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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 β -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., Competing Interests: The authors declare there are no competing interests., (©2023 Thaotumpitak et al.)

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 × 10 4 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., Competing Interests: The authors declare that they have no competing interests., (© 2021 Atwill and Jeamsripong.)

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 10 2 , 10 4 , 10 6 , 10 8 , and 10 10 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 10 4 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., Competing Interests: The authors declare that they have no competing interests., (© 2021 Antaki-Zukoski et al.)

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., Competing Interests: Sharif S. Aly is an Academic Editor for PeerJ., (© 2021 Pandit et al.)

Epidemiological studies of low prevalence disease problems are often hindered by the high cost of diagnostic testing. The objective of this study was to evaluate PCR screening of both individual and pooled fecal samples from culled dairy cows for the inv A gene of Salmonella followed by culture to determine if the sensitivity and specificity were comparable to the results from traditional culture methods applied to individual samples. Cows from six different dairies were sampled in all four seasons. A total of 240 individual cow fecal samples, 24 fecal pools and 24 pools of 24-hour tetrathionate enrichment broth were tested. Diagnostic sensitivity of PCR screening followed by culture of PCR positive or indeterminate samples (i.e PCR-CUL method) was lower than that of culture (CUL) when applied to individual fecal samples (94.8%, 99.5%), however the specificity was comparable (99.6% and 97.7% respectively). For pools of five fecal samples and pools of five, 24 h tetrathionate broth samples, the specificity of both tests were comparable (∼98%); however, their sensitivity was only comparable in pooled fecal samples (∼93%) but greater for culture compared to PCR-CUL in pooled broth samples (∼99% versus ∼93%). Compared to culture results from testing of individual fecal samples, testing pooled fecal samples by culture had a relative sensitivity of 74% and relative specificity of 96%, testing pooled fecal samples by PCR-CUL resulted in relative sensitivity of 90% and relative specificity of 96%. Testing of pooled 24-hour enrichment broth by PCR-CUL increased the relative sensitivity and specificity to 100%. PCR testing followed by culture of positive or indeterminate samples is a time saving alternative to traditional methods. In addition, pooling of samples may be a useful method for decreasing cost if study aims can accommodate a moderate loss of relative sensitivity., Competing Interests: The authors declare there are no competing interests., (©2020 Adaska et al.)

Background: In this study cull dairy cows from six California dairy herds were sampled seasonally over the course of a year. The objectives were to determine the prevalence of antimicrobial resistant (AMR) Salmonella spp. shed in cull cow feces, and the factors associated with fecal shedding of AMR and multidrug resistant (MDR) Salmonella ., Methods: Six dairy farms located in the San Joaquin Valley of California were identified and enrolled as a convenience sample. On each dairy, and once during each of the four seasons, 10 cull cows were randomly selected for fecal sampling on the day of their removal from the herd. In addition, study personnel completed a survey based on responses of the herd manager to questions related to the previous 4 month's herd management and the specific cattle sampled. Fecal samples were submitted to the California Animal Health and Food Safety laboratory for Salmonella isolation. Antimicrobial resistance was evaluated using broth microdilution method and a gram-negative assay plate following Clinical Laboratory Standards Institute (CLSI) guidelines and breakpoint references. All statistical models were survey adjusted for number of animals on sampling day., Results: A total of 62 Salmonella were isolated from 60 of the 239 fecal samples collected. For 12% (95% confidence interval (CI) [3-20]) of fecal samples a multidrug resistant Salmonella was isolated. The survey-weighted results for the two most common drug classes for which isolates were resistant were tetracycline (39%; 95% CI [27-51]) and ampicillin (18%; 95% CI [9-27]). An important finding was the identification of cephalosporin as the third most common drug class for which isolates were resistant, with ceftriaxone (10%; 95% CI [2-17]) being the most common drug associated with resistance in that class. At the cow-level, reason for culling, prior treatment with antimicrobial drugs as the reason for culling was associated with higher odds of isolating an AMR Salmonella isolate. At the herd-level, percent of animals monthly culled on the farm as well as number of milking cows in the herd were associated with isolation of antimicrobial resistant Salmonella in cull cows., Discussion: Salmonella isolated from fecal samples from cull cows were resistant to important antimicrobials, such as ceftriaxone. The most common drug classes for which isolates were resistant were tetracyclines and beta-lactams, with ampicillin, ceftriaxone and ceftiofur being the three most common drugs within the latter. Cow and herd level factors were associated with isolating antimicrobial resistant Salmonella that should be further investigated for their potential role in promoting occurrence of AMR Salmonella . Our results also highlight the importance of monitoring dairy cattle sent to slaughter for shedding of Salmonella resistant to medically important antimicrobial drugs., Competing Interests: The authors declare that they have no competing interests.

