Phenotypic and genotypic characterization of antimicrobial resistance of non-typhoidal Salmonella from retail meat in California.

Antimicrobial resistance (AMR) is a global emerging problem for food safety and public health. Retail meat is one of the vehicles that may transmit antimicrobial resistant bacteria to humans. Here we assessed the phenotypic and genotypic resistance of non-typhoidal Salmonella from retail meat collected in California in 2019 by the National Antimicrobial Resistance Monitoring System (NARMS) Retail Food Surveillance program. A total of 849 fresh meat samples were collected from randomly selected grocery stores in Northern and Southern California from January to December 2019. The overall prevalence of Salmonella was 15.31 %, with a significantly higher occurrence in Southern (28.38%) than in Northern (5.22 %) California. The prevalence of Salmonella in chicken (24.01 %) was higher (p < 0.001) compared to ground turkey (5.42 %) and pork (3.08 %) samples. No Salmonella were recovered from ground beef samples. The prevalence of Salmonella in meat with reduced antibiotic claim (20.35 %) was higher (p < 0.001) than that with conventional production (11.96 %). Salmonella isolates were classified into 25 serotypes with S. Kentucky (47.73 %), S. typhimurium (11.36 %), and S. Alachua (7.58 %) as predominant serotypes. Thirty-two out of 132 (24.24 %) Salmonella isolates were susceptible to all tested antimicrobial drugs, while 75.76 % were resistant to one or more drugs, 62.88 % to two or more drugs, and 9.85 % to three or more drugs. Antimicrobials that Salmonella exhibited high resistance to were tetracycline (82/132, 62.12 %) and streptomycin (79/132, 59.85 %). No significant difference was observed between reduced antibiotic claim and conventional production in the occurrence of single and multidrug resistance. A total of 23 resistant genes, a D87Y mutation of gyrA, and 23 plasmid replicons were identified from resistant Salmonella isolates. Genotypic and phenotypic results were well correlated with an overall sensitivity of 96.85 %. S. infantis was the most resistant serotype which also harbored the IncFIB (pN55391) plasmid replicon and gyrA (87) mutation. Data from Northern and Southern California in this study helps us to understand the AMR trends in Salmonella from retail meat sold in the highly populous and demographically diverse state of California.

Distribution of bacteria and antimicrobial resistance in retail Nile tilapia (Oreochromis spp.) as potential sources of foodborne illness.

This study aimed to investigate AMR profiles of Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae isolated from Nile tilapia (Oreochromis spp.) (n = 276) purchased from fresh markets and supermarkets in Bangkok, Thailand. A sample of tilapia was divided into three parts: fish intestine (n = 276), fish meat (n = 276), and liver and kidney (n = 276). The occurrence of A. hydrophila, Salmonella, and V. cholerae was 3.1%, 7.4%, and 8.5%, respectively. A high prevalence of these pathogenic bacteria was observed in fresh market tilapia compared to those from supermarkets (p < 0.05). The predominant Salmonella serovars were Paratyphi B (6.4%), followed by Escanaba (5.7%), and Saintpaul (5.7%). All isolates tested positive for the virulence genes of A. hydrophila (aero and hly), Salmonella (invA), and V. cholerae (hlyA). A. hydrophila (65.4%), Salmonella (31.2%), and V. cholerae (2.9%) showed multidrug resistant isolates. All A. hydrophila isolates (n = 26) exhibited resistant to ampicillin (100.0%) and florfenicol (100.0%), and often carried sul1 (53.8%) and tetA (50.0%). Salmonella isolates were primarily resistant to ampicillin (36.9%), with a high incidence of blaTEM (26.2%) and qnrS (25.5%). For V. cholerae isolates, resistance was observed against ampicillin (48.6%), and they commonly carried qnrS (24.3%) and tetA (22.9%). To identify mutations in the quinolone resistance determining regions (QRDRs), a single C248A point mutation of C248A (Ser-83-Tyr) in the gyrA region was identified in six out of seven isolates of Salmonella isolates. This study highlighted the presence of antimicrobial-resistant pathogenic bacteria in Nile tilapia at a selling point. It is important to rigorously implement strategies for AMR control and prevention.

Occurrence and molecular characteristics of antimicrobial resistance, virulence factors, and extended-spectrum ß-lactamase (ESBL) producing Salmonella enterica and Escherichia coli isolated from the retail produce commodities in Bangkok, Thailand.

