Investigation and characterization of Aliarcobacter spp. isolated from cattle slaughterhouse in Türkiye.

Aliarcobacter spp. have been isolated from numerous food products at retail and from animal carcasses and feces at slaughter. The objectives of this study were as follows: (i) to isolate Aliarcobacter species from different slaughterhouses' samples and (ii) to detect genetic diversity, antibiotic resistance, biofilm ability, and putative virulence gene profiles of the isolates. A molecular investigation of antibiotic resistance and virulence factors was also conducted using polymerase chain reaction (PCR). Among 150 samples, a total of 22 (14.6%) Aliarcobacter spp. isolates were obtained, with varying levels of antibiotic resistance observed. The genes tetO, tetW, and gyrA were detected in 0%, 31.8%, and 27.2% of the isolates, respectively. All isolates were resistant to ampicillin, rifampin, and erythromycin, while tetracycline was found to be the most effective antibiotic, with 81.8% of the isolates showing susceptibility to it. All isolates (100%) harbored more than one of the nine putative virulence genes tested, with 18.1% of isolates carrying more than three. Regarding biofilm formation, 7 (31.8%) and 4 (18.1%) isolates were found to form strong and moderate biofilms, respectively, while one (4.5%) isolate was classified as a weak biofilm producer. ERIC-PCR band patterns suggested that the isolated Aliarcobacter spp. from slaughterhouses had different sources of contamination. These findings highlight the potential risk posed by pathogenic and multidrug-resistant Aliarcobacter spp. in food and the need for control measures throughout the food chain to prevent the spread of these strains. The results indicate that foods of animal origin and cattle slaughterhouses are significant sources of antimicrobial resistant Aliarcobacter.

Emergence and Characterization of Tigecycline Resistance Gene tet(X4) in ST609 Escherichia coli Isolates from Wastewater in Turkey.

Emergence of pathogens harboring tigecycline resistance genes incurs great concerns. Wastewater is recognized as the important reservoir of antimicrobial resistance genes. Here we characterized the phenotypes and genotypes of bacteria carrying tet(X4) from wastewater in Turkey for the first time. Four tet(X4)-positive Escherichia coli isolates were identified and characterized by PCR, Sanger sequencing, antimicrobial susceptibility testing, conjugation assays, Illumina sequencing, nanopore sequencing and bioinformatic analysis. Four tet(X4)-harboring isolates were multidrug-resistant (MDR) bacteria and the tet(X4) gene was nontransferable in four isolates. Genetic analysis revealed that tet(X4) genes in four isolates were located on plasmids co-harboring two replicons IncFIA(HI1) and IncFIB(K). However, none of the four plasmids carried genes associated with horizontal transfer of plasmids. The coexistence of blaSHV-12-bearing IncX3-type plasmid and tet(X4)-harboring plasmid was also found in one isolate. These findings indicate that continuous surveillance of the tet(X4)-bearing isolates in different environments worldwide should be strengthened. IMPORTANCE The emergence of tigecycline resistance genes in humans and animals in China seriously threatens the clinical utility of tigecycline, but the molecular epidemiology of tigecycline-resistant bacteria in other countries remained largely unknown. Therefore, it is necessary to learn the prevalence and molecular characteristics of bacteria carrying tigecycline resistance genes, particularly the mobilizable tet(X4), in other countries. In the study, we first described the presence and molecular characteristics of the tet(X4)-positive E. coli isolates from wastewater in Turkey. Four tet(X4)-bearing isolates belonged to ST609, an E. coli clone commonly found from humans, animals and the environment. These findings highlight the importance of monitoring the tet(X4) gene in different settings globally.

Escherichia coli serogroups in slaughterhouses: Antibiotic susceptibility and molecular typing of isolates.

This study aimed to investigate the contamination of carcasses and slaughterhouse environment with Escherichia coli O157:H7 and non-O157 serogroups (O45:H2, O103:H2, O121:H19, O145:H28, O26:H11, O111:H8). For this purpose, a total of 150 samples (30 carcasses, 30 shredding units, 30 knives, 30 slaughterhouse waste water and 30 wall surfaces) were collected from 5 different slaughterhouses in Kayseri, Turkey. The conventional and molecular methods were performed in order to detect Escherichia coli and its serogroups. Of the 150 samples, 55 (36%) were found to be contaminated with E. coli. Among isolates, E. coli serogroup (O157:H7) were detected in 2 (11%) carcass and 2 (11%) wastewater samples. None of the E. coli isolates harbored tested genes (stx1, stx2, eaeA, and hylA). Effective infection control measures and antibiotic stewardship programs should be adopted to limit the spread of multidrug-resistant bacteria. It was also deduced that these isolates resistance to different antibiotics could be hazardous for public health.

Prevalence and molecular characterization of Listeria monocytogenes isolated from wastewater of cattle slaughterhouses in Turkey.

