Phenotypic and Genotypic Characterizations of Antimicrobial-Resistant Escherichia coli Isolates from Diverse Retail Meat Samples in North Carolina During 2018-2019.

Surveillance of antimicrobial-resistant pathogens in U.S. retail meats is conducted to identify potential risks of foodborne illness. In this study, we conducted a phenotypic and genotypic analysis of Escherichia coli recovered from a diverse range of retail meat types during 2018-2019 in North Carolina. The investigation was conducted as part of the National Antimicrobial Resistance Monitoring System (NARMS). Retail meat sampling and E. coli isolation were performed in accordance with NARMS retail meat isolation protocols. We used the Sensititre™ broth microdilution system to determine phenotypic resistance to 14 antimicrobial agents and the Illumina next-generation sequencing platform for genotypic resistance profiling. The highest prevalence of E. coli isolates was found in ground turkey (n = 57, 42.9%) and chicken (n = 27, 20.3%), followed by ground beef (n = 25, 18.9%) and pork (n = 24, 18%). The isolates were divided into seven different phylogroups using the Clermont typing tool, with B1 (n = 59, 44.4%) and A (n = 39, 29.3%) being the most dominant, followed by B2 (n = 14, 10.5%), D (n = 7, 5.3%), F (n = 6, 4.5%), E (n = 3, 2.3%), and C (n = 2, 1.5%). Using multilocus sequence typing (MLST), 128 Sequence types (STs) were identified indicating high diversity. Phenotypic and genotypic resistance was observed toward aminoglycosides, sulfonamides, beta-lactams, macrolides, tetracyclines, phenicols, and fluoroquinolones. Ground turkey samples were more resistant to the panel of tested antimicrobials than chicken, beef, or pork (p < 0.05). All isolates were found to be susceptible to meropenem. A high percentage of turkey isolates (n = 16, 28%) were multidrug-resistant (MDR) compared with 18.5% of chicken (n = 5), 8.4% of pork (n = 2), and 8% of beef isolates (n = 2). This study highlights the benefit of surveillance to identify MDR E. coli for epidemiologic tracking and is a comprehensive report of the phenotypic and genotypic characterization of E. coli isolated from retail meats in North Carolina.

Megaplasmid Dissemination in Multidrug-Resistant Salmonella Serotypes from Backyard and Commercial Broiler Production Systems in the Southeastern United States.

Over the past decade, there has been a rise in U.S. backyard poultry ownership, raising concern for residential area antimicrobial-resistant (AMR) Salmonella contamination. This study aims to lay the groundwork to better understand the persistence of AMR Salmonella in residential broiler production systems and make comparisons with commercial systems. Ten backyard and 10 commercial farms were sampled at three time points across bird production. Both fecal (n = 10) and environmental (soil, n = 5, litter/compost, n = 5, feeder, and waterer swabs, n = 6) samples were collected at each visit on days 10, 31, and 52 of production for backyard farms and days 10, 24, and 38 of production for commercial farms. AMR Salmonella was characterized phenotypically by broth microdilution and genotypically by whole-genome sequencing. Overall, Salmonella was more prevalent in commercial farm samples (52.31%) over backyard farms (19.10%). Kentucky (sequence type (ST) 152) was the most common serotype found in both backyard and commercial farms. Multidrug-resistant (MDR, resistance to ≥3 or more antimicrobial classes) isolates were found in both production systems, while ciprofloxacin- and nalidixic acid-resistant and intermediate isolates were more prevalent in commercial (33%) than backyard samples (1%). Plasmids that have been associated with MDR were found in Kentucky and Infantis isolates, particularly IncFIB(K)_1_Kpn3 megaplasmid (Infantis). Our study emphasizes the need to understand the selection pressures in disseminating megaplasmids in MDR Salmonella in distinct broiler production systems.

Detection of resistance and virulence plasmids in Campylobacter coli and Campylobacter jejuni isolated from North Carolina food animal production, 2018-2019.

Campylobacter remains the leading cause of bacterial foodborne illness in the U.S. and worldwide. Campylobacter plasmids may play a significant role in antimicrobial resistance (AMR) and virulence factor distribution, and potentially drive rapid adaptation. C. coli (n = 345) and C. jejuni (n = 199) isolates collected from live cattle, swine, turkey, and chickens, poultry carcasses at production, and retail meat in N.C. were analyzed to determine plasmid prevalence, extrachromosomal virulence and AMR genes, and the phylogeny of assembled plasmids. Putative plasmids ranging from <2 kb to 237kb were identified with virulence factors present in 66.1% (228/345) C. coli and 88.4% (176/199) C. jejuni plasmids (promoting adherence, invasion, exotoxin production, immune modulation, chemotaxis, mobility, and the type IV secretion system). AMR genes were identified in 21.2% (73/345) C. coli and 28.1% C. jejuni plasmids (conferring resistance to tetracyclines, aminoglycosides, beta-lactams, nucleosides, and lincosamides). Megaplasmids (>100 kb) were present in 25.7% (140/544) of the isolates and carried genes previously recognized to be involved with interspecies recombination. Our study highlights the extensive distribution and diversity of Campylobacter plasmids in food animal production and their role in the dissemination of biomedically important genes. Characterizing Campylobacter plasmids within the food animal production niche is important to understanding the epidemiology of potential emerging strains.

