Getahun E. Agga

Antimicrobial-Resistant Bacterial Populations and Antimicrobial Resistance Genes Obtained from Environments Impacted by Livestock and Municipal Waste

This study compared the populations of antimicrobial-resistant bacteria and the repertoire of antimicrobial resistance genes in four environments: effluent of three municipal wastewater treatment facilities, three cattle feedlot runoff catchment ponds, three swine waste lagoons, and two “low impact” environments (an urban lake and a relict prairie). Multiple liquid and solid samples were collected from each environment. The prevalences and concentrations of antimicrobial-resistant (AMR) Gram-negative (Escherichia coli and Salmonella enterica) and Gram-positive (enterococci) bacteria were determined from individual samples (n = 174). The prevalences of 84 antimicrobial resistance genes in metagenomic DNA isolated from samples pooled (n = 44) by collection date, location, and sample type were determined. The prevalences and concentrations of AMR E. coli and Salmonella were similar among the livestock and municipal sample sources. The levels of erythromycin-resistant enterococci were significantly higher in liquid samples from cattle catchment ponds and swine waste lagoons than in liquid samples from municipal wastewater treatment facilities, but solid samples from these environments did not differ significantly. Similarly, trimethoprim/sulfamethoxazole-resistant E. coli concentrations were significantly higher in swine liquid than in municipal liquid samples, but there was no difference in solid samples. Multivariate analysis of the distribution of antimicrobial resistance genes using principal coordinate analysis showed distinct clustering of samples with livestock (cattle and swine), low impact environment and municipal samples forming three separate clusters. The numbers of class A beta-lactamase, class C beta-lactamase, and fluoroquinolone resistance genes detected were significantly higher (P < 0.05) in municipal samples than in cattle runoff or swine lagoon samples. In conclusion, we report that AMR is a very widespread phenomenon and that similar prevalences and concentrations of antimicrobial-resistant bacteria and antimicrobial resistance genes exist in cattle, human, and swine waste streams, but a higher diversity of antimicrobial resistance genes are present in treated human waste discharged from municipal wastewater treatment plants than in livestock environments.

Occurrence of Antimicrobial-Resistant Escherichia coli and Salmonella enterica in the Beef Cattle Production and Processing Continuum

ABSTRACT Specific concerns have been raised that third-generation cephalosporin-resistant (3GCr) Escherichia coli, trimethoprim-sulfamethoxazole-resistant (COTr) E. coli, 3GCr Salmonella enterica, and nalidixic acid-resistant (NALr) S. enterica may be present in cattle production environments, persist through beef processing, and contaminate final products. The prevalences and concentrations of these organisms were determined in feces and hides (at feedlot and processing plant), pre-evisceration carcasses, and final carcasses from three lots of fed cattle (n = 184). The prevalences and concentrations were further determined for strip loins from 103 of the carcasses. 3GCr Salmonella was detected on 7.6% of hides during processing and was not detected on the final carcasses or strip loins. NALr S. enterica was detected on only one hide. 3GCr E. coli and COTr E. coli were detected on 100.0% of hides during processing. Concentrations of 3GCr E. coli and COTr E. coli on hides were correlated with pre-evisceration carcass contamination. 3GCr E. coli and COTr E. coli were each detected on only 0.5% of final carcasses and were not detected on strip loins. Five hundred and 42 isolates were screened for extraintestinal pathogenic E. coli (ExPEC) virulence-associated markers. Only two COTr E. coli isolates from hides were ExPEC, indicating that fed cattle products are not a significant source of ExPEC causing human urinary tract infections. The very low prevalences of these organisms on final carcasses and their absence on strip loins demonstrate that current sanitary dressing procedures and processing interventions are effective against antimicrobial-resistant bacteria.

Effects of chlortetracycline and copper supplementation on antimicrobial resistance of fecal Escherichia coli from weaned pigs

