Yilmaz Emre Gencay

Prevalence and molecular characterization of sorbitol fermenting and non-fermenting Escherichia coli O157:H7(+)/H7(-) isolated from cattle at slaughterhouse and slaughterhouse wastewater.

The prevalence and seasonal distribution of E. coli O157:H7(+)/H7(-) in an array of aged cattle at slaughter and its dissemination with slaughterhouse wastewater over a two year period in Turkey were investigated. For this purpose, a total of 720 samples (240 rectoanal mucosal swap [RAMS], 240 carcass sponge and 240 bile samples) of 240 cattle categorized according to age, gender, breed and sampling site were collected along with additional 24 wastewater samples and were subjected to immunomagnetic separation based cultivation technique to efficiently isolate E. coli O157 from the background flora. Identification (rfbEO157, fliCh7), detection of major virulence factors (stx1, stx2, eaeA, hly, lpfA1-3 and espA), intimin variants (eae-α1, eae-α2, eae-β, eae-β1, eae-β2, eae-γ1 and eae-γ2/θ) and shiga toxin variants (stx1c, stx1d, stx2c, stx2d, stx2e, stx2f and stx2g) of all the isolates were assessed by PCR. From 10 (4.2%) of RAMS and 11 (4.6%) of carcass sponge samples and 5 (20.8%) of slaughterhouse wastewater samples, a total of 102 colonies (99 sorbitol negative and 3 sorbitol positive) were isolated. Overall, 17 (7.1%) and 15 (6.3%) of 240 sampled cattle were shown to harbor E. coli O157 and E. coli O157:H7, respectively either in their RAMS or carcass sponge samples analyzed. Statistically significant differences between categories; season, age, gender and breed of cattle were not observed (p>0.05). None of the isolated E. coli O157:H7(+)/H7(-) strains harbored any of the investigated intimin types other than eaeγ1 or shiga toxin variants stx1d, stx2e, stx2f or stx2g while all were lpfA1-3(+) except 5 E. coli O157:H7(-) strains. Intimin variant eaeγ1 and shiga toxin 1 variant stx1c were detected from all of the eaeA(+) (97/102, 95.1%) and stx1(+) (32/102, 31.3%) strains, respectively while from stx2(+) (80/102, 78.4%) isolates, both stx2c (68/80, 85.0%) and stx2d (12/80, 15.0%) variants were determined. In the last decade, prevalence of E. coli O157:H7 has an increasing trend in cattle. Slaughterhouses are the significant sources of environmental contamination with E. coli O157:H7. Isolation and molecular characterization of sorbitol fermenting E. coli O157:H7 are a novel finding and may lead to a revision of reference isolation procedure of E. coli O157:H7 in future.

Serotype identification and antimicrobial resistance profiles of Salmonella spp. isolated from chicken carcasses

In this study, 32 Salmonella strains isolated from 400 chicken carcasses were serotyped, and antibiotic resistance profiles were detected against 12 selected antimicrobial agents using disc diffusion method. Thirty-two isolates were identified as follows; 22 (68.7%) Salmonella Enteritidis, five (15.6%) Salmonella Virchow, three (9.3%) Salmonella Typhimurium and two (6.2%) Salmonella Hadar. In all Salmonella isolates, antibiotic resistance were detected. Out of 32 Salmonella strains, 22 (68.75%) displayed multi-drug resistance. Thirty-two (100.0%) of the isolates were found to be resistant to penicillin G, 20 (62.5%) to nalidixic acid, four (12.5%) to cephalothin, two (6.2%) to streptomycin and two (6.2%) to tetracycline. Fifteen (68.1%) Salmonella Enteritidis, one (33.3%) Salmonella Typhimurium, two (100.0%) Salmonella Hadar and two (40.0%) Salmonella Virchow were shown to be resistant to nalidixic acid. Cephalothin resistance was detected in 9.0%, 33.3%, and 20.0% for Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Virchow, respectively. The results indicate that Salmonella recovered from chicken carcasses were resistant to multiple antimicrobials and that resistance among these isolates varies by serotype. Also, this emerged as a significant public health problem.