Field trials were conducted in July-August and October 2012 to quantify the inactivation rate of Escherichia coli O157:H7 when mixed with fecal slurry and applied to romaine lettuce leaves. Lettuce was grown under commercial conditions in Salinas Valley, California. One-half milliliter of rabbit, chicken, or pig fecal slurry, containing an average of 4.05 × 10 7 CFU E. coli O157:H7 (C 0 ), was inoculated onto the upper (adaxial) surface of a lower leaf on 288 heads of lettuce per trial immediately following a 2.5 h irrigation event. To estimate the bacterial inactivation rate as a function of time, fecal matrix, irrigation and seasonal climate effects, sets of lettuce heads ( n = 28) were sampled each day over 10 days and the concentration of E. coli O157:H7 (C t ) determined. E. coli O157:H7 was detected on 100% of heads during the 10-day duration, with concentrations ranging from ≤340 MPN/head (∼5-log reduction) to >3.45 × 10 12 MPN/head (∼5-log growth). Relative to C 0 , on day 10 (C t = 12 ) we observed an overall 2.6-log and 3.2-log mean reduction of E. coli O157:H7 in July and October, respectively. However, we observed relative maximum concentrations due to bacterial growth on day 6 (maximum C t = 8 ) apparently stimulated by foliar irrigation on day 5. From this maximum there was a mean 5.3-log and 5.1-log reduction by day 10 (C t = 12 ) for the July and October trials, respectively. This study provides insight into the inactivation and growth kinetics of E. coli O157:H7 on romaine lettuce leaves under natural field conditions. This study provides evidence that harvesting within 24 h post irrigation has the potential to increase the concentration of E. coli O157:H7 contamination, if present on heads of romaine lettuce; foliar irrigation can temporarily stimulate substantial regrowth of E. coli O157:H7., Competing Interests: The authors declare that they have no competing interests.

Background: Although a wealth of literature points to the importance of social factors on health, a detailed understanding of the complex interplay between social and biological systems is lacking. Social status is one aspect of social life that is made up of multiple structural (humans: income, education; animals: mating system, dominance rank) and relational components (perceived social status, dominance interactions). In a nonhuman primate model we use novel network techniques to decouple two components of social status, dominance rank (a commonly used measure of social status in animal models) and dominance certainty (the relative certainty vs. ambiguity of an individual's status), allowing for a more complex examination of how social status impacts health., Methods: Behavioral observations were conducted on three outdoor captive groups of rhesus macaques (N = 252 subjects). Subjects' general physical health (diarrhea) was assessed twice weekly, and blood was drawn once to assess biomarkers of inflammation (interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and C-reactive protein (CRP))., Results: Dominance rank alone did not fully account for the complex way that social status exerted its effect on health. Instead, dominance certainty modified the impact of rank on biomarkers of inflammation. Specifically, high-ranked animals with more ambiguous status relationships had higher levels of inflammation than low-ranked animals, whereas little effect of rank was seen for animals with more certain status relationships. The impact of status on physical health was more straightforward: individuals with more ambiguous status relationships had more frequent diarrhea; there was marginal evidence that high-ranked animals had less frequent diarrhea., Discussion: Social status has a complex and multi-faceted impact on individual health. Our work suggests an important role of uncertainty in one's social status in status-health research. This work also suggests that in order to fully explore the mechanisms for how social life influences health, more complex metrics of social systems and their dynamics are needed., Competing Interests: The authors declare that they have no competing interests.