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Journal of Food Protection, Vol. 70, No. 2, 2007, Pages 450–455 Copyright ᮊ, International Association for Food Protection

Enterococci from Appenzeller and Raw Milk : Antibiotic Resistance, Virulence Factors, and Persistence of Particular Strains in the Products

S. P. TEMPLER AND A. BAUMGARTNER*

Section of Microbiology and Biotechnology, Swiss Federal Office of Public Health, Schwarzenburgstrasse 165, 3097 Liebefeld,

MS 06-298: Received 31 May 2006/Accepted 29 September 2006 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/2/450/1678809/0362-028x-70_2_450.pdf by guest on 25 September 2021 ABSTRACT

Enterococci are natural residents of human and animal intestinal tracts, and grow to high numbers in a variety of . The aim of this study was to determine the diversity of enterococci in two types of artisanal raw milk cheese (Schabziger and Appenzeller) and to investigate whether particular strains with triple resistance against chloramphenicol (Chl), tetracycline (Tet), and erythromycin (Ery) persist in the production system. Of 46 cheese samples, a total of 312 Enterococcus strains were isolated over a 5-month period on selective agar plates containing Chl, Tet, or, Ery. Enterococcus faecalis was the predominant species (80.7%), followed by Enterococcus faecium (5.1%), and Enterococcus durans (11.7%). According to the phenotypic resistance patterns, a selection of 150 strains was analyzed with PCR for the presence of genes encoding resistance to Ery (ereA, ereB, mphA, ermA, ermB, ermC, mrsA/mrsB, mefA/mefE), and Tet (tetM, tetL). Because virulence factors have been linked to the pathogenicity of enterococci, the strain selection was also tested for the presence of the following virulence factors: Agg, GelE, Cyl, Esp, EfaAfs, EfaAfm, Cpd, Cob, and Ccf. All tested strains contained at least two of the nine virulence genes taken into analysis. Pulsed-field gel electrophoresis patterns of the isolates showed a limited persistence of several strains over a period of 1 to 2 months in Schabziger, and more than 2 months in Appenzeller. Finally, the enterococcal flora in the two types of cheeses seems to be rather unrelated. Within 150 strains from 25 different cheese samples (11 Appenzeller and 14 Schabziger), 41 pulsed-field gel electrophoresis patterns could be identified, and only 1 of these was found in enterococci from both .