Genomic Screening of Antimicrobial Resistance Markers in UK and US Campylobacter Isolates Highlights Stability of Resistance over an 18-Year Period.

Campylobacter jejuni and Campylobacter coli are important bacterial causes of human foodborne illness. Despite several years of reduced antibiotics usage in livestock production in the United Kingdom (UK) and United States (US), a high prevalence of antimicrobial resistance (AMR) persists in Campylobacter. Both countries have instigated genome sequencing-based surveillance programs for Campylobacter, and in this study, we have identified AMR genes in 32,256 C. jejuni and 8,776 C. coli publicly available genome sequences to compare the prevalence and trends of AMR in Campylobacter isolated in the UK and US between 2001 and 2018. AMR markers were detected in 68% of C. coli and 53% of C. jejuni isolates, with 15% of C. coli isolates being multidrug resistant (MDR), compared to only 2% of C. jejuni isolates. The prevalence of aminoglycoside, macrolide, quinolone, and tetracycline resistance remained fairly stable from 2001 to 2018 in both C. jejuni and C. coli, but statistically significant differences were observed between the UK and US. There was a statistically significant higher prevalence of aminoglycoside and tetracycline resistance for US C. coli and C. jejuni isolates and macrolide resistance for US C. coli isolates. In contrast, UK C. coli and C. jejuni isolates showed a significantly higher prevalence of quinolone resistance. Specific multilocus sequence type (MLST) clonal complexes (e.g., ST-353/464) showed >95% quinolone resistance. This large-scale comparison of AMR prevalence has shown that the prevalence of AMR remains stable for Campylobacter in the UK and the US. This suggests that antimicrobial stewardship and restricted antibiotic usage may help contain further expansion of AMR prevalence in Campylobacter but are unlikely to reduce it in the short term.

Tracking the Transmission of Antimicrobial-Resistant Non-O157 Escherichia coli and Salmonella Isolates at the Interface of Food Animals and Fresh Produce from Agriculture Operations Using Whole-Genome Sequencing.

An increasing number of outbreaks are caused by foodborne pathogens such as Escherichia coli and Salmonella, which often harbor antimicrobial resistance (AMR) genes. We previously demonstrated the transmission of pathogens from animal operations to produce fields on sustainable farms, which illustrated an urgent need to develop and implement novel prevention methods and remediation practices such as the vegetative buffer zone (VBZ) to prevent this movement. The focus of this study was to use whole-genome sequencing (WGS) to characterize the AMR, virulence, and single-nucleotide polymorphism profile of 15 Salmonella and 128 E. coli isolates collected from small-scale dairy and poultry farms on a research station in North Carolina. Phenotypically, seven E. coli and three Salmonella isolates displayed resistance to antibiotics such as tetracycline (n = 4), ampicillin (n = 4), nalidixic acid (n = 3), chloramphenicol (n = 2), sulfisoxazole (n = 1), and streptomycin (n = 1). A single E. coli isolate was found to be resistant to five different antibiotic class types and possessed the blaTEM-150 resistance gene. Virulence genes that facilitate toxin production and cell invasion were identified. Mauve analysis of the E. coli isolates identified seven clusters (dairy-six and poultry-one) indicating that transmission is occurring from animal operations to fresh produce fields and the surrounding environment when the VBZ is denudated. This suggests that the VBZ is a useful barrier to reducing the transmission of enteric pathogens in agricultural systems. Our study demonstrates the prevalence of AMR and virulence genes on small-scale sustainable farms and highlights the advantage of using WGS to assess the impact of the VBZ to reduce the transmission of E. coli and Salmonella.

Toward the Adoption of Loop-Mediated Isothermal Amplification for Salmonella Screening at the National Antimicrobial Resistance Monitoring System's Retail Meat Sites.

