<I>Listeria Monocytogenes</I>

531

Journal of Food Protection, Vol. 72, No. 3, 2009, Pages 531–538 Copyright ᮊ, International Association for Food Protection

Fate of Listeria monocytogenes on Fully Ripened Greek Graviera Cheese Stored at 4, 12, or 25؇C in Air or Vacuum Packages: In Situ PCR Detection of a Cocktail of Bacteriocins Potentially Contributing to Pathogen Inhibition

ELENI GIANNOU,1 ATHANASIA KAKOURI,1 BOJANA BOGOVICˇ MATIJASˇIC´ ,2 IRENA ROGELJ,2 AND JOHN SAMELIS1*

1National Agricultural Research Foundation, Dairy Research Institute, Katsikas, 45221 Ioannina, Greece; and 2University of Ljubljana, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/3/531/1679719/0362-028x-72_3_531.pdf by guest on 25 September 2021 Biotechnical Faculty, Chair of Dairy Science, SI-1230 Domzˇale, Slovenia

MS 08-408: Received 20 August 2008/Accepted 3 October 2008

ABSTRACT

The behavior of Listeria monocytogenes on fully ripened Greek Graviera cheese was evaluated. Three batches (A, B, and C) were tested. Batches A and C were prepared with a commercial starter culture, while in batch B the starter culture was combined with an enterocin-producing Enterococcus faecium Graviera isolate. Cheese pieces were surface inoculated with a ﬁve-strain cocktail of L. monocytogenes at ca. 3 log CFU/cm2, packed under air or vacuum conditions, stored at 4, 12, or 25ЊC, and analyzed after 0, 3, 7, 15, 30, 60, and 90 days. L. monocytogenes did not grow on the cheese surface, regardless of storage conditions. However, long-term survival of the pathogen was noted in all treatments, being the highest (P Ͻ 0.05) at 4ЊC under vacuum conditions. Overall, the lower the storage temperature, the higher and longer the survival of L. monocytogenes was. Although enterocin A–speciﬁc PCR products were detected in situ in cheese batch B, inhibition of L. monocytogenes by the enterocin-producing strain was not enhanced compared with batches A and C, which also contained enterocin A, but in lower amounts. Additionally enterocins B, P, L50A, and L50B; lactococcin G; and plantaricin A genes were detected in all batches, suggesting that indigenous bacteriocin-producing lactic acid bacteria might contribute to Listeria inhibition in cheese. In conclusion, Graviera cheeses that may be accidentally contaminated in retail at the European Union maximal allowable level of 100 CFU/cm2 or g are at low risk regarding a potential outgrowth of L. monocytogenes, which, however, may survive for a long period during cheese storage.

Listeria monocytogenes has been detected in various with the exception of protected designation of origin Gra- retail cheese products (21, 24, 25, 41, 43). The public health viera Naxos, which is made from cows’ milk. Milk curdling concerns associated with the presence of L. monocytogenes is initiated at 32 to 36ЊC, and then the curd is cut down in in cheese have been demonstrated by several listeriosis out- cubes and ‘‘cooked’’ to 48 to 52ЊC before molding. Molds breaks, mostly linked to soft cheeses (11, 15, 21, 41). Con- are pressed, drained, brined, and ripened at 12 to 18ЊC and tamination occurs either as a result of the use of raw con- 85 to 95% relative humidity for at least 90 days before taminated milk (5, 24, 29) or as a result of contamination consumption. In general, Gravieras are hard, fully ripened during and after cheese processing (21, 39, 41, 43). Fresh cheeses with whitish to yellowish rind, symmetric and or mold-ripened soft cheeses of pH 5.5 to 7.0 show a higher evenly distributed ‘‘shiny eyes’’ in the core, and have a incidence (11, 15, 25, 40, 41) and potential for survival or pleasant, mild, sweet taste and rich aroma. When ready for growth of L. monocytogenes (3, 17, 36, 41), due to their sale, the cheese molds (ca. 13 to 15 kg in weight) should overall lower hurdle effect compared with other cheese have a maximal permitted water content of 38 to 40% and types. However, acid-soft, semi-hard, or hard cheeses may a minimal permitted fat content in dry matter of 40% (1). also harbor L. monocytogenes or may be postprocess con- Although Graviera is the ﬁnest and most popular taminated during retail operations, such as cutting, portion- cheese among the Greek hard cheeses, research data on its ing, and wrapping and packaging (21, 41, 43). microbial ecology and quality are scarce (20, 23), particu- Gravieras are among the most popular cheese varieties larly with regard to safety and L. monocytogenes. The new in Greece, and are traditionally produced in many Greek microbiological criteria that have been incorporated in E.U. regions. Three of them, namely Graviera Kritis, Naxou, and Regulation 2073/2005 specify a maximal allowable con- Agrafon, have protected designation of origin recognition centration of 100 CFU/g or ml in food products ready to (1). Gravieras are produced from ewes’ milk, pure or mixed be placed on the market and during their shelf lives (13). with 10 to 30% goats’ milk, depending on the cheese type, This criterion has replaced the zero-tolerance requirement in ready-to-eat foods unable to support the growth of L. * Author for correspondence: Tel: ϩ302-651-094789; Fax: ϩ302-651- monocytogenes, and as such are considered products with Յ Յ Յ 092523; E-mail address: [email protected]. pH 4.4 or water activity (aw) 0.92, or pH 5.0 and 532 GIANNOU ET AL. J. Food Prot., Vol. 72, No. 3