Enterococci are found in a variety of cheeses made associated protein that is involved in the immune evasion. from raw or pasteurized cow’s milk (3, 13). For a long time, It is possible that esp and cylABM genes are associated on the presence of these bacteria in foods was associated with a pathogenicity island (25). Agg is a surface-localized pro- fecal contamination, but nowadays they are considered as tein encoded by pheromone-responsive, self-transmissible normal part of food microflora (19). Enterococci seem to plasmids, which mediates binding of donor bacterial cells improve the flavor development and cheese quality (13). with plasmid-free recipients, allowing efficient conjugal Because of antilisterial activity based on bacteriocin pro- transfer in a liquid environment (9). Another adhesin-like duction, they are also used in food preservation (13, 14, virulence factor is the E. faecalis and E. faecium antigen A 16). (EfaAfs and EfaAfm, respectively) that are expressed in the In spite of the beneficial activities of enterococci, they serum (21). Gelatinase is an extracellular metalloendopep- have become important over the past decade as one of the tidase that hydrolyzes gelatin, collagen, hemoglobin, and most frequently encountered nosocomial pathogens and ap- other bioactive compounds (31). The sex pheromones (Cpd, pear to have increasing resistance to antimicrobials (20, 22, Cob, Ccf ) are thought to be involved in eliciting an inflam- 23). An even greater threat is the transfer of resistance to matory response. These pheromones are chemotactic for vancomycin or other antimicrobials within and over the human leukocytes and induce production and secretion of species border (9–11, 20). To cause infection, enterococci lysosomal enzymes and thereby facilitate conjugation (8). must be able to colonize the host tissue, resist host-specific Previous studies showed that enterococci can be found and -unspecific defense mechanisms, and cause pathologi- in a variety of ready-to-eat products in Switzerland (3, 5). cal changes. Thus they have to be virulent, and virulence Further, in a high amount of enterococcal isolates from raw factors including cytolysin (CylABM), enterococcal surface milk cheese, triple resistance against chloramphenicol protein (Esp), aggregation substance (Agg), and gelatinase (Chl), tetracycline (Tet), and erythromycin (Ery) could be (GelE) should be taken into consideration as well (12, 13, demonstrated. The aim of this study was to determine the 22, 25). CylABM causes ruptures of a variety of mem- diversity of enterococci in two types of artisan raw milk branes, including those of bacterial cells, erythrocytes, and cheese (Schabziger and Appenzeller), and to investigate other mammalian cells (29). Esp (30) produces a cell wall– whether particular strains with triple resistance against Chl, * Author for correspondence. Tel: ϩ41 31 322 95 82; Fax: ϩ41 31 322 Tet, and Ery persist in the production system. 95 74; E-mail: [email protected]. Appenzeller is a hard-type cheese, produced from raw J. Food Prot., Vol. 70, No. 2 PERSISTENCE OF ANTIBIOTIC-RESISTANT ENTEROCOCCI IN RAW MILK CHEESE 451 cow’s milk. Milk is inoculated with a starter culture con- Phenotypic and genotypic assessment of virulence traits. taining lactic acid bacteria. The milk coagulation is started The phenotypic assessment of hemolysis was examined by streak- with addition of rennet. Cheese is normally ripened for ing the isolates on 5% sheep blood Colombia agar (bioMe´rieux) Њ about 3 to 4 months. Schabziger cheese (also known as and incubated at 37 C for 24 h in order to determine the hemolytic Sapsago) is a creamery, hard, fat-free, pungent grating activity. The gelatinase activity of the isolates was tested with brain heart infusion broth (Oxoid) containing 12% of gelatin. The cheese. It is spiced with fenugreek. During the ripening brain heart infusion–gelatin broth was liquefied at 37ЊC and in- process the cheese is grated to avoid formation of rind. oculated with the isolates. After incubation (37ЊC, 48 h) the tubes Schabziger cheese is produced in only one production plant were kept at 4ЊC for 30 min. Gelatinase-positive isolates could be in Switzerland and is therefore an ideal model system to identified by the liquid medium. PCR assays were performed us- show persistence of particular strains of enterococci. ing pairs of previously reported primers (12) for the detection of the following genes: agg, gelE, cylM, cylB, cylA, esp, efaAfs, MATERIALS AND METHODS efaAfm, cpd, cob, and ccf.