The National Antimicrobial Resistance Monitoring System (NARMS) is a One Health program in the United States that collects data on antimicrobial resistance in enteric bacteria from humans, animals, and the environment. Salmonella is a major pathogen tracked by the NARMS retail meat arm but currently lacks a uniform screening method. We evaluated a loop-mediated isothermal amplification (LAMP) assay for the rapid screening of Salmonella from 69 NARMS retail meat and poultry samples. All samples were processed side by side for culture isolation using two protocols, one from NARMS and the other one described in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM). Overall, 10 (14.5%) samples screened positive by the Salmonella LAMP assay. Of those, six were culture-confirmed by the NARMS protocol and six by the BAM method with overlap on four samples. No Salmonella isolates were recovered from samples that screened negative with LAMP. These results suggested 100% sensitivity for LAMP in reference to culture. Antimicrobial susceptibility testing and whole-genome sequencing analysis confirmed identities of these isolates. Using the BAM protocol, all Salmonella isolates were recovered from samples undergoing Rappaport-Vassiliadis medium selective enrichment and presumptive colonies (n = 130) were dominated by Hafnia alvei (44.6%), Proteus mirabilis (22.3%), and Morganella morganii (9.9%) based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This method comparison study clearly demonstrated the benefit of a rapid, robust, and highly sensitive molecular screening method in streamlining the laboratory workflow. Fourteen NARMS retail meat sites further verified the performance of this assay using a portion of their routine samples, reporting an overall specificity of 98.8% and sensitivity of 90%. As of July 2022, the vast majority of NARMS retail meat sites have adopted the Salmonella LAMP assay for rapid screening of Salmonella in all samples.

Impact of the COVID-19 pandemic on the surveillance, prevention and control of antimicrobial resistance: a global survey.

OBJECTIVES: The COVID-19 pandemic has had a substantial impact on health systems. The WHO Antimicrobial Resistance (AMR) Surveillance and Quality Assessment Collaborating Centres Network conducted a survey to assess the effects of COVID-19 on AMR surveillance, prevention and control. METHODS: From October to December 2020, WHO Global Antimicrobial Resistance and Use Surveillance System (GLASS) national focal points completed a questionnaire, including Likert scales and open-ended questions. Data were descriptively analysed, income/regional differences were assessed and free-text questions were thematically analysed. RESULTS: Seventy-three countries across income levels participated. During the COVID-19 pandemic, 67% reported limited ability to work with AMR partnerships; decreases in funding were frequently reported by low- and middle-income countries (LMICs; P < 0.01). Reduced availability of nursing, medical and public health staff for AMR was reported by 71%, 69% and 64%, respectively, whereas 67% reported stable cleaning staff availability. The majority (58%) reported reduced reagents/consumables, particularly LMICs (P < 0.01). Decreased numbers of cultures, elective procedures, chronically ill admissions and outpatients and increased ICU admissions reported could bias AMR data. Reported overall infection prevention and control (IPC) improvement could decrease AMR rates, whereas increases in selected inappropriate IPC practices and antimicrobial prescribing could increase rates. Most did not yet have complete data on changing AMR rates due to COVID-19. CONCLUSIONS: This was the first survey to explore the global impact of COVID-19 on AMR among GLASS countries. Responses highlight important actions to help ensure that AMR remains a global health priority, including engaging with GLASS to facilitate reliable AMR surveillance data, seizing the opportunity to develop more sustainable IPC programmes, promoting integrated antibiotic stewardship guidance, leveraging increased laboratory capabilities and other system-strengthening efforts.

The role of Enterococcus faecalis during co-infection with avian pathogenic Escherichia coli in avian colibacillosis.

Enterococcus spp. (ENT) are frequently co-isolated with avian pathogenic E. coli (APEC) from poultry with colibacillosis, a leading cause of flock mortality. Although largely overlooked, ENT may play an active role in these infections. To assess the frequency of ENT co-isolation in colibacillosis, cultures were collected from birds with gross lesions of omphalitis, polyserositis, and septicaemia over a 3-year period from three turkey flocks and three broiler flocks. In birds diagnosed with colibacillosis based on gross findings and isolation of E. coli, ENT were co-isolated with APEC in 35.7% (n = 41/115) of colibacillosis mortality and 3.7% of total mortality (n = 41/1122). Co-isolated APEC and ENT pairs (n = 41) were further characterized using antimicrobial resistance phenotyping and in vitro co-culture assays. E. faecalis (EF) was the most commonly co-isolated species (68% n = 28/41) and tetracycline resistance was the resistance phenotype most commonly found among APEC (51% n = 21/41) and ENT (93% n = 38/41). Under iron-restricted conditions, EF enhanced APEC growth in a proximity-dependent manner and APEC grown in mixed culture with EF exhibited a significant growth and survival advantage (P ≤ 0.01). In an embryo lethality assay, APEC co-infection with EF resulted in decreased survival of broiler embryos compared to mono-infections (P ≤ 0.05). These data demonstrate that EF augmented APEC survival and growth under iron limiting conditions, possibly translating to the increased virulence of APEC in broiler embryos. Thus, ENT co-infections may be a previously unrecognized contributor to colibacillosis-related mortality. Further investigations into the mechanism of this interaction are warranted. RESEARCH HIGHLIGHTS Enterococcus is frequently co-isolated with avian pathogenic E. coli (APEC). Enterococcus faecalis (EF) enhances survival of APEC in iron restricted conditions. EF co-infection increases APEC virulence in broiler embryos.

