Virulence Determinants and Genetic Diversity of Yersinia Species Isolated from Retail Meat

Yersinia enterocolitica is an important foodborne pathogen, and the determination of its virulence factors and genetic diversity within the food chain could help understand the epidemiology of yersiniosis. The aim of the present study was to detect the prevalence, and characterize the virulence determinants and genetic diversity, of Yersinia species isolated from meat. A total of 330 samples of retailed beef (n = 150) and pork (n = 180) in Latvia were investigated with culture and molecular methods. Whole genome sequencing (WGS) was applied for the detection of virulence and genetic diversity. The antimicrobial resistance of pathogenic Y. enterocolitica isolates was detected in accordance with EUCAST. Yersinia species were isolated from 24% (79/330) of meats, and the prevalence of Y. enterocolitica in pork (24%, 44/180) was significantly higher (p < 0.05) than in beef (13%, 19/150). Y. enterocolitica pathogenic bioserovars 2/O:9 and 4/O:3 were isolated from pork samples (3%, 6/180). Only resistance to ampicillin was confirmed in Y. enterocolitica 4/O:3 and 2/O:9 isolates, but not in other antimicrobials. Major virulence determinants, including ail, inv, virF, ystA and myfA, were confirmed with WGS in Y. enterocolitica 2/O:9 and 4/O:3. MLST typing revealed 15 STs (sequence types) of Y. enterocolitica with ST12 and ST18, which were associated with pathogenic bioserovars. For Y. enterocolitica 1A, Y. kristensenii, Y. intermedia and Y. frederiksenii, novel STs were registered (ST680-688). The presence of virulence genes and genetic characteristics of certain Y. enterocolitica STs confirm the common knowledge that pork could be an important source of pathogenic Yersinia.

Chromosomally Located fosA7 in Salmonella Isolates From China

This study aimed to investigate the prevalence of fosfomycin fosA7 in Salmonella enterica isolates from food animals and retail meat products in China and the impact of fosA7 on bacterial fitness. A total of 360 Salmonella isolates collected from 11 provinces and cities in China were detected for fosA7. All fosA7-positive Salmonella isolates were determined minimum inhibitory concentrations (MICs) and sequenced by Illumina Hiseq. The fosA7 gene of S. Derby isolate HA2-WA5 was knocked out. The full length of fosA7 was cloned into vector pBR322 and then transformed into various hosts. MICs of fosfomycin, growth curves, stability, and fitness of fosA7 were evaluated. The fosA7 gene was identified in S. Derby (ST40, n = 30) and S. Reading (ST1628, n = 5). MICs to fosfomycin of 35 fosA7-positive isolates were 1 to 32 mg/L. All fosA7 were located on chromosomes of Salmonella. The deletion of fosA7 in HA2-WA5 decreased fosfomycin MIC by 16-fold and slightly affected its fitness. The acquisition of plasmid-borne fosA7 enhanced MICs of fosfomycin in Salmonella (1,024-fold) and Escherichia coli (16-fold). The recombinant plasmid pBR322-fosA7 was stable in Salmonella Typhimurium, S. Pullorum, S. Derby, and E. coli, except for Salmonella Enteritidis, and barely affected on the growth of them but significantly increased biological fitness in Salmonella. The spread of specific Salmonella serovars such as S. Derby ST40 will facilitate the dissemination of fosA7. fosA7 can confer high-level fosfomycin resistance and enhance bacterial fitness in Salmonella if transferred on plasmids; thus, it has the potential to be a reservoir of the mobilized fosfomycin resistance gene.

Characterization of Carbapenem-Resistant Enterobacteriaceae Cultured From Retail Meat Products, Patients, and Porcine Excrement in China