Cheese sampling and isolation of enterococci. Over a pe- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/2/450/1678809/0362-028x-70_2_450.pdf by guest on 25 September 2021 PCR assay. For all detection assays, a common PCR core riod of 5 months, a cheese sample of every new batch production mix (total volume of 50 ␮l) was used consisting of 1ϫ PCR buffer of Schabziger cheese (21 samples) and Appenzeller cheese (26 (Promega, Madison, Wis.), 200-␮M concentrations of deoxynu- samples) was analyzed. The samples were diluted 1:10 in 0.85% cleoside triphosphates (Promega), 1 U of Taq DNA polymerase (wt/vol) NaCl plus 0.01% (wt/vol) tryptone casein peptone (pH (Promega), 4 mM MgCl , and 20 pmol of the corresponding prim- 7.0) and homogenized for 2 min in a laboratory blender (Stom- 2 ers (Thermo Electron GmbH, Ulm, Germany). A 50-ng portion of acher 400, Seward, PBI International, Milano, Italy), and 100 ␮l intact total DNA was used as PCR template. of a 10-fold dilution series were spread plated on kanamycin aes- culin azide agar base (Oxoid, Ltd., Hampshire, England) to de- PFGE typing. The chromosomal DNA was prepared as pre- termine the number of enterococci per gram of product. To deter- viously described (4) by growing the cells on brain heart infusion mine the number of Chl-, Ery-, and Tet-resistant enterococci per agar for 48 h at 37ЊC. Cells were suspended in 10 mM Tris–100 gram of product, 100 ␮l of a 10-fold dilution series was spread mM EDTA, pH 8.0 (TE-100 buffer), and consequently the sus- plated on kanamycin aesculin azide agar base containing 20 ␮g/ pension’s OD was adjusted to 2.5. This suspension was mixed ml of Chl, 10 ␮g/ml of Ery, or 10 ␮g/ml of Tet. Plates were 600 with an equal volume of 1.5% SeaKem Gold Agarose (Cambrex incubated at 37ЊC for 48 h. Bio Science Rockland, Rockland, Maine) in 10 mM Tris–1 mM Up to 10 colonies were randomly selected from antibiotic- EDTA, pH 8.0 (TE-buffer), to pour plugs. The solidified agar containing agar plates used for plate counting of a cheese sample. plugs were transferred in 1.5 ml TE-100 buffer with 25 mg/ml The colonies were purified twice on brain heart infusion agar (Ox- lysozyme (Sigma-Aldrich, Buchs, Switzerland) and rotated over- oid). The strain labeling was done as follows. The cheese type night at 10 rpm (Hybaid Mini 10, MWG-Biotech, Ebersber, Ger- was abbreviated with ‘‘SCH’’ for Schabziger and ‘‘APP’’ for Ap- many) at 37ЊC. After washing the plugs twice in TE-100 buffer penzeller, respectively. This abbreviation was followed by the iso- for 15 min, 1.5 ml of 0.5 M EDTA–1% N-lauroylsarcosine (pH late number (consecutively numbered) and the hyphenated cheese 8.0) and 2 mg/ml of proteinase K (Roche, Basel, Switzerland) sample number (example: SCH003-1, indicating the third entero- were added and incubated overnight at 50ЊC, under rotation. Sub- coccal isolate coming from the Schabziger cheese sample no. 1). sequently, the plugs were washed five times in TE-buffer for 1 h, Phenotypic characterization. All 216 isolates from Schab- under rotation. Subtyping of strains was performed by using ziger and 100 isolates from Appenzeller were characterized using pulsed-field gel electrophoresis (PFGE) with SmaI-digested (Fer- the Gram stain method. Gram-positive and coccal-shaped isolates mentas, Inc., Hannover, Md.) chromosomal DNA. PFGE was per- were further characterized with API 20 Strep (bioMe´rieux, Inc., formed with a 1.0% agarose gel by using a CHEF-DR III appa- Lyon, France). ratus (BioRad Laboratories, Hercules, Calif.) in 0.5ϫ Tris borate– EDTA buffer at 12ЊC at 6 V/cm, with an angle of 120Њ. A linearly Phenotypic and genotypic assessment of antibiotic suscep- ramped switching time from 1 to 16 s was applied for 16.5 h. tibility. Strains were screened for phenotypic resistance to 12 an- tibiotics with the disc diffusion method and Mueller-Hinton agar RESULTS (Oxoid). The following antibiotic discs (Difco, Becton Dickinson, Le Pont de Claix, France) were used: penicillin (10 ␮g), ampicillin Enterococcal populations in Schabziger and Appen- (10 ␮g), amoxicillin/clavulanic acid (20/10 ␮g), Chl (30 ␮g), Tet zeller cheese. Twenty-three of 26 tested Appenzeller and (30 ␮g), Ery (15 ␮g), vancomycin (30 ␮g), teicoplanin (30 ␮g), 17 of the 21 tested Schabziger cheeses were positive for imipenem (10 ␮g), ciprofloxacin (5 ␮g), nitrofurantoin (300 ␮g), enterococci. The counts varied from 3.2 ϫ 104 to 1.6 ϫ and rifampin (5 ␮g). Inhibition zones were interpreted following 106 CFU/g in Schabziger cheese and from 2.0 ϫ 102 to 2.5 the guideline tables of the NCCLS (24). In addition, the pheno- ϫ 106 CFU/g in Appenzeller cheese, which is in accor- typic resistance to vancomycin (MIC) was further tested with the dance with published data (3, 17). The screening for Chlr, E-test (AB BIODISC, Solna, Sweden), following the manufactur- Tetr,orEryr enterococcal strains resulted in 100 isolates er’s guidelines. from Appenzeller and 216 from Schabziger, respectively. It Total genomic DNA was prepared using a protocol based on was noticeable that Schabziger more frequently harbored the method of Pospiech and Neumann (26). PCR assays were performed using pairs of previously reported primers (1, 32, 33, resistant enterococci than did Appenzeller (Table 1). 36) for the detection of resistance genes: Ery drug inactivation All tested isolates were gram positive and coccal- (ereA, ereB, mphA), methylation mechanism (ermA, ermB, ermC), shaped. Two hundred seventy of the 312 isolates were and efflux systems (mrsA/mrsB, mefA/mefE); Tet resistance by ri- shown to be E. faecalis by biochemical genus and species bosomal protection mechanism (tetM); and an efflux system (tetL). identification, followed by 37 E. durans, and 16 E. faecium 452 TEMPLER AND BAUMGARTNER J. Food Prot., Vol. 70, No. 2

