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• entA f: AAA TAT TAT GGA AAT GGA GTG TAT r: GCA CTT CCC TGG AAT TGC TC 56 126 (15) entB f: GAA AAT GAT CAC AGA ATG CCT A r: GTT GCA TTT AGA GTA TAC ATT TG 50 162 (15) entP f: TAT GGT AAT GGT GTT TAT TGT AAT r: ATG TCC CAT ACC TGC CAA AC 50 120 (15) entL50A/B f: TGG GAG CAA TCG CAA AAT TAG r: ATT GCC CAT CCT TCT CCA AT 52 98 (11) bac31 f: TAT TAC GGA AAT GGT TTA TAT TGT r: TCT AGG AGC CCA AGG GCC 50 123 (15) entAS48 f: GAG GAG TTT CAT GAT TTA AAG A r: CAT ATT GTT AAA TTA CCA AGC AA 50 340 (15) entQ f: ATG AAT TTT CTT CTT AAA AAT GGT ATC GCA r: TTA ACA AGA AAT TTT TTC CCA TGG CAA 56 105 (11) ent1071A/B f: CCT ATT GGG GGA GAG TCG GT r: ATA CAT TCT TCC ACT TAT TTT T 51 343 (11)
• Haemolytic and gelatinase activity The E. faecium strains were grown overnight in MRS broth at 37 °C. For determination of haemolytic activity, strains were streaked onto Columbia Agar plates (Laboratorios Conda S.A., Madrid, Spain) containing 5% (v/v) of either sheep blood or human blood. The plates were incubated at 37 °C for 48 h under aerobic conditions. Presence of zone of clearing around the colonies was interpreted as β-haemolysis (8). Production of gelatinase was determined on ToddHewitt agar (Merck) containing 30 g of gelatine (Merck) per litre as described by Eaton and Gasson (9). PCR detection of virulence genes The virulence factors in 3 bacteriocin-producer E. faecium strains were investigated by PCR amplification, revealing the presence of genes encoding for gelatinase (gelE), cell wall adhesin (efaAfm and efaAfs), sex pheromone (cpd, cob, ccf, and cad), collagen adhesin (ace), enterococcal surface protein (espfm and espfs), aggregation substance (agg), and cytolysin (cylM, cylB, and cylA). The primers used in this assay are listed in Table 2. PCR for virulence genes was performed as an initial cycle of denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 54 °C (56 °C for gene agg) for 30 s, and elongation at 72 °C for 1 min (16). The amplification products were analysed by electrophoresis on 1.5% agarose gel at 120 V for 1.5 h in Tris-acetate-EDTA buffer and revealed in ethidium bromide (20 µg/mL). The gel was photographed under UV light. Antibiotic resistance The antibiotic susceptibility patterns of the E. faecium strains were detected by the disc diffusion method on Muller-Hinton agar (Merck) as described by Cariolato et al. (8). The antibiotics used were ampicillin (10 µg), chloramphenicol (30 µg), erythromycin (15 µg), gentamicin (120 µg), norfloxacin (10 µg), penicillin G (10 U), streptomycin (300 µg), tetracycline (30 µg), sulphamethoxazole/trimethoprim (30 µg), and vancomycin (30 µg). All antibiotic discs were obtained from Oxoid (Basingstoke, UK). Susceptibility or resistance was determined according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) (17). Table Polymerase chain reaction primers and product sizes for detection of virulence determinants. Gene Primer sequences (5’ to 3’) Product size (bp) Reference gelE f: ACC CCG TAT CAT TGG TTT r: ACG CAT TGC TTT TCC ATC 419 (16) efaAfm f: AAC AGA TCC GCA TGA ATA r: CAT TTC ATC ATC TGA TAG TA 735 (16) efaAfs f: GAC AGA CCC TCA CGA ATA r: AGT TCA TCA TGC TGT AGT A 705 (16) cpd f: TGG TGG GTT ATT TTT CAA TTC r: TAC GGC TCT GGC TTA CTA 782 (16) cob f: AAC ATT CAG CAA ACA AAG C r: TTG TCA TAA AGA GTG GTC AT 1405 (16) ccf f: GGG AAT TGA GTA GTG AAG AAG r: AGC CGC TAA AAT CGG TAA AAT 543 (16) cad f: TGC TTT GTC ATT GAC AAT CCG r: ACT TTT TCC CAA CCC CTC AA 1299 (16) ace f: AAA GTA GAA TTA GAT CCA CAC r: TCT ATC ACA TTC GGT TGC G 350 (11) espfm f: TTG CTA ATG CAA GTC ACG TCC r: GCA TCA ACA CTT GCA TTA CCG AA 955 (16) espfs f: TTG CTA ATG CTA GTC CAC GAC C r: GCG TCA ACA CTT GCA TTG CCG AA 933 (16) agg f: AAG AAA AAG AAG TAG ACC AAC r: AAA CGG CAA GAC AAG TAA ATA 1553 (9) cylM f: CTG ATG GAA AGA AGA TAG TAT r: TGA GTT GGT CTG ATT ACA TTT 742 (16) cylB f: ATT CCT ACC TAT GTT CTG TTA r: AAT AAA CTC TTC TTT TCC AAC 843 (16) cylA f: TGG ATG ATA GTG ATA GGA AGT r: TCT ACA GTA AAT CTT TCG TCA 517 (16)