The incidence of antimicrobial resistance (AMR) in the environment is often overlooked and leads to serious health threats under the One Health paradigm. Infection with extended-spectrum ß-lactamase (ESBL) producing bacteria in humans and animals has been widely examined, with the mode of transmission routes such as food, water, and contact with a contaminated environment. The purpose of this study was to determine the occurrence and molecular characteristics of resistant Salmonella enterica (S. enterica) (n = 59) and Escherichia coli (E. coli) (n = 392) isolated from produce commodities collected from fresh markets and supermarkets in Bangkok, Thailand. In this study, the S. enterica isolates exhibited the highest prevalence of resistance to tetracycline (11.9%) and streptomycin (8.5%), while the E. coli isolates were predominantly resistant to tetracycline (22.5%), ampicillin (21.4%), and sulfamethoxazole (11.5%). Among isolates of S. enterica (6.8%) and E. coli (15.3%) were determined as multidrug resistant (MDR). The prevalence of ESBL-producing isolates was 5.1% and 1.0% in S. enterica and E. coli, respectively. A minority of S. enterica isolates, where a single isolate exclusively carried blaCTX-M-55 (n = 1), and another isolate harbored both blaCTX-M-55 and blaTEM-1 (n = 1); similarly, a minority of E. coli isolates contained blaCTX-M-55 (n = 2) and blaCTX-M-15 (n = 1). QnrS (11.9%) and blaTEM (20.2%) were the most common resistant genes found in S. enterica and E. coli, respectively. Nine isolates resistant to ciprofloxacin contained point mutations in gyrA and parC. In addition, the odds of resistance to tetracycline among isolates of S. enterica were positively associated with the co-occurrence of ampicillin resistance and the presence of tetB (P = 0.001), while the E. coli isolates were positively associated with ampicillin resistance, streptomycin resistance, and the presence of tetA (P < 0.0001) in this study. In summary, these findings demonstrate that fresh vegetables and fruits, such as cucumbers and tomatoes, can serve as an important source of foodborne AMR S. enterica and E. coli in the greater Bangkok area, especially given the popularity of these fresh commodities in Thai cuisine.

Impact of zinc supplementation on phenotypic antimicrobial resistance of fecal commensal bacteria from pre-weaned dairy calves.

The objective of this study was to evaluate the impact of dietary zinc supplementation in pre-weaned dairy calves on the phenotypic antimicrobial resistance (AMR) of fecal commensal bacteria. A repository of fecal specimens from a random sample of calves block-randomized into placebo (n = 39) and zinc sulfate (n = 28) groups collected over a zinc supplementation clinical trial at the onset of calf diarrhea, calf diarrheal cure, and the last day of 14 cumulative days of zinc or placebo treatment were analyzed. Antimicrobial susceptibility testing was conducted for Enterococcus spp. (n = 167) and E. coli (n = 44), with one representative isolate of each commensal bacteria tested per sample. Parametric survival interval regression models were constructed to evaluate the association between zinc treatment and phenotypic AMR, with exponentiated accelerated failure time (AFT) coefficients adapted for MIC instead of time representing the degree of change in AMR (MIC Ratio, MR). Findings from our study indicated that zinc supplementation did not significantly alter the MIC in Enterococcus spp. for 13 drugs: gentamicin, vancomycin, ciprofloxacin, erythromycin, penicillin, nitrofurantoin, linezolid, quinupristin/dalfopristin, tylosin tartrate, streptomycin, daptomycin, chloramphenicol, and tigecycline (MR = 0.96-2.94, p > 0.05). In E. coli, zinc supplementation was not associated with resistance to azithromycin (MR = 0.80, p > 0.05) and ceftriaxone (MR = 0.95, p > 0.05). However, a significant reduction in E. coli MIC values was observed for ciprofloxacin (MR = 0.17, 95% CI 0.03-0.97) and nalidixic acid (MR = 0.28, 95% CI 0.15-0.53) for zinc-treated compared to placebo-treated calves. Alongside predictions of MIC values generated from these 17 AFT models, findings from this study corroborate the influence of age and antimicrobial exposure on phenotypic AMR.

Antimicrobial resistance, virulence profile, and genetic analysis of ESBL-producing Escherichia coli isolated from Nile tilapia in fresh markets and supermarkets in Thailand.

This study investigated the prevalence and antimicrobial resistance (AMR) of Escherichia coli (E. coli) in Nile tilapia from fresh markets and supermarkets. A total of samples (n = 828) were collected from Nile tilapia including fish flesh (n = 276), liver and kidney (n = 276), and intestine (n = 276). Overall prevalence of fecal coliforms (61.6%) and E. coli (53.0%) were observed. High prevalence of E. coli was found in the intestine (71.4%), followed by the liver and kidney (45.7%). The highest prevalence of resistance was commonly found against tetracycline (78.5%), ampicillin (72.8%), and sulfamethoxazole (45.6%) with resistance to only tetracycline (15.2%) as the most common antibiogram. The prevalence of multidrug resistance (MDR) (54.4%) and Extended-spectrum beta-lactamases (ESBLs) (5.7%) were examined. The predominant virulence genes (n = 158) were st (14.6%), followed by eaeA (0.6%). The blaTEM (73.4%), tetA (65.2%), and qnrS (57.6%). There is statistical significance between Nile tilapia from fresh markets and supermarkets. Based on logistic regression analysis, ampicillin-resistant E. coli was statistically associated with the phenotypic resistance to tetracycline and trimethoprim, and the presence of blaTEM and tetA (p < 0.05). Further investigation of AMR transference and their mechanisms is needed for AMR control.