TABLE 1. Enterococci isolated from 21 Schabziger and 26 Appenzeller cheese samples recovered from kanamycin aesculin azide agar plates containing Chl, Tet, or Ery No. (%) of samples:

Analyzed Positive for enterococci With Chlr strains With Tetr strains With Eryr strains

Appenzeller 26 23 (88.5) 4 (15.4) 11 (42.3) 6 (23.1) Schabziger 21 17 (81.0) 9 (42.9) 14 (66.7) 13 (62.0) isolates. Eight isolates could only be characterized as en- 102 Tetr isolates showing tetM and tetL genes together. terococci on the genus level. Only 6 isolates had the tetL resistance alone, and the re- maining 38 isolates were positive for the tetM resistance Antibiotic susceptibility: disc test, E-test, and PCR. alone. Most of the Eryr isolates showed their resistance be- One of the most important characteristics for the evaluation Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/2/450/1678809/0362-028x-70_2_450.pdf by guest on 25 September 2021 ing based on the methylation mechanism of ermB. Eleven of enterococci is their susceptibility to different antibiotics. ermC, mph, and msrA/msrB genes could be amplified with All 312 enterococcal isolates were tested with 12 different PCR. Within the 150 isolates, 114 were positive for the antibiotics, using the disc method. According to the zone transposon integrase gene (int) of the Tn916–Tn1545 fam- diameter interpretive standards for Enterococcus spp., all ily. the tested strains were sensitive to vancomycin. This find- ing was confirmed with the E-test. The disc test method Strain typing with PFGE. PFGE was performed on showed 94 (30%) isolates resistant against Chl, 157 (50%) 150 selected isolates and all additional 50 isolates from the against Ery, and 212 (68%) against Tet. Arranging the 312 resistance profiles ‘‘Chl Tet Ery’’ and ‘‘Chl Tet Ery Rif.’’ isolated enterococci according to their phenotypic resis- The resulting 200 PFGE band patterns were clustered and tance resulted in 16 different patterns (Table 2). compared visually and with BioNumerics (BioSysematica, For PFGE typing, analysis of virulence factors and re- Ceredigion, UK) software. Thirty-four different PFGE pat- sistance genes, a selection was done as follows. From each terns were found among the 200 isolates tested. With one cheese sample, at most two isolates representing 1 of the exception, there were no strains with common patterns in 16 resistance patterns were chosen, resulting in a selection Appenzeller and Schabziger cheese. All tested Schabziger containing 150 isolates from 25 different cheese samples samples showed at least two different PFGE patterns. Six (11 Appenzeller and 14 Schabziger). Within this selection Schabziger samples harbored strains that showed the same 146 were resistant against Tet and 92 against Ery. Further PFGE pattern but had different resistance profiles, whereas characterization of the resistance mechanisms resulted in seven showed the same profile within one pattern. Four Schabziger samples were each dominated by a particular single clone. Only four Appenzeller samples showed het- TABLE 2. Enterococci from Appenzeller and Schabziger cheese with resistance to Chl, Ery, or Teta erogeneous PFGE patterns, whereof two samples harbored strains with a single resistance profile. In six cheese sam- No. (%) ples (two Appenzeller and four Schabziger) several strains r r r E. faecalis E. durans E. faecium with a triple resistance (Chl ,Ery,Tet) showed completely Resistance (n ϭ 253) (n ϭ 39) (n ϭ 20) different PFGE patterns (Fig. 1A). As previously shown (3), enterococci with the resistance profile ‘‘Chl Ery Tet’’ Tet 57 (22.5) 4 (10.3) 1 (5.0) are commonly found in Swiss raw milk cheese. In our Ery Tet 55 (21.7) 2 (5.1) 4 (20.0) study, the resulting PFGE patterns of the triple-resistant Chl Tet 5 (2.0) 0 (0) 0 (0) strains (Chlr,Eryr,Tetr) from six cheese samples were clus- Ery Nit 0 (0) 2 (5.1) 0 (0) tered in order to show whether enterococci with this com- Rif Tet 21 (8.0) 0 (0) 0 (0) Chl Ery Tet 74 (29.2) 0 (0) 0 (0) bination of resistance persist in cheese samples produced Chl Rif Tet 17 (6.7) 0 (0) 0 (0) over a certain period of time. Consequently, five E. faecalis Ery Nit Tet 1 (0.4) 29 (74.4) 2 (0) clones from an Appenzeller sample and four Schabziger Ery Rif Tet 1 (0.4) 0 (0) 0 (0) samples could be found throughout 1 to 3 months of the Cip Rif Tet 1 (0.4) 0 (0) 0 (0) whole screening period (Fig. 1B). Nit Rif Tet 0 (0) 0 (0) 3 (15.0) Ery Pen Tet 0 (0) 0 (0) 1 (5.0) PCR analysis for virulence factors. The prevailing Ery Nit Pen Tet 0 (0) 2 (5.1) 0 (0) species in Appenzeller and Schabziger cheese is E. faecalis, Chl Ery Rif Tet 21 (8.0) 0 (0) 0 (0) which is known to harbor multiple virulence factors (12). Cip Nit Rif Tet 0 (0) 0 (0) 8 (40.0) The presence of virulence factors (Fig. 2) in enterococci Cip Ery Imi Pen Tet 0 (0) 0 (0) 1 (5.0) isolated from Appenzeller and Schabziger varied between 2 and 10. The sex pheromone genes (cpd, cob, ccf) were a Resistance patterns by testing with 11 other antimicrobial sub- present in 72 to 98% of the strains tested. Further, no tested stances: penicillin (Pen), ampicillin (Amp), amoxicillin (Amo), enterococcal strain possessed all 11 virulence factors. The chloramphenicol (Chl), tetracycline (Tet), erythromycin (Ery), vancomycin (Van), teicoplanin (Tec), imipenem (Imi), ciproflox- gelE gene was detected with 76% of the isolates, and cylM acin (Cip), nitrofurantoin (Nit), and rifampin (Rif). and esp genes occurred with the frequency of 6 and 64%, J. Food Prot., Vol. 70, No. 2 PERSISTENCE OF ANTIBIOTIC-RESISTANT ENTEROCOCCI IN RAW MILK CHEESE 453 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/2/450/1678809/0362-028x-70_2_450.pdf by guest on 25 September 2021

