The ARIMA model approach for the biofilm-forming capacity prediction of Listeria monocytogenes recovered from carcasses.

The present study aimed to predict the biofilm-formation ability of L. monocytogenes isolates obtained from cattle carcasses via the ARIMA model at different temperature parameters. The identification of L. monocytogenes obtained from carcass samples collected from slaughterhouses was determined by PCR. The biofilm-forming abilities of isolates were phenotypically determined by calculating the OD value and categorizing the ability via the microplate test. The presence of some virulence genes related to biofilm was revealed by QPCR to support the biofilm profile genotypically. Biofilm-formation of the isolates was evaluated at different temperature parameters (37 °C, 22 °C, 4 °C and - 20 °C). Estimated OD values were obtained with the ARIMA model by dividing them into eight different estimation groups. The prediction performance was determined by performance measurement metrics (ME, MAE, MSE, RMSE, MPE and MAPE). One week of incubation showed all isolates strongly formed biofilm at all controlled temperatures except - 20 °C. In terms of the metrics examined, the 3 days to 7 days forecast group has a reasonable prediction accuracy based on OD values occurring at 37 °C, 22 °C, and 4 °C. It was concluded that measurements at 22 °C had lower prediction accuracy compared to predictions from other temperatures. Overall, the best OD prediction accuracy belonged to the data obtained from biofilm formation at -20 °C. For all temperatures studied, especially after the 3 days to 7 days forecast group, there was a significant decrease in the error metrics and the forecast accuracy increased. When evaluating the best prediction group, the lowest RMSE at 37 °C (0.055), 22 °C (0.027) and 4 °C (0.024) belonged to the 15 days to 21 days group. For the OD predictions obtained at -20 °C, the 15 days to 21 days prediction group had also good performance (0.011) and the lowest RMSE belongs to the 7 days to 15 days group (0.007). In conclusion, this study will guide in using indicator parameters to evaluate biofilm forming ability to predict optimum temperature-time. The ARIMA models integrated with this study can be useful tools for industrial application and risk assessment studies using different parameters such as pH, NaCl concentration, and especially temperature applied during food processing and storage on the biofilm-formation ability of L. monocytogenes.

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.

Campylobacter jejuni and Campylobacter coli in human stool samples: antibiotic resistance profiles, putative virulence determinants and molecular characterization of the isolates.

Campylobacters, especially C. jejuni and C. coli, have become one of the leading causes of acute gastroenteritis in humans worldwide in recent years. We aimed to investigate the presence, antimicrobial resistance, putative virulence genes, and molecular characterization of C. jejuni and C. coli recovered from human acute gastroenteritis cases in the study. In the study, suspected Campylobacter spp. isolates were obtained in 43 (5%) feces samples collected from a total of 850 patients who applied to the Erciyes University Medical Faculty acute clinic between March 2019 and February 2020. As a result of the phenotypic tests, these isolates were determined to be Campylobacter spp. According to the multiplex PCR, 33 of 43 Campylobacter spp. isolates were identified as C. jejuni (76%) and ten isolates were as C. coli (24%). In the disc diffusion test, the highest resistance rate was found in the trimethoprim/sulfamethoxazole (90.1%) and ciprofloxacin (90.1%), and the lowest rate was found in the amoxicillin-clavulanic acid (9.3%). In Campylobacter spp. isolates, the virulence genes cdtA, virB11, cdtB, cadF, iam, ceu, and flaA were found to be positive at rates of 26 (60%), 28 (65.6%), 13 (30%), 4 (9%), 27 (62%), 17 (39%), and 7 (16%), respectively. However, the cdtC gene was not detected in any of the isolates. The study searched tetO gene to examine the genetic aspect of tetracycline resistance, which was found in all Campylobacter spp. isolates. In the PCR reactions to investigate A2074C and A2075G mutations of macrolide resistance, it was determined as 7 (16%) and 21 (48%) of the isolates. To detect quinolone resistance, the rates of quinolone resistance-determining regions (QRDR) were 20 (45.4%) and the gyrA gene mutations in the mismatch amplification mutation assay PCR (MAMA-PCR), were 19 (43.1%) of isolates. In addition, the quinolone resistance gene (qnr) carried by plasmid in Campylobacter isolates was not found in the study. BlaOXA-61 and CmeB (multi-drug efflux pump) genes were detected as 28 (63.6%) and 30 (68.1), respectively. The Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR) used for typing the isolates revealed that the band profiles obtained from the isolates were different. In conclusion, this was a very comprehensive study revealing the presence of antibiotic-resistant C. jejuni and C. coli with various virulence genes in patients admitted to a university hospital with acute gastroenteritis. This is of utmost significance for public health. Since campylobacteria are foodborne, zoonotic pathogens and transmission occurs mostly through food. People should have detailed information about the transmission routes of campylobacteria and risky foods. In addition, staff, food processors and caterers, should be trained in hygiene.