Prevalence and antimicrobial resistance profiles of Vibrio spp. and Enterococcus spp. in retail shrimp in Northern California.

Shrimp is one of the most consumed seafood products globally. Antimicrobial drugs play an integral role in disease mitigation in aquaculture settings, but their prevalent use raises public health concerns on the emergence and spread of antimicrobial resistant microorganisms. Vibrio spp., as the most common causative agents of seafood-borne infections in humans, and Enterococcus spp., as an indicator organism, are focal bacteria of interest for the monitoring of antimicrobial resistance (AMR) in seafood. In this study, 400 samples of retail shrimp were collected from randomly selected grocery stores in the Greater Sacramento, California, area between September 2019 and June 2020. The prevalence of Vibrio spp. and Enterococcus spp. was 60.25% (241/400) and 89.75% (359/400), respectively. Subsamples of Vibrio (n = 110) and Enterococcus (n = 110) isolates were subjected to antimicrobial susceptibility testing (AST). Vibrio isolates had high phenotypic resistance to ampicillin (52/110, 47.27%) and cefoxitin (39/110, 35.45%). Enterococcus were most frequently resistant to lincomycin (106/110, 96.36%), quinupristin-dalfopristin (96/110, 87.27%), ciprofloxacin (93/110, 84.55%), linezolid (86/110, 78.18%), and erythromycin (58/110, 52.73%). For both Vibrio and Enterococcus, no significant associations were observed between multidrug resistance (MDR, resistance to ≥3 drug classes) in isolates from farm raised and wild caught shrimp (p > 0.05) and in isolates of domestic and imported origin (p > 0.05). Whole genome sequencing (WGS) of a subset of Vibrio isolates (n = 42) speciated isolates as primarily V. metschnikovii (24/42; 57.14%) and V. parahaemolyticus (12/42; 28.57%), and detected 27 unique antimicrobial resistance genes (ARGs) across these isolates, most commonly qnrVC6 (19.05%, 8/42), dfrA31 (11.90%, 5/42), dfrA6 (9.5%, 4/42), qnrVC1 (9.5%, 4/42). Additionally, WGS predicted phenotypic resistance in Vibrio isolates with an overall sensitivity of 11.54% and specificity of 96.05%. This study provides insights on the prevalence and distribution of AMR in Vibrio spp. and Enterococcus spp. from retail shrimp in California which are important for food safety and public health and exemplifies the value of surveillance in monitoring the spread of AMR and its genetic determinants.

Dispersal and Risk Factors for Airborne Escherichia coli in the Proximity to Beef Cattle Feedlots.

California Leafy Green Products Handler Marketing Agreement (LGMA) established food safety metrics with guidance recommendations of 366 m (1,200 feet) and 1,609 m (1 mile) distances between production fields of leafy greens and a concentrated animal feeding operation (CAFO) containing >1,000 and >80,000 head of cattle, respectively. This study evaluated the effect of these distance metrics and environmental factors on the occurrence of airborneEscherichia coliin proximity to seven commercial beef cattle feedlots located in Imperial Valley, California. A total of 168 air samples were collected from seven beef cattle feedlots during March and April 2020, which were the months implicated in the 2018 Yuma Arizona lettuce outbreak of E. coli O157:H7. The distance between air sampling sites and the edge of the feedlot ranged from ∼0 to ∼2,200 m (∼1.3 mile), with each sample comprised of 1,000 L of processed air taken at a 1.2 m elevation over a 10-minute duration. E. colicolonies were enumerated on CHROMagar ECC selective agar and confirmed with conventional PCR. Meteorological data (air temperature, wind speed, wind direction, relative humidity) were collectedin situ. The prevalence and mean concentration ofE. coliwere 6.55% (11/168) and 0.09 CFU per 1,000 L of air, with positive samples limited to within 37 m (120 ft) of the feedlot.Based on logistic regression, the odds of airborne E. coli detection were associated with little to no wind and close proximity to a feedlot. This pilot study found limited dispersal of airborne E. coli in proximity to commercial feedlots in Imperial Valley, with light-to-no wind and proximity within 37 m of a feedlot significant factor-associated airborne E. coli in this produce-growing region of California.

Assessment of Prevalence and Diversity of Antimicrobial Resistant Escherichia coli from Retail Meats in Southern California.

