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.

Coliform and Escherichia coli Contamination on External and Internal Surfaces of Beef Carcasses with and without Tissue Adhesion Excision.

ABSTRACT: Following removal of hides and viscera during beef processing, carcasses are inspected for tissue adhesions that can affect meat quality or harbor bacteria. Carcasses with pleural or abdominal adhesions may be diverted from the production line for manual excision and then returned to the line. No published data indicate whether adhesion excision is associated with bacterial contamination. Therefore, our objective was to determine the presence and concentration of generic Escherichia coli and non-E. coli coliforms from the internal and external surfaces of carcasses that were, or were not, diverted for adhesion excision. During 9 processing days over a 4-month period in a large commercial beef processing facility, 1,738 carcass sponge samples from 2,730 cm2 areas on both the internal and the external surfaces of carcasses with and without tissue adhesions were collected. Coliforms and E. coli were cultured and enumerated using Petrifilm procedures, and data were analyzed with mixed models. Coliforms were present at higher concentrations than E. coli, and prevalence and mean log concentrations of both coliforms and E. coli were significantly higher for samples from the external than from the internal surfaces of carcasses. However, differences in prevalence and concentration of coliforms between external and internal surfaces varied significantly based on whether carcasses had adhesions excised. The difference was greatest for coliforms present on the external (2.06 log CFU/100 cm2) versus the internal (0.93 log CFU/100 cm2) carcass surfaces without adhesions, whereas the difference in concentrations from the external (1.80 log CFU/100 cm2) and the internal (1.31 log CFU/100 cm2) surfaces of carcasses with adhesions was not as large. These results indicate that surveillance of carcass bacteria may be affected by whether the external versus the internal surfaces are sampled and whether carcasses are diverted for excision of adhesions.

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.

Associations Between Season, Processing Plant, and Hide Cleanliness Scores with Prevalence and Concentration of Major Shiga Toxin-Producing Escherichia coli on Beef Cattle Hides.

The objectives of this study were (1) to estimate the prevalence and concentration of the seven major Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, O145, and O157), collectively called STEC-7, on cattle hides collected in different seasons and beef processing plants; and (2) to determine associations of season, plant, and hide cleanliness scores with the prevalence and concentration of STEC-7. A total of 720 hide surface samples (240/season) were collected over three seasons (summer and fall 2015 and spring 2016) from beef cattle carcasses in four commercial processing plants in the United States. Samples were subjected to selective culture and spiral plating methods. Overall model-adjusted mean prevalence (95% confidence interval) was 0.3% (0.03-2.3%) for STEC O26; 0.05% (<0.01-8.5%) for STEC O45; 0.2% (0.02-1.9%) for STEC O103; 0.05% (<0.01-8.5%) for STEC O145; and 3.1% (0.6-15.2%) for STEC O157. Four percent of hide samples were enumerable for STEC O157; mean concentration (standard deviation) = 2.1 (0.7) log10 colony-forming units (CFUs)/100 cm2. No samples were enumerable for non-O157 STEC. Hide-on prevalence of STEC O157 and STEC non-O157 (specifically of STEC O103) was higher in summer and spring, respectively. Across seasons and plants, the most common STEC non-O157 serogroups in this study (O26 and O103) were associated with a higher prevalence of STEC O157. Season and plant played a role in prevalence and concentration of STEC in beef cattle hides, varying by serogroup. Tailoring mitigation strategies at the plant can be challenging and processors would benefit from supplementary preharvest interventions to reduce overall contamination pressure at the plant, especially in fall and spring months when hide-on prevalence of STEC non-O157 is higher.

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.

Quantification of Bacteria Indicative of Fecal and Environmental Contamination from Hides to Carcasses.