The emergence and dissemination of carbapenem-resistant Enterobacteriaceae (CRE) is a growing concern to animal and public health. However, little is known about the spread of CRE in food and livestock and its potential transmission to humans. To identify CRE strains from different origins and sources, 53 isolates were cultured from 760 samples including retail meat products, patients, and porcine excrement. Antimicrobial susceptibility testing was carried out, followed by phylogenetic typing, whole-genome sequencing, broth mating assays, and plasmids analyses. Forty-three Escherichia coli, nine Klebsiella pneumoniae, and one Enterobacter cloacae isolates were identified, each exhibiting multidrug-resistant phenotypes. Genetically, the main sequence types (STs) of E. coli were ST156 (n = 7), ST354 (n = 7), and ST48 (n = 7), and the dominant ST of K. pneumoniae is ST11 (n = 5). blaNDM–5 (n = 40) of E. coli and blaKPC–2 (n = 5) were the key genes that conferred carbapenem resistance phenotypes in these CRE strains. Additionally, the mcr-1 gene was identified in 17 blaNDM-producing isolates. The blaNDM–5 gene from eight strains could be transferred to the recipients via conjugation assays. Two mcr-1 genes in the E. coli isolates could be co-transferred along with the blaNDM–5 genes. IncF and IncX3 plasmids have been found to be predominantly associated with blaNDM gene in these strains. Strains isolated in our study from different sources and regions tend to be concordant and overlap. CRE strains from retail meat products are a reservoir for transition of CRE strains between animals and humans. These data also provide evidence of the dissemination of CRE strains and carbapenem-resistant genes between animal and human sources.

Third-Generation Cephalosporin Resistance in Intrinsic Colistin-Resistant Enterobacterales Isolated from Retail Meat

Consumption of retail meat contaminated with antimicrobial-resistant (AMR) bacteria is a common route for transmitting clinically relevant resistant bacteria to humans. Here, we investigated the genotypic and phenotypic resistance profiles of intrinsic colistin-resistant (ICR) Enterobacterales isolated from retail meats. ICR Enterobacterales were isolated from 103 samples of chicken, 103 samples of pork, and 104 samples of beef purchased from retail shops in Japan, using colistin-containing media, and their antimicrobial susceptibility was examined. Serratia spp. (440 isolates) showed resistance to cefotaxime (19 isolates, 4.3%), tetracycline (15 isolates, 3.4%), and other antimicrobials (<1%). Hafnia spp. (136) showed resistance to cefotaxime (12 isolates, 8.6%), ceftazidime (four isolates, 2.9%), and tetracycline (two isolates, 1.4%). Proteus spp. (39) showed resistance to chloramphenicol (four isolates, 10.3%), sulfamethoxazole-trimethoprim (four isolates, 10.3%), cefotaxime (two isolates, 5.1%), kanamycin (two isolates, 5.1%), and gentamicin (one isolate, 2.6%). Cedecea spp. (22) were resistant to tetracycline (two isolates, 9.1%) whereas Morganella spp. (11) were resistant to tetracycline (four isolates, 36.4%) and chloramphenicol (one isolate, 9.2%). The resistance genes blafonA, blaACC, and blaDHA were detected in cefotaxime-resistant Serratia spp., Hafnia spp., and Morganella spp. isolates, respectively. This emergence of antimicrobial resistance in ICR Enterobacterales may pose a public health risk.

Quality and safety of fresh beef in retail: a review

This study aimed to review the scientific literature on the quality and safety of beef sold in retail, methodologies used for quality evaluation, and proposed interventions for the sector. Bibliographical research was performed in order to identify scientific articles using the electronic databases Ovid, PubMed, Scielo, LILACS, Scopus and Embase. Thirty-three articles were included in the review. Given the different dimensions of the studies, the results were organized into three categories, according to the following approaches: food, environment and handlers; consumers and selection criteria; and intervention strategies for the segment. The applied methodological strategies revealed inadequate conditions and neglect in several processing and operation stages that are fundamental to the maintenance of quality standards. The results confirm a worrying scenario and the presence of risks to consumers. Based on the studies evaluated, it is necessary to emphasize the consumer concern about the importance of the integrated information chain - all stages involved in the production process and meat distribution for retail meat stores. The reported conditions on different studies indicate the need for interventions, with a view to prevention of health risks, continued training of food handlers and transformation of evidenced realities. In this context, it is possible to point out the predominance of diagnostic studies, in contrast with interventional studies focused on the training / qualification of food handlers.