Retail meat products may serve as reservoirs and conduits for antimicrobial resistance, which is frequently monitored using Escherichia coli as indicator bacteria. In this study, E. coli isolation was conducted on 221 retail meat samples (56 chicken, 54 ground turkey, 55 ground beef, and 56 pork chops) collected over a one-year period from grocery stores in southern California. The overall prevalence of E. coli in retail meat samples was 47.51% (105/221), with E. coli contamination found to be significantly associated with meat type and season of sampling. From antimicrobial susceptibility testing, 51 isolates (48.57%) were susceptible to all antimicrobials tested, 54 (51.34%) were resistant to at least 1 drug, 39 (37.14%) to 2 or more drugs, and 21 (20.00%) to 3 or more drugs. Resistance to ampicillin, gentamicin, streptomycin, and tetracycline were significantly associated with meat type, with poultry counterparts (chicken or ground turkey) exhibiting higher odds for resistance to these drugs compared to non-poultry meats (beef and pork). From the 52 E. coli isolates selected to undergo whole-genome sequencing (WGS), 27 antimicrobial resistance genes (ARGs) were identified and predicted phenotypic AMR profiles with an overall sensitivity and specificity of 93.33% and 99.84%, respectively. Clustering assessment and co-occurrence networks revealed that the genomic AMR determinants of E. coli from retail meat were highly heterogeneous, with a sparsity of shared gene networks.

Emergence of colistin resistance and characterization of antimicrobial resistance and virulence factors of Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae isolated from hybrid red tilapia cage culture.

Background: Tilapia is a primary aquaculture fish in Thailand, but little is known about the occurrence of antimicrobial resistance (AMR) in Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae colonizing healthy tilapia intended for human consumption and the co-occurrence of these AMR bacteria in the cultivation water. Methods: This study determined the phenotype and genotype of AMR, extended-spectrum ß-lactamase (ESBL) production, and virulence factors of A. hydrophila, Salmonella spp., and V. cholerae isolated from hybrid red tilapia and cultivation water in Thailand. Standard culture methods such as USFDA's BAM or ISO procedures were used for the original isolation, with all isolates confirmed by biochemical tests, serotyping, and species-specific gene detection based on PCR. Results: A total of 278 isolates consisting of 15 A. hydrophila, 188 Salmonella spp., and 75 V. cholerae isolates were retrieved from a previous study. All isolates of A. hydrophila and Salmonella isolates were resistance to at least one antimicrobial, with 26.7% and 72.3% of the isolates being multidrug resistant (MDR), respectively. All A. hydrophila isolates were resistant to ampicillin (100%), followed by oxytetracycline (26.7%), tetracycline (26.7%), trimethoprim (26.7%), and oxolinic acid (20.0%). The predominant resistance genes in A. hydrophila were mcr-3 (20.0%), followed by 13.3% of isolates having floR, qnrS, sul1, sul2, and dfrA1. Salmonella isolates also exhibited a high prevalence of resistance to ampicillin (79.3%), oxolinic acid (75.5%), oxytetracycline (71.8%), chloramphenicol (62.8%), and florfenicol (55.3%). The most common resistance genes in these Salmonella isolates were qnrS (65.4%), tetA (64.9%), bla TEM (63.8%), and floR (55.9%). All V. cholerae isolates were susceptible to all antimicrobials tested, while the most common resistance gene was sul1 (12.0%). One isolate of A. hydrophila was positive for int1, while all isolates of Salmonella and V. cholerae isolates were negative for integrons and int SXT. None of the bacterial isolates in this study were producing ESBL. The occurrence of mcr-3 (20.0%) in these isolates from tilapia aquaculture may signify a serious occupational and consumer health risk given that colistin is a last resort antimicrobial for treatment of Gram-negative bacteria infections. Conclusions: Findings from this study on AMR bacteria in hybrid red tilapia suggest that aquaculture as practiced in Thailand can select for ubiquitous AMR pathogens, mobile genetic elements, and an emerging reservoir of mcr and colistin-resistant bacteria. Resistant and pathogenic bacteria, such as resistance to ampicillin and tetracycline, or MDR Salmonella circulating in aquaculture, together highlight the public health concerns and foodborne risks of zoonotic pathogens in humans from cultured freshwater fish.

Low to Zero Concentrations of Airborne Bacterial Pathogens and Indicator E. coli in Proximity to Beef Cattle Feedlots in Imperial Valley, California.