Phenotypic and Genotypic Characterization of Escherichia coli and Salmonella enterica from Dairy Cattle Farms in the Wakiso District, Uganda: A Cross-Sectional Study.

Enterobacteriaceae producing ß-lactamases have spread rapidly worldwide and pose a serious threat to human-animal-environment interface. In this study, we present the presence of Salmonella enterica (1.3%) and commensal Escherichia coli (96.3%) isolated from 400 environmental fecal dairy cattle samples over 20 farms in Uganda. Among E. coli isolates, 21% were resistant to at least one antimicrobial tested and 7% exhibited multidrug resistance. Four E. coli isolates displayed extended-spectrum beta-lactamase (ESBL)-producing genes, including blaCTX-M-15 (n = 2/4), blaCTX-M-27 (n = 1/4), blaSHV-12 (n = 1/4), and blaTEM-1B (n = 2/4). Whole genome sequencing confirmed the presence of the plasmid-mediated quinolone resistance qnrS1 gene among three ESBL isolates. No statistically significant differences in seasonal prevalence for E. coli and S. enterica among dairy cattle sampling periods were observed. Furthermore, to our knowledge, this is the first report of E. coli carrying blaCTX-M-15, blaCTX-M-27, blaSHV-12, or qnrS1 isolated from dairy cattle in Uganda. We conclude that the presence of globally disseminated blaCTX-M-15 and blaCTX-M-27 warrants further study to prevent further spread. In addition, the presence of fluoroquinolone resistant ESBL-producing E. coli on dairy farms highlights the potential risk among the human-livestock-environment interaction. This study can be used as a baseline for implementation of a more robust national integrated surveillance system throughout Uganda.

Whole genome sequencing analysis of multiple Salmonella serovars provides insights into phylogenetic relatedness, antimicrobial resistance, and virulence markers across humans, food animals and agriculture environmental sources.

BACKGROUND: Salmonella enterica is a significant foodborne pathogen, which can be transmitted via several distinct routes, and reports on acquisition of antimicrobial resistance (AMR) are increasing. To better understand the association between human Salmonella clinical isolates and the potential environmental/animal reservoirs, whole genome sequencing (WGS) was used to investigate the epidemiology and AMR patterns within Salmonella isolates from two adjacent US states. RESULTS: WGS data of 200 S. enterica isolates recovered from human (n = 44), swine (n = 32), poultry (n = 22), and farm environment (n = 102) were used for in silico prediction of serovar, distribution of virulence genes, and phylogenetically clustered using core genome single nucleotide polymorphism (SNP) and feature frequency profiling (FFP). Furthermore, AMR was studied both by genotypic prediction using five curated AMR databases, and compared to phenotypic AMR using broth microdilution. Core genome SNP-based and FFP-based phylogenetic trees showed consistent clustering of isolates into the respective serovars, and suggested clustering of isolates based on the source of isolation. The overall correlation of phenotypic and genotypic AMR was 87.61% and 97.13% for sensitivity and specificity, respectively. AMR and virulence genes clustered with the Salmonella serovars, while there were also associations between the presence of virulence genes in both animal/environmental isolates and human clinical samples. CONCLUSIONS: WGS is a helpful tool for Salmonella phylogenetic analysis, AMR and virulence gene predictions. The clinical isolates clustered closely with animal and environmental isolates, suggesting that animals and environment are potential sources for dissemination of AMR and virulence genes between Salmonella serovars.

Environmental Dissemination of Multidrug Methicillin-Resistant Staphylococcus sciuri After Application of Manure from Commercial Swine Production Systems.

The deposition of manure originating from food animal farms in the environment can lead to the dissemination of antimicrobial-resistant (AMR) bacterial foodborne pathogens, thereby potentially impacting human health. The objective of our study was to determine the dissemination of multidrug methicillin-resistant Staphylococcus sciuri (MDR-MRSS) in the environment after land application of manure on commercial swine farms. A total of 400 environmental samples (40 manure and 360 soil) were collected after repeated sampling from four commercial swine farms located in North Carolina (n = 1) and Iowa (n = 3) in the United States. At each farm, we collected 10 manure and 40 soil samples (20 samples before and after 2 h of manure application) from four plots (five soil samples/plot) on day 0. Subsequently, 20 soil samples were collected on day 7, 14, and 21 from the same plots. A total of 67 (16.75%) MRSS were isolated from the 400 samples. The prevalence in soil and manure was 13.33% (48/360) and 47.5% (19/40), respectively. Prevalence was highest in the soil samples collected after 2 h of manure application on day 0 and decreased subsequently on 7, 14, and 21 days. Antimicrobial susceptibility testing was done against a panel of 12 antibiotics. A majority of S. sciuri isolates exhibited resistance against ampicillin (AMP; 95.5%), penicillin (PEN; 95.5%), clindamycin (CLI; 95.5%), cefoxitin (FOX; 92.5%), ceftiofur (XNL; 92.5%), tetracycline (TET; 86.56%), and erythromycin (ERY; 50.74%). The MDR pattern AMP FOX CLI PEN TET XNL (n = 24; 35.8%) was the most commonly observed. We detected multiple AMR genes, including mecA, aac(6'), Ie-aph(2″)Ia, tetM, tetK, mphC, ermA, ermB, and ermC. Pulsed-field gel electrophoresis clustered isolates from different sample collection days from the same farm into one group. Overall, our study identifies swine manure as an important reservoir of MDR-MRSS and highlights its dissemination in the environment upon spreading of manure.

