Ngome et al. AMB Expr (2018) 8:105 https://doi.org/10.1186/s13568-018-0634-z ORIGINAL ARTICLE Open Access Linalool, citral, eugenol and thymol: control of planktonic and sessile cells of Shigella fexneri Moisés Tomás Ngome1, José Guilherme Lembi Ferreira Alves1* , Ana Cristina Freitas de Oliveira1, Patrícia da Silva Machado2, Olga Lucía Mondragón‑Bernal1 and Roberta Hilsdorf Piccoli3 Abstract The antimicrobial activity of linalool, citral, eugenol and thymol was determined in growth studies of both planktonic (PC) and bioflm cells (BC) Shigella fexneri. These components were evaluated either in isolation or in combinations using a sequential experimental strategy with Plackett & Burman and central composite rotational designs totaling 1 1 47 treatments. The minimum inhibitory concentration for PC was 0.125% (v v− ) for linalool and 0.5% (v v− ) for citral, 1 eugenol and thymol. The bioflm minimum bactericidal concentration was 3 and 1% (v v− ) for linalool and citral, 1 respectively, and 2% (v ­v− ) for eugenol and thymol. In the mixtures, the minimum concentrations in the efcient 1 assays for PC growth inhibition were 0.0003, 0.0443 and 0.0443% (v ­v− ), for linalool, citral and thymol, respectively. 1 In the BC, only two assays with concentrations of 0.0558, 0.0558 and 0.319% (v v− ) and 0.035, 0.035 and 0.3999% 1 (v v− ) for linalool, citral and thymol, respectively, inhibited Shigella growth. Synergism was observed among the components, where PC and BC growth inhibition occurred at lower concentrations than those noted individually. The bactericidal efect of the components in microplate was diferent from the observed in stain steel coupons. Therefore, the obtained model can describe and predict the PC count of S. fexneri in medium with the tested compounds and they could be an alternative for the use in microbiological control in food industry. Keywords: Antimicrobial activity, Essential oils, Synergism, Pathogenic bacteria Introduction large intestine in humans, causing infammation and Shigella spp. are rod-shaped Gram-negative bacteria damage to the epithelium, thus giving rise to the dis- belonging to the family Enterobacteriaceae (Kane and ease called shigellosis (Arena et al. 2015). Te number of Dorman 2012). Te genus Shigella comprises four sub- deaths caused by this disease was estimated at 40,000 in groups traditionally known as species: Shigella boydii, 2010, whereas in 2013, 34,400 deaths of children under S. sonnei, S. dysenteriae and S. fexneri (Cruz et al. 2014; the age of fve worldwide were estimated due to Shi- Nüesch-inderbinen et al. 2016; Grimont et al. 2007). gella infections (Mani et al. 2016). In the United States, Among the enteric pathogenic bacteria transmitted by approximately 500,000 cases of shigellosis are annually food, Shigella is one of the most common (Hu and Wai reported (CDC 2013). 2017). Tey are pathogens responsible for severe diar- Foodborne illness have always been a threat to human rheal diseases in young children worldwide, especially health. Tey bring the emerging concern of public health in developing countries (Kane and Dorman 2012; Mani in all continents, with numerous cases associated with et al. 2016). Shigella spp. can invade the mucosa of the the presence of bioflms (Srey et al. 2013). Bioflms are associations of microorganisms attached to a surface and involved in extracellular matrix of poly- *Correspondence: [email protected] meric substances (Simões et al. 2010). Any type of micro- 1 Laboratory of Bioprocess Engineering, Department of Food Science, Federal University of Lavras, B.O.: 3037, Lavras, MG, 37.200.000, Brazil organism can form bioflm in inhospitable environments Full list of author information is available at the end of the article in order to survive and play an important role in several © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Ngome et al. AMB Expr (2018) 8:105 Page 2 of 10 infections. Te formation of bioflms is a systematic and Microorganism, storage and inoculum standardization dynamic process divided into fve stages: (i) initial attach- Te strain used was S. fexneri INCQS 00152 (ATCC ment, (ii) irreversible attachment, (iii) initial develop- 12022) supplied by the Collection of Reference Micro- ment of the bioflm architecture, (iv) maturation and (v) organisms in Sanitary Surveillance of the Oswaldo Cruz dispersion. Te initial attachment stage is very important Foundation (FIOCRUZ/CMRVS—WDCM 575). Stock once it is reversible (Srey et al. 2013). In this stage, cells cultures were stored in freezing medium (15 mL glycerol, are called planktonic, i.e., non-adherent cells diferent 0.5 g bacterial peptone, 0.3 g yeast extract, 0.5 g sodium from bioflms that have sessile cells (Costa et al. 2017). chloride, 100 mL distilled water, and pH adjusted to 7.0). In the food industry bioflms are generally found inside Cultures were reactivated by inoculating 100 μL