This study characterized the effect of distance from beef cattle feedlots, environmental factors, and climate on the occurrence of airborne bacterial indicators and pathogens. Three hundred air samples were collected over 6 months from five feedlots, with each air sample comprising 6000 L of air. Air samples were processed onto TSB-enriched air filters, qPCR-screened, and then qPCR-confirmed for suspect positive colonies of E. coli O157, non-O157-Shiga-toxin-producing E. coli (STEC), Salmonella, and E. coli. Direct enumeration of E. coli was also collected. Although no bacterial pathogens were qPCR-confirmed for the 300 samples, E. coli was detected in 16.7% (50/300) of samples, with an overall mean concentration of 0.17 CFU/6000 L air. Logistic regression analyses revealed a higher odds of E. coli for samples in close proximity compared to >610 m (2000 ft) distance from feedlots, along with significant associations with meteorological factors, sampling hour of day, and the presence of a dust-generating activity such as plowing a field or nearby vehicular traffic. The lack of bacterial pathogen detection suggests airborne deposition from nearby feedlots may not be a significant mechanism of leafy green bacterial pathogen contamination; the result of our study provides data to inform future revisions of produce-safety guidance.

Molecular Epidemiology of Antimicrobial Resistance and Virulence Profiles of Escherichia coli, Salmonella spp., and Vibrio spp. Isolated from Coastal Seawater for Aquaculture.

The occurrence of waterborne antimicrobial-resistant (AMR) bacteria in areas of high-density oyster cultivation is an ongoing environmental and public health threat given the popularity of shellfish consumption, water-related human recreation throughout coastal Thailand, and the geographical expansion of Thailand's shellfish industry. This study characterized the association of phenotypic and genotypic AMR, including extended-spectrum ß-lactamase (ESBL) production, and virulence genes isolated from waterborne Escherichia coli (E. coli) (n = 84), Salmonella enterica (S. enterica) subsp. enterica (n = 12), Vibrio parahaemolyticus (V. parahaemolyticus) (n = 249), and Vibrio cholerae (V. cholerae) (n = 39) from Thailand's coastal aquaculture regions. All Salmonella (100.0%) and half of V. cholerae (51.3%) isolates harbored their unique virulence gene, invA and ompW, respectively. The majority of isolates of V. parahaemolyticus and E. coli, ~25% of S. enterica subsp. enterica, and ~12% of V. cholerae, exhibited phenotypic AMR to multiple antimicrobials, with 8.9% of all coastal water isolates exhibiting multidrug resistance (MDR). Taken together, we recommend that coastal water quality surveillance programs include monitoring for bacterial AMR for food safety and recreational water exposure to water for Thailand's coastal water resources.

Diverse Genotypes of Cryptosporidium in Sheep in California, USA.

Cryptosporidium spp. is a parasite that can infect a wide variety of vertebrate species. The parasite has been detected in sheep worldwide with diverse species and genotypes of various levels of zoonotic potential and public health concern. The purpose of this study was to determine the distribution of genotypes of Cryptosporidium in sheep in California, USA. Microscopic positive samples from individual sheep from central and northern California ranches were genotyped by sequencing a fragment of the 18S rRNA gene and BLAST analysis. Eighty-eight (63.8%) of the microscopic positive samples were genotyped, and multiple genotypes of Cryptosporidium were identified from sheep in the enrolled ranches. Approximately 89% of isolates (n = 78) were C. xiaoi or C. bovis, 10% of isolates (n = 9) were C. ubiquitum, and 1% of isolates (n = 1) were C. parvum. The C. parvum and C. ubiquitum isolates were detected only from lambs and limited to four farms. Given that the majority of Cryptosporidium species (i.e., C. xiaoi and C. bovis) were of minor zoonotic concern, the results of this study suggest that sheep are not a reservoir of major zoonotic Cryptosporidium in California ranches.

Impact of joint interactions with humans and social interactions with conspecifics on the risk of zooanthroponotic outbreaks among wildlife populations.

Pandemics caused by pathogens that originate in wildlife highlight the importance of understanding the behavioral ecology of disease outbreaks at human-wildlife interfaces. Specifically, the relative effects of human-wildlife and wildlife-wildlife interactions on disease outbreaks among wildlife populations in urban and peri-urban environments remain unclear. We used social network analysis and epidemiological Susceptible-Infected-Recovered models to simulate zooanthroponotic outbreaks, through wild animals' joint propensities to co-interact with humans, and their social grooming of conspecifics. On 10 groups of macaques (Macaca spp.) in peri-urban environments in Asia, we collected behavioral data using event sampling of human-macaque interactions within the same time and space, and focal sampling of macaques' social interactions with conspecifics and overall anthropogenic exposure. Model-predicted outbreak sizes were related to structural features of macaques' networks. For all three species, and for both anthropogenic (co-interactions) and social (grooming) contexts, outbreak sizes were positively correlated to the network centrality of first-infected macaques. Across host species and contexts, the above effects were stronger through macaques' human co-interaction networks than through their grooming networks, particularly for rhesus and bonnet macaques. Long-tailed macaques appeared to show intraspecific variation in these effects. Our findings suggest that among wildlife in anthropogenically-impacted environments, the structure of their aggregations around anthropogenic factors makes them more vulnerable to zooanthroponotic outbreaks than their social structure. The global features of these networks that influence disease outbreaks, and their underlying socio-ecological covariates, need further investigation. Animals that consistently interact with both humans and their conspecifics are important targets for disease control.

