

4.2. A highly variable effectiveness against planktonic cells and biofilms formed on stainless steel was found among EOs tested, but no EO removed biofilms completely. Thyme oil was found to be the most effective in both cases, but high concentrations were needed to reduce significantly the number of viable biofilm cells. Alternatively, the presence of sub-lethal doses of thyme oil slowed down biofilm formation, and enhanced the efficiency of thyme oil and BAC against biofilms. Therefore, EOs should be aimed to be a preventive strategy against

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General Conclusions biofilms rather than to remove biofilms formed. However, some adaptation to thyme oil was detected, which could be a drawback for this intended use. The rotation and combination of different EOs or with other biocides should be therefore employed to prevent the emergence of antimicrobial-resistant strains.

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Wertheim, H.F.L., Melles, D.C., Vos, M.C., van Leeuwen, W., van Belkum, A., Verbrugh, H.A., Nouwen, J.L., 2005. The role of nasal carriage in Staphylococcus aureus infections. Lancet Infect. Dis. 5, 751–762. White, G.C., 1999. Handbook of chlorination and alternative disinfectants, 4th ed. John Wiley and Sons, New York. Wieneke, a a, Roberts, D., Gilbert, R.J., 1993. Staphylococcal food poisoning in the United Kingdom , 1969-90. Epidemiol. Infect. 110, 519–531. Wirtanen, G., Salo, S., 2003. Disinfection in food processing – efficacy testing of disinfectants. Rev. Environ. Sci. Bio/Technology 2, 293–306. Woolaway, M.C., Bartlett, C.L., Wieneke, A.A., Gilbert, R.J., Murrell, H.C., Aureli, P., 1986. International outbreak of staphylococcal food poisoning caused by contaminated lasagne. J. Hyg. (Lond). 96, 67–73. Xing, K., Chen, X.G., Kong, M., Liu, C.S., Cha, D.S., Park, H.J., 2009. Effect of oleoyl- chitosan nanoparticles as a novel antibacterial dispersion system on viability, membrane permeability and cell morphology of Escherichia coli and Staphylococcus aureus. Carbohydr. Polym. 76, 17–22. Xing, M., Shen, F., Liu, L., Chen, Z., Guo, N., Wang, X., Wang, W., Zhang, K., Wu, X., Li, Y., Sun, S., Yu, L., 2012. Antimicrobial efficacy of the alkaloid harmaline alone and in combination with chlorhexidine digluconate against clinical isolates of Staphylococcus aureus grown in planktonic and biofilm cultures. Lett. Appl. Microbiol. 54, 475–482. Xu, H., Zou, Y., Lee, H.-Y., Ahn, J., 2010. Effect of NaCl on the biofilm formation by foodborne pathogens. J. Food Sci. 75, M580–585. Yang, Z.-Q., Jiao, X.-A., Zhou, X.-H., Cao, G.-X., Fang, W.-M., Gu, R.-X., 2008. Isolation and molecular characterization of Vibrio parahaemolyticus from fresh, low-temperature preserved, dried, and salted seafood products in two coastal areas of eastern China. Int. J. Food Microbiol. 125, 279–285. Yu, D., Zhao, L., Xue, T., Sun, B., 2012. Staphylococcus aureus autoinducer-2 quorum sensing decreases biofilm formation in an icaR-dependent manner. BMC Microbiol. 12, 288. Zabala, A.J., Font, M.O., Gallastegi, P.M., Ibarbia, E.S., Juaristi, A., Santa, L., Rodríguez, M., 2011. Situación de los desinfectantes de uso ambiental y en industria alimentaria registrados en España tras la publicación de la Directiva 98/8/CE. Rev. Esp. Salud Publica 85, 175–188. Zeng, X., Tang, W., Ye, G., Ouyang, T., Tian, L., Ni, Y., Li, P., 2010. Studies on disinfection mechanism of electrolyzed oxidizing water on E. coli and Staphylococcus aureus. J. Food Sci. 75, M253–260. Zhang, L., Pornpattananangku, D., Hu, C.-M.J., Huang, C.-M., 2010. Development of nanoparticles for antimicrobial drug delivery. Curr. Med. Chem. 17, 585–594. Zmantar, T., Kouidhi, B., Miladi, H., Mahdouani, K., Bakhrouf, A., 2010. A microtiter plate assay for Staphylococcus aureus biofilm quantification at various pH levels and hydrogen peroxide supplementation. New Microbiol. 33, 137–145. Zwietering, M.H., Jongenburger, I., Rombouts, F.M., Riet, K., 1990. Modeling of the bacterial growth curve. Appl. Environ. Microbiol. 56, 1875–81.

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List of original publications

The thesis is based on the following original publications:

1. Vázquez-Sánchez, D., López-Cabo, M., Saá-Ibusquiza, P., Rodríguez-Herrera, J.J., 2012. Incidence and characterization of Staphylococcus aureus in fishery products marketed in Galicia (Northwest Spain). Int. J. Food Microbiol. (IF: 3.425; Q: Q1; Year: 2012, JCR) 157, 286-296. ISSN: 0168-1605

2. Vázquez-Sánchez, D., Habimana, O., Holck, A., 2013. Impact of food-related environmental factors on the adherence and biofilm formation of natural Staphylococcus aureus isolates. Curr. Microbiol. (IF: 1.520; Q: Q4; Year: 2012, JCR) 66, 110-121. ISSN: 0343-8651

3. Vázquez-Sánchez, D., Cabo, M.L., Ibusquiza, P.S., Rodríguez-Herrera, J.J., 2014. Biofilm-forming ability and resistance to industrial disinfectants of Staphylococcus aureus isolated from fishery products. Food Control (IF: 2.738; Q: Q1; Year: 2012, JCR) 39, 8-16. ISSN: 0956-7135

4. Vázquez-Sánchez, D., Cabo, M.L., Rodríguez-Herrera, J.J. Single and sequential application of electrolyzed water with benzalkonium chloride or peracetic acid for removal of Staphylococcus aureus biofilms. J. Food Saf. (under review).

