Temporal dynamics of the fecal microbiome in female pigs from early life through estrus, parturition, and weaning of the first litter of piglets.

BACKGROUND: Age-associated changes in the gastrointestinal microbiome of young pigs have been robustly described; however, the temporal dynamics of the fecal microbiome of the female pig from early life to first parity are not well understood. Our objective was to describe microbiome and antimicrobial resistance dynamics of the fecal microbiome of breeding sows from early life through estrus, parturition and weaning of the first litter of piglets (i.e., from 3 to 53 weeks of age). RESULTS: Our analysis revealed that fecal bacterial populations in developing gilts undergo changes consistent with major maturation milestones. As the pigs progressed towards first estrus, the fecal bacteriome shifted from Rikenellaceae RC9 gut group- and UCG-002-dominated enterotypes to Treponema- and Clostridium sensu stricto 1-dominated enterotypes. After first estrus, the fecal bacteriome stabilized, with minimal changes in enterotype transition and associated microbial diversity from estrus to parturition and subsequent weaning of first litter piglets. Unlike bacterial communities, fecal fungal communities exhibited low diversity with high inter- and intra-pig variability and an increased relative abundance of certain taxa at parturition, including Candida spp. Counts of resistant fecal bacteria also fluctuated over time, and were highest in early life and subsequently abated as the pigs progressed to adulthood. CONCLUSIONS: This study provides insights into how the fecal microbial community and antimicrobial resistance in female pigs change from three weeks of age throughout their first breeding lifetime. The fecal bacteriome enterotypes and diversity are found to be age-driven and established by the time of first estrus, with minimal changes observed during subsequent physiological stages, such as parturition and lactation, when compared to the earlier age-related shifts. The use of pigs as a model for humans is well-established, however, further studies are needed to understand how our results compare to the human microbiome dynamics. Our findings suggest that the fecal microbiome exhibited consistent changes across individual pigs and became more diverse with age, which is a beneficial characteristic for an animal model system.

An Experimental Field Trial Investigating the Use of Bacteriophage and Manure Slurry Applications in Beef Cattle Feedlot Pens for Salmonella Mitigation.

Post-harvest Salmonella mitigation techniques are insufficient at addressing Salmonella harbored in cattle lymph nodes, necessitating the exploration of pre-harvest alternatives that reduce Salmonella prior to dissemination to the lymph nodes. A 2 × 2, unbalanced experiment was conducted to determine the effectiveness of pre-harvest treatments applied to the pen surface for Salmonella mitigation in cattle. Treatments included manure slurry intended to mimic pen run-off water (n = 4 pens), a bacteriophage cocktail (n = 4), a combination of both treatments (n = 5), and a control group (n = 5) that received no treatment. Environment samples from 18 feedlot pens and fecal grabs, hide swabs, and subiliac lymph nodes from 178 cattle were collected and selectively enriched for Salmonella, and Salmonella isolates were sequenced. The combination treatment was most effective at reducing Salmonella, and the prevalence was significantly lower compared with the control group for rump swabs on Days 14 and 21. The treatment impact on Salmonella in the lymph nodes could not be determined due to low prevalence. The reduction on cattle hides suggests that bacteriophage or water treatments applied to the feedlot pen surface may reduce Salmonella populations in cattle during the pre-harvest period, resulting in reduced contamination during slaughter and processing.

Global Estimate of Human Brucellosis Incidence.

Brucellosis is a major public health concern worldwide, especially for persons living in resource-limited settings. Historically, an evidence-based estimate of the global annual incidence of human cases has been elusive. We used international public health data to fill this information gap through application of risk metrics to worldwide and regional at-risk populations. We performed estimations using 3 statistical models (weighted average interpolation, bootstrap resampling, and Bayesian inference) and considered missing information. An evidence-based conservative estimate of the annual global incidence is 2.1 million, significantly higher than was previously assumed. Our models indicate Africa and Asia sustain most of the global risk and cases, although areas within the Americas and Europe remain of concern. This study reveals that disease risk and incidence are higher than previously suggested and lie mainly within resource-limited settings. Clarification of both misdiagnosis and underdiagnosis is required because those factors will amplify case estimates.

A Longitudinal Study on the Dynamics of Salmonella enterica Prevalence and Serovar Composition in Beef Cattle Feces and Lymph Nodes and Potential Contributing Sources from the Feedlot Environment.