Dynamic changes in fecal bacterial microbiota of dairy cattle across the production line.

BACKGROUND: Microbiota play important roles in the gastrointestinal tract (GIT) of dairy cattle as the communities are responsible for host health, growth, and production performance. However, a systematic characterization and comparison of microbial communities in the GIT of cattle housed in different management units on a modern dairy farm are still lacking. We used 16S rRNA gene sequencing to evaluate the fecal bacterial communities of 90 dairy cattle housed in 12 distinctly defined management units on a modern dairy farm. RESULTS: We found that cattle from management units 5, 6, 8, and 9 had similar bacterial communities while the other units showed varying levels of differences. Hutch calves had a dramatically different bacterial community than adult cattle, with at least 10 genera exclusively detected in their samples but not in non-neonatal cattle. Moreover, we compared fecal bacteria of cattle from every pair of the management units and detailed the number and relative abundance of the significantly differential genera. Lastly, we identified 181 pairs of strongly correlated taxa in the community, showing possible synergistic or antagonistic relationships. CONCLUSIONS: This study assesses the fecal microbiota of cattle from 12 distinctly defined management units along the production line on a California dairy farm. The results highlight the similarities and differences of fecal microbiota between cattle from each pair of the management units. Especially, the data indicate that the newborn calves host very different gut bacterial communities than non-neonatal cattle, while non-neonatal cattle adopt one of the two distinct types of gut bacterial communities with subtle differences among the management units. The gut microbial communities of dairy cattle change dramatically in bacterial abundances at different taxonomic levels along the production line. The findings provide a reference for research and practice in modern dairy farm management.

Antimicrobial Resistance Profiles of Non-typhoidal Salmonella From Retail Meat Products in California, 2018.

Non-typhoidal Salmonella remains a leading cause of foodborne illness in the United States, with food animal products serving as a key conduit for transmission. The emergence of antimicrobial resistance (AMR) poses an additional public health concern warranting better understanding of its epidemiology. In this study, 958 retail meat samples collected from January to December 2018 in California were tested for Salmonella. From multivariable logistic regression, there was a 6.47 (90% CI 2.29-18.27), 3.81 (90% CI 1.29-11.27), and 3.12 (90% CI 1.03-9.45) higher odds of contamination in samples purchased in the fall, spring, and summer than in winter months, respectively, and a 3.70 (90% CI 1.05-13.07) higher odds in ground turkey compared to pork samples. Fourteen distinct serotypes and 17 multilocus sequence types were identified among the 43 isolates recovered, with S. Kentucky (25.58%), S. Reading (18.60%), S. Infantis (11.63%), and S. Typhimurium (9.30%) comprising the top serotypes. High prevalence of resistance was observed in retail chicken isolates for streptomycin (12/23, 52.17%) and tetracycline (12/23, 52.17%), in ground turkey isolates for ampicillin (8/15, 53.34%), and in ground beef isolates for nalidixic acid (2/3, 66.67%). Fourteen (32.56%) were susceptible to all antimicrobials tested, 11 (25.58%) were resistant to one drug, and 12 (27.91%) were resistant to two drugs. The remaining six isolates (13.95%) were multidrug-resistant (MDR, ≥3 drug classes) S. Infantis (n = 4), S. Reading (n = 1), and S. Kentucky (n = 1). Whole-genome sequencing (WGS) identified 16 AMR genes and 17 plasmid replicons, including bla CTX-M-65 encoding ceftriaxone resistance and a D87Y mutation in gyrA conferring resistance to nalidixic acid and reduced susceptibility to ciprofloxacin. The IncFIB(pN55391) replicon previously identified in connection to the worldwide dissemination of pESI-like mega plasmid carriage in an emerged S. Infantis clone was detected in four of the six MDR isolates. Genotypes from WGS showed high concordance with phenotype with overall sensitivity and specificity of 95.31% and 100%, respectively. This study provides insight into the AMR profiles of a diversity of Salmonella serotypes isolated from retail meat products in California and highlights the value of routine retail food surveillance for the detection and characterization of AMR in foodborne pathogens.

Antibiotic Resistance Genes and Associated Phenotypes in Escherichia coli and Enterococcus from Cattle at Different Production Stages on a Dairy Farm in Central California.