FIGURE 1. PFGE profiles of SmaI-digested E. faecalis strains from Schabziger and Appenzeller samples. Clustered with BioNumerics software (band position tolerance 1%). (A) Three E. faecalis strains from one Schabziger sample showing the same resistance profile (Chlr, Eryr, Terr) and different PFGE patterns. Lane 1, SCH086-23; lane 2, SCH092-23; lane 3, SCH089-23; lane 4, MidRange PFG Marker I (New England BioLabs, Ipswich, Mass.). (B) Five enterococcal clones repeatedly isolated throughout 1 to 3 months from Appenzeller and Schabziger cheese. X, samples taken. respectively. The presence of cylAB factors only (no cyto- and further characterization of the isolates. Enterococcal lysin production) occurred in 4% of the isolates. counts in Schabziger and Appenzeller cheese were as ex- pected. The higher level of enterococci in Schabziger can DISCUSSION be explained with the shorter ripening period and the lower In this study two types of raw milk cheese were sam- salt content of the cheese. Preliminary experiments have pled for enterococci within a period of 5 months. Both phe- shown that the enterococcal counts of young (ripening at notypic and genotypic analyses were used for identification least 3 months, lower salt content) compared with older

FIGURE 2. Incidence of virulence factors among E. faecalis strains isolated from Ap- penzeller and Schabziger cheese. 454 TEMPLER AND BAUMGARTNER J. Food Prot., Vol. 70, No. 2