Salmonella can persist in the feedlot pen environment, acting as a source of transmission among beef cattle. Concurrently, cattle that are colonized with Salmonella can perpetuate contamination of the pen environment through fecal shedding. To study these cyclical dynamics, pen environment and bovine samples were collected for a 7-month longitudinal comparison of Salmonella prevalence, serovar, and antimicrobial resistance profiles. These samples included composite environment, water, and feed from the feedlot pens (n = 30) and cattle (n = 282) feces and subiliac lymph nodes. Salmonella prevalence across all sample types was 57.7%, with the highest prevalence in the pen environment (76.0%) and feces (70.9%). Salmonella was identified in 42.3% of the subiliac lymph nodes. Based on a multilevel mixed-effects logistic regression model, Salmonella prevalence varied significantly (P < 0.05) by collection month for most sample types. Eight Salmonella serovars were identified, and most isolates were pansusceptible, except for a point mutation in the parC gene, associated with fluoroquinolone resistance. There was a proportional difference in serovars Montevideo, Anatum, and Lubbock comparing the environment (37.2, 15.9, and 11.0%, respectively), fecal (27.5, 22.2, and 14.6%, respectively), and lymph node (15.6, 30.2, and 17.7%, respectively) samples. This suggests that the ability of Salmonella to migrate from the pen environment to the cattle host-or vice versa-is serovar specific. The presence of certain serovars also varied by season. Our results provide evidence that Salmonella serovar dynamics differ when comparing environment and host; therefore, developing serovar-specific preharvest environmental Salmonella mitigation strategies should be considered. IMPORTANCE Salmonella contamination of beef products, specifically from the incorporation of bovine lymph nodes into ground beef, remains a food safety concern. Current postharvest Salmonella mitigation techniques do not address Salmonella bacteria that are harbored in the lymph nodes, nor is it well understood how Salmonella invades the lymph nodes. Alternatively, preharvest mitigation techniques that can be applied to the feedlot environment, such as moisture applications, probiotics, or bacteriophage, may reduce Salmonella before dissemination into cattle lymph nodes. However, previous research conducted in cattle feedlots includes study designs that are cross-sectional, are limited to point-in-time sampling, or are limited to sampling of the cattle host, making it difficult to assess the Salmonella interactions between environment and hosts. This longitudinal analysis of the cattle feedlot explores the Salmonella dynamics between the feedlot environment and beef cattle over time to determine the applicability of preharvest environmental treatments.

Combined Quantification and Deep Serotyping for Salmonella Risk Profiling in Broiler Flocks.

Despite a reduction of Salmonella contamination on final poultry products, the level of human salmonellosis cases attributed to poultry has remained unchanged over the last few years. There needs to be improved effort to target serovars which may survive antimicrobial interventions and cause illness, as well as to focus on lessening the amount of contamination entering the processing plant. Advances in molecular enumeration approaches allow for the rapid detection and quantification of Salmonella in pre- and postharvest samples, which can be combined with deep serotyping to properly assess the risk affiliated with a poultry flock. In this study, we collected a total of 160 boot sock samples from 20 broiler farms across four different integrators with different antibiotic management programs. Overall, Salmonella was found in 85% (68/80) of the houses, with each farm having at least one Salmonella-positive house. The average Salmonella quantity across all four complexes was 3.6 log10 CFU/sample. Eleven different serovars were identified through deep serotyping, including all three key performance indicators (KPIs; serovars Enteritidis, Infantis, and Typhimurium) defined by the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS). There were eight multidrug resistant isolates identified in this study, and seven which were serovar Infantis. We generated risk scores for each flock based on the presence or absence of KPIs, the relative abundance of each serovar as calculated with CRISPR-SeroSeq (serotyping by sequencing the clustered regularly interspaced palindromic repeats), and the quantity of Salmonella organisms detected. The work presented here provides a framework to develop directed processing approaches and highlights the limitations of conventional Salmonella sampling and culturing methods. IMPORTANCE Nearly one in five foodborne Salmonella illnesses are derived from chicken, making it the largest single food category to cause salmonellosis and indicating a need for effective pathogen mitigation. Although industry has successfully reduced Salmonella incidence in poultry products, there has not been a concurrent reduction in human salmonellosis linked to chicken consumption. New efforts are focused on improved control at preharvest, which requires improved Salmonella surveillance. Here, we present a high-resolution surveillance approach that combines quantity and identity of Salmonella in broiler flocks prior to processing which will further support improved Salmonella controls in poultry. We developed a framework for this approach, indicating that it is possible and important to harness deep serotyping and molecular enumeration to inform on-farm management practices and to minimize risk of cross-contamination between flocks at processing. Additionally, this framework could be adapted to Salmonella surveillance in other food animal production systems.

Age influences the temporal dynamics of microbiome and antimicrobial resistance genes among fecal bacteria in a cohort of production pigs.