Յ Њ aw 0.94, or products with a shelf life less than 5 days batches were manufactured from thermized (63 C for 30 s) ewes’: (13). Theoretically, Graviera cheeses, which have pH 5.2 goats’ (90:10) milk under the same manufacturing protocol. Be- to 5.6 and a 0.94 to 0.96, do not belong to these ‘‘safe- fore the rennin process (40 g rennin powder per 1,000 liter of w Њ food’’ categories. However, the above criterion applies to milk at 34 C for 35 min), a commercial freeze-dried lactic starter other ready-to-eat foods if the manufacturer is able to dem- culture consisting of Streptococcus thermophilus, Lactococcus lactis subsp. lactis, L. lactis subsp. diacetylactis, and leuconostocs onstrate to the satisfaction of the competent authority that (GR02, Mofin Alce Group, Italy) was added (50 U/1,000 liters of the product will not exceed the limit of 100 CFU/g through- milk) to all batches. In addition, in batch B the above starter out its shelf life (13). Such scientific justification is cur- culture (SC) was supplemented with E. faecium KE82, an Entϩ rently lacking, and thus is required, particularly for the tra- strain previously isolated from Graviera cheese produced in the ditionally manufactured Graviera cheeses from raw or ther- same plant, without addition of commercial starters (18). One liter mized milk in small-scale Greek plants. These cheeses may of an overnight culture of the Entϩ strain KE82 in sterile 10% contain a few L. monocytogenes survivors post-ripening, skim milk (LAB M) cultivated at 37ЊC was poured into 1,000 thus failing to comply with the alternative criterion ‘‘absent liters of cheese milk to achieve an inoculation level of ca. 6 log Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/3/531/1679719/0362-028x-72_3_531.pdf by guest on 25 September 2021 in 25 g’’ when ready for sale (13). Moreover, the large CFU/ml. Cooking, molding, brining, fermentation, and ripening Graviera molds (13 to 15 kg) are either portioned in smaller of the cheeses were carried out under standard in-plant operations. pieces in retail outlets, such as groceries, supermarkets, del- After ripening, one Graviera cheese mold (14 to 14.5 kg) from each of the batches A, B, and C was transported to our laboratory icatessens, for immediate sale, or wrapped after portioning for portioning and use in the challenge experiments. in air-permeable films or vacuum bags for retail distribu- tion, and stored usually under refrigeration temperatures. Preparation of L. monocytogenes inoculum. L. monocyto- During these operations, cheese portions may be surface genes strains were cultivated in 10 ml of brain heart infusion broth contaminated with L. monocytogenes, which is a safety at 30ЊC for 20 to 22 h, and the cells were harvested by centrifu- concern because this pathogen is psychrotrophic, may grow gation (3,200 ϫ g for 15 min). The pelleted cells of each of the five strains were resuspended in 5 ml sterile of Ringer’s solution at pH and aw values as low as 4.5 and 0.92, respectively, and is very adaptive and resistant to food-related stresses (Merck, Darmstadt, Germany), combined, and serially diluted to a concentration estimated to yield approximately 103 CFU/cm2 of (4, 7, 14, 26, 42). Thus, L. monocytogenes may survive for Graviera when a 50-␮l aliquot of the inoculum was applied to (15, long times under stressful conditions in various foods one side of a cheese piece. The concentration of L. monocytogenes 26), including cheeses with much lower pH than that of inoculum was determined on tryptic soy agar (LAB M) plus 0.6% Graviera (17, 32, 33, 36, 39, 41), and may even grow to yeast extract and Listeria Ottaviani Agosti agar (AES Laboratoire, exceed the 100 CFU/g limit. This study was therefore un- Bruz, France) plates, incubated at 30ЊC for 48 h. dertaken to evaluate the behavior of L. monocytogenes when introduced as a postprocess contaminant on fully rip- Product inoculation. The cylindrical cheese molds (A, B, ened Graviera cheese portions packaged in air or under vac- and C) were first cut in parallel slices of approximately 1-cm uum and stored under refrigeration (4ЊC) or temperature- thickness with the aid of a hand-operated cheese cutter disinfected with 70% ethanol, under a biological safety cabinet. The cheese abuse (12 and 25ЊC) conditions. Potential differences in rind (ca. 0.5 cm) was removed, and each slice was cut into small survival or growth of L. monocytogenes in the presence or pieces of 3.5 by 6.0 cm. Afterward, 50 ␮l of composite inoculum ϩ absence of an enterocin (Ent )–producing Enterococcus was deposited on one side of a piece (21 cm2) and spread over faecium strain as protective adjunct in the cheeses used the cheese surface with a sterile bent glass rod. Inoculated pieces were also evaluated. Ultimately, the behavior of the path- were left to stand individually at 4ЊC for 15 min, for inoculum ogen prompted a PCR analysis for in situ detection in the attachment. Then, the same procedure was repeated on the other tested cheeses of 23 known lactic acid bacteria bacteriocin side of each piece, to give a total inoculated surface of 42 cm2. genes. After inoculation, the cheese pieces were individually inserted into separate polyethylene bags (AlexPak, Ioannina, Greece); half of MATERIALS AND METHODS the bags were vacuum packaged at 80 mmHg, and the other half were air sealed in an MVS (Dalmine BG, Italy) 45L vacuum pack- L. monocytogenes strains. Five strains of L. monocytogenes aging machine. Afterward, the slices were divided into six groups were used in this study: Scott A (clinical isolate, serotype 4b), for different treatments (two packaging conditions [air or vacuum] ISS G79 (soft cheese isolate, serotype 1/2b), and ISS G185 (blue at three storage temperatures [4, 12, or 25 Ϯ 0.5ЊC]) and stored veined cheese isolate, serotype 1/2a), kindly provided by Dr. Paolo in three cooling incubators for up to 90 days unless spoilage oc- Aureli (Instituto Superiore di Sanita, Rome, Italy); and WSLC curred earlier. 1294 (goat cheese isolate; serotype 3b) and WSLC 1482 (soft cheese isolate, serotype 1/2b), kindly provided by Dr. Klaus Neu- Microbiological analyses. Samples from different treatments haus (Technical University of Munich, Germany). All strains were were evaluated microbiologically immediately after inoculation stored as frozen (Ϫ30ЊC) stock cultures in tryptic soy broth (LAB (day 0) and after 3, 7, 15, 30, 60, and 90 days of storage, unless M, Bury, Lancanshire, UK) with 0.6% yeast extract (LAB M) plus visible spoilage of the samples was earlier. Cheese samples (one 20% (wt/vol) glycerol, and were activated and subcultured twice piece each) were transferred to stomacher bags, mixed with 50 ml (30ЊC for 18 to 22 h) in 10 ml of brain heart infusion broth (LAB of 0.1% (wt/vol) buffer peptone water (LAB M), and homoge- M) before use in the experiments. nized in a stomacher (Lab Blender, Seward 400, London, UK) for 1 min at room temperature. On each sampling day, duplicate Graviera cheeses. Three batches (A, B, and C) of fully rip- cheese samples from two individual packs per treatment were an- ened (Ͼ90 days of age) Graviera cheeses produced in a local dairy alyzed. Appropriate decimal dilutions in buffer peptone water plant (Pappas Bros., Filippiada, Epirus, Greece) were used. All were prepared and plated by spreading 0.1 ml on agar plates in J. Food Prot., Vol. 72, No. 3 BEHAVIOR OF L. MONOCYTOGENES IN GREEK GRAVIERA CHEESE 533