High Prevalence of Vancomycin Non-Susceptible and Multi-Drug Resistant Enterococci in Farmed Animals and Fresh Retail Meats in Bangladesh

The emergence of antimicrobial resistant Enterococcus spp., a leading cause of untreatable nosocomial infection, in food animals and dissemination to humans is a public health concern. The study was conducted to determine the prevalence and antimicrobial resistance, and virulence characteristics of Enterococcus faecalis and Enterococcus faecium in food animals and meats in Bangladesh. Enterococcus spp., were confirmed using sodA gene specific PCR, and antimicrobial resistance and virulence properties were characterized by PCR. Enterococcus spp. were recovered from 57% of the collected samples (n=201/352). Farm samples yielded significantly higher (p≤0.05) prevalence (62%) compared to retail meat samples (41%) and E. faecalis (52%) was most frequently isolated species. High proportions of isolates exhibited resistance to tetracycline (74%), erythromycin (65%) and ciprofloxacin (34%). Fifty-one isolates were vancomycin non-susceptible enterococci (VNSE), of which forty-seven were MDR and 20 were linezolid resistant, a last line drug for VNSE. Virulence factors such as gelatinase (gelE), aggregation factor (asa1) and sex pheromone (cpd) were detected along with vancomycin resistance gene (vanA, vanB and vanC2/C3) in VNSE isolates. The high prevalence of MDR enterococci in food animals and retail meats may lead to infection in consumers with concomitant reduced therapeutic options available for treatment.

230. Nontyphoidal Salmonella from Clinical and Retail Meat Sources Reveal Antimicrobial Resistance Genes for Ceftriaxone and Ciprofloxacin

Background Pennsylvania participates in the National Antimicrobial Resistance Monitoring System (NARMS), which includes monitoring of Nontyphoidal Salmonella (NTS), a leading cause of bacterial foodborne illnesses in the United States. Methods Clinical NTS isolates submitted to the Pennsylvania Department of Health (2015-18) were tested for susceptibility to 15 antimicrobial agents and analyzed by whole-genome sequencing (WGS). Concurrently, we conducted a prospective microbiological survey of NTS in retail meat products (chicken breasts, ground turkey, and pork chops) with susceptibility testing and WGS. Results Of a sample of 426 clinical Salmonella isolates from humans analyzed for antimicrobial susceptibility, 65 (15.3%) had decreased susceptibility to ciprofloxacin (DSC). Ampicillin resistance was observed in 39 (9.2%) and 15 (3.5%) were ceftriaxone-resistant. Ten ceftriaxone-resistant isolates had genetic elements that confer resistance to third generation extended-spectrum cephalosporins (ESC) [blaCMY−2, n=8 and blaCTX-M-65, n=2]. The blaCTX-M-65- positive isolates had a mutation in gyrA that confers fluoroquinolone resistance. Thirteen clinical isolates carried plasmid-mediated fluoroquinolone resistance genes (PMQR) [qnrB19, qnrS1, qnrA1]. We detected NTS in 131 (3.8%) of 3480 meat samples tested. 7 (5.3%) had DSC, while 38 (29%) and 21 (16%) were resistant to ampicillin and ceftriaxone, respectively. Four S. Infantis isolates had DSC and a blaCTX-M-65 gene plus a mutation in gyrA. Thirteen meat isolates had the blaCMY-2 gene. One additional blaCTX-M-65-positive S. Infantis without gyrA from ground turkey (SRR6351119) differed from four clinical isolates by ≤10 single-nucleotide polymorphisms. Percent of isolates from patients and meat sources that demonstrated resistance to amoxicillin-clavulanate (AMC), ceftriaxone, and decreased susceptibility to ciprofloxacin (DSC) to nine antimicrobial classes tested. Among isolates from patients, resistance to ceftriaxone, a third-generation cephalosporin preferred for severe infections in children, increased from zero in 2015 to 5.8% in 2017. Overall, DSC increased in isolates from human sources while in strains from meat sources, DSC increased from zero in 2015 to over five percent in 2018. Conclusion NTS isolated from human and meat sources were multi-drug resistant. Demonstration of similar resistance genes in meat and in ill humans may be consistent with spread of antibiotic-resistant pathogens from food sources. Dissemination of genes that confer resistance to third generation cephalosporins and fluoroquinolones, including some on mobile plasmids, may undermine recommended treatment for severe NTS infections. These results underscore the need for antimicrobial stewardship efforts in both agriculture and human medicine. Disclosures All Authors: No reported disclosures