Fecal bacteria, which reside in the gastrointestinal tract of cattle, can contaminate beef carcasses during processing. In beef cattle slaughter plants, the presence and concentrations of generic Escherichia coli, coliforms, Enterobacteriaceae (EB), and total aerobic bacteria are monitored as indicator organisms of fecal and environmental contamination. The objectives of this study were as follows: (1) to determine the concentrations of generic E. coli, coliforms, EB, and aerobic bacteria on beef carcasses at different processing points in Midwestern commercial beef slaughter plants during the summer, spring, and fall seasons; and (2) to estimate bacterial transfer on carcasses during the hide removal and evisceration processes. Hide and carcass surface sample swabs were collected from slaughtered cattle at four large commercial processing plants. At each plant visit (3 visits to each of the 4 plants) and during 3 seasons, 20 samples were collected at 5 points: hide-on (hide of animal near exsanguination pit), hide-off carcass, pre-evisceration carcass, postevisceration carcass, and postintervention carcass, for a total of 3600 samples. Bacterial concentrations were determined using 3M™ Petrifilm™ plates. Associations between season and processing plant with concentrations of E. coli, coliforms, EB, and total aerobic bacteria, overall, between hide-on and hide-off, and between pre- and post-evisceration, were evaluated using multilevel mixed-effects linear regression models. Bacterial concentrations on beef carcasses significantly decreased throughout processing. Moreover, hide removal was an important source of carcass contamination, given bacterial concentrations detected on hide-off carcass samples were the highest, and bearing in mind that carcass muscle surfaces should be sterile. Results from this study indicate that the interventions applied by the processing plants were effective, as they probably contributed to the significant reduction of bacterial concentrations of carcasses.

Detection and Quantification of Seven Major Serogroups of Shiga Toxin-Producing Escherichia coli on Hides of Cull Dairy, Cull Beef, and Fed Beef Cattle at Slaughter†.

Dehiding during beef cattle processing can introduce fecal contaminants, including Shiga toxin-producing Escherichia coli (STEC), from hides onto carcass surfaces, creating the potential for contaminated beef. Fecal shedding of major STEC serogroups (O26, O45, O103, O111, O121, O145, and O157; STEC-7) may differ among cattle populations, yet no study has been conducted to isolate STEC-7 on hides of multiple cattle types on the same production days at the same processing plant. Our objective was to estimate and compare prevalence and concentrations of STEC-7 on hides of cull dairy, cull beef, and fed beef cattle from the same date and processing plant. Overall, 1,500 cattle hides were sponge sampled from cull dairy ( n = 500), cull beef ( n = 500) and fed beef cattle ( n = 500) over 10 processing days. To determine prevalence, samples were subjected to an immunomagnetic separation culture method, and presumptive STEC isolates were tested by PCR for serogroup and major virulence genes. A spiral plate method was used to enumerate STEC-7 from hide samples. Data were analyzed with linear mixed models. All STEC-7 serogroups except O121 were detected and quantified on cattle hides in this study population. Slightly more fed beef hides (77 of 500; 15.4%) and cull beef hides (76 of 500; 15.2%) were positive for at least one STEC-7 strain compared with cull dairy hides (57 of 500; 11.4%), but cattle type was not significantly associated ( P = 0.19) with STEC-7 prevalence. Fed beef hides had a significantly higher prevalence ( P < 0.05) of STEC O103, O145, and O157 serogroups than did either of the other cattle types. The highest proportions of quantifiable samples were for STEC O145 (32 of 1,500 samples; 2.1%) and O157 (31 of 1,500 samples; 2.1%) serogroups, with the majority of concentrations at 3 to 5 and 2 to 4 log CFU/100 cm2 of hide, respectively. Results indicate that hide contamination with some major STEC serogroups differs significantly among cattle types at harvest, even within the same day and location.

Feedlot- and Pen-Level Prevalence of Enterohemorrhagic Escherichia coli in Feces of Commercial Feedlot Cattle in Two Major U.S. Cattle Feeding Areas.

The objective of this study was to determine feedlot- and pen-level fecal prevalence of seven enterohemorrhagic Escherichia coli (EHEC) belonging to serogroups (O26, O45, O103, O111, O121, O145, and O157, or EHEC-7) in feces of feedlot cattle in two feeding areas in the United States. Cattle pens from four commercial feedlots in each of the two major U.S. beef cattle areas were sampled. Up to 16 pen-floor fecal samples were collected from each of 4-6 pens per feedlot, monthly, for a total of three visits per feedlot, from June to August, 2014. Culture procedures including fecal enrichment in E. coli broth, immunomagnetic separation, and plating on selective media, followed by confirmation through polymerase chain reaction (PCR) testing, were conducted. Generalized linear mixed models were fitted to estimate feedlot-, pen-, and sample-level fecal prevalence of EHEC-7 and to evaluate associations between potential demographic and management risk factors with feedlot and within-pen prevalence of EHEC-7. All study feedlots and 31.0% of the study pens had at least one non-O157 EHEC-positive fecal sample, whereas 62.4% of pens tested positive for EHEC O157; sample-level prevalence estimates ranged from 0.0% for EHEC O121 to 18.7% for EHEC O157. Within-pen prevalence of EHEC O157 varied significantly by sampling month; similarly within-pen prevalence of non-O157 EHEC varied significantly by month and by the sex composition of the pen (heifer, steer, or mixed). Feedlot management factors, however, were not significantly associated with fecal prevalence of EHEC-7. Intraclass correlation coefficients for EHEC-7 models indicated that most of the variation occurred between pens, rather than within pens, or between feedlots. Hence, the potential combination of preharvest interventions and pen-level management strategies may have positive food safety impacts downstream along the beef chain.