duplicate. L. monocytogenes was selectively enumerated on Lis- MgCl2, and dNTP were purchased from Promega. The reaction Њ ϫ teria Ottaviani Agosti agar incubated at 37 C for 24 to 48 h. For mixtures contained 5 GoTaq buffer (including 1.5 mM MgCl2 L. monocytogenes determination, 1 ml of the original 50-ml or additional MgCl2 when required), GoTaq DNA polymerase (0.5 cheese sample slurry was also plated in tetraplicate on Listeria U), dNTP (100 ␮M each), 1 ␮M of appropriate primers, and 1.5 Ottaviani Agosti agar plates to achieve lowest detection limit of ␮l of DNA samples. Amplified fragments were resolved by elec- 0.08 log CFU/cm2. Total mesophilic lactic acid bacteria (LAB) trophoresis on 1.8% (wt/vol) agarose gels and stained with SYBR were enumerated on M-17 agar (LAB M) incubated at 30ЊC for Safe DNA gel stain (Invitrogen Molecular Probes, Eugene, OR). 48 h; enterococci were selectively enumerated on kanamycin aesculin azide agar (LAB M) incubated at 37ЊC for 48 h. Statistical analysis. The microbiological counts of all sam- ϫ ϭ In addition, the fully ripened cheese batches (A, B, and C) ples (92 per batch 3 batches 276) were converted to log were evaluated microbiologically before portioning for use in the CFU per square centimeter, and the means and standard deviations experiments (day 0). Duplicate samples were analyzed for total were calculated. Data analysis was statistically performed using mesophilic bacteria, mesophilic and thermophilic LAB, meso- SPSS for Windows (version 15.0, Microsoft, Redmond, WA). philic and thermophilic dairy cocci, enterococci, staphylococci Data relating to storage time, type of packaging, and temperature and other catalase-positive cocci, pseudomonads, total enterobac- were subjected to analysis of variance, using the time as a factor. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/3/531/1679719/0362-028x-72_3_531.pdf by guest on 25 September 2021 teria, yeasts and molds, and coagulase-positive staphylococci by In addition multiple comparisons and post hoc tests according to direct plating (CFU per gram). The media and methods used were Tukey and the least-significance difference procedures were per- as described by Bontinis et al. (8). Also, the presence of Listeria formed to determine differences between the means at the level Ͻ spp. and L. monocytogenes was evaluated by homogenizing 25 g of significance P 0.05. of cheese in 225 ml of Listeria enrichment broth (Merck). After RESULTS AND DISCUSSION incubation at 30ЊC for 48 h, the enriched cultures were streaked on Listeria Ottaviani Agosti agar plates for detection of charac- Characteristics of fully ripened Graviera cheeses teristic Listeria colonies after incubation at 37ЊC for 24 to 48 h. before inoculation. Mean populations of mesophilic LAB (deMan Rogosa Sharpe agar at 30ЊC), mesophilic cocci Determination of pH, moisture, and aw. The pH of all (M-17 agar at 22ЊC), and thermophilic streptococci (M-17 cheese treatments was measured throughout storage with a Jenway agar at 42ЊC) of cheese samples before inoculation were 3510 digital pH meter (Essex, UK) equipped with a glass elec- Ϯ Ϯ Ϯ trode, which was immersed in cheese samples homogenized with 8.1 0.1, 7.7 0.1, and 7.7 0.2 log CFU/g, respectively. Populations of naturally occurring enterococci (kana- distilled water. The aw and moisture of the cheeses were determined on day 0 samples, according to Bontinis et al. (8). mycin aesculin azide agar at 37ЊC) in batches A (6.8 log CFU/g) and C (5.9 log CFU/g) were lower than were en- Bacteriocin gene detection in Graviera cheeses and their terococcal populations in batch B (7.1 log CFU/g), which microbial consortia. The presence of 23 known LAB bacteriocin had been inoculated with