• Results and discussion Genetic identification of strains Bacteriocin-producer EYT17, EYT31, and EYT39 strains were identified previously as E. faecium based on phenotypic characteristics (12). In this study, these bacteriocin-producer strains were identified at the species level by 16S rDNA sequence homology. PCR product of an approximately 900-bp DNA fragment was amplified from all 3 strains. Sequencing of PCR amplification products of the EYT17, EYT31, and EYT39 strains showed 99% homology with the E. faecium genome deposited in GenBank. These results confirmed the biochemical identification of these strains. Detection of enterocin structural genes The purified DNAs of bacteriocin-producer E. faecium EYT17, EYT31, and EYT39 strains were used as a template in PCR amplifications to determine the existence of structural genes encoding 8 enterocins (Table 1). All 3 E. faecium strains carried the enterocin A and B structural genes, while E. faecium EYT17 and EYT31 strains also carried the enterocin P structural gene. None of the evaluated strains showed PCR amplification fragments for enterocin L50A/B, bacteriocin 31, enterocin AS48, enterocin 1071A/1071B, and enterocin Q structural genes
• (Table 3). Previous studies showed that PCR detection of more than one bacteriocin encoding gene in the Enterococcus strains isolated from fermented foods is not unusual (6,11,15), as confirmed in this study. According to the classification scheme of Franz et al. (18), enterocin A and enterocin P are grouped in class II.1 (pediocinlike bacteriocins), which has a very effective antilisterial activity, and enterocin B is grouped into class II.3 (linear nonpediocin-like bacteriocins). Casaus et al. (19) reported that enterocin B exhibited synergistic activity with enterocin A. The number of survivors was drastically reduced when a mixture of the 2 bacteriocins (enterocin A and B) was added to the cells. In a previous study, Tuncer (12) reported that E. faecium EYT17 and EYT31 strains (enterocin A, B, and P producers) showed the same antibacterial activity spectrum and these strains exhibited broader inhibitory activity spectrum than the E. faecium EYT39 strain (enterocin A and B producer). Enterocins produced in E. faecium EYT17, EYT31, and EYT39 strains probably act in a synergistic mode of action, as reported by Casaus et al. (19). Further investigations are also needed for the determination of the synergistic mode of action of these bacteriocins. Haemolytic and gelatinase activity Haemolytic activity of bacteriocin-producer E. faecium strains was tested on sheep blood and human blood agars. None of the E. faecium strains showed β-haemolytic activity on either of the 2 blood agars. Different researchers reported that Enterococcus strains isolated from a variety of fermented food products exhibited no β-haemolytic activity, similar to our results (10,20). Haemolysin production can increase the severity of enterococcal infections, and the presence of genes involved in haemolysin/cytolysin production is also considered a risk factor (21). The β-haemolytic enterococci isolates are considered undesirable in foods, and their use as starter cultures in food fermentations is not recommended (22). None of our strains exhibiting β-haemolytic phenotype might be considered as advantageous for the safety of the strains. Gelatinase activity of E. faecium strains was tested on Todd-Hewitt agar containing gelatine (30 g/L). Gelatinase activity was not detected in E. faecium strains. The same findings were reported by Eaton and Gasson (9) and Franz et al. (23). None of the E. faecium strains involved in either study showed gelatinase activity. On the other hand, a high number of E. faecalis strains isolated from food were detected to be producing gelatinase in these 2 studies. Gelatinase production by E. faecium species is not common (23), as confirmed in this study. Table Detection of enterocin structural genes in 3 bacteriocin-producer Enterococcus faecium strains from Turkish tulum cheese. Enterocin structural genes (PCR amplification) Enterococcus faecium strains EYT17 EYT31 EYT39 entA + + + entB + + + entP + + entL50A/B bac31 entAS48 entQ ent1071A/B