The objectives of this study were to characterize overall genomic antibiotic resistance profiles of fecal Escherichia coli and Enterococcus spp. from dairy cattle at different production stages using whole-genome sequencing and to determine the association between antimicrobial resistance (AMR) phenotypes and their corresponding genotypes. The Comprehensive Antibiotic Resistance Database (CARD) and ResFinder, two publicly available databases of antimicrobial resistance genes, were used to annotate isolates. Based on the ResFinder database, 27.5% and 20.0% of tested E. coli isolates (n = 40) harbored single and ≥3 antimicrobial resistance genes, respectively; for Enterococcus spp., we observed 87.8% and 8.2%, respectively. The highest prevalence of AMR genes in E. coli was for resistance to tetracycline (27.5%), followed by sulphonamide (22.5%) and aminoglycoside (20.0%); the predominant antimicrobial resistance genes in Enterococcus spp. targeted macrolide drugs (77.6%). Based on the CARD database, resistance to ≥3 antimicrobial classes was observed in all E. coli and 77.6% in Enterococcus spp. isolates. A high degree of agreement existed between the resistance phenotype and the presence of resistance genes for various antimicrobial classes for E. coli but much less so for isolates of Enterococcus. Consistent with prior work, fecal E. coli and Enterococcus spp. isolates from calves harbored a wide spectrum of resistance genes, compared to those from cattle at other production stages, based on the cross-sectional samples from the studied farm.

Dairy management practices associated with multi-drug resistant fecal commensals and Salmonella in cull cows: a machine learning approach.

BACKGROUND: Understanding the effects of herd management practices on the prevalence of multidrug-resistant pathogenic Salmonella and commensals Enterococcus spp. and Escherichia coli in dairy cattle is key in reducing antibacterial resistant infections in humans originating from food animals. Our objective was to explore the herd and cow level features associated with the multi-drug resistant, and resistance phenotypes shared between Salmonella, E. coli and Enterococcus spp. using machine learning algorithms. METHODS: Randomly collected fecal samples from cull dairy cows from six dairy farms in central California were tested for multi-drug resistance phenotypes of Salmonella, E. coli and Enterococcus spp. Using data on herd management practices collected from a questionnaire, we built three machine learning algorithms (decision tree classifier, random forest, and gradient boosting decision trees) to predict the cows shedding multidrug-resistant Salmonella and commensal bacteria. RESULTS: The decision tree classifier identified rolling herd average milk production as an important feature for predicting fecal shedding of multi-drug resistance in Salmonella or commensal bacteria. The number of culled animals, monthly culling frequency and percentage, herd size, and proportion of Holstein cows in the herd were found to be influential herd characteristics predicting fecal shedding of multidrug-resistant phenotypes based on random forest models for Salmonella and commensal bacteria. Gradient boosting models showed that higher culling frequency and monthly culling percentages were associated with fecal shedding of multidrug resistant Salmonella or commensal bacteria. In contrast, an overall increase in the number of culled animals on a culling day showed a negative trend with classifying a cow as shedding multidrug-resistant bacteria. Increasing rolling herd average milk production and spring season were positively associated with fecal shedding of multidrug- resistant Salmonella. Only six individual cows were detected sharing tetracycline resistance phenotypes between Salmonella and either of the commensal bacteria. DISCUSSION: Percent culled and culling rate reflect the increase in culling over time adjusting for herd size and were associated with shedding multidrug resistant bacteria. In contrast, number culled was negatively associated with shedding multidrug resistant bacteria which may reflect producer decisions to prioritize the culling of otherwise healthy but low-producing cows based on milk or beef prices (with respect to dairy beef), amongst other factors. Using a data-driven suite of machine learning algorithms we identified generalizable and distant associations between antimicrobial resistance in Salmonella and fecal commensal bacteria, that can help develop a producer-friendly and data-informed risk assessment tool to reduce shedding of multidrug-resistant bacteria in cull dairy cows.

Diverse Genotypes and Species of Cryptosporidium in Wild Rodent Species from the West Coast of the USA and Implications for Raw Produce Safety and Microbial Water Quality.

Cryptosporidium spp. are protozoan parasites that infect perhaps all vertebrate animals, with a subset of species and genotypes that function as food- and waterborne pathogens. The objective of this work was to collate the Cryptosporidium species and genotypes from common wild rodents on the west coast of the USA and update the information regarding the zoonotic potential of Cryptosporidium from these ubiquitous wild species. Representative sequences of the 18S rRNA gene for a unique set of Cryptosporidium isolates obtained from deer mice, house mice, mountain beavers, yellow-bellied marmot, long-tailed vole, California ground squirrels, Belding's ground squirrels, and a golden-mantled ground squirrel in GenBank were selected for phylogenetic analysis. Phylogenetic and BLAST analysis indicated that 4 (18%) of the 22 unique Cryptosporidium sequences from these wild rodent species were 99.75% to 100% identical to known zoonotic species (C. parvum, C. ubiquitum, C. xiaoi), suggesting that a minority of these representative Cryptosporidium isolates could have a public health impact through food and waterborne routes of human exposure. These zoonotic isolates were shed by deer mice and a yellow-bellied marmot from California, and from a mountain beaver trapped in Oregon. In addition, the group of unique Cryptosporidium isolates from deer mice and ground dwelling squirrels exhibited considerable DNA diversity, with multiple isolates appearing to be either host-limited or distributed throughout the various clades within the phylogenetic tree representing the various Cryptosporidium species from host mammals. These results indicate that only a subset of the unique Cryptosporidium genotypes and species obtained from wild rodents on the US west coast are of public health concern; nevertheless, given the geographic ubiquity of many of these host species and often high density at critical locations like municipal watersheds or produce production fields, prudent pest control practices are warranted to minimize the risks of water- and foodborne transmission to humans.