(ripening at least 4 months, higher salt content) Appenzeller broadly eliminate microorganisms in the producing facility. cheese (data not shown) are higher. E. faecalis was the most With a next production batch, new strains can establish in frequently isolated species, which is in accordance with the production system until they are eliminated again. As similar observations reported by other European research- for Appenzeller cheese, the situation cannot be explained ers. Andrighetto et al. (2) stated that most of the strains in a similar way. Since there are several production plants isolated in Italian cheeses were identified as E. faecalis; we could not show whether the limited persistence was due likewise, E. faecalis dominated in the milk and Irish Ched- to a cleaning concept in the producing facilities. dar–type cheese as reported by Gelsomino et al. (15) and Nearly all strains included in this study contained one did so in Slovenian Tolmic cheese as well (6). or more virulence genes that have previously been found The level of enterococci resistant to Chl, Tet, or Ery in human isolates (12). Over 75% of all tested isolates car- was higher than expected. Resistance to Tet was widespread ried the gelE gene, although phenotypic GelE activity could in enterococci isolated from Schabziger and Appenzeller, not be detected. Similar results were obtained from hemo- which is in accordance to findings of previous studies (18). lysis testing. Collectively, these results seem to indicate that Downloaded from http://meridian.allenpress.com/jfp/article-pdf/70/2/450/1678809/0362-028x-70_2_450.pdf by guest on 25 September 2021 Chl and Ery resistance occurred at a lower level, but could the investigated strains contain silent gelE and cylABM often be found in combination with Tet resistance. In the genes. According to Eaton and Gasson (12), there are sev- last years, this multiresistance was described by several re- eral environmental or temporal factors that may account for searchers (3, 18, 28, 35). In order to assess the potential the apparent lack of phenotypic expression of enterococcal risk associated with the presence and its possible ability of virulence genes. Similar to GelE, the presence of the conjugative exchange of this multiresistance in the cheese cylABM gene is not always linked to the phenotypic ex- investigated in this study, detailed analyses of the potential pression of hemolysin activity. This phenomenon may be mobility of the detected resistances are required. The tetM due to low-level gene expression or to the presence of an gene was found in all but 11 Tetr E. faecalis isolates, and inactive gene product. Fifty-three percent of the strains con- 110 of 146 isolates showed the efflux system tetL. A similar tained the agg gene responsible for the clumping factor situation was found during a major survey of 229 entero- aggregation substance. This virulence factor was in most coccal isolates collected in 10 hospitals in France, where cases associated with the presence of pheromone determi- tetM and tetL were the dominant Tetr determinants (7). In nants which is in accordance with findings published by many enterococci and streptococci of clinical or food ori- Eaton and Gasson (12). The incidence of Esp-positive en- gin, drug resistance genes occur more frequently on con- terococci was in the same range (64%) as previously pub- jugative transposons than on plasmids. Also in this study, lished (12). Since pheromone determinants are thought to 78% of the tetM-containing isolates (106 of 136) were pos- facilitate conjugation, their high level (72 to 98% of all 150 itive by PCR for the integrase gene int, indicating that they isolates) could indicate possible gene exchange during contain a member of the broad host range Tn916–Tn1545 cheese production. conjugative transposon family (Table 2). Further character- The results of the current study indicate that the en- ization of the 87 Eryr isolates resulted in 83 containing the terococci present in Schabziger and Appenzeller cheese can erm(B) gene, which is considered to be the most wide- harbor various antibiotic resistance and virulence traits. All spread macrolide resistance gene among enterococci from virulence traits found in the strains examined have also food animals or foods and from clinical isolates (34). Fur- been found in human clinical isolates. However, these re- thermore, 69 of these erm(B)-containing strains were also sults as such do not allow conclusions with regard to safety positive for a Tn916–Tn1545 element. erm(B) genes in en- of the cheese itself. Complete risk assessment would require terococci can also occur on other mobile elements, such as further research on the potential source and the in vitro and conjugative multiresistance plasmids (35) or members of in situ transferability of these resistance and virulence prop- the Tn917 family (27). The findings of PFGE typing erties. showed that enterococcal isolates with a very similar PFGE profile can have completely different resistance patterns. ACKNOWLEDGMENT Furthermore, identical resistance pattern could be found in This work was granted by the Swiss Federal Office of Public Health completely different PFGE profile. These results support (SFOPH). the idea of possible conjugative gene exchange during cheese production. 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