Feed-grade chlortetracycline (CTC) and copper are both widely utilized in U.S. pig production. Cluster randomized experiment was conducted to evaluate the effects of CTC and copper supplementation in weaned pigs on antimicrobial resistance (AMR) among fecal Escherichia coli. Four treatment groups: control, copper, CTC, or copper plus CTC were randomly allocated to 32 pens with five pigs per pen. Fecal samples were collected weekly from three pigs per pen for six weeks. Two E. coli isolates per fecal sample were tested for phenotypic and genotypic resistance against antibiotics and copper. Data were analyzed with multilevel mixed effects logistic regression, multivariate probit analysis and discrete time survival analysis. CTC-supplementation was significantly (99% [95% CI=98-100%]) associated with increased tetracycline resistance compared to the control group (95% [95% CI=94-97%]). Copper supplementation was associated with decreased resistance to most of the antibiotics tested, including cephalosporins, over the treatment period. Overall, 91% of the E. coli isolates were multidrug resistant (MDR) (resistant to ≥3 antimicrobial classes). tetA and blaCMY-2 genes were positively associated (P<0.05) with MDR categorization, while tetB and pcoD were negatively associated with MDR. tetA and blaCMY-2 were positively associated with each other and in turn, these were negatively associated with both tetB and pcoD genes; which were also positively associated with one another. Copper minimum inhibitory concentration was not affected by copper supplementation or by pcoD gene carriage. CTC supplementation was significantly associated with increased susceptibilities of E. coli to copper (HR=7 [95% CI=2.5-19.5]) during treatment period. In conclusion, E. coli isolates from the nursery pigs exhibited high levels of antibiotic resistance, with diverse multi-resistant phenotypic profiles. The roles of copper supplementation in pig production, and pco-mediated copper resistance among E. coli in particular, need to be further explored since a strong negative association of pco with both tetA and blaCMY-2 points to opportunities for selecting a more innocuous resistance profile.

Effects of chlortetracycline and copper supplementation on the prevalence, distribution, and quantity of antimicrobial resistance genes in the fecal metagenome of weaned pigs

Use of in-feed antibiotics such as chlortetracycline (CTC) in food animals is fiercely debated as a cause of antimicrobial resistance in human pathogens; as a result, alternatives to antibiotics such as heavy metals have been proposed. We used a total community DNA approach to experimentally investigate the effects of CTC and copper supplementation on the presence and quantity of antimicrobial resistance elements in the gut microbial ecology of pigs. Total community DNA was extracted from 569 fecal samples collected weekly over a 6-week period from groups of 5 pigs housed in 32 pens that were randomized to receive either control, CTC, copper, or copper plus CTC regimens. Qualitative and quantitative PCR were used to detect the presence of 14 tetracycline resistance (tet) genes and to quantify gene copies of tetA, tetB, blaCMY-2 (a 3rd generation cephalosporin resistance gene), and pcoD (a copper resistance gene), respectively. The detection of tetA and tetB decreased over the subsequent sampling periods, whereas the prevalence of tetC and tetP increased. CTC and copper plus CTC supplementation increased both the prevalence and gene copy numbers of tetA, while decreasing both the prevalence and gene copies of tetB. In summary, tet gene presence was initially very diverse in the gut bacterial community of weaned pigs; thereafter, copper and CTC supplementation differentially impacted the prevalence and quantity of the various tetracycline, ceftiofur and copper resistance genes resulting in a less diverse gene population.

Evaluation of Bacteriophage Application to Cattle in Lairage at Beef Processing Plants to Reduce Escherichia coli O157:H7 Prevalence on Hides and Carcasses

Escherichia coli O157:H7 is a major food safety concern for the beef industry. Several studies have provided evidence that cattle hides are the main source of beef carcass contamination during processing and that reductions in the E. coli O157:H7 load on the hides of cattle entering processing facilities will lead to reductions in carcass contamination. Bacteriophages have been proposed as a novel preharvest antimicrobial intervention to reduce the levels of E. coli O157:H7 on cattle hides. The objective of this study was to evaluate a commercialized phage application administered in the lairage area of commercial beef processing plants for the ability to reduce E. coli O157:H7 contamination of cattle hides and carcasses. Cattle lots either received phage spray treatment (n = 289) or did not (n = 301), as they entered the lairage environments in two separate experiments at two different commercial beef processing plants. Hide and carcass samples were collected and analyzed for E. coli O157:H7 prevalence and concentration. Cattle hides receiving phage treatment had an E. coli O157:H7 prevalence of 51.8%, whereas untreated hides had a prevalence of 57.6%. For carcass samples, the E. coli O157 prevalence in treated and untreated samples was 17.1% and 17.6%, respectively. The results obtained from these experiments demonstrated that the treatment of cattle hides with bacteriophages before processing did not produce a significant reduction of E. coli O157:H7 on cattle hides or beef carcasses during processing.