Understanding the transmission dynamics of Escherichia coli O157:H7 super-shedding infections in feedlot cattle.

BACKGROUND: The presence of Escherichia coli O157:H7 (E. coli O157:H7) super-shedding cattle in feedlots has the potential to increase the overall number (bio-burden) of E. coli O157:H7 in the environment. It is important to identify factors to reduce the bio-burden of E. coli O157 in feedlots by clarifying practices associated with the occurrence of super-shedders in feedlot cattle. METHODS: The objective of this study is to (1) identify host, pathogen, and management risk factors associated with naturally infected feedlot cattle excreting high concentrations of E. coli O157:H7 in their feces and (2) to determine whether the ingested dose or the specific strain of E. coli O157:H7 influences a super-shedder infection within experimentally inoculated feedlot cattle. To address this, (1) pen floor fecal samples and herd parameters were collected from four feedlots over a 9-month period, then (2) 6 strains of E. coli O157:H7, 3 strains isolated from normal shedder steers and 3 strains isolated from super-shedder steers, were inoculated into 30 one-year-old feedlot steers. Five steers were assigned to each E. coli O157:H7 strain group and inoculated with targeted numbers of 102, 104, 106, 108, and 1010 CFU of bacteria respectively. RESULTS: In the feedlots, prevalence of infection with E. coli O157:H7 for the 890 fecal samples collected was 22.4%, with individual pen prevalence ranging from 0% to 90% and individual feedlot prevalence ranging from 8.4% to 30.2%. Three samples had E. coli O157:H7 levels greater than 104 MPN/g feces, thereby meeting the definition of super-shedder. Lower body weight at entry to the feedlot and higher daily maximum ambient temperature were associated with increased odds of a sample testing positive for E. coli O157:H7. In the experimental inoculation trial, the duration and total environmental shedding load of E. coli O157:H7 suggests that the time post-inoculation and the dose of inoculated E. coli O157:H7 are important while the E. coli O157:H7 strain and shedding characteristic (normal or super-shedder) are not. DISCUSSION: Under the conditions of this experiment, super-shedding appears to be the result of cattle ingesting a high dose of any strain of E. coli O157:H7. Therefore strategies that minimize exposure to large numbers of E. coli O157:H7 should be beneficial against the super-shedding of E. coli O157:H7 in feedlots.

Bacterial diversity and potential risk factors associated with Salmonella contamination of seafood products sold in retail markets in Bangkok, Thailand.

Consumption of contaminated food causes 600 million cases, including 420,000 of fatal infections every year. Estimated cost from food-borne illnesses is USD 110 billion per year, which is an economic burden to low- and middle-income countries. Thailand is a leading producer and consumer of seafood, but little is known about bacterial contamination in seafood. In particular, public health agencies need to know the relationship between Salmonella contamination in seafood and risk factors, as assessed with readily available culture-dependent and bacterial phenotyping methods. To address this, levels of indicator bacteria, Salmonella and Vibrio in various seafood products were determined to identify risk factors associated with Salmonella contamination. A total of 335 samples were collected from October 2018 to July 2019 at seafood markets throughout Bangkok, Thailand; overall sample composition was Pacific white shrimp (n = 85), oysters (n = 82), blood cockles (n = 84), and Asian seabass (n = 84). Prevalence was 100% for fecal coliforms and 85% for E. coli. In contrast, prevalence was 59% for V. parahaemolyticus, 49% for V. cholerae, 19% for V. alginolyticus, 18% for V. vulnificus, and 36% for Salmonella. Highest concentrations of fecal coliforms and E. coli were in oysters. Highest concentrations of Salmonella with Matopeni (31%) being the predominant serotype were in shrimp. Salmonella contamination was significantly associated with type of seafood, sampling location, retail conditions, and the presence of E. coli, V. alginolyticus and V. vulnificus. A cutoff value for E. coli concentration of 1.3 × 104 MPN/g predicted contamination of Salmonella, with a sensitivity of 84% and specificity of 61%. Displaying seafood products on ice, presence of E. coli and Vibrio, and seafood derived from Eastern Thailand were associated with an increased risk of Salmonella contamination.