BACKGROUND: The pig gastrointestinal tract hosts a diverse microbiome, which can serve to select and maintain a reservoir of antimicrobial resistance genes (ARG). Studies suggest that the types and quantities of antimicrobial resistance (AMR) in fecal bacteria change as the animal host ages, yet the temporal dynamics of AMR within communities of bacteria in pigs during a full production cycle remains largely unstudied. RESULTS: A longitudinal study was performed to evaluate the dynamics of fecal microbiome and AMR in a cohort of pigs during a production cycle; from birth to market age. Our data showed that piglet fecal microbial communities assemble rapidly after birth and become more diverse with age. Individual piglet fecal microbiomes progressed along similar trajectories with age-specific community types/enterotypes and showed a clear shift from E. coli/Shigella-, Fusobacteria-, Bacteroides-dominant enterotypes to Prevotella-, Megaspheara-, and Lactobacillus-dominated enterotypes with aging. Even when the fecal microbiome was the least diverse, the richness of ARGs, quantities of AMR gene copies, and counts of AMR fecal bacteria were highest in piglets at 2 days of age; subsequently, these declined over time, likely due to age-related competitive changes in the underlying microbiome. ARGs conferring resistance to metals and multi-compound/biocides were detected predominately at the earliest sampled ages. CONCLUSIONS: The fecal microbiome and resistome-along with evaluated descriptors of phenotypic antimicrobial susceptibility of fecal bacteria-among a cohort of pigs, demonstrated opposing trajectories in diversity primarily driven by the aging of pigs.

Long-Term Effects of Single-Dose Cephalosporin or Macrolide Use on the Prevalence of AmpC and Extended-Spectrum ß-Lactamase Producing Escherichia coli in the Feces of Beef Cattle.

Extended-spectrum-ß-lactamase (ESBL) and AmpC-lactamase-producing Enterobacteriaceae are serious public health threats. Due to an increasing number of reports of ESBL and AmpC producing Escherichia coli in agricultural settings, it is critical to understand the relationship between the use of two of the highest priority critically important human antibiotics (e.g., third generation cephalosporins [3GC] and macrolides) in food animals and their potential contribution to the selection of ESBL/AmpC E. coli. The objective of our randomized controlled feedlot trial was to measure the effects of ceftiofur crystalline-free acid and tulathromycin on 3GC resistant fecal E. coli populations in cattle before and at various time points after treatment up to and including at slaughter. Multi-level mixed-effects linear regression showed no effect of ceftiofur and tulathromycin on 3GC E. coli CFU counts at slaughter (Day 99); however, a significant (p < 0.05) population shift was observed from susceptible to 3GC resistant E. coli immediately after ceftiofur administration (Day 7). Among 799 fecal samples screened using selective media, 17.7% were ESBL/AmpC E. coli positive, which were further tested for phenotypic antibiotic susceptibility. The majority of the isolates from these plates were multidrug-resistant (94.3%) and expressed either AmpC (78.1%) or ESBL (28.1%) phenotype. A subset of isolates was whole-genome sequenced (n = 20) and identified to harbor chromosomal and/or plasmidal bla genes such as CMY-2, CTX-M, and TEM. Our findings show a time-dependent selection of antibiotics on 3GC-resistant E. coli. High prevalence of multidrug-resistant ESBL/AmpC E. coli found in cattle feces highlights the importance of prudent use of antibiotics in livestock.

A longitudinal investigation of the effects of age, dietary fiber type and level, and injectable antimicrobials on the fecal microbiome and antimicrobial resistance of finisher pigs.