Evaluation of Rectoanal Mucosal Swab Sampling for Molecular Detection of Enterohemorrhagic Escherichia coli in Beef Cattle

Cattle are a primary reservoir of enterohemorrhagic Escherichia coli (EHEC), and contaminated beef products are a source of human infections. The U.S. Department of Agriculture Food Safety and Inspection Service declared seven EHEC serogroups (O26, O45, O103, O111, O121, O145, and O157) as adulterants in raw ground beef. Sampling a large number of animals for EHEC surveillance or evaluations of EHEC-focused preharvest interventions requires a convenient and robust sampling method. We evaluated the diagnostic performance of rectoanal mucosal swab (RAMS) for the detection of the top seven EHEC serogroups. Paired fecal grab (FG) and RAMS samples were collected from 176 beef cattle and tested using the NeoSEEK Shiga toxin-producing E. coli (STEC) confirmation method. The prevalence of virulence-associated genes (stx1, stx2, stx2c, eae, and nleB) was higher in RAMS than in FG samples. The results of the two methods had poor agreement, as indicated by kappa statistics, for the detection of the seven serogroups. When FG and RAMS results were combined for comparison, RAMS was more sensitive than FG for the detection of serogroups O103 (82% versus 39%), O157 (75% versus 67%), and O45 (79% versus 73%) with similar sensitivity for the detection of serogroup O145 (67%). Serogroups O111 and O121 were detected from one and two samples, respectively, by FG and were not detected by RAMS. Serogroup O26 was not detected with either method. RAMS appears to be equivalent or superior to FG sampling for detection of the top seven EHEC serogroups in the feces of beef cattle with the NeoSEEK STEC confirmation test.

Prevalence of Escherichia coli O157:H7 in beef cattle at slaughter and beef carcasses at retail shops in Ethiopia.

BackgroundThere is paucity of information regarding the epidemiology of Escherichia coli O157: H7 in developing countries. In this study, we investigated the occurrence of E. coli O157: H7 associated with beef cattle at processing plants and at retail shops in Ethiopia.MethodsVarious samples were collected from beef cattle at slaughter/processing plants, carcass at retail shops and humans at health centers. E. coli O157: H7 was isolated, identified and characterized for antimicrobial resistance, using standard microbiological methods.ResultsAt the processing plants E. coli O157: H7 was detected in 1.89% of fecal, 0.81% of intestinal mucosal swab, 0.54% of skin swab and 0.54% of carcass internal swab samples. At retail shops it was detected in 0.8% of carcass and 0.8% of cutting board swab samples, while all samples from utensils, hands from workers, and fecal and stool samples were negative. All isolates were resistant to Amoxicillin, moderately resistant to Cefoxitine and Nitrofurantoins but susceptible to other antimicrobials tested.ConclusionsE. coli O157: H7 occurs at low prevalence in beef cattle, and the current sanitary dressing procedures in the processing plants and storage conditions in the retail shops are effective against E. coli O157: H7.

Effects of In-Feed Chlortetracycline Prophylaxis in Beef Cattle on Animal Health and Antimicrobial-Resistant Escherichia coli

ABSTRACT Concerns have been raised that in-feed chlortetracycline (CTC) may increase antimicrobial resistance (AMR), specifically tetracycline-resistant (TETr) Escherichia coli and third-generation cephalosporin-resistant (3GCr) E. coli. We evaluated the impact of a 5-day in-feed CTC prophylaxis on animal health, TETr E. coli, and 3GCr E. coli. A control group of cattle (n = 150) received no CTC, while a CTC group (n = 150) received in-feed CTC (10 mg/lb of body weight/day) from the 5th to the 9th day after feedlot arrival. Over 25% (38/150) of the animals in the control group developed illnesses requiring therapeutic treatment with antimicrobials critically important to human medicine. Only two animals (1.3%) in the CTC group required such treatments. Fecal swab and pen surface occurrences of generic E. coli (isolated on media that did not contain antimicrobials of interest and were not isolated based on any specific resistance), TETr E. coli, and 3GCr E. coli were determined on five sampling occasions: arrival at the feedlot, 5 days posttreatment (5 dpt), 27 dpt, 75 dpt, and 117 dpt. On 5 dpt, TETr E. coli concentrations were higher for the CTC group than the control group (P < 0.01). On 27 dpt, 75 dpt, and 117 dpt, TETr E. coli concentrations did not differ between groups. 3GCr E. coli occurrences did not differ between control and CTC groups on any sampling occasion. For both groups, generic, TETr, and 3GCr E. coli occurrences were highest on 75 dpt and 117 dpt, suggesting that factors other than in-feed CTC contributed more significantly to antimicrobial-resistant E. coli occurrence. IMPORTANCE The occurrence of human bacterial infections resistant to antimicrobial therapy has been increasing. It has been postulated that antimicrobial resistance was inevitable, but the life span of the antimicrobial era has been prematurely compromised due to the misuse of antimicrobials in clinical and agricultural practices. Direct evidence relating the use of antimicrobials in livestock production to diminished human health outcomes due to antimicrobial resistance is lacking, and the U.S. Food and Drug Administration has taken an approach to maximize therapeutic efficacy and minimize the selection of resistant microorganisms through judicious use of antimicrobials. This study demonstrated that prophylactic in-feed treatment of chlortetracycline administered for 5 days to calves entering feedlots is judicious, as this therapy reduced animal morbidity, reduced the use of antimicrobials more critical to human health, and had no long-term impact on the occurrence of antimicrobial-resistant E. coli.