Primary Isolation Strain Determines Both Phage Type and Receptors Recognised by Campylobacter jejuni Bacteriophages

In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated using NCTC12662 as the indicator strain, which may have biased the selection of phages. A large group of C. jejuni phages rely on the highly diverse capsular polysaccharide (CPS) for infection and recent work identified the O-methyl phosphoramidate modification (MeOPN) of CPS as a phage receptor. We therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages were identified based on host range analysis and genome restriction profiles. Most phages were isolated using C. jejuni strains NCTC12662 and RM1221 and interestingly phage genome size (140 kb vs. 190 kb), host range and morphological appearance correlated with the isolation strain. Thus, according to C. jejuni phage grouping, NCTC12662 and NCTC12658 selected for CP81-type phages, while RM1221 selected for CP220-type phages. Furthermore, using acapsular ∆kpsM mutants we demonstrated that phages isolated on NCTC12658 and NCTC12662 were dependent on the capsule for infection. In contrast, CP220-type phages isolated on RM1221 were unable to infect non-motile ∆motA mutants, hence requiring motility for successful infection. Hence, the primary phage isolation strain determines both phage type (CP81 or CP220) as well as receptors (CPS or flagella) recognised by the isolated phages.

Sheep as an important source of E. coli O157/O157:H7 in Turkey

Escherichia coli O157:H7 is a globally important foodborne pathogen and has been mainly associated with cattle as the reservoir. However, accumulating data shows the importance of sheep as an E. coli O157:H7 vehicle. The presence of E. coli O157/O157:H7 in recto-anal mucosal swap and carcass sponge samples of 100 sheep brought to the slaughterhouse in Kirikkale were analyzed over a year. Molecular characteristics (stx1, stx2, eaeA, hly, lpfA1-3, espA, eae-α1, eae-α2, eae-β, eae-β1, eae-β2, eae-γ1, eae-γ2/θ, stx1c, stx1d, stx2c, stx2d, stx2e, stx2f, stx2g, blaampC, tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), sul1, sul2, floR, cmlA, strA, strB and aadA) of 79 isolates were determined and minimum inhibitory concentrations of 20 different antibiotics were investigated. E. coli O157/O157:H7 was found in 18% of sheep included in the study and was more prevalent in yearlings than lambs and mature sheep, and male than female sheep, though none of the categories (season, sex or age range) had significant effect on prevalence. Furthermore, Shiga-toxigenic E. coli (STEC) O157:H7 was determined in 2% and 8% of sheep feces and carcasses, respectively. Additionally, lpfA1-3 and eae-γ1 were detected in all isolates. None of the isolates showed resistance against investigated antibiotics, even though 4 sorbitol fermenting E. coli O157 isolates were positive for tet(A), sul1 and aadA. This is the first study in Turkey that reveals the potential public health risk due to the contamination of sheep carcasses with potentially highly pathogenic STEC O157:H7 strains.

The relationship between seropositivity and tissue cysts in sheep naturally infected with Toxoplasma gondii

Skeletal muscles (tongue, masseter, leg, intercostal, and diaphragmatic muscles) and brain samples of 100 sheep at slaughter were analysed for the presence of T. gondii tissue cysts along with serum IgG titres. Two methods of isolation by percoll gradient centrifugation and tissue microarray (TMA) technique with immunoperoxidase staining were used. Seropositivity was detected in 88% (88/100) of sheep sera analysed by indirect fluorescent antibody test. Tissue cysts were observed in 46 (52.3%, 46/88) and 15 (17%, 15/88) of the seropositive sheep with the isolation technique and TMA and immunoperoxidase staining, respectively. The diameters of the tissue cysts were 25–58 × 25–62 (mean 34 × 36) µm. The relationship between the presence of tissue cysts and seropositivity in sheep was statistically significant at 1/16 (P < 0.01) and at 1/64 and 1/128 (P < 0.001) serum dilutions.