Age and diet are among the factors that influence the community composition of the fecal microbiome. Additionally, antimicrobial use can alter the composition of bacterial communities. An 86-d study with finisher pigs aimed to evaluate age-related dynamics (day 98 to 177 of age), effects of types and levels of dietary fiber, and injectable antimicrobials on the fecal microbiome and antimicrobial resistance (AMR) was conducted. A total of 287 pigs, housed in 36 pens, with 7 to 8 pigs per pen, fed a corn grain and soybean meal-based basal diet, formulated to contain 8.7% neutral detergent fiber (NDF), were randomly assigned to one of three treatments: 1) basal diet with no supplement, 2) basal diet supplemented with 20% distillers dried grains with solubles (DDGS) formulated to contain 13.6% NDF, or 3) basal diet supplemented with 14.5% sugar beet pulp (SBP) formulated to contain 13.6% NDF. Five finisher pigs from each treatment group were selected randomly, and fecal samples were collected on days 98, 110, 144, and 177 of age. In addition, fecal samples were collected from pigs that were injected intramuscularly ceftiofur hydrochloride or penicillin G on days 1 and 3 along with pen-mate-untreated controls on day 1. Fecal samples were subjected to 16S rRNA amplicon-based microbiome analysis and culture methods to quantify the abundance of total AMR coliforms and enterococci populations. The alpha-diversity, such as species richness, increased with age, and the overall bacterial composition changed with age (P =0.001) and diet (P = 0.001). Diet-associated shifts in the specific bacterial taxa were observed. The richness, diversity, and evenness of bacterial taxa did not differ between pigs that were injected with ceftiofur vs. their untreated pen mates or by dietary treatments but differed in pigs that received penicillin G injection. Both antimicrobial treatments contributed to changes in the overall fecal bacterial composition at the genus level. Collectively, the data demonstrate that both age and the diet (control vs. DDGS-, control vs. SBP-, or DDGS- vs. SBP-based diets) were associated with the overall bacterial community composition, and the impact of age on variations in fecal microbiome composition was greater than the diet. Antibiotic treatment had minimal effect on bacterial diversity and relative abundance of taxa. Furthermore, diets and antimicrobial treatment had minimal impact on the overall counts of AMR coliforms and enterococci populations in feces.

Bacterial communities in the gut and the feces are strongly influenced by a number of factors, particularly the age of the animal and the diet. In addition, antibiotic administration routinely used to treat bacterial diseases can also affect the community composition. A study with finisher pigs was conducted to evaluate age-related changes, effects of types­distiller's dried grains with solubles (DGGS) or sugar beet pulp (SBP)­and levels of dietary fiber, and injectable antibiotics on the fecal bacterial composition and antibiotic resistance in fecal bacteria. Fecal samples were collected from five pigs in each of the three dietary treatment groups, control diet with no supplement or supplemented with DDGS or SBP, on days 98, 110, 144, and 177 of age and on days 1 and 3 after the first injection of antibiotics, ceftiofur or penicillin G. Samples were analyzed to identify the bacterial community composition and prevalence of antibiotic resistance in fecal bacteria. Data generated suggested that the overall bacterial composition changed with age and diet, and age appeared to have a greater impact than diet. Antibiotics had only a modest impact on the bacterial community and had minimum impact on antibiotic resistance of fecal bacteria.

Patterns of antimicrobial drug use in veterinary primary care and specialty practice: A 6-year multi-institution study.

BACKGROUND: Combatting antimicrobial resistance requires a One Health approach to antimicrobial stewardship including antimicrobial drug (AMD) use evaluation. Current veterinary AMD prescribing data are limited. OBJECTIVES: To quantify companion animal AMD prescribing in primary care and specialty practice across 3 academic veterinary hospitals with particular focus on third-generation cephalosporins, fluoroquinolones, and carbapenems. ANIMALS: Dogs and cats presented to 3 academic veterinary hospitals from 2012 to 2017. METHODS: In this retrospective study, AMD prescribing data from 2012 to 2017 were extracted from electronic medical records at each hospital and prescriptions classified by service type: primary care, specialty practice or Emergency/Critical Care (ECC). Hospital-level AMD prescribing data were summarized by species, service type, AMD class, and drug. Multivariable logistic full-factorial regression models were used to estimate hospital, year, species, and service-type effects on AMD prescribing. Estimated marginal means and confidence intervals were plotted over time. RESULTS: The probability of systemic AMD prescribing for any indication ranged between 0.15 and 0.28 and was higher for dogs than cats (P < .05) apart from 2017 at hospital 1. Animals presented to primary care were least likely to receive AMDs (dogs 0.03-0.15, cats 0.03-0.18). The most commonly prescribed AMD classes were aminopenicillins/ß-lactamase inhibitors (0.02-0.15), first-generation cephalosporins (0.00-0.09), fluoroquinolones (0.00-0.04), nitroimidazoles (0.01-0.06), and tetracyclines (0.00-0.03). Among the highest priority classes, fluoroquinolones (dogs 0.00-0.09, cats 0.00-0.08) and third-generation cephalosporins (dogs 0.00-0.04, cats 0.00-0.05) were most frequently prescribed. CONCLUSIONS AND CLINICAL IMPORTANCE: Antimicrobial drug prescribing frequencies were comparable to previous studies. Additional stewardship efforts might focus on fluoroquinolones and third-generation cephalosporins.

Antibiotic resistance among Escherichia coli and Salmonella isolated from dairy cattle feces in Texas.