Diagnostic Accuracy of Rectoanal Mucosal Swab of Feedlot Cattle for Detection and Enumeration of Salmonella enterica.

Cattle are noted carriers of the foodborne pathogen Salmonella enterica. The perceived need to decrease the potential human health risk posed by excretion of this pathogen has resulted in numerous studies examining the factors that influence Salmonella shedding in cattle. Fecal grab (FG) samples have been the predominant method used to identify cattle colonized or infected with Salmonella; however, FG sampling can be impractical in certain situations, and rectoanal mucosal swabs (RAMS) are a more convenient sample type to collect. Despite a lack of studies comparing FG and RAMS for the detection and enumeration of Salmonella fecal shedding, RAMS is perceived as less sensitive because a smaller amount of feces is cultured. In a cross-sectional study to address these concerns, paired RAMS and FG samples were collected from 403 adult feedlot cattle approximately 90 days prior to harvest. Samples were processed for Salmonella enumeration (direct plating) and detection (enrichment and immunomagnetic separation). In all, 89.6% of RAMS and 98.8% of FG samples were positive for Salmonella, and concordant prevalence outcomes were observed for 90.8% of samples. Mean enumeration values were 3.01 and 3.12 log CFU/ml for RAMS and FG, respectively. The sensitivity and specificity of RAMS were 91% (95% confidence interval [CI]: 87.5 to 93%) and 100% (95% CI: 48 to 100%), respectively, for Salmonella detection. Furthermore, RAMS Salmonella enumeration was substantially concordant (ρc = 0.89; 95% CI: 0.86 to 0.91) with FG values. We conclude that RAMS are a reliable alternative to FG for assessing cattle Salmonella fecal shedding status, especially for cattle shedding high levels of Salmonella.

Use of generalized ordered logistic regression for the analysis of multidrug resistance data

Statistical analysis of antimicrobial resistance data largely focuses on individual antimicrobials binary outcome (susceptible or resistant). However, bacteria are becoming increasingly multidrug resistant (MDR). Statistical analysis of MDR data is mostly descriptive often with tabular or graphical presentations. Here we report the applicability of generalized ordinal logistic regression model for the analysis of MDR data. A total of 1,152 Escherichia coli, isolated from the feces of weaned pigs experimentally supplemented with chlortetracycline (CTC) and copper, were tested for susceptibilities against 15 antimicrobials and were binary classified into resistant or susceptible. The 15 antimicrobial agents tested were grouped into eight different antimicrobial classes. We defined MDR as the number of antimicrobial classes to which E. coli isolates were resistant ranging from 0 to 8. Proportionality of the odds assumption of the ordinal logistic regression model was violated only for the effect of treatment period (pre-treatment, during-treatment and post-treatment); but not for the effect of CTC or copper supplementation. Subsequently, a partially constrained generalized ordinal logistic model was built that allows for the effect of treatment period to vary while constraining the effects of treatment (CTC and copper supplementation) to be constant across the levels of MDR classes. Copper (Proportional Odds Ratio [Prop OR]=1.03; 95% CI=0.73-1.47) and CTC (Prop OR=1.1; 95% CI=0.78-1.56) supplementation were not significantly associated with the level of MDR adjusted for the effect of treatment period. MDR generally declined over the trial period. In conclusion, generalized ordered logistic regression can be used for the analysis of ordinal data such as MDR data when the proportionality assumptions for ordered logistic regression are violated.