Methods for Isolation, Purification, and Propagation of Bacteriophages of Campylobacter jejuni

Here, we describe the methods for isolation, purification, and propagation of Campylobacter jejuni bacteriophages from samples expected to contain high number of phages such as chicken feces. The overall steps are (1) liberation of phages from the sample material; (2) observation of plaque-forming units on C. jejuni lawns using a spot assay; (3) isolation of single plaques; (4) consecutive purification procedures; and (5) propagation of purified phages from a plate lysate to prepare master stocks.

Species distribution, molecular characteristics and vancomycin resistance gene profiles of Enterococcus sp. isolates from farmhouse cheeses in western Turkey

Species distribution, virulence traits and vancomycin resistance gene profiles of Enterococcus isolated from 43 home-made artisan cheese samples collected from open markets, located in Aydin region of Turkey, were investigated. Of the 129 isolates, 95 were identified as Enterococcus sp.; Enterococcus faecium being the most prevalent species (82.1%), followed by Enterococcus faecalis (13.6%) and Enterococcus durans (1.0%). None of the enterococci were harbouring vanA or vanC, while seven isolates (7.3%) were shown to harbour vanB gene by multiplex PCR. gelE (49.4%) being the most prevalent virulence factor was followed by asa1 (27.3%), esp (22.1%), cylA (4.2%) and hyl (3.1%).

Phase Variable Expression of a Single Phage Receptor in Campylobacter jejuni NCTC12662 Influences Sensitivity Toward Several Diverse CPS-Dependent Phages

Campylobacter jejuni NCTC12662 is sensitive to infection by many Campylobacter bacteriophages. Here we used this strain to investigate the molecular mechanism behind phage resistance development when exposed to a single phage and demonstrate how phase variable expression of one surface component influences phage sensitivity against many diverse C. jejuni phages. When C. jejuni NCTC12662 was exposed to phage F207 overnight, 25% of the bacterial cells were able to grow on a lawn of phage F207, suggesting that resistance develops at a high frequency. One resistant variant, 12662R, was further characterized and shown to be an adsorption mutant. Plaque assays using our large phage collection showed that seven out of 36 diverse capsular polysaccharide (CPS)-dependent phages could not infect 12662R, whereas the remaining phages formed plaques on 12662R with reduced efficiencies. Analysis of the CPS composition of 12662R by high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) showed a diminished signal for O-methyl phosphoramidate (MeOPN), a phase variable modification of the CPS. This suggested that the majority of the 12662R population did not express this phase variable modification in the CPS, indicating that MeOPN serves as a phage receptor in NCTC12662. Whole genome analysis of 12662R showed a switch in the length of the phase variable homopolymeric G tract of gene 06810, encoding a putative MeOPN-transferase located in the CPS locus, resulting in a non-functional protein. To confirm the role of 06810 in phage resistance development of NCTC12662, a 06810 knockout mutant in NCTC12662 was constructed and analyzed by HR-MAS NMR demonstrating the absence of MeOPN in the CPS of the mutant. Plaque assays using NCTC12662Δ06810 demonstrated that seven of our CPS-dependent Campylobacter phages are dependent on the presence of MeOPN for successful infection of C. jejuni, whereas the remaining 29 phages infect independently of MeOPN, although with reduced efficiencies. Our data indicate that CPS-dependent phages uses diverse mechanisms for their initial interaction with their C. jejuni host.

Methods for Initial Characterization of Campylobacter jejuni Bacteriophages

Here we describe an initial characterization of Campylobacter jejuni bacteriophages by host range analysis, genome size determination by pulsed-field gel electrophoresis, and receptor-type identification by screening mutants for phage sensitivity.

Whole-Genome Sequence of the Bacteriophage-Sensitive Strain Campylobacter jejuni NCTC12662

ABSTRACT Campylobacter jejuni NCTC12662 has been the choice bacteriophage isolation strain due to its susceptibility to C. jejuni bacteriophages. This trait makes it a good candidate for studying bacteriophage–host interactions. We report here the whole-genome sequence of NCTC12662, allowing future elucidation of the molecular mechanisms of phage–host interactions in C. jejuni.