Entϩ E. faecium KE82 at manu- genes (Table 1) in situ in cheese and in microbial consortia was facture. Thermophilic LAB populations (deMan Rogosa determined by PCR. Total DNA was extracted from cheese sam- Sharpe agar at 45ЊC) in batches A (6.2 log CFU/g) and C ples of batches A, B, and C on day 0, and from consortia of mesophilic LAB, i.e., mixture of colonies grown on M-17 agar at (6.1 log CFU/g) also were lower than in batch B (7.6 log 30ЊC. For DNA extraction from cheese, aliquots (10 g) of each CFU/g), since the later populations were mostly enterococci sample were resuspended in trisodium citrate dihydrate (Kemika, selected for growth on deMan Rogosa Sharpe at 45ЊC. Zagreb, Croatia) buffer (90 g) prepared according to the Interna- Cheese samples also contained 3.0 Ϯ 0.9 log CFU/g of tional Dairy Federation (19), and homogenized in a Stomacher catalase-positive cocci, 2.2 Ϯ 1.0 log CFU/g of total en- Lab-Blender (Bagmixer R400, Interscience, St. Nom, France). Af- terobacteria, and less than 2 log CFU/g of pseudomonads, ter centrifugation (6,000 ϫ g for 10 min at 4ЊC) of 10 ml of coliforms, coagulase-positive staphylococci and yeasts, suspension, the pellet was resuspended in 1 ml of sterile-filtered whereas they did not contain Listeria spp. in 25-g samples water. After lysozyme treatment of pelleted cells in 500 ␮l of Tris- (data not shown). The mean pH and aw values and moisture EDTA buffer containing 10 mg/ml lysozyme and 25 U/ml mutan- content of the ripened cheeses were 5.6 Ϯ 0.1, 0.948 Ϯ olysin (Sigma-Aldrich Chemie GmbH, Steiheim, Germany) at 0.006, and 34.8% Ϯ 0.4%, respectively; no major differ- 37ЊC for 1 h, DNA extraction was carried out with a Maxwell 16 tissue DNA purification kit and a Maxwell 16 Instrument (Pro- ences were observed between batches. These results indi- mega, Madison, WI), and finally resuspended in 300 ␮l of elution cated that the hygienic quality of the cheeses used was in buffer. For DNA extraction from consortia, approximately 300 compliance with the microbiological criteria of European colonies were rinsed from the surface of M-17 agar plates with 2 Union Regulation 2073/2005, and confirmed that Graviera ml of quarter-strength Ringer’s solution. DNA was isolated from cheese may theoretically support growth of L. monocyto- 1 ml of each suspension by the Maxwell system, as described genes, based on its final pH and aw values (13). Also, the above. PCR amplification of structural bacteriocin genes was per- above differences in enterococcal counts of batches A and formed as described in previous studies, which are listed in Table C from B indicated that the 6-log inoculum of Entϩ E. 1. Oligonucleotide primers (PA 49 RF) were purchased from In- faecium KE82 in batch B was able to increase by ca. 1 log vitrogen (Paisley, UK). Primers for lactococcin G were designed CFU/g in the presence of SC during cheese fermentation based on published data (31) and I. F. Nes, who provided the and ripening. This is the first report on the use of an Entϩ nucleotide sequence (30). PCR amplifications were carried out in a Mastercycler Gradient thermal cycler (Eppendorf, Hamburg, Enterococcus strain as protective adjunct in cooked hard ϩ Germany) in a final volume of 20 ␮l. The program used was 95ЊC Greek cheeses. However, previous use of Ent E. faecium for 3 min for the first cycle; 95ЊC for 30 s, 64ЊC for 30 s, and FAIR-E 198 strain as an adjunct in soft Greek Feta cheese 72ЊC for 30 s for the next 35 cycles; and 72ЊC for 5 min for the also resulted in 1- to 2-log higher enterococcal populations last cycle. GoTaq DNA polymerase with appropriate buffer, compared with the control cheeses after 60 days of ripening 534 GIANNOU ET AL. J. Food Prot., Vol. 72, No. 3