In several developing countries, studies on antimicrobial resistance among bacteria from food animals are rare mostly because of under-resourced laboratories. The objective of this study was to develop and field-test a low cost protocol to estimate the isolate- and sample-level prevalence of resistance to critically important antibiotics among Escherichia coli and Salmonella isolated from dairy cattle feces. Using a predesigned protocol, fecal samples were collected to isolate non-type-specific E. coli and Salmonella using selective media without antibiotic supplements. Besides, samples were screened for E. coli and Salmonella isolates not susceptible to third-generation cephalosporins and quinolones using selective media supplemented with cefotaxime (1.0 µg/mL) and ciprofloxacine (0.5 µg/mL), respectively. All bacterial isolates were further tested for antibiotic susceptibility using disk diffusion. Bacterial isolates not susceptible to third-generation cephalosporins were tested for extended spectrum beta-lactamase (ESBL) phenotype using the combination disk test. Molecular methods were performed on selected bacterial isolates to identify and distinguish genetic determinants associated with the observed phenotypes. Among 85 non-type-specific E. coli isolated from MacConkey agar without antibiotics, the isolate-level prevalence of resistance to tetracycline was the highest (8.2%). Among 37 E. coli recovered from MacConkey agar with cefotaxime, 56.8% were resistant ceftriaxone. Among 22 E. coli isolates recovered from MacConkey agar with ciprofloxacin, 77.3% and 54.5% were resistant to nalidixic acid and ciprofloxacin, respectively. Sixteen Salmonella were isolated and only one demonstrated any resistance (i.e., single resistance to streptomycin). Among E. coli isolates not susceptible to ceftriaxone, an AmpC phenotype was more common than an ESBL phenotype (29 versus 10 isolates, respectively). Whole genome sequencing showed that phenotypic profiles of antibiotic resistance detected were generally substantiated by genotypic profiles. The tested protocol is suited to detecting and estimating prevalence of antimicrobial resistance in bacteria isolated from food animal feces in resource-limited laboratories in the developing world.

Antibiotic-Resistant Escherichia coli and Salmonella from the Feces of Food Animals in the East Province of Rwanda.

In Rwanda, information on antibiotic resistance in food animals is scarce. This study was conducted to detect and phenotypically characterize antibiotic-resistant Escherichia coli and Salmonella in feces of cattle, goats, pigs, and poultry in the East province of Rwanda. We isolated non-type-specific (NTS) E. coli and Salmonella using plain culture media. In addition, we used MacConkey agar media supplemented with cefotaxime at 1.0 µg/mL and ciprofloxacin at 0.5 µg/mL to increase the probability of detecting E. coli with low susceptibility to third-generation cephalosporins and quinolones, respectively. Antibiotic susceptibility testing was performed using the disk diffusion test. Among 540 NTS E. coli isolates, resistance to tetracycline was the most frequently observed (35.6%), followed by resistance to ampicillin (19.6%) and streptomycin (16.5%). Percentages of NTS E. coli resistant to all three antibiotics and percentages of multidrug-resistant strains were higher in isolates from poultry. All isolated Salmonella were susceptible to all antibiotics. The sample-level prevalence for resistance to third-generation cephalosporins was estimated at 35.6% with all third-generation cephalosporin-resistant E. coli, expressing an extended-spectrum beta-lactamase phenotype. The sample-level prevalence for quinolone resistance was estimated at 48.3%. These results provided a baseline for future research and the development of integrated surveillance initiatives.

High-Resolution Genomic Comparisons within Salmonella enterica Serotypes Derived from Beef Feedlot Cattle: Parsing the Roles of Cattle Source, Pen, Animal, Sample Type, and Production Period.

