Role of biofilms in antimicrobial resistance of the bacterial bovine respiratory disease complex.

An increase in chronic, non-responsive bovine respiratory disease (BRD) infections in North American feedlot cattle is observed each fall, a time when cattle are administered multiple antimicrobial treatments for BRD. A number of factors are responsible for BRD antimicrobial treatment failure, with formation of biofilms possibly being one. It is widely accepted that biofilms play a role in chronic infections in humans and it has been hypothesized that they are the default lifestyle of most bacteria. However, research on bacterial biofilms associated with livestock is scarce and significant knowledge gaps exist in our understanding of their role in AMR of the bacterial BRD complex. The four main bacterial species of the BRD complex, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis are able to form biofilms in vitro and there is evidence that at least H. somni retains this ability in vivo. However, there is a need to elucidate whether their biofilm-forming ability contributes to pathogenicity and antimicrobial treatment failure of BRD. Overall, a better understanding of the possible role of BRD bacterial biofilms in clinical disease and AMR could assist in the prevention and management of respiratory infections in feedlot cattle. We review and discuss the current knowledge of BRD bacteria biofilm biology, study methodologies, and their possible relationship to AMR.

Improving the detection of integrative conjugative elements in bovine nasopharyngeal swabs using multiplex recombinase polymerase amplification.

Bovine respiratory disease (BRD) is an important health and economic burden to the cattle industry worldwide. Three bacterial pathogens frequently associated with BRD (Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni) can possess integrative and conjugative elements (ICEs), a diverse group of mobile genetic elements that acquire antimicrobial resistance (AMR) genes (ARGs) and decrease the therapeutic efficacy of antimicrobial drugs. We developed a duplex recombinase polymerase amplification (RPA) assay to detect up to two variants of ICEs in these Pasteurellaceae. Whole genome sequence analysis of M. haemolytica, P. multocida, and H. somni isolates harbouring ICEs revealed the presence of tnpA or ebrB next to tet(H), a conserved ARG that is frequently detected in ICEs within BRD-associated bacteria. This real-time multiplex RPA assay targeted both ICE variants simultaneously, denoted as tetH_tnpA and tetH_ebrB, with a limit of detection (LOD) of 29 (95% CI [23, 46]) and 38 genome copies (95% CI [30, 59]), respectively. DNA was extracted from 100 deep nasopharyngeal swabs collected from feedlot cattle on arrival. Samples were tested for ICEs using a real-time multiplex RPA assay, and for M. haemolytica, P. multocida, H. somni, and Mycoplasma bovis using both culture methods and RPA. The assay provided sensitive and accurate identification of ICEs in extracted DNA, providing a useful molecular tool for timely detection of potential risk factors associated with the development of antimicrobial-resistant BRD in feedlot cattle.

Evaluating the liver abscess microbiota of beef cattle during a reduction in tylosin supplementation shows differences according to abscess size and fraction.

Liver abscesses (LA) resulting from bacterial infection in cattle pose a significant global challenge to the beef and dairy industries. Economic losses from liver discounts at slaughter and reduced animal performance drive the need for effective mitigation strategies. Tylosin phosphate supplementation is widely used to reduce LA occurrence, but concerns over antimicrobial overuse emphasize the urgency to explore alternative approaches. Understanding the microbial ecology of LA is crucial to this, and we hypothesized that a reduced timeframe of tylosin delivery would alter LA microbiomes. We conducted 16S rRNA sequencing to assess severe liver abscess bacteriomes in beef cattle supplemented with in-feed tylosin. Our findings revealed that shortening tylosin supplementation did not notably alter microbial communities. Additionally, our findings highlighted the significance of sample processing methods, showing differing communities in bulk purulent material and the capsule-adhered material. Fusobacterium or Bacteroides ASVs dominated LA, alongside probable opportunistic gut pathogens and other microbes. Moreover, we suggest that liver abscess size correlates with microbial community composition. These insights contribute to our understanding of factors impacting liver abscess microbial ecology and will be valuable in identifying antibiotic alternatives. They underscore the importance of exploring varied approaches to address LA while reducