Developing Innolysins against Campylobacter jejuni using a novel prophage receptor-binding protein

Campylobacter contaminated poultry remains the major cause of foodborne gastroenteritis worldwide, calling for novel antibacterials. We previously developed the concept of Innolysin composed of an endolysin fused to a phage receptor binding protein (RBP) and provided the proof-of-concept that Innolysins exert bactericidal activity against Escherichia coli. Here, we have expanded the Innolysin concept to target Campylobacter jejuni. As no C. jejuni phage RBP had been identified so far, we first showed that the H-fiber originating from a CJIE1-like prophage of C. jejuni CAMSA2147 functions as a novel RBP. By fusing this H-fiber to phage T5 endolysin, we constructed Innolysins targeting C. jejuni (Innolysins Cj). Innolysin Cj1 exerts antibacterial activity against diverse C. jejuni strains after in vitro exposure for 45 min at 20°C, reaching up to 1.30 ± 0.21 log reduction in CAMSA2147 cell counts. Screening of a library of Innolysins Cj composed of distinct endolysins for growth inhibition, allowed us to select Innolysin Cj5 as an additional promising antibacterial candidate. Application of either Innolysin Cj1 or Innolysin Cj5 on chicken skin refrigerated to 5°C and contaminated with C. jejuni CAMSA2147 led to 1.63 ± 0.46 and 1.18 ± 0.10 log reduction of cells, respectively, confirming that Innolysins Cj can kill C. jejuni in situ. The receptor of Innolysins Cj remains to be identified, however, the RBP component (H-fiber) recognizes a novel receptor compared to lytic phages binding to capsular polysaccharide or flagella. Identification of other unexplored Campylobacter phage RBPs may further increase the repertoire of new Innolysins Cj targeting distinct receptors and working as antibacterials against Campylobacter

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

Gencay, Yilmaz Emre/0000-0002-2154-9663WOS: 000340692900034PubMed: 25042529Escherichia 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 (stx(1), stx(2), eaeA, hly, lpfA1-3, espA, eae-alpha 1, eae-alpha 2, eae-beta, eae-beta 1, eae-beta 2, eae-gamma 1, eae-gamma 2/theta, stx1(c),stx1(d), Stx2(c), stx2(d), stx2(e), stx2(f), stx2(g), bla(ampC), 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. colt 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. colt (STEC) O157:H7 was determined in 2% and 8% of sheep feces and carcasses, respectively. Additionally, lpfA1-3 and eae-gamma 1 were detected in all isolates. None of the isolates showed resistance against investigated antibiotics, even though 4 sorbitol fermenting E. colt 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. (C) 2014 Elsevier B.V. All rights reserved

Phenotypic and genotypic antibiotic resistance profiles of Escherichia coli O157 from cattle and slaughterhouse wastewater isolates

Gencay, Yilmaz Emre/0000-0002-2154-9663WOS: 000354724300054The aims of this study were to determine the minimal inhibition concentration of 20 different antibiotics on cattle and slaughterhouse wastewater Escherichia coli O157, including both Shiga toxigenic E. coli O157 (STEC O157) and non-Shiga toxigenic strains (non-STEC O157) by the Epsilometer test, and to determine the antibiotic resistance gene profiles of the isolates by PCR. A total of 102 cattle and slaughterhouse wastewater E. coli O157 isolates including 96 E. coli O157:H7(+) (81 non-sorbitol fermenting [NSF] STEC O157:H7, 12 NSF non-STEC O157:H7, and three sorbitol fermenting [SF] non-STEC O157:H7) and six non-STEC O157:H7(-) isolated from 744 cattle and slaughterhouse wastewater samples collected within a 2-year period were assessed. Of 93 NSF E. coli O157:H7 isolates, 19 were resistant to tetracycline and sulfamethoxazole, 14 to trimethoprim, 13 to cefoxitin, 11 to streptomycin, 10 to ampicillin, eight to chloramphenicol, six to cephalothin, four to cefaclor, four to aztreonam, and four to nalidixic acid. In six of the E. coli O157:H7(-) isolates, tetracycline resistance was detected while five of them were also resistant to ampicillin, sulfamethoxazole, and trimethoprim. In PCR analysis, 26.0 % (25/96) of the NSF E. coli O157:H7(+) and all of the E. coli O157:H7(-) isolates harbored one or more antibiotic resistance genes. While tetA, tetB, tetC, strA, strB, and sulI genes were detected from a number of the isolates, tetD, tetE, tetG, cmlA, floR, sulII, aadA, and ampC genes were not detected in any of the isolates. Results suggest a high antibiotic resistance in E. coli O157:H7(+)/H7(-) cattle and wastewater isolates. The majority of our resistant isolates, antibacterial resistance genes did not correlate with observed phenotypic resistance. Other resistance traits and regulatory factors that mediate antibiotic resistance should be included in further antimicrobial resistance investigations.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [110R013]This study was supported by The Scientific and Technological Research Council of Turkey (TUBITAK, project no: 110R013)