Salmonella enterica is a major foodborne pathogen, and contaminated beef products have been identified as one of the primary sources of Salmonella-related outbreaks. Pathogenicity and antibiotic resistance of Salmonella are highly serotype and subpopulation specific, which makes it essential to understand high-resolution Salmonella population dynamics in cattle. Time of year, source of cattle, pen, and sample type (i.e., feces, hide, or lymph nodes) have previously been identified as important factors influencing the serotype distribution of Salmonella (e.g., Anatum, Lubbock, Cerro, Montevideo, Kentucky, Newport, and Norwich) that were isolated from a longitudinal sampling design in a research feedlot. In this study, we performed high-resolution genomic comparisons of Salmonella isolates within each serotype using both single-nucleotide polymorphism-based maximum-likelihood phylogeny and hierarchical clustering of core-genome multilocus sequence typing. The importance of the aforementioned features in clonal Salmonella expansion was further explored using a supervised machine learning algorithm. In addition, we identified and compared the resistance genes, plasmids, and pathogenicity island profiles of the isolates within each subpopulation. Our findings indicate that clonal expansion of Salmonella strains in cattle was mainly influenced by the randomization of block and pen, as well as the origin/source of the cattle, i.e., regardless of sampling time and sample type (i.e., feces, lymph node, or hide). Further research is needed concerning the role of the feedlot pen environment prior to cattle placement to better understand carryover contributions of existing strains of Salmonella and their bacteriophages. IMPORTANCESalmonella serotypes isolated from outbreaks in humans can also be found in beef cattle and feedlots. Virulence factors and antibiotic resistance are among the primary defense mechanisms of Salmonella, and are often associated with clonal expansion. This makes understanding the subpopulation dynamics of Salmonella in cattle critical for effective mitigation. There remains a gap in the literature concerning subpopulation dynamics within Salmonella serotypes in feedlot cattle from the beginning of feeding up until slaughter. Here, we explore Salmonella population dynamics within each serotype using core-genome phylogeny and hierarchical classifications. We used machine learning to quantitatively parse the relative importance of both hierarchical and longitudinal clustering among cattle host samples. Our results reveal that Salmonella populations in cattle are highly clonal over a 6-month study period and that clonal dissemination of Salmonella in cattle is mainly influenced spatially by experimental block and pen, as well by the geographical origin of the cattle.

Antimicrobial Resistance Hidden within Multiserovar Salmonella Populations.

Salmonella enterica can exist in food animals as multiserovar populations, and different serovars can harbor diverse antimicrobial resistance (AMR) profiles. Conventional Salmonella isolation assesses AMR only in the most abundant members of a multiserovar population, which typically reflects their relative abundance in the initial sample. Therefore, AMR in underlying serovars is an undetected reservoir that can readily be expanded upon antimicrobial use. CRISPR-SeroSeq profiling demonstrated that 60% of cattle fecal samples harbored multiple serovars, including low levels of Salmonella serovar Reading in 11% of samples, which were not found by culture-based Salmonella isolation. An in vitro challenge revealed that Salmonella serovar Reading was tetracycline resistant, while more abundant serovars were susceptible. This study highlights the importance of AMR surveillance in multiserovar populations.

Can the use of older-generation beta-lactam antibiotics in livestock production over-select for beta-lactamases of greatest consequence for human medicine? An in vitro experimental model.

Though carbapenems are not licensed for use in food animals in the U.S., carbapenem resistance among Enterobacteriaceae has been identified in farm animals and their environments. The objective of our study was to determine the extent to which older-generation ß-lactam antibiotics approved for use in food animals in the U.S. might differentially select for resistance to antibiotics of critical importance to human health, such as carbapenems. Escherichia coli (E. coli) strains from humans, food animals, or the environment bearing a single ß-lactamase gene (n = 20 each) for blaTEM-1, blaCMY-2, and blaCTX-M-* or else blaKPC/IMP/NDM (due to limited availability, often in combination with other bla genes), were identified, along with 20 E. coli strains lacking any known beta-lactamase genes. Baseline estimates of intrinsic bacterial fitness were derived from the population growth curves. Effects of ampicillin (32 µg/mL), ceftriaxone (4 µg/mL) and meropenem (4 µg/mL) on each strain and resistance-group also were assessed. Further, in vitro batch cultures were prepared by mixing equal concentrations of 10 representative E. coli strains (two from each resistance gene group), and each mixture was incubated at 37°C for 24 hours in non-antibiotic cation-adjusted Mueller-Hinton II (CAMH-2) broth, ampicillin + CAMH-2 broth (at 2, 4, 8, 16, and 32 µg/mL) and ceftiofur + CAMH-2 broth (at 0.5, 1, 2, 4, and 8µg/mL). Relative and absolute abundance of resistance-groups were estimated phenotypically. Line plots of the raw data were generated, and non-linear Gompertz models and multilevel mixed-effect linear regression models were fitted to the data. The observed strain growth rate distributions were significantly different across the groups. AmpC strains (i.e., blaCMY-2) had distinctly less robust (p < 0.05) growth in ceftriaxone (4 µg/mL) compared to extended-spectrum beta-lactamase (ESBL) producers harboring blaCTX-M-*variants. With increasing beta-lactam antibiotic concentrations, relative proportions of ESBLs and CREs were over-represented in the mixed bacterial communities; importantly, this was more pronounced with ceftiofur than with ampicillin. These results indicate that aminopenicillins and extended-spectrum cephalosporins would be expected to propagate carbapenemase-producing Enterobacteriaceae in food animals if and when Enterobacteriaceae from human health care settings enter the food animal environment.