Arcobacter species isolated from various seafood and water sources; virulence genes, antibiotic resistance genes and molecular characterization.

Arcobacter spp. has gained clinical significance as an emerging diarrheagenic pathogen associated with water reservoirs in recent years. The complete clinical significance of Arcobacter remains rather speculative due to the virulence and antibiotic susceptibility of individual strains. This study aimed to assess the prevalence of Arcobacter spp. in fish, water, and shellfish. A total of 150 samples were collected from the Adana, Kayseri and Kahramanmaras provinces in Turkey. Arcobacter spp. was isolated from 32 (21%) of the 150 samples. The most prevalent species was A. cryaerophilus, 17 (56%), A. butzleri 13 (37%) and A. lacus 2 (6%). As a result, the ratios of the mviN, irgA, pldA, tlyA and hecA target genes were found as 17 (51%), 1 (3%), 7 (23%), 7 (23%), 1 (3%), respectively. While bla OXA-61, tetO and tetW were positive in all isolates, were found as mcr1/2/6, mcr3/7, and mcr5, genes %37.5, %25, and %34.3, respectively. Although in A. butzleri was found 10 (58%), 1 (3%), 3 (43%), 2 (28%) (mviN, irgA, pldA, and tlyA, respectively) virulence genes 7 (42%), 4 (57%), 5 (72%), 1 (3%) was found (mviN, irgA, tlyA, and hecA, respectively) virulence genes in A. cryoaerophilus. Moreover, was found for the mcr 1/2/6 7 (58%) genes, for the mcr 3/7 genes 3 (38%) in A. butzleri. In A. cryoaerophilus was found for the mcr 1/2/6 genes 5 (42%), for the mcr 3/7 genes 5 (62%), and for the mcr 5 gene 10 (100%). Thus, the current study indicated that the existence of Arcobacter spp. isolated from fish and mussel samples may pose a potential risk to public health.

Biofilm formation and antibiotic resistance profiles of water-borne pathogens.

Water sources (surface water, drinking water, rivers, and ponds) are significant reservoirs for transmitting antibiotic-resistant bacteria. In addition, these waters are an important public health problem because they are suitable environments for transferring antibiotic resistance genes between bacterial species. Our study aimed to assess the prevalence of Extended-spectrum beta-lactamase (ESBL) producing isolates in water samples, the susceptibility of the isolates to the specified antibiotics, the determination of biofilm ability, antibiotic resistance genes, and the molecular typing of the isolates. For this purpose, Polymerase chain reaction (PCR) and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses were used. Out of 70 isolates, 15 (21%) were ESBL producing, and sent for the MALDI-TOF analysis, where Escherichia coli, Acinetobacter calcoaceticus, Enterobacter bugandensis, Acinetobacter pittii, Pseudomonas aeruginosa, Acinetobacter junii, Pseudomonas oleovorans, and Enterobacter ludwigigii were identified. Moreover, colistin resistance genes (mcr 1/2/6, mcr 4, mcr 5, mcr 3/7, and mcr 8), ESBL-encoding genes (blaSHV, blaTEM, and blaCTX-M) and carbapenemase genes (blaNDM, blaOXA-48, and blaKPC) using molecular analysis (PCR) were confirmed. The colistin resistance gene was detected at 80% (12/15) in the isolates obtained. The distribution of these isolates according to resistance genes was found as mcr 1/2/6 4 (20%), mcr 3/7 3 (13%), and mcr 5 (40%). Additionally, the isolates harbored blaSHV(6.6%) and blaTEM (6.6%) genes. However, blaNDM, blaOXA-48, blaKPC, and blaCTX-M genes were not detected in any isolates. According to the Congo red agar method, seven (46.6%) isolates showed negative biofilm ability, and eight (53.3%) showed moderate biofilm ability. However, the microplate method detected weak biofilm in 53.3% of the isolates. In conclusion, this study provides evidence for the existence of multidrug-resistant bacteria that co-exist with mcr and ESBL genes in water sources. These bacteria can migrate to other environments and pose increasing threats to public health.