AIM: The study aimed to investigate the role of cattle slaughterhouse wastewater as a possible source for the environmental distribution of Listeria monocytogenes. METHODS AND RESULTS: Listeria spp. isolation was performed by collecting 117 wastewater samples from four different cattle slaughterhouses in Turkey. Species-specific identification was performed biochemically, and L. monocytogenes isolates were confirmed with polymerase chain reaction (PCR). In all, 71 (62.2%) of the wastewater samples were found to be positive for Listeria spp., and 17 (14.9%) of these samples were contaminated with L. monocytogenes. Pulsed field gel electrophoresis (PFGE) analysis revealed that all L. monocytogenes isolates were of different pulsotypes and isolates belonged to seven different phylogenetic clusters. Multiplex PCR analysis for genoserotypes and lineage determination showed that the isolates were divided into genoserotypes IVb and IIc in Lineages I and II. Also, it has been investigated with SYBR-Green Real-time PCR whether the L. monocytogenes isolates harboured virulence genes (hly, sigB, plcA, plcB, inlA, inlB, inlC and inlJ), and it was found that all isolates were substantially positive. Antibiotic resistance profiles and MIC values of the isolates were determined, and all L. monocytogenes isolates were found susceptible to ampicillin. In contrast, two isolates were resistant to meropenem and erythromycin, and three isolates were resistant to trimethoprim/sulfamethoxazole. CONCLUSION: L. monocytogenes, which pose a threat to public health and resists to antibiotics effectively used in treatments, can environmentally spread via wastewater of cattle slaughterhouses. The wastewater of the food industry, which has rich microbiota, should be treated carefully, and possible environmental contamination should be prevented. SIGNIFICANCE AND IMPACT OF STUDY: This is the first study that investigates the molecular characterization of L. monocytogenes isolated from cattle slaughterhouse wastewater and the findings represent the importance of cattle wastewater in the epidemiology of L. monocytogenes in Turkey.

[Virulence Genes, Antibiotic Susceptibility Profiles and Molecular Characterization of Campylobacter Species Isolated from Different Slaughterhouses]. / Farkli Kesimhanelerden Izole Edilen Campylobacter Türlerinin Virülans Genleri, Antibiyotik Duyarlilik Profilleri ve Moleküler Karakterizasyonu.

The aim of this study was to investigate the frequency of Campylobacter species, to detect the antibiotic resistance profiles and the virulence genes and to determine the clonal proximity of the isolates in the samples of cutting board, slaughterhouse waste water, wall, knife and carcass from three different slaughterhouses in Kayseri region. For this purpose, a total of 150 samples, 10 of each from knife, wall, cutting board, carcass smear sample and slaughterhouse wastewater were collected from each of the three types of slaughterhouses in 2018 in Kayseri. For the isolation of the Campylobacter species, following preenrichment, the suspensions were inoculated onto modified charcoal cefoperazone desoxycholate (CCD) agar and were incubated at 37°C under microaerophilic condition for 48-72 hours. Suspicious colonies with gray-white color were recovered and subjected to phenotypical (Gram staining, oxidase, catalase test, and motion test) tests. Multiplex polymerase chain reaction (mPCR) was used for the molecular identification of the Campylobacter species. Antimicrobial susceptibilities of the isolates identified at the species level were detected by using the disk diffusion test and antibiotic gradient test. Virulence genes (iam, cadF, cdtA, flaA, ceuE, cdtC, cdtB and virB11) among the isolates were evaluated by PCR. The molecular typing of the isolates determined at species level was performed by Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR). In the study, 17 (11.3%) of the 150 samples taken from the slaughterhouse were found to be suspicious in terms of Campylobacter spp. and as a result of phenotypic identification tests, all of the isolates were verified as Campylobacter spp.. As a result of mPCR; eight of the isolates were identified as Campylobacter jejuni, eight as Campylobacter fetus and one as Campylobacter coli. The isolation of the Campylobacter species from different sources was found to be higher in slaughterhouse wastewater than those of others (p<0.001) and the difference in the proportional distribution of the Campylobacter species obtained from various sources was statistically significant (p<0.05). As a result of the disk diffusion test, while, all C.jejuni isolates were resistant to ciprofloxacin, 87.5%, 25%, 25% and 12.5% of C.jejuni isolates were resistant to enrofloxacin, neomycin, amoxicillin/clavulanic acid, and erythromycin, respectively. In addition, 25%, 25% and 12.5% of C.fetus isolates were resistant to amoxicillin/clavulanic acid, neomycin and gentamicin, respectively. C.coli isolate was not resistant to any of the antibiotics tested. Antibiotic gradient test results were found to be compatible with the disc diffusion test results. One of the virulence genes examined, virB11, was not detected in any of the isolates. Moreover, iam gene was not present in C.fetus and C.coli isolates, but only in one C.jejuni isolate. The flaA gene was detected in six C.jejuni isolates. C.coli isolate and seven C.jejuni and seven C.fetus isolates were positive in terms of the cdtC gene. The cdtA, cdtB, ceuE and cadF genes were found to be positive in all C.jejuni isolates. All isolates analyzed in the study demonstrated different ERIC-PCR profiles. In conclusion, it was shown that Campylobacter strains isolated from slaughterhouses were resistant to the most of the current antibiotics. Moreover, the presence of highly virulent Campylobacters in the slaughterhouse environment threatens public health due to the risk of contamination of the humans via carcasses and foods. Therefore, it is recommended that strict hygiene rules should be followed to reduce Campylobacter species contamination in slaughterhouses.