TABLE 1. PCR primers used for the detection of 23 known LAB bacteriocins in fully ripened Graviera cheese Primer sequence (5Ј to 3Ј) Target bacteriocin Abbreviated name Reference

Forward: 5Ј-GGT ACC ACT CAT AGT GGA AA-3Ј Enterocin A A De Vuyst et al. (12) Reverse: 5Ј-CCC TGG AAT TGC TCC ACC TAA-3Ј Forward: 5Ј-CAA AAT GTA AAA GAA TTA AGT ACG-3Ј Enterocin B B De Vuyst et al. (12) Reverse: 5Ј-AGA GTA TAC ATT TGC TAA CCC-3Ј Forward: 5Ј-GCT ACG CGT TCA TAT GGT AAT-3Ј Enterocin P P De Vuyst et al. (12) Reverse: 5Ј-TCC TGC AAT ATT CTC TTT AGC-3Ј Forward: 5Ј-CCT ACG TAT TAC GGA AAT GGT-3Ј Enterocin 31 31 De Vuyst et al. (12) Reverse: 5Ј-GCC ATG TTG TAC CCA ACC ATT-3Ј Forward: 5Ј-GAG GAG TAT CAT GGT TAA AGA-3Ј Enterocin AS-48 AS48 De Vuyst et al. (12) Reverse: 5Ј-ATA TTG TTA AAT TAC CAA-3Ј Ј Ј

Forward: 5 -ATG GGA GCA ATC GCA AAA TTA-3 Enterocin L50A L50A De Vuyst et al. (12) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/3/531/1679719/0362-028x-72_3_531.pdf by guest on 25 September 2021 Reverse: 5Ј-TTT GTT AAT TGC CCA TCC TTC-3Ј Forward: 5Ј-ATG GGA GCA ATC GCA AAA TTA-3Ј Enterocin L50B L50B De Vuyst et al. (12) Reverse: 5Ј-TAG CCA TTT TTC AAT TTG ATC-3Ј Forward: 5Ј- GGC GGT ATT TTT ACT GGA GTN-3Ј Cytolysin Cyl De Vuyst et al. (12) Reverse: 5Ј- CCT ACT CCT AAG CCT ATG GTA-3Ј Forward: 5Ј- CCT ATT GGG GGA GAG TCG GT-3Ј Enterocin 1071A and 1071A and 1071 B Ben Omar et al. (6) Reverse: 5Ј- ATA CAT TCT TCC ACT TAT TTT T-3Ј Enterocin 1071B Forward: 5Ј-AAG AAT CTC TCA TGA GT-3Ј Nisin Nis Rodrı´guez et al. (38) Reverse: 5Ј-CCA TGT CTG AAC TAA CA-3Ј Forward: 5Ј-TCT GCA CTC ACT TCA TTA GTT A-3Ј Lacticin 481 Lac481 Rodrı´guez et al. (38) Reverse: 5Ј-AAG GTA ATT ACA CCT CTT TTA-T3Ј Forward: 5Ј-CAA TCA GTA GAG TTA TTA ACA TTT G-3Ј Lactococcin A LccA Rodrı´guez et al. (38) Reverse: 5Ј-GAT TTA AAA AGA CAT TCG ATA ATT AT-3Ј Forward: 5Ј-AGA ATT ACG AGA ATG CGT TGG-3Ј Lactococcin G LccG This study Reverse: 5Ј-GAA GCT TGA TTA ACA TCG CTC A-3Ј Forward: GCT TGC AGT ATG TTA TGA GTG Lactococcin LCNB LccLCNB Villani et al. (46) Reverse: CCT ACC ATC CAG GAT TTT CTT Forward: GCT CCA AAA AGC GCT AGA TC Lactococcin 513 Lcc513 Villani et al. (46) Reverse: GCT GGC TAC GAT ATT GCT AG Forward: ATC CTA TCC GAT ACC GTC AG Lactococcin RM LccRM Villani et al. (46) Reverse: GTT TTC CCT GAA CCA TTG GG Forward: GCG CTC TCT TGC ATA GTG AG Lactococcin 972 Lcc972 Villani et al. (46) Reverse: ACT CCT CCA TTA GTA CCA GC Forward: 5Ј-TGG TGT GCA TTG TAC T-3Ј Acidocin A AcidA Majhenicˇ et al. (27) Reverse: 5Ј-TTG ATC GGC AAC GAT T-3Ј Forward: 5Ј-AGA TGC AGT GGC TTC T-3Ј Acidocin B AcidB Majhenicˇ et al. (27) Reverse: 5Ј-CCA TGC AGG TAA TGT C-3Ј Forward: 5Ј-TGA AAA TTC AAA TTA AAG GTA TGA AGC-3Ј Plantaricin A PlnA Maldonado et al. (28) Reverse: 5Ј-TTA CCA TCC CCA TTT TTT AAA CAG TTT C-3Ј Forward: 5Ј-ATG GAA AAG TTT ATT GAA TTA-3Ј Sakacin P SakP Remiger et al. (37) Reverse: 5Ј-TTA TTT ATT CCA GCC AGC GTT-3Ј Forward: 5Ј-AGA CAT GGG AAT TTG CTG GT-3Ј Helveticin J HelvJ Trmcˇic´ et al. (45) Reverse: 5Ј- GGC GCG ATT CAA GTA GGA TA-3Ј

