Investigation of virulence factors of enterococci strains isolated from sewater

Enterococcus bacteria, which classified in the group of Lactic Acid Bacteria, are the natural inhabitants of the intestinal flora of humans and animals and causing various infections in humans by spread to extra-intestinal regions. Enterococci, which is known as fecal pollution indicator in marine waters where environmental pollution is high and where sewage and sewage waters are mixed, are important for public health. Enterococci, which have intrinsic and extrinsic antibiotic resistance mechanisms and virulence factors by chromosomal, plasmid or conjugative transposons, are pathogenic microorganisms. In our study, 66 Enterococcus strains isolated from a previous study on sea water samples taken from Marmara Sea and Black Sea (Kilyos, Rumelikavağı, Yeşilyurt and Üsküdar), which are important in tourism, fishing, irrigation and utility water, were confirmed with API®20 Strep test kit at species level. Antibiotic resistance profile [nalidixic acid (NA), streptomycin (S), penicillin (P), chloramphenicol (C), vancomycin (VA), tetracycline (TE), ciprofloxacin (CIP), canamycin (K), amikacin (AM), erythromycin (E), gentamicin (CN), rifampicin (RIF)], hemolysis production activity, gelatinase and caseinase activity, biofilm formation capacity, serum resistance activity, hemagglutination and bacteriocin activity of the Enterococcus strains used in the study were examined phenotypically. Chromosomal DNA isolations of the strains were made and antibiotic resistance genes and virulence factors were examined by PCR method. Binding to extracellular matrix proteins (ace), gelatinase activity (gelE), aggregation factor (agg), adherence to host tissues and biofilm formation (esp), regulation of gelatinase and serine protease expression (fsr), cytolysin production (cylM, cylB and cylA) The genes responsible for the presence of enterococcal adhesion protein (efaAfs and efaAfm), sex pheromones (cpd, cob and ccf) and virulence factors [vancomycin (vanA and vanB), streptomycin (ant (6)-la), gentamicin (acc(6')-aph (2'')), canamycin (aphA-3), rifampicin (rpoB1 and rpoB2), ampicillin (bla(Z)), erythromycin (ermB), chloramphenicol (catpIP), tetracycline (tetM and tetL)] related to antibiotic resistance were examined by PCR method. On the other hand, plasmid profiles of Enterococcus strains were determined by comparing their molecular size with the known marker. İn this study, the resistance profile of 66 Enterococci isolates against 13 different antibiotics were examined according to Kirby-Bauer disc diffusion method and the intraspecific antibiotic resistance rates of E. faecalis and E. faecium strains were 60.8% and 84.2% for penicillin, 10.86% and 31.57% for chloramphenicol, 41.3% and 57.9% for vancomycin, 52.7% and 63.15% for tetracycline, 17.4% and 36.84% for ciprofloxacin, 97.82% and 94.7% for kanamycin, 39.13% and %57.9 for ampicillin, 50% and 84.2% for erythromycin, 100% and 94.7% for gentamicin, respectively. All strains were also resistant to nalidixic acid, amikacin and rifampicin and susceptible to streptomycin. When multiple antibiotic resistance profiles were examined phenotypically, it was found that multiple antibiotic resistance ranged between 5-11, 3 (6.52%) E. faecalis and 5 (26.31%) E. faecium strains were up to 11 antibiotics and 3 (6.52%) E. faecalis strains multiple resistance to at least 5 antibiotics. On the other hand, when compared the phenotypic virulence factors of E. faecalis and E. faecium strains, 91.3% and 94.7% for gelatinase activity, 67.4% and 31.57% for caseinase activity, 41.3% and 47.36% for strong retention in biofilm formation capacity, 52.7% and 31.57% for serum resistance activity, 32.6% and 0% for very strong reaction in hemagglutination activity, bacteriocin activity against Staphylococcus aureus ATCC 25923 was 32.6% and 5.26%, and bacteriocin activity against Escherichia coli ATCC 25922 bacteria was 23.4% and 0% determined, respectively. Genotypic analysis of the antibiotic resistance genes of E. faecalis and E. faecium strains examined by PCR method in the study resulted in the in vitro antibiotic resistance gene ratios for 32.6% and 31.57% vanA and 21.73% and 31.57% for vanB genes, 100% and 94.73% for the acc(6')-aph (2''), gene, 97.82% and 89.47% for the aphA-3 gene, 97.82% and 100% for the rpoB2 gene, 93.47% and 89.47% for the bla(Z) gene, 50% and 84.2% for ermB, 71.73% and 63.15% for catpIP, 56.52% and 21.21% for tetM and 43.47% and 36.84% for tetL genes were identified. All strains had the rpoB1 gene but not the ant(6)-la gene. When the virulence genes were examined by PCR method, the intra-species virulence gene rates were 86.9% and 0% for the ace gene, 91.3% and 89.4% for gelE gene, 95.6% and 0% for efaAfs, 58.7% and 63.1% for efaAfm and 41.3% and 26.3% for cpd gene, 30.4% and 63.1% for ccf gene, 56.5% and 63.1% for cob gene, 84.7% and 57.9% for agg gene, 60.8% and 63.1% for fsr gene, respectively. All strains were found to have the genes responsible for the production of cytolysin, cylM, cylB, and cylA genes. When the multiple antibiotic resistance gene profile and virulence gene profile of 66 Enterococci isolates examined in the study were analysed, one (2.17%) E. faecalis and one (100%) E. gallinarum strain had at least 4, and 1 (2.17%) E. faecalis strain had up to 11 of 12 different antibiotic resistance genes was determined. At the same time, 2 (3.03%) E. faecalis strains from 12 different virulence genes had the highest virulence profile with 12 gene regions and 1 (1.51%) E. gallinarum strain had the lowest virulence profile with 3 gene regions. Finally, in the study, plasmid profiles of 66 Enterococci isolates were determined and 16 (24.24%) strains, including 13 (28.26%) E. faecalis and 3 (15.78%) E. faecium, contained 26 different sized plasmids ranging from 340 to 32600 bp. The results show that Enterococcus strains isolated from seawater samples have antibiotic resistance and virulence genes and may cause problems for public health. It is thought that the data obtained from this study will contribute to the awareness of virulence of Enterococcus bacteria, which are indicators of faecal pollution in sea water and to determine possible approaches in treatment.