Comprehensive phenotypic and genotypic characterization and comparison of virulence, biofilm, and antimicrobial resistance in urinary Escherichia coli isolated from canines.

Urinary tract infections (UTIs) affect nearly half of women and an estimated 14 % of the canine companion animal population at least once in their lifetime. As with humans, Escherichia coli is the most commonly isolated bacteria from canine UTIs and infections are dominated by specific phylogenetic groups with notable virulence attributes. In this study, we evaluated uropathogenic E. coli (UPEC) (n = 69) isolated from canine UTIs phenotypically and genotypically for virulence factors, biofilm formation and antimicrobial resistance profiles. Biofilm formation in UPEC strains was positively associated with common virulence factors including papG (p = 0.006), fimH (p < 0.0001), sfaS (p = 0.004), focA (p = 0.004), cnf-1 (p = 0.009) and hlyA (p = 0.006). There was a negative association between biofilm formation and phenotypic antimicrobial resistance for ampicillin (p < 0.0004), ciprofloxacin (p < 0.0001), and trimethoprim-sulfamethoxazole (p < 0.02), as well as multidrug resistance (isolates resistant to ≥ 3 classes of antimicrobials) (p < 0.0002), and the presence of extended spectrum beta-lactamase (ESBL)-producing genes (p < 0.05). In conclusion, UPECs isolated from clinical cases of canine UTIs show a broad negative association between antimicrobial resistance and biofilm formation, and this observation is supported both by phenotypic and genotypic endpoints. As the biofilm formation may result in antimicrobial tolerance, this could be a secondary evasive tactic of UPEC lacking traditional antimicrobial resistance traits. This observation is important for veterinary practitioners to consider when treating puzzling chronic intractable and/or recurrent cases of UTI that appear to be susceptible to antimicrobial therapy via traditional antimicrobial susceptibility testing (AST) methods.

Scoping review of brucellosis in Cameroon: Where do we stand, and where are we going?

Brucellosis is a zoonotic disease known to be endemic to parts of western and sub-Saharan Africa. However, the epidemiology for humans and animals remains largely unknown in many of these countries with Cameroon being a typical example. Despite common knowledge that brucellosis affects livestock, the actual number of infected animals remains unknown. Through a scoping review, the current known status of the disease is described. The aim is to ascertain relevant and publicly accessible research and knowledge of human and animal brucellosis in the country, and to provide an overview of the factors associated with its known persistence. Seroprevalence has been estimated and published in 12 separate instances (1 human; 9 cattle; 1 human and cattle; and 1 that includes cattle, pigs, and small ruminants), between 1982 and 2020, in 9 of the country's 10 geopolitical regions. In 1983, Brucella abortus and B. melitensis were isolated in cattle, but no further bacterial isolation has been published since. The seroprevalence from 196 total humans has ranged between 5.6% and 28.1%, and between 3.0% and 30.8% for 14,044 total cattle. As there is no ongoing surveillance program, it is not currently possible to identify the specific Brucella spp. that are endemic to the country and its regions. There are sufficient agricultural systems of cattle, pigs, goats, and sheep to sustain the presence of multiple Brucella spp. Surveillance information is the cornerstone of epidemiologic decision making, and is needed to direct policy makers, public health authorities, and veterinary services to appropriate actions. A combination of serological and molecular based diagnostics for surveillance is necessary to identify, quantify, and direct the appropriate public health interventions. Cameroon has an opportunity to build public and animal health infrastructure, leading the way for central Africa in the management and future eradication of brucellosis.

The effect of tylosin on antimicrobial resistance in beef cattle enteric bacteria: A systematic review and meta-analysis.