(44). Moreover, the prevalence of indigenous milk entero- Behavior of inoculated L. monocytogenes on fully cocci, especially of E. faecium, in traditional hard Greek ripened Graviera cheese. Statistical evaluation of the data Kefalotyri cheese made without starters (22), and more re- relating to L. monocytogenes behavior in the three batches cently in Graviera cheeses from a sample of which the Entϩ showed no signiﬁcant (P Ͼ 0.05) differences in pathogen’s KE82 strain was isolated (18), is well documented. Al- survival between batches A and C manufactured with the though the use of commercial starters reduced indigenous commercial SC alone and batch B manufactured with the enterococcal populations in matured protected designation SC plus the Entϩ E. faecium KE82. Therefore, survival data of origin Graviera Kritis cheese compared with control of L. monocytogenes in the three batches were pooled (Ta- cheese without starters (20), the present and previous (44) ble 2). The results showed that L. monocytogenes was un- data indicate that Entϩ E. faecium adjunct strains proliferate able to grow on the surface of fully ripened Graviera pieces and compete well in cheese niches of their natural origin. under all conditions tested. However, the pathogen was able Whether these strains would produce enterocin in situ re- to survive for long periods with a low death rate. In all mains questionable (44), and is discussed below. treatments, there were no signiﬁcant (P Ͼ 0.05) reductions J. Food Prot., Vol. 72, No. 3 BEHAVIOR OF L. MONOCYTOGENES IN GREEK GRAVIERA CHEESE 535

in the populations of inoculated L. monocytogenes during b a

0.05). the ﬁrst 15 days, although the pathogen tended to decline C in air Њ B A Њ Њ Ͻ faster at 25 C than at 4 or 12 C. Indeed, after 30 days of c P storage, viable counts of L. monocytogenes in each of the 25ЊC treatments were signiﬁcantly (P Ͻ 0.05) reduced, and thus were much lower than counts at 4 and 12ЊC, especially those packed under vacuum conditions (P Ͻ 0.05). After 60 days of storage at 25ЊC, surviving populations of L. c 1.48 (0.71) cbc 2.43 (0.44) NA bNA monocytogenes fell below the detection limit, regardless of aNA aNA AB A B A

B C packaging (air or vacuum) conditions. Conversely, survival d) are the means (SD) of the three was the highest (Ͻ1 log reduction) at 4ЊC under vacuum 0.08 0.08 Ͻ Ͻ conditions after 60 days, as well as after 90 days, when the samples stored at 12 and 25ЊC had spoiled. Overall, the Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/3/531/1679719/0362-028x-72_3_531.pdf by guest on 25 September 2021 lower the storage temperature, the higher and longer the bc 2.17 (0.55) b 1.58 (0.31) a survival of L. monocytogenes was. In addition, survival at cbc 2.70 (0.52) 2.12 (0.83) a

AB A AB A B BC a given temperature was generally greater under vacuum than under air conditions (Table 2), indicating that storage of Graviera cheese in air enhances pathogen’s death. The inability of L. monocytogenes to grow on fully ripened Gra- viera cheese was in agreement with its growth stasis on several market hard cheeses (pH 5.0 to 5.6), i.e., Cotija, a 2.66a (0.55) 2.28 (0.51) a 1.09 (0.84)

aa 2.93 (0.56) a 2.72 (0.57) 1.26 (0.33) Monterey Jack, Swiss, Cheddar, and Provolone, during aer-

AB A AB A A AB obic storage at 4, 8, and 30ЊC for 36 days (17). Overall, consistent with results in Table 2, previous research has shown that L. monocytogenes was able to survive with strong ‘‘tailing’’ in hard cheeses with pH values Ͼ5.0 and Storage time (days): even in soft cheeses with pH 4.0 to 4.7, for 1 to 3 months (17, 32, 33, 36, 39, 41). Thus, the acid pH–aw combined

aaa 3.02 (0.57) a 3.16 (0.48) a 2.72 (0.57) a 2.94 (0.56) 2.45 (0.48) 2.31 (0.29) hurdle effect is sufficient to inhibit growth, but may be on the surface of fully ripened Graviera cheese pieces during storage at 4, 12, and 25 A A A A A A insufficient to eliminate L. monocytogenes in matured hard cheeses, especially when stored under refrigeration in retail outlets or homes. Long-term survival of L. monocytogenes 0.05). Mean values in columns bearing different lowercase letters are significantly different ( on market cheeses is a safety concern, particularly due to Ͻ