Prevalence and molecular characterization of sorbitol fermenting and non-fermenting Escherichia coli 0157:H7(+)/H7(-) isolated from cattle at slaughterhouse and slaughterhouse wastewater

Gencay, Yilmaz Emre/0000-0002-2154-9663WOS: 000332436800005PubMed: 24448275The prevalence and seasonal distribution of E. colt 0157: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 0157 from the background flora. Identification (rfbE(0157),fliC(h7)), detection of major virulence factors (sbc7, sU2, eaeA, hly, lpfA1-3 and espA), intimin variants (eae-alpha 1, eae-alpha 2, eae-beta 3, eae-beta 1, eae-beta 2, eae-gamma l and eae-gamma 2/0) and shiga toxin variants (stx70 stx(1d), stx(2c), stx(2d), stx(2e), stx(2f)and stx(2g)) of all the isolates were assessed by PCR. From 10(42%) 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 0157 and E. colt 0157: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 0157:H7+/H7- strains harbored any of the investigated intimin types other than eaeyi or shiga toxin variants stx(1d), StX(2e), six(2f) or StX(2g) while all were lpfA1-3+ except 5 E. coli 0157:H7- strains. Intimin variant eaey, and shiga toxin 1 variant stx/c were detected from all of the eaeA+ (97/102, 95.1%) and stxt (32/102,313%) strains, respectively while from sbd" (80/102, 78.4%) isolates, both stx(2c) (68/80, 85.0%) and 5tx(2d) (12/80, 15.0%) variants were determined. In the last decade, prevalence of E. coli 0157:H7 has an increasing trend in cattle. Slaughterhouses are the significant sources of environmental contamination with E. coli 0157:H7. Isolation and molecular characterization of sorbitol fermenting E. coli 0157:H7 are a novel finding and may lead to a revision of reference isolation procedure of E. coli 0157:H7 in future. (C) 2014 Elsevier B.V. All rights reserved.TUBITAK (The Scientific and Technological Research Council of Turkey)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [110R013]This work was supported by TUBITAK (The Scientific and Technological Research Council of Turkey) grant no. 110R013. We are grateful to J. Osek and C. Garcia-Aljaro for kindly supplying the reference strains

A potential infection source for humans: Frozen buffalo meat can harbour tissue cysts of Toxoplasma gondii

Gencay, Yilmaz Emre/0000-0002-2154-9663; YILDIZ, Kader/0000-0001-5802-6156WOS: 000311175500014Buffaloes are considered resistant to toxoplasmosis. Tissue cysts of Toxoplasma gondii found rarely in skeletal muscles of buffaloes. However, in the present study, we found tissue cyst of T gondii in frozen boneless buffalo meat illegally imported to Turkey. Spherical tissue cysts of T. gondii. 27-34 to 30-32 (mean 31 x 30) mu m in diameter, were found in 3 out of 20 (15%) spontaneously thawed meat samples analyzed by light microscopy of percoll dilutions. All positive samples were also confirmed by observation of 97 bp gene products of T. gondii repetitive B1 gene by nested PCR. The tissue cysts however, were not found following a preservation of two days at -18 degrees C. Present study shows that frozen buffalo meat can be a potential new infection source for human toxoplasmosis. (C) 2012 Elsevier Ltd. All rights reserved

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

Gencay, Yilmaz Emre/0000-0002-2154-9663;WOS: 000279713700014PubMed: 19946796In 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

Comparison of Virulence Gene Profiles of Enterococcus faecium and Enterococcus faecalis Chicken Neck Skin and Faeces Isolates

Gencay, Yilmaz Emre/0000-0002-2154-9663WOS: 000281800200022The objective of this study was to find out the distribution of major virulence determinants asa1, gelE, cylA, esp, and hyl by multiplex PCR in 132 Enterococcus faecium and 67 Enterococcus faecalis isolates originated from chicken neck skin samples at slaughterhouse and faeces samples from intensive broiler enterprises and rural poultry establishments. In the study, 31.2% (62/199) of the enterococcal strains harbored at least one virulence determinant. The gelE gene was the predominant (30.2%) virulence trait among the enterococci investigated followed by asa1 (15.6%). Both gelE and asa1 genes were significantly higher in E. faecalis than E. faecium. The hyl, esp and cylA genes were detected with percentages of 1.5%, 1.5% and 0.8% in E. faecium isolates. None of the E. faecalis strains harbored cylA, esp and hyl genes. The results indicate that a clear difference was observed in the kind of virulence factor present in strains between faecal samples and skin samples. Also. E. faecium strains isolated from both chicken skin samples and faeces presented lower pathogenicity potential than did E. faecalis