• Detection of virulence genes Bacteriocin-producer E. faecium EYT17, EYT31, and EYT39 strains were screened for the presence of 14 known virulence determinants. PCR analysis revealed that these strains were clear of potential virulence determinants, except for ccf and efaAfm. The structural genes of 12 virulence factors, namely gelatinase (gelE), cell wall adhesin (efaAfs), sex pheromone (cpd, cob, and cad), collagen adhesin (ace), enterococcal surface protein (espfm and espfs), aggregation substance (agg), and cytolysin (cylM, cylB, and cylA) were not found in any of the multiple enterocin-producer strains (Table 4). The ccf gene was found in all 3 strains. The PCR amplification of the ccf gene yielded an amplicon of the expected 543-bp size (Figure, lane 5). The adhesion-like E. faecium antigen A gene (efaAfm) was only found in the EYT17 strain, amplifying a 735-bp fragment (Figure, lane 2). The sex pheromones are not considered a virulence factor. On the other hand, the enterococci strains with the sex pheromone (cpd, cob, ccf, and cad) determinants have the potential to acquire the respective sex pheromone plasmids and, hence, the associated virulence determinants (9). The exact role of efaAfm as a virulence factor is still unknown (11). The efaAfm gene has not yet been conclusively shown to contribute to pathogenesis in animal studies, in contrast to the adhesion-like E. faecalis antigen A (efaAfs) (15). Furthermore, the absence of cylMBA and gelE genes in E. faecium EYT17, EYT31, and EYT39 strains agrees with our observation that these strains lack β-haemolytic and gelatinase activity. Recently, observations similar to our results were reported by different researchers. These studies showed that a large number of E. faecium strains of food origin had ccf and efaAfm genes (9,11,18,24,25). Basanta et al. (25) showed that the only virulence genes in the multiple enterocin-producer E. faecium L50 strain are ccf and efaAfm, as confirmed in this study. The low amounts of virulence factors contained in E. faecium EYT17, EYT31, and EYT39 strains are advantages. Antibiotic resistance In this study, the disc diffusion method was used to determine the antibiotic resistance patterns of the E. faecium EYT17, EYT31, and EYT39 strains. All of the strains were sensitive to ampicillin, chloramphenicol, gentamicin, norfloxacin, penicillin, streptomycin, tetracycline, sulphamethoxazole/trimethoprim, and vancomycin. The E. faecium strains only exhibited intermediary resistance to erythromycin (15 µg). The resistance against antibiotics is an important factor for the evaluation of the safety of enterococci (16). These bacteria have natural and acquired resistance to antibiotics. Antibiotic-resistant enterococci are widespread in meat products, dairy products, and ready-to-eat foods (26). Susceptibility to clinically relevant antibiotics of enterococci of food origin is very important for consumer health. Thus, the complete susceptibility to clinically important antibiotics of the multiple enterocinproducing E. faecium EYT17, EYT31, and EYT39 strains is advantageous. In conclusion, the multiple enterocin encoding genes were detected in bacteriocinogenic E. faecium EYT17, EYT31, and EYT39 strains. These strains were found Table Incidence of virulence factors in 3 bacteriocin-producer Enterococcus faecium strains from Turkish tulum cheese. Virulence determinants (PCR amplification) Enterococcus faecium strains EYT17 EYT31 EYT39 gelE efaAfm + efaAfs cpd cob ccf + + + cad ace espfm espfs agg cylM cylB cylA clear of potential virulence determinants, except for ccf and efaAfm. In addition, all of these strains were found completely susceptible to clinically important antibiotics. These findings suggest that multiple enterocin-producing E. faecium EYT17, EYT31, and EYT39 strains have low risk factors for consumer health and these strains may be used for food preservation. Further studies should be made to evaluate the application of these strains as adjunct cultures in the dairy fermentation process. Acknowledgements Part of this study was presented as a poster presentation at the 11th Food Congress of Turkey in Hatay, Turkey, and the abstract was published in the Congress’s book of abstracts. 1000 bp 500 bp 100 bp
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