ali- closed surfaces, such as pipes where liquid fows on solid quots into tubes containing 10 mL brain–heart infusion surfaces. On open surfaces, fouling allows microbial (BHI) broth and incubated at 37 °C for 24 h. Te inocu- retention (Whitehead and Verran 2015). Moreover, it is lum standardization performed by using a growth curve well reported that bioflm has become a problem in the with absorbance monitoring (OD600nm) of the tryptone food industry because it makes its population resistant soy broth (TSB) culture in a spectrophotometer (BEL to antimicrobial agents and to cleaning (Srey et al. 2013) SP-200) and plating in tryptic soy agar (TSA). Te plates due to their particular intrinsic characteristics, thus were incubated at 37 °C for 24 h and cultures were stand- resulting in an increasingly negative impact on the food ardized at 108 CFU mL−1. sector (Srey et al. 2013). In literature few studies with S. fexneri bioflm were done. Planktonic cell death curve of S. fexneri exposed to major Tere is a trend of reducing the use of chemical sanitiz- components ers with antimicrobial activity in the food industry due to Exposure time infuence of S. fexneri at diferent concen- their negative efects (Souza et al. 2014; Moradi and Sad- trations of linalool, citral, eugenol and thymol was deter- eghi 2017). Moreover, much pressure has been imposed mined through the absorbance (OD600 nm) monitoring in by consumers and legal authorities linked to the food sec- a spectrophotometer (uv BEL SP-200) every 1.0 h. Ali- tor mainly focused on adopting more natural alternatives quots of 1 mL of standard cultures were inoculated into a in the food production chain (Beyki et al. 2014; Souza fask containing 100 mL TSB plus 0.5% Tween 80 and dif- et al. 2014). In this sense, the use of essential oils and ferent concentrations of major components [0.0, 0.0625, their components, which are natural products, arises as 0.125 and 0.5% (v v−1)]. Cultures were incubated at 37 °C an alternative for the control of many foodborne patho- for 8 h. Te experiment was performed in triplicate with gens and degrading microorganisms (Bassolé and Juliani three repetitions. 2012). Te essential oils and their components, such as linalool, citral, eugenol and thymol (Goldbeck et al. 2014; Selection of the main major components Ait-Ouazzou et al. 2011; Chauhan and Kang 2014), e.g., against planktonic cells of S. fexneri have already shown to play an important role in the dis- Te selection of the major components with greater anti- covery of new antibacterial agents (Araújo et al. 2017; microbial efectiveness against planktonic cells (PC) of Hyldgaard et al. 2012; Mahdavi et al. 2017). Although S. fexneri was performed using the Plackett & Burman the mechanism of the antimicrobial activity of oils has design (Rodrigues and Lemma 2012), with eight treat- not been fully understood, it is known that these make ments (PB 8) and 3 central points, according to Table 1. the membrane of bacterial cells more permeable, causing Te solutions were prepared in TSB added with 0.5% leakage of cytoplasmic components and then cell inacti- Tween 80 and diferent concentrations of linalool, citral, vation (Chai et al. 2016). eugenol and thymol (Table 1). Aliquots of 10 μL of stand- Terefore, the aim of the present study was to evalu- ardized culture were transferred into 150 μL of solutions ate the growth inhibition of planktonic cell and bioflm of contained in the polystyrene microplates and incubated Shigella fexneri INCQS 00152 through both isolated and at 37 °C for 24 h. Subsequently, aliquots of each culture combined use of linalool, citral, eugenol and thymol. were plated in TSA then incubated at 37 °C for 24 h. Determination of the minimum bactericidal concentration Materials and methods (MBC) of mixtures of the major components on planktonic Te major essential oils (EO) p.a. grade components; lin- cells alool (97% v v−1), citral (95% v v−1), eugenol (99% v v−1) Te MBC of the mixtures of linalool, thymol and citral and thymol (99% v v−1), were purchased from the Sigma- were determined using the central composite rotational Aldrich company. 3 design (CCRD) of 2 with four center points and six Ngome et al. AMB Expr (2018) 8:105 Page 3 of 10 Table 1 Plackett & Burman 8 matrix containing coded and real values 1 Test Coded values Real values (v v− ) X1 X2 X3 X4 Linalool Citral Eugenol Thymol 1 1 1 1 1 0.0156 0 0 0.0625 + − − + 2 1 1 1 1 0.0156 0.0625 0 0 + + − − 3 1 1 1 1 0.0156 0.0625 0.0313 0 + + + − 4 1 1 1 1 0 0.0625 0.0313 0.0625 − + + + 5 1 1 1 1 0.0156 0 0.0313 0.0625 + − + + 6 1 1 1 1 0 0.0625 0 0.0625 − + − + 7 1 1 1 1 0 0 0.0313 0 − − + − 8 1 1 1 1 0 0 0 0 − − − − 9 0 0 0 0 0.0078 0.0313 0.01563 0.0313 10 0 0 0 0 0.0078 0.0313 0.01563 0.0313 11 0 0 0 0 0.0078 0.0313 0.01563 0.0313 x1, x2, x3 e x4—codifed values of linalool, citral, eugenol and thymol respectively axial points, totaling 18 assays.