P the risk of pathogen transmittance via common food contact surfaces such as slicers, to other ready-to-eat foods that are aaa 3.04 (0.55) a 3.10 (0.41) a 3.02 (0.51) a 2.98 (0.55) 2.82 (0.43) 2.84 (0.49) supportive for its growth (17, 21). Thus, additional hurdles, A A A A A A

Listeria monocytogenes such as bacteriocin-producing LAB protective adjuncts, should be assessed for their ability to enhance gradual inactivation of L. monocytogenes caused by the combined

3.14 (0.54) pH-aw hurdle effect in cheese, as assessed for Graviera in this study. ϩ

b Although populations of the Ent E. faecium strain a aaa 3.20 (0.45) a 3.13 (0.53) a 3.08 (0.58) 3.09 (0.34) 3.06 (0.42)

A A A A A A KE82 in batch B were sufﬁciently high to have produced

0 3 7signiﬁcant amounts 15 of enterocin, 30 neither inhibition 60 of 90 growth nor gradual rate of inactivation of L. monocytogenes were enhanced compared with batches A and C processed with the SC only (Table 2). Lack of the enhancement of L. monocytogenes growth inhibition in batch B was not sur- prising, since there was no growth in the other two batches anyway. The inability of the Entϩ KE 82 strain to enhance in situ inactivation of L. monocytogenes populations in Vacuum 3.22 (0.41) Vacuum 3.22 (0.41) Vacuum 3.22 (0.41) batch B was consistent with the strain’s mode of antilisterial

a activity in vitro (18). Strain KE82, which constantly caused large Entϩ-mediated antilisterial zones of inhibition in well C) Packaging condition Њ diffusion assays, completely inhibited growth of the same Changes in populations of inoculated (ﬁve-strain cocktail) L. monocytogenes strains (5 log CFU/ml) in co-culture in 4 Air 3.22 (0.41)

1225 Air Air 3.22 (0.41) 3.22 (0.41) deMan Rogosa Sharpe broth at 30ЊC for 24 h; however, reductions in L. monocytogenes populations after 24 h were batches. There were no differences seen for cheese batchesMean A, values B, in and rows C, bearing so different data capital were letters pooled are for signiﬁcantly analysis; different thus, ( values (expressed in log CFU per centimeter square NA, not analyzed due to sample spoilage.

TABLE 2. or vacuum packages Storage temp ( a b c minor (Ͻ0.2 log CFU/ml), indicating that the net inhibitory 536 GIANNOU ET AL. J. Food Prot., Vol. 72, No. 3

TABLE 3. Detection by PCR ampliﬁcation of different bacteriocin genesa in total DNA extracted from samples of three Graviera cheese batches and their respective mesophilicb microbial consortia Type of bacteriocinsd:

Enterocins: Other LAB bacteriocins:

Sample typec A B P L50A L50B Cyl Lactococcin G Plantaricin A

GA-ch ϩϩϩϩϩϪ ϩϩ GA-co ϩ! ϩ! ϩϩϩϪ Ϫϩ GB-ch ϩ! ϩ! ϩϩϩϪ ϩϩ GB-co ϩ! ϩ! ϩϪϪϪ Ϫϩ GC-ch ϩϩϩϩϩϪ ϩϩ GC-co ϩ! ϩ! ϩϩϩϩ Ϫϩ Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/3/531/1679719/0362-028x-72_3_531.pdf by guest on 25 September 2021 a The following bacteriocin genes were not detected in any sample and are therefore not presented in the table: enterocins 31, AS-48, 1071A, and 1071B; nisin; lacticin 481; lactococcins A, LCNB, 513, RM, and 972; acidocin A; acidocin B; helveticin J; and sakacin P. b M-17 agar at 30ЊC. c GA, Graviera batch A; GB, Graviera batch B; GC, Graviera batch C; ch, cheese samples; co, consortia samples. d ϩ, gene determinants detected; Ϫ, gene determinants not detected; !, very strong band. effect was strongly listeriostatic rather than listericidal (18). E. faecium cells, which were still present in cheese sample Nevertheless, it was reasonable to validate in situ enterocin B. Cytolysin on the other hand, the only E. faecalis enter- production in cheese batch B. For this purpose, samples of ocin (16) found, was amplified only in the case of DNA cheese batches A, B, and C were comparatively analyzed from consortia of batch C (Table 3), indicating the low by PCR for the content of 23 LAB bacteriocin genes (Table amount of cytolysin-containing E. faecalis cells in this 1). cheese. Another interesting phenomenon was that the band containing PCR products of amplification of DNA extracted Bacteriocin detection in cheese. Surprisingly, genes from cheese sample B with enterocin A–specific primers of enterocins A, B, P, L50A, and L50B, all produced by E. was stronger compared with cheese samples A and C (Fig. faecium (2, 9, 10, 16), were detected in all batches of Gra- 1). This could be a result of a higher number of target viera cheese, as well as in respective bacterial consortia enterocin A genes amplified in cheese sample B, most prob- recovered after plating of cheese samples on M-17 agar ably derived from the cells of Entϩ E. faecium KE82 that (Table 3). The only exception was the consortium derived were present in high concentrations in this batch (Fig. 1). from batch B, in which enterocins L50A and L50B genes Since, however, enterocins A, B, and P genes were detected were not detected although found in the respective cheese in all cheese batches and their consortia (Table 3), the nat- sample. This observation can be explained by the amplifi- ural co-presence of several E. faecium strains producing cation of DNA derived from nonviable, enterocin-L50A/Bϩ these enterocins and maintaining viability in fully ripened Graviera was evident. To our further surprise, in addition to the above enterocin-gene cocktails, lactococcin G and plantaricin A genes were found in all three cheese batches (Table 3). Notably, while lactococcin G genes were detected in the total DNA extracted from the cheese samples only, plantaricin A genes were detected in all cheese samples and also in their respective viable consortia (Table 3). Thus, plantaricin A–producing Lactobacillus plantarum strains appeared to be numerous within the mesophilic LAB populations predominating (Ͼ8 log CFU/g) in fully ripened Graviera cheeses, and therefore, might have contributed to the observed growth inhibition of L. monocytogenes on the samples during storage (Table 2). Conversely, lactococcin G–producing lactococci appeared to be important during FIGURE 1. PCR products amplified in PCR with enterocin A– early processing steps of Graviera cheese. Since lactococcin specific primers. Lane M, GeneRuler 100-bp DNA ladder; lanes G genes were not detected in any of the viable LAB con- 1 through 5, DNA extracted from different cheese consortia; lanes sortia from the ripened cheeses (Table 3), these lactococci 6 through 8, DNA extracted from day 0 samples of Graviera cheese batches A, B, and C, respectively; lane 9, negative control; were probably outnumbered by mesophilic lactobacilli and lane 10, positive control; lanes 11 through 13, DNA extracted enterococci during ripening. from the respective bacterial consortia collected from M-17 agar When all the above results are seen in combination, it plates incubated at 30ЊC after plating of Graviera cheese samples appears that the complete growth inhibition of L. monocy- A, B, and C. togenes on the surface of fully ripened Graviera cheese may J. Food Prot., Vol. 72, No. 3 BEHAVIOR OF L. MONOCYTOGENES IN GREEK GRAVIERA CHEESE 537 be attributed to the combined hurdle effect of several fac- 8. Bontinis, T. G., H. Mallatou, E. Alichanidis, A. Kakouri, and J. Sa- tors. They include the pH, a , and organic acid content of melis. 2008. Physicochemical, microbiological and sensory changes w during ripening and storage of Xinotyri, a traditional Greek cheese cheese plus various enterocins and plantaricins produced in from raw goat’s milk. Int. J. Dairy Technol. 61:229–236. situ by indigenous nonstarter LAB, such as Enterococcus 9. Casaus, P., T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hernandez, and spp., mainly E. faecium and L. plantarum. Consequently, H. Holo. 1997. Enterocin B, a new bacteriocin from Enterococcus the use of Entϩ E. faecium KE82 as protective adjunct to faecium T136 which can act synergistically with enterocin A. Mi- cheese batch B was essentially supplementary to in situ crobiology 143:2287–2294. production of antilisterial bacteriocins. The biochemical 10. Cintas, L. M., P. Casaus, H. Holo, P. E. Hernandez, I. F. Nes, and L. S. Havarstein. 1998. Enterocins L50A and L50B, two novel bacte- properties of naturally occurring enterococci in Graviera, riocins from Enterococcus faecium L50, are related to staphylococcal ϩ and particularly those of KE82 or other Ent adjunct hemolysins. J. Bacteriol. 180:1988–1994. strains, require careful evaluation given their controversial 11. De Buyser, M., B. Dufour, M. Maire, and V. Lafarge. 2001. Impli- role in food and health (6, 16, 34). Indeed, certain non- cation of milk and milk products in food-borne diseases in France harmful strains of E. faecium evaluated for probiotic prop- and different industrialized countries. Int. J. Food Microbiol. 67:1– 17. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/3/531/1679719/0362-028x-72_3_531.pdf by guest on 25 September 2021 erties rather than the entire species currently have the status 12. De Vuyst, L., M. R. Foulquie Moreno, and H. Revets. 2003. Screen- of generally recognized as safe (35, 47). Studies on these ing for enterocins and detection of hemolysin and vancomycin re- issues are in progress. sistance in enterococci of different origins. Int. J. Food Microbiol. In conclusion, Graviera cheeses that may be acciden- 84:299–318. tally contaminated in retail with L. monocytogenes are at 13. European Commission. 2005. Commission Regulation no 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. Off. low risk regarding a potential outgrowth of the pathogen J. Eur. Union L338:1–26. 2 above the E.U. maximum allowable level of 100 CFU/cm 14. Faleiro, M. L., P. W. Andrew, and D. Power. 2003. Stress response or g, but may support its survival for long periods during of Listeria monocytogenes isolated from cheese and other foods. Int. storage. A cocktail of bacteriocins naturally present in J. Food Microbiol. 84:207–216. cheese and consisting mainly of enterocins associated with 15. Farber, J. M., and P. I. Peterkin. 1991. 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&lt;I&gt;Listeria Monocytogenes&lt;/I&gt;

<I>Listeria Monocytogenes</I>