5. Vázquez-Sánchez, D., Cabo, M.L., Rodríguez-Herrera, J.J. Antimicrobial activity of essential oils against Staphylococcus aureus biofilms. J. Food Saf. (under review).

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Agradecimientos / Acknowledgements

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Agradecimientos / Acknowledgements

Primero, quiero agradecer la labor del Dr. Juan José Rodríguez Herrera y de la Dra. Marta López Cabo en la supervisión de esta tesis, así como en mi propia formación científica en el ámbito de la Microbiología y Seguridad Alimentaria. Hacéis un tándem perfecto del que tomé buena nota. Agradecer también a la Dra. Paloma Morán Martínez su colaboración y ayuda en la publicación de esta tesis en la Universidad de Vigo.

Además, quiero darles mi más sincera gratitud a todos los compañeros y compañeras que conocí en el IIM durante la realización de esta tesis. Gracias por vuestro apoyo durante todos estos años. Sonia, Eva e Vanessa, foi un pracer traballar ó voso carón, compartindo campá, chisqueiro, pipetas e emisora de radio: sempre facedes máis sinxelo o traballo dos demais. Paula, gracias por todos los momentos inolvidables en el laboratorio. Marta, Alberto, Teresa, Graciela y Ana, fue una suerte trabajar juntos y espero tomar más cafés con vosotros. Pedro, ànim amb la teva tesis, que als propers pintxos convides tu. Gabriel, Laura, Lola, Javier y Pilar, gracias por vuestra agradable (aunque breve) compañía. Diana, Natalia, Adelaida, Lidia, Noemí, Maider y Jesús, aprendimos mucho juntos y vuestra presencia en el laboratorio fue muy gratificante.

Gracias también al resto del personal del IIM por vuestra ayuda durante mi estancia, especialmente a los departamentos de Ecología y Biodiversidad Marina y Biología y Fisiología Larvaria de Peces, por dejarnos usar amablemente vuestro transiluminador; a Juan Luis, por su excelente asesoramiento informático; a Luisa, por dejarme participar en actividades de divulgación científica tan satisfactorias; a Frank y demás compañeros de las clases de inglés; a Estrella, por su compañía y generosidad, y por mantener el laboratorio siempre limpio y ordenado; y a todos los colegas de pachanga que siempre me dieron su mejor pase (Elsi, Quitos, Alhambra, Miguel, Javi, Fer, Waldo, Jorge, Gabriel, Mar, etc.).

Special thanks to Dr. Askild Holck and Dr. Olivier Habimana for their contributions to the present doctoral thesis. It was a pleasure work together and I will always appreciate your excellent advices. Thanks also to all people that I met at Nofima,

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Agradecimientos / Acknowledgements particularly to Monika, Diego, Nebojsa, Natthorn, Anne, Ulrike, etc., for all the unforgettable moments that you have given me, and to my laboratory colleagues Tone Mari, Signe, Birgitte, etc., for their inestimable assistance during my experiments.

Por otra parte, agradecer la inmensa paciencia y cariño de mi familia durante esta larga travesía. Especialmente a mis padres Miguel y Fevi, a los que nunca seré capaz de recompensar por su esfuerzo en darme unos principios y una educación digna, y por su infinita paciencia. También a mi hermano Isaac, que siempre me sirvió de referencia para crecer como persona. A mis abuelos Toncho y Lila, porque de mayor quisiera llegar a ser como vosotros, gracias por vuestro apoyo y generosidad. A mi abuela María, a la que siempre recordaré con cariño cuando vaya por Entrimo. Ó meu tío Xurxo, por tódalas revistas do National Geographic e coleccións de A Nosa Terra das que tanto gocei aprendendo. A mi madrina-bióloga Yadira, que me dio una infancia inolvidable. A toda la primada coruñesa, que no son pocos y que a cada cual mejor. A mi familia de Mallorca, especialmente a mi ahijada Ángela, que tanto eché de menos durante estos años de tesis. A Liliana, por regalarme esta maravillosa portada para mi tesis. Y también a la familia de Vero, por acogerme como uno más y tratarme siempre tan bien.

Muchas gracias a mis amigos y amigas de Vigo (y comarca) por todas las comidas y cenas, fiestas y conciertos, excursiones y viajes, o simples paseos y quedadas. Óscar, Paula, Raquel, Marta, Nuria, José, Plato, Carmen, Alba, Sandra, Roi, Adri, Gene, Almu, María... ¡¡sois los mejores!! Siempre sabéis como hacerme pasar momentos legen...darios. Y Óscar, ve preparando la mochila y los variformes que pronto saldremos a hacer el Camino de nuevo.

Y finalmente quería agradecerle eternamente a la persona responsable de que tenga hoy esta Tesis Doctoral entre mis manos. Vero, mi amor, gracias por quererme todos estos años, por animarme, por apoyarme, por hacer que cada día a tu lado sea inolvidable. Sin ti, esto nunca hubiese sucedido. Gracias, preciosa.

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