Efficacy of a Salmonella Siderophore Receptor Protein Vaccine on Fecal Shedding and Lymph Node Carriage of Salmonella in Commercial Feedlot Cattle.

The efficacy of a Salmonella vaccine for reducing fecal shedding of Salmonella during the finishing period and lymph node (LN) carriage at harvest was investigated in commercial feedlot cattle. The study was designed as a pen-level randomized complete block with two treatment groups, a Salmonella Newport siderophore receptor and porin proteins-based vaccine (VAC) and a nonvaccinated control (CON). Cattle were randomly allocated into 24 pens within 12 blocks based on the time of allocation. Twenty to 25 fecal pats were collected from each of the study pen floors once a month from June to August 2013. During harvest, a minimum of 25 sub-iliac LN were collected from carcasses within each study pen. Fecal and pulverized LN samples were cultured for Salmonella quantification and detection. Mixed models were used to analyze the effect of vaccination on fecal shedding and LN carriage of Salmonella. Montevideo and Anatum were the predominant Salmonella serotypes among fecal samples and LNs; no Newport isolates were recovered. Vaccination was not significantly associated (p = 0.57) with the prevalence of Salmonella in feces over time; the mean within-pen prevalence was 62.3% and 66.0% among VAC and CON, respectively. Sampling month was significantly associated (p < 0.01) with fecal prevalence; mean prevalence was 71.4% for June, 48.6% for July, and 70.8% for August. Across all pens, the cumulative prevalence of Salmonella in LN was 86.4%. Vaccination resulted in no significant reduction in LN prevalence (p = 0.52); mean prevalence was 85.7% for VAC and 87.4% for CON groups. Although vaccinated cattle had numerically fewer Salmonella LN and fecal positives, there were no statistically significant vaccine effects. Potential reasons for the lack of vaccine efficacy could include an overwhelming Salmonella exposure, a lack of cross-protection against non-Newport serotypes, and insufficient duration of immunity relative to harvest.

Use of Antimicrobial Metaphylaxis for the Control of Bovine Respiratory Disease in High-Risk Cattle.

Despite research and increased availability of antimicrobials, the prevalence and challenges associated with BRD in stocker and feedlot operations remain. Preconditioned calves can better handle the transition from the origin ranch to the feedlot, yet there is incentive for buyers to purchase high-risk cattle at a reduced cost, and this is influenced by the proven efficacy and availability of antimicrobial metaphylaxis. The poor sensitivity of current BRD field diagnostic methods, typical pathogenesis of BRD, and labor issues are additional reasons to use metaphylaxis. Nevertheless, practitioners should consider comprehensive and novel approaches to judiciously guide decisions on metaphylactic use of antimicrobials.

Potential associations between fecal shedding of Salmonella in feedlot cattle treated for apparent respiratory disease and subsequent adverse health outcomes.