Profile of Aliarcobacter spp. from edible giblets: Genetic diversity, antibiotic resistance, biofilm formation.

Aliarcobacter spp. are recognized as emerging foodborne pathogens and consumption of foods contaminated with them can be a hazard to human and animal health. This study was conducted to investigate the prevalence of Aliarcobacter spp. in edible internal organs of different animal species from retail markets and giblet sellers. Additionally, this study was focused on the antimicrobial resistance, virulence profiles, biofilm-forming capabilities, and phylogenetic relationships of obtained isolates. A total of 270 samples were analyzed from which, 28 (10.4 %) were isolated as Aliarcobacter spp. by conventional methods. Within the 28 Aliarcobacter spp. isolates, 17 (60.7 %) were identified as A. butzleri, 10 (35.7 %) were A. cryaerophilus and one (3.5 %) was A. skirrowii by PCR method. The disc diffusion method showed that the highest resistance rate of Aliarcobacter spp. was seen against oxacillin (78.5 %), and 20 (71.4 %) out of the 28 isolates exhibited multidrug resistance (MDR). Out of the 28 isolates, mviN, pldA, tlyA, and hecB virulence genes were detected in 85.7 %, 46.4 %, 46.4 %, and 3.5 %, respectively, but irgA, Cj1349, ciaB, cadF, and hecA genes were not detected. According to the microplate test, 27 (96.4 %) isolates had weak biofilm ability while one A. cryaerophilus isolate (3.6 %) exhibited strong biofilm formation. ERIC-PCR band patterns suggested that isolated Aliarcobacter spp. from giblets, have different contamination sources. The presence of pathogenic and multidrug-resistant Aliarcobacter spp. in food poses a potential risk to public health and control measures throughout the food chain are necessary to prevent the spread of these strains.

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.

Mycotoxigenic and phylogenetic perspective to the yeasts and filamentous moulds in mould-matured Turkish cheese.

This study was conducted to determine the diversity of yeasts and filamentous moulds in mould-matured cheese (MMC) consumed in Turkey. Overall, 120 samples were collected from 12 different geographical locations between March 2016 and April 2017. The morphological observation was applied in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and molecular analyses to determine yeasts and filamentous moulds in the cheeses. High-performance liquid chromatography (HPLC) technique was used to evaluate the ability of mycotoxins production of fungal isolates and the presence of mycotoxins in cheese samples. A total of 241 fungi (81 filamentous moulds and 160 yeast) were recovered, and Penicillium roqueforti and Debaryomyces hansenii were the most frequently isolated species in all cheese samples. The rep-PCR results indicated a high level of genetic diversity among fungal isolates, regardless of isolation source or geographical origin. Filamentous mould strains isolated from MMC were found to synthesize at least one mycotoxin (Aflatoxin B1, B2, G1 and G2, citrinine, cyclopiazonic acid, mycophenolic acid, ochratoxin A, penicillic acid and roquefortine C). Although mycotoxin producing ability was observed from all isolates, none of the cheese samples were found positive for these mycotoxins. AFM1 was detected in 8 (6.6%) MMC samples from which 2 (1.6%) were above the legal limits (0.05 µg/kg) set by the Turkish Food Codex (TFC) and European Commission (EC). In conclusion, Turkish MMCs were found to be contaminated with toxigenic fungi, so a potential public health risk, while low, exists. Therefore, the selection of nontoxigenic filamentous mould strains for cheese manufacturing and control of the ripening conditions is a critical need to ensure the quality and safety of Turkish MMC.

[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.