Salmonella from Farm to Table: Isolation, Characterization, and Antimicrobial Resistance of Salmonella from Commercial Broiler Supply Chain and Its Environment

Antimicrobial resistance (AMR) in poultry production chain is one of the major food safety concerns due to indiscriminate usage of antibiotics and the presence of pathogens such as Salmonella which causes infections in various stages of production. In the present study, 182 samples were collected from commercial broiler supply chain, viz., three hatcheries ( n = 29 ), three commercial broiler farms (CBF; n = 99 ), and three retail meat shops (RMS; n = 54 ), and used for isolation and identification of Salmonella using three different selective agar media and a selective enrichment medium followed by PCR confirmation targeting the hilA gene. The overall prevalence of Salmonella was 47/182 (25.82%), and a significantly higher ( P < 0.05 ) prevalence was observed in retail meat shops (46.29%), CBF (19.19%), and hatcheries (10.34%). Comparison of three agar media for isolation of Salmonella revealed that all the media were equally selective. However, PCR amplification of hilA gene fragment was significantly higher ( P < 0.01 ) in selective enrichment culture tetrathionate brilliant green bile broth (TTB) as compared to all solid (agar-based) media. Susceptibility pattern against most frequently used antibiotics revealed that 100% of the isolates were resistant to at least one antibiotic. High resistance was observed for doxycycline (94.34%), followed by cefpodoxime (84.91%), ciprofloxacin (72.64%), gentamicin (65.09%), enrofloxacin (61.32%), colistin sulphate (40.42%), amikacin (34.91%), ampicillin (33.96%), neomycin (33.02), cefotaxime (30.19%), ceftazidime (29.25%), trimethoprim-sulfamethoxazole (23.58%), amoxicillin+clavulanic acid (21.70%), and chloramphenicol (12.26%); 16.98% of the isolates were ex-tended spectrum β-lactamase (ESBL) producers, and 76.41% were multidrug resistant (MDR). MDR Salmonella were significantly higher ( P < 0.01 ) in RMS (91.66%) followed by CBF (82.75%), whereas no MDR isolates were present in the isolates from hatcheries. The results indicated a higher prevalence of Salmonella and AMR for commonly used antibiotics in the complete broiler supply chain, especially RMS and CBF. Also, this study idicated that TTB enrichment followed by PCR and colony PCR was found to be rapid, specific and time-saving method.

A one health genomic investigation of gentamicin resistance in Salmonella from human and chicken sources in Canada, 2014-2017

Objectives: We investigated whether the increased prevalence of gentamicin resistance in Salmonella from human infections was related to a similar increased prevalence in isolates from broiler chickens and whether this increase may have been due to co-selection from use of lincomycin-spectinomycin in chickens on farms. Methods: Whole genome sequencing was performed on gentamicin-resistant (gen-R) Salmonella isolates from human and chicken sources collected from 2014-2017 by the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). We determined the genomic relatedness of strains and characterized resistance genes and plasmids. Results: From 2014-2017, 247 isolates of gen-R Salmonella were identified by CIPARS: 188 were from humans and 59 from chicken sources (26 from live animals on farm and 33 from retail meat). The five most common gen-R serovars were Heidelberg (n=93, 31.5%), 4,[5],12:i:- (n=42, 14.2%), Kentucky (n=37, 12.5%), Infantis (n=33, 11.2%), and Typhimurium (n=23, 7.8%). Phylogenomic analysis revealed that for S. Heidelberg and S. Infantis, there were closely related isolates from human and chicken sources. In both sources, resistance to gentamicin and spectinomycin was most frequently conferred by aac(3)-VIa and ant(3’’)-Ia , respectively. Plasmid closure confirmed linkages of gentamicin and spectinomycin resistance genes and revealed instances of similar plasmids from both sources. Conclusions: Gentamicin and spectinomycin resistance genes were linked on the same plasmids, and some plasmids and isolates from humans and chickens were genetically similar, suggesting that the use of lincomycin-spectinomycin in chickens may be selecting for gentamicin-resistant Salmonella in broiler chickens and that these resistant strains may be acquired by humans.