Multilaboratory Survey To Evaluate Salmonella Prevalence in Diarrheic and Nondiarrheic Dogs and Cats in the United States between 2012 and 2014.

Eleven laboratories collaborated to determine the periodic prevalence of Salmonella in a population of dogs and cats in the United States visiting veterinary clinics. Fecal samples (2,965) solicited from 11 geographically dispersed veterinary testing laboratories were collected in 36 states between January 2012 and April 2014 and tested using a harmonized method. The overall study prevalence of Salmonella in cats (3 of 542) was <1%. The prevalence in dogs (60 of 2,422) was 2.5%. Diarrhea was present in only 55% of positive dogs; however, 3.8% of the all diarrheic dogs were positive, compared with 1.8% of the nondiarrheic dogs. Salmonella-positive dogs were significantly more likely to have consumed raw food (P = 0.01), to have consumed probiotics (P = 0.002), or to have been given antibiotics (P = 0.01). Rural dogs were also more likely to be Salmonella positive than urban (P = 0.002) or suburban (P = 0.001) dogs. In the 67 isolates, 27 unique serovars were identified, with three dogs having two serovars present. Antimicrobial susceptibility testing of 66 isolates revealed that only four of the isolates were resistant to one or more antibiotics. Additional characterization of the 66 isolates was done using pulsed-field gel electrophoresis and whole-genome sequencing (WGS). Sequence data compared well to resistance phenotypic data and were submitted to the National Center for Biotechnology Information (NCBI). This study suggests an overall decline in prevalence of Salmonella-positive dogs and cats over the last decades and identifies consumption of raw food as a major risk factor for Salmonella infection. Of note is that almost half of the Salmonella-positive animals were clinically nondiarrheic.

Horizontal Dissemination of Antimicrobial Resistance Determinants in Multiple Salmonella Serotypes following Isolation from the Commercial Swine Operation Environment after Manure Application.

The aim of this study was to characterize the plasmids carrying antimicrobial resistance (AMR) determinants in multiple Salmonella serotypes recovered from the commercial swine farm environment after manure application on land. Manure and soil samples were collected on day 0 before and after manure application on six farms in North Carolina, and sequential soil samples were recollected on days 7, 14, and 21 from the same plots. All environmental samples were processed for Salmonella, and their plasmid contents were further characterized. A total of 14 isolates including Salmonella enterica serotypes Johannesburg (n = 2), Ohio (n = 2), Rissen (n = 1), Typhimurium var5- (n = 5), Worthington (n = 3), and 4,12:i:- (n = 1), representing different farms, were selected for plasmid analysis. Antimicrobial susceptibility testing was done by broth microdilution against a panel of 14 antimicrobials on the 14 confirmed transconjugants after conjugation assays. The plasmids were isolated by modified alkaline lysis, and PCRs were performed on purified plasmid DNA to identify the AMR determinants and the plasmid replicon types. The plasmids were sequenced for further analysis and to compare profiles and create phylogenetic trees. A class 1 integron with an ANT(2″)-Ia-aadA2 cassette was detected in the 50-kb IncN plasmids identified in S Worthington isolates. We identified 100-kb and 90-kb IncI1 plasmids in S Johannesburg and S Rissen isolates carrying the blaCMY-2 and tet(A) genes, respectively. An identical 95-kb IncF plasmid was widely disseminated among the different serotypes and across different farms. Our study provides evidence on the importance of horizontal dissemination of resistance determinants through plasmids of multiple Salmonella serotypes distributed across commercial swine farms after manure application.IMPORTANCE The horizontal gene transfer of antimicrobial resistance (AMR) determinants located on plasmids is considered to be the main reason for the rapid proliferation and spread of drug resistance. The deposition of manure generated in swine production systems into the environment is identified as a potential source of AMR dissemination. In this study, AMR gene-carrying plasmids were detected in multiple Salmonella serotypes across different commercial swine farms in North Carolina. The plasmid profiles were characterized based on Salmonella serotype donors and incompatibility (Inc) groups. We found that different Inc plasmids showed evidence of AMR gene transfer in multiple Salmonella serotypes. We detected an identical 95-kb plasmid that was widely distributed across swine farms in North Carolina. These conjugable resistance plasmids were able to persist on land after swine manure application. Our study provides strong evidence of AMR determinant dissemination present in plasmids of multiple Salmonella serotypes in the environment after manure application.