A prospective cohort study was used to assess whether Salmonella fecal shedding in commercial feedlot cattle treated with antimicrobials for respiratory disease was associated with subsequent adverse health outcomes. Feces were collected per rectum from cattle that were examined for apparent respiratory disease, had a rectal temperature > or = 40 degrees C, and subsequently received antimicrobial treatment. Salmonella were recovered from 918 (73.7%) of 1 245 fecal samples and weekly prevalence estimates ranged from 49 to 100% over the 3-month study. Genotypic and phenotypic characteristics of Salmonella strains in the population were determined. Serogroup E Salmonella were most common (73.3%), followed by C1 (11.0%), C3 (8.6%), and B (1.1%). Predominant serotypes were Orion (46.5%), Anatum (19.8%), Kentucky (8.7%), Montevideo (7.5%), and Senftenberg (4.9%). Few isolates (36/918) were positive for antimicrobial resistance-associated integron gene intI1. Phenotypic susceptibility was associated with isolate intI1 status. Crude re-pull, re-treatment and case fatality risks were higher for cattle that were Salmonella-positive versus -negative at initial treatment, but not statistically different on multivariable analysis. However, case fatality risk was higher for cattle shedding Group B Salmonella than for cattle shedding other serogroups. Lots (groups) with a higher Salmonella prevalence at first treatment had a higher proportion of mortalities occur in a hospital pen, higher overall re-treatment risks, and were more likely to be sampled later in the study. Results indicate a high prevalence of Salmonella in this population of cattle treated for apparent respiratory disease, but that effects associated with clinical outcomes may depend on the Salmonella strain.

Antimicrobial susceptibility of enteric bacteria recovered from feedlot cattle administered chlortetracycline in feed.

OBJECTIVE: To evaluate administration of chlortetracycline in feed of cattle as a method to select for tetracycline resistance among enteric bacteria in feedlot settings. ANIMALS: 20 steers. PROCEDURES: Steers were randomly assigned to an exposed cohort (n = 10) or an unexposed cohort (control cohort; 10). Chlortetracycline (22 mg/kg) in cottonseed meal was administered to the exposed cohort on days 0 through 4, 6 through 10, and 12 through 16. The control cohort was administered only cottonseed meal. Fecal samples were collected from 16 steers on days -7, 0, 2, 6, 8, 12, 14, 19, 22, 26, and 33, and Escherichia coli and Enterococcus spp were isolated. Minimum inhibitory concentration (MIC) of selected antimicrobials was estimated. RESULTS: Overall, 56.0% and 31.4% of E coli and Enterococcus isolates, respectively, were resistant to tetracycline. Exposure to chlortetracycline was associated with a significant temporary increase in log(2) MIC for both genera but returned to preexposure values by day 33. Averaged across time, the proportion of tetracycline-resistant E coli and Enterococcus isolates was significantly greater in exposed than in unexposed steers. Although all ceftiofur-resistant E coli isolates were coresistant to tetracycline, exposure to chlortetracycline led to a significant decrease in the proportion of E coli resistant to ceftiofur during exposure. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure to chlortetracycline was associated with a temporary increase in the likelihood of recovering resistant bacteria. Exposure to chlortetracycline decreased the likelihood of recovering ceftiofur-resistant E coli isolates, even though isolates were coresistant to tetracycline. These findings warrant further investigation.

Changes in antimicrobial susceptibility in a population of Escherichia coli isolated from feedlot cattle administered ceftiofur crystalline-free acid.

OBJECTIVE: To determine effects of administration of ceftiofur crystalline-free acid (CCFA) on antimicrobial susceptibility of Escherichia coli in feedlot cattle. ANIMALS: 61 feedlot steers. PROCEDURES: A cohort study was conducted. Steers were housed in pens (5 pens with 10 steers and 1 pen with 11 steers). Five steers in each pen were administered CCFA, and 5 served as control steers (1 pen had 6 control steers). The CCFA administration included a single-dose regimen (6.6 mg/kg, SC, on day 0), two-thirds-dose regimen (4.4 mg/kg, SC, on day 0), and 3-dose regimen (6.6 mg/kg, SC, on days 0, 6, and 13). Fecal samples were collected on days 0, 2, 6, 9, 13, 16, 20, and 28. Fecal samples were collected immediately before CCFA administration. Minimum inhibitory concentrations of 15 antimicrobials were determined for 3 E coli isolates/fecal sample. Escherichia coli were enumerated by use of direct-plating techniques. RESULTS: Resistance to 1 or more antimicrobials was detected in 986 of 1,441 (68.4%) isolates recovered. Administration of CCFA was associated with a transient increase in the population of ceftiofur-resistant isolates. Susceptibility returned to day 0 values (ie, samples collected immediately before CCFA administration) approximately 2 weeks after completion of CCFA administration. Agreement between ceftiofur resistance and co-resistance to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, and tetracycline was almost perfect (kappa 0.97). We did not detect variation in susceptibility of E coli recovered from commingled control steers. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of CCFA provided selection pressure that favored transient expansion of multiple-resistant variants.