BACKGROUND: Tylosin is a commonly used in-feed antimicrobial and is approved in several countries to reduce the incidence of liver abscesses in beef cattle. Macrolides are critically important antimicrobials in human health and used to treat some foodborne bacterial diseases, such as Campylobacter jejuni and Salmonella. Feeding tylosin could select for resistant enteric bacteria in cattle, which could contaminate beef products at slaughter and potentially cause foodborne illness. We conducted a systematic review and meta-analysis to evaluate the impact of feeding tylosin to cattle on phenotypic and genotypic resistance in several potential zoonotic enteric bacteria: Enterococcus species, Escherichia coli, Salmonella enterica subspecies enterica, and Campylobacter species. This review was registered with PROSPERO (#CRD42018085949). RESULTS: Eleven databases were searched for primary research studies that fed tylosin at approved doses to feedlot cattle and tested bacteria of interest for phenotypic or genotypic resistance. We screened 1,626 citations and identified 13 studies that met the inclusion criteria. Enterococcus species were tested in seven studies, Escherichia coli was isolated in five studies, three studies reported on Salmonella, and two studies reported on Campylobacter species. Most studies relied on phenotypic antimicrobial susceptibility testing and seven also reported resistance gene testing. A random-effects meta-analyses of erythromycin-resistant enterococci from four studies had significant residual heterogeneity. Only two studies were available for a meta-analysis of tylosin-resistant enterococci. A semi-quantitative analysis demonstrated an increase in macrolide-resistant enterococci after long durations of tylosin administration (>100 days). Semi-quantitative analyses of other bacteria-antimicrobial combinations revealed mixed results, but many comparisons found no effect of tylosin administration. However, about half of these no-effect comparisons did not record the cumulative days of tylosin administration or the time since the last dose. CONCLUSIONS: When fed at approved dosages for typical durations, tylosin increases the proportion of macrolide-resistant enterococci in the cattle gastrointestinal tract, which could pose a zoonotic risk to human beef consumers. Feeding tylosin for short durations may mitigate the impact on macrolide-resistant enterococci and further studies are encouraged to determine the effect of minimizing or eliminating tylosin use in beef cattle. There may also be an impact on other bacteria and other antimicrobial resistances but additional details or data are needed to strengthen these comparisons. We encourage authors of antimicrobial-resistance studies to follow reporting guidelines and publish details of all comparisons to strengthen future meta-analyses.

Strategic Priorities for Research on Antibiotic Alternatives in Animal Agriculture-Results From an Expert Workshop.

The emergence, spread, and expansion of antibiotic resistance and increasing restrictions on the use of antibiotics in animal agriculture have created a need for efficacious alternatives that remains unmet. Prioritizing research needs in the development of alternatives is key to ensuring that scarce research resources are dedicated to the most promising approaches. However, frameworks to enable a consistent, systematic, and transparent evaluation of antibiotic alternative candidates are lacking. Here, we present such an evaluation framework.

Population Dynamics of Salmonella enterica within Beef Cattle Cohorts Followed from Single-Dose Metaphylactic Antibiotic Treatment until Slaughter.

Antibiotic use in cattle can select for multidrug-resistant Salmonella enterica, which is considered a serious threat by the U.S. Centers for Disease Control and Prevention. A randomized controlled longitudinal field trial was designed to determine the long-term effects of a single dose of ceftiofur or tulathromycin on Salmonella population characteristics in cattle feces and peripheral lymph nodes and on hides. A total of 134 beef cattle from two sources were divided among 12 pens, with cattle in each of the 3-pen blocks receiving a single dose of either ceftiofur or tulathromycin or neither (control) on day 0. Fecal samples were collected before treatment (day 0) and repeatedly following treatment until slaughter (day 99+). Hide and lymph node samples were collected at slaughter age. Salmonella prevalence, phenotypic antimicrobial resistance, serotype, and phylogenetic relationships were examined. Multilevel mixed logistic regression models indicated no significant effects (P ≥ 0.218) of metaphylactic antibiotics on the prevalence of Salmonella across sample types. However, there was a significant time effect observed, with prevalence increasing from spring through the midsummer months (P < 0.0001) in feces. The majority of Salmonella isolates were pansusceptible to a panel of 14 antibiotics both before and after treatment. Highly prevalent Salmonella serotypes were Salmonella enterica serovar Montevideo, Salmonella enterica serovar Anatum, Salmonella enterica serovar Cerro, and Salmonella enterica serovar Lubbock across all sample types. Strong pen and cattle source serotype clustering effects were observed among Salmonella isolates originating from fecal, lymph node, and hide samples; however, the potential role of Salmonella isolates from the pen environment prior to animal placement was not assessed in this study.IMPORTANCESalmonella is a leading bacterial foodborne pathogen, causing a significant number of human infections and deaths every year in the United States. Macrolides and 3rd-generation cephalosporins play critical roles in the treatment of human salmonellosis. Use of these antibiotics in beef cattle can select for resistant bacteria that may enter the food chain or spread from the farm via manure. There is a lack of longitudinal research concerning the long-term effects of metaphylactic antibiotic administration. Here, we assessed Salmonella population dynamics during the feeding period until slaughter following single-dose antibiotic treatment. We found no long-term effects of antibiotic use early in the cattle-feeding period on Salmonella prevalence and antimicrobial resistance at slaughter. We identified the pens in which cattle were housed as the factor that contributed most to Salmonella serotypes being shared; importantly, the dominant strain in each pen changed repeatedly over the entire feeding period.