Biofilm Formation by Environmental Isolates of Salmonella and Their Sensitivity to Natural Antimicrobials.

The objective of this study was to determine reduction of Salmonella in biofilms by essential oils. Biofilm formation of 15 Salmonella isolates from conventional swine farm environment was evaluated by 96-well microtiter plate crystal violet and minimum biofilm eradication concentration (MBEC) assays. Only one of the 15 isolates was a strong biofilm producer as classified by crystal violet assay. All Salmonella isolates formed biofilm on MBEC assay. The curli expression was robust among strains S322 and S435 (Salmonella Infantis), S644, S777, S931, S953, and S977 (Salmonella Typhimurium) as observed by Congo red dye binding assay. The cell hydrophobicity varied with strains and growth phase of the strain; however, there was no significant difference in hydrophobicity of these strains. Natural antimicrobials were evaluated with MBEC assay for their bactericidal efficacy in reducing Salmonella in biofilms. Cinnamaldehyde and sporran at 1000 ppm significantly reduced Salmonella in biofilms. The bactericidal effect of these antimicrobials increased with their concentrations. Salmonella were reduced by 6 log CFU from their initial populations of 7-7.5 log CFU/cm(2) when 2000 ppm concentration of these antimicrobials were used. Salmonella were undetectable when 3000 ppm of cinnamaldehyde or sporran was used. Natural antimicrobials such as cinnamaldehyde and sporran can be used to reduce Salmonella in biofilms.

Comparative phenotypic and genotypic characterization of temporally related nontyphoidal Salmonella isolated from human clinical cases, pigs, and the environment in North Carolina.

Nontyphoidal Salmonella infections caused by antimicrobial-resistant (AMR) strains are of great public health concern. We compared the phenotypic and genotypic relationships among temporally and spatially related AMR Salmonella isolates (n=1058) representing several predominant serovars, including Salmonella Typhimurium, Salmonella Typhimurium var. 5-, Salmonella Derby, Salmonella Heidelberg, Salmonella Muenchen, Salmonella Schwarzengrund, and Salmonella Rissen of human clinical cases (n=572), pig (n=212), and farm environment (n=274) origin in North Carolina. Antimicrobial susceptibility testing was performed using the broth microdilution method, and genotypic resistance determinants, including class I and II integrons, were identified. Overall, Salmonella isolates exhibited the highest frequency of resistance to tetracycline (50%), followed by sulfisoxazole (36%) and streptomycin (27%). We identified 16 different antimicrobial resistance genes, including extended spectrum and AmpC ß-lactamases-producing genes (bla(TEM), bla(PSE), and bla(CMY-2)), in all the ß-lactam- and cephalosporin-resistant Salmonella isolates from humans, pigs, and the environment. Class I integrons of 1-kb and 1.2-kb size were identified from all the three sources (humans, 66%; pigs, 85%; environment, 58%), while Class II integrons of 2-kb size were identified only in pig (10%) and environmental (19%) isolates. We detected genotypic similarity between Salmonella Typhimurium isolated from humans, pigs, and the environment while serovars Derby, Heidelberg, and Muenchen exhibited genotypic diversity. Detection of AMR Salmonella isolates from humans, pigs, and the environment is a concern for clinicians and veterinarians to mitigate the dissemination of AMR Salmonella strains.

Serovar-level identification of bacterial foodborne pathogens from full-length 16S rRNA gene sequencing.

The resolution of variation within species is critical for interpreting and acting on many microbial measurements. In the key foodborne pathogens Salmonella and Escherichia coli, the primary subspecies classification scheme used is serotyping: differentiating variants within these species by surface antigen profiles. Serotype prediction from whole-genome sequencing (WGS) of isolates is now seen as comparable or preferable to traditional laboratory methods where WGS is available. However, laboratory and WGS methods depend on an isolation step that is time-consuming and incompletely represents the sample when multiple strains are present. Community sequencing approaches that skip the isolation step are, therefore, of interest for pathogen surveillance. Here, we evaluated the viability of amplicon sequencing of the full-length 16S rRNA gene for serotyping Salmonella enterica and E. coli. We developed a novel algorithm for serotype prediction, implemented as an R package (Seroplacer), which takes as input full-length 16S rRNA gene sequences and outputs serovar predictions after phylogenetic placement into a reference phylogeny. We achieved over 89% accuracy in predicting Salmonella serotypes on in silico test data and identified key pathogenic serovars of Salmonella and E. coli in isolate and environmental test samples. Although serotype prediction from 16S rRNA gene sequences is not as accurate as serotype prediction from WGS of isolates, the potential to identify dangerous serovars directly from amplicon sequencing of environmental samples is intriguing for pathogen surveillance. The capabilities developed here are also broadly relevant to other applications where intraspecies variation and direct sequencing from environmental samples could be valuable.IMPORTANCEIn order to prevent and stop outbreaks of foodborne pathogens, it is important that we can detect when pathogenic bacteria are present in a food or food-associated site and identify connections between specific pathogenic bacteria present in different samples. In this work, we develop a new computational technology that allows the important foodborne pathogens Escherichia coli and Salmonella enterica to be serotyped (a subspecies level classification) from sequencing of a single-marker gene, and the 16S rRNA gene often used to surveil bacterial communities. Our results suggest current limitations to serotyping from 16S rRNA gene sequencing alone but set the stage for further progress that we consider likely given the rapid advance in the long-read sequencing technologies and genomic databases our work leverages. If this research direction succeeds, it could enable better detection of foodborne pathogens before they reach the public and speed the resolution of foodborne pathogen outbreaks.

Characteristics of antimicrobial resistance in Escherichia coli isolated from retail meat products in North Carolina.

BACKGROUND: Escherichia coli is commonly used as an indicator for antimicrobial resistance (AMR) in food, animal, environment, and human surveillance systems. Our study aimed to characterize AMR in E. coli isolated from retail meat purchased from grocery stores in North Carolina, USA as part of the National Antimicrobial Resistance Monitoring System (NARMS). MATERIALS AND METHODS: Retail chicken (breast, n = 96; giblets, n = 24), turkey (n = 96), and pork (n = 96) products were purchased monthly from different counties in North Carolina during 2022. Label claims on packages regarding antibiotic use were recorded at collection. E. coli was isolated from meat samples using culture-based methods and isolates were characterized for antimicrobial resistance using whole genome sequencing. Multi-locus sequence typing, phylogroups, and a single nucleotide polymorphism (SNP)-based maximum-likelihood phylogenic tree was generated. Data were analyzed statistically to determine differences between antibiotic use claims and meat type. RESULTS: Of 312 retail meat samples, 138 (44.2%) were positive for E. coli, with turkey (78/138; 56.5%) demonstrating the highest prevalence. Prevalence was lower in chicken (41/138; 29.7%) and pork (19/138;13.8%). Quality sequence data was available from 84.8% (117/138) of the E. coli isolates, which included 72 (61.5%) from turkey, 27 (23.1%) from chicken breast, and 18 (15.4%) from pork. Genes associated with AMR were detected in 77.8% (91/117) of the isolates and 35.9% (42/117) were defined as multidrug resistant (MDR: being resistant to ≥3 distinct classes of antimicrobials). Commonly observed AMR genes included tetB (35%), tetA (24.8%), aph(3'')-lb (24.8%), and blaTEM-1 (20.5%), the majority of which originated from turkey isolates. Antibiotics use claims had no statistical effect on MDR E. coli isolates from the different meat types (X2 = 2.21, p = 0.33). MDR was observed in isolates from meat products with labels indicating "no claims" (n = 29; 69%), "no antibiotics ever" (n = 9; 21.4%), and "organic" (n = 4; 9.5%). Thirty-four different replicon types were observed. AMR genes were carried on plasmids in 17 E. coli isolates, of which 15 (88.2%) were from turkey and two (11.8%) from chicken. Known sequence types (STs) were described for 81 E. coli isolates, with ST117 (8.5%), ST297 (5.1%), and ST58 (3.4%) being the most prevalent across retail meat types. The most prevalent phylogroups were B1 (29.1%) and A (28.2%). Five clonal patterns were detected among isolates. CONCLUSIONS: E. coli prevalence and the presence of AMR and MDR were highest in turkey retail meat. The lack of an association between MDR E. coli in retail meat and antibiotic use claim, including those with no indication of antimicrobial use, suggests that additional research is required to understand the origin of resistance. The presence of ST117, an emerging human pathogen, warrants further surveillance. The isolates were distinctly diverse suggesting an instability in population dynamics.

Widespread prevalence of plasmid-mediated blaCTX-M type extended-spectrum beta-lactamase Escherichia coli in backyard broiler production systems in the United States.

Extended-spectrum beta-lactamase (ESBL) Escherichia coli (E. coli) is an emerging pathogen of high concern given its resistance to extended-spectrum cephalosporins. Broiler chicken, which is the number one consumed meat in the United States and worldwide, can be a reservoir of ESBL E. coli. Backyard poultry ownership is on the rise in the United States, yet there is little research investigating prevalence of ESBL E. coli in this setting. This study aims to identify the prevalence and antimicrobial resistance profiles (phenotypically and genotypically) of ESBL E. coli in some backyard and commercial broiler farms in the U.S. For this study ten backyard and ten commercial farms were visited at three time-points across flock production. Fecal (n = 10), litter/compost (n = 5), soil (n = 5), and swabs of feeders and waterers (n = 6) were collected at each visit and processed for E. coli. Assessment of ESBL phenotype was determined through using disk diffusion with 3rd generation cephalosporins, cefotaxime and ceftazidime, and that with clavulanic acid. Broth microdilution and whole genome sequencing were used to investigate both phenotypic and genotypic resistance profiles, respectively. ESBL E. coli was more prevalent in backyard farms with 12.95% of samples testing positive whereas 0.77% of commercial farm samples were positive. All isolates contained a blaCTX-M gene, the dominant variant being blaCTX-M-1, and its presence was entirely due to plasmids. Our study confirms concerns of growing resistance to fourth generation cephalosporin, cefepime, as roughly half (51.4%) of all isolates were found to be susceptible dose-dependent and few were resistant. Resistance to non-beta lactams, gentamicin and ciprofloxacin, was also detected in our samples. Our study identifies prevalence of blaCTX-M type ESBL E. coli in U.S. backyard broiler farms, emphasizing the need for interventions for food and production safety.

Longitudinal study of distributions of similar antimicrobial-resistant Salmonella serovars in pigs and their environment in two distinct swine production systems.

The aim of this longitudinal study was to determine and compare the prevalences and genotypic profiles of antimicrobial-resistant (AR) Salmonella isolates from pigs reared in antimicrobial-free (ABF) and conventional production systems at farm, at slaughter, and in their environment. We collected 2,889 pig fecal and 2,122 environmental (feed, water, soil, lagoon, truck, and floor swabs) samples from 10 conventional and eight ABF longitudinal cohorts at different stages of production (farrowing, nursery, finishing) and slaughter (postevisceration, postchill, and mesenteric lymph nodes [MLN]). In addition, we collected 1,363 carcass swabs and 205 lairage and truck samples at slaughter. A total of 1,090 Salmonella isolates were recovered from the samples; these were isolated with a significantly higher prevalence in conventionally reared pigs (4.0%; n = 66) and their environment (11.7%; n = 156) than in ABF pigs (0.2%; n = 2) and their environment (0.6%; n = 5) (P < 0.001). Salmonella was isolated from all stages at slaughter, including the postchill step, in the two production systems. Salmonella prevalence was significantly higher in MLN extracted from conventional carcasses than those extracted from ABF carcasses (P < 0.001). We identified a total of 24 different serotypes, with Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Anatum, Salmonella enterica serovar Infantis, and Salmonella enterica serovar Derby being predominant. The highest frequencies of antimicrobial resistance (AR) were exhibited to tetracycline (71%), sulfisoxazole (42%), and streptomycin (17%). Multidrug resistance (resistance to ≥ 3 antimicrobials; MDR) was detected in 27% (n = 254) of the Salmonella isolates from the conventional system. Our study reports a low prevalence of Salmonella in both production systems in pigs on farms, while a higher prevalence was detected among the carcasses at slaughter. The dynamics of Salmonella prevalence in pigs and carcasses were reciprocated in the farm and slaughter environment, clearly indicating an exchange of this pathogen between the pigs and their surroundings. Furthermore, the phenotypic and genotypic fingerprint profile results underscore the potential role played by environmental factors in dissemination of AR Salmonella to pigs.

Yersinia enterocolitica of porcine origin: carriage of virulence genes and genotypic diversity.

Yersinia enterocolitica is an important foodborne pathogen, and pigs are recognized as a major reservoir and potential source of pathogenic strains to humans. A total of 172 Y. enterocolitica recovered from conventional and antimicrobial-free pig production systems from different geographic regions (North Carolina, Ohio, Michigan, Wisconsin, and Iowa) were investigated to determine their pathogenic significance to humans. Phenotypic and genotypic diversity of the isolates was assessed using antibiogram, serogrouping, and amplified fragment length polymorphism (AFLP). Carriage of chromosomal and plasmid-borne virulence genes were investigated using polymerase chain reaction. A total of 12 antimicrobial resistance patterns were identified. More than two-thirds (67.4%) of Y. enterocolitica were pan-susceptible, and 27.9% were resistant against ß-lactams. The most predominant serogroup was O:3 (43%), followed by O:5 (25.6%) and O:9 (4.1%). Twenty-two of 172 (12.8%) isolates were found to carry Yersinia adhesion A (yadA), a virulence gene encoded on the Yersinia virulence plasmid. Sixty-nine (40.1%) isolates were found to carry ail gene. The ystA and ystB genes were detected in 77% and 26.2% of the strains, respectively. AFLP genotyping of isolates showed wide genotypic diversity and were grouped into nine clades with an overall genotypic similarity of 66.8-99.3%. AFLP analysis revealed that isolates from the same production system showed clonal relatedness, while more than one genotype of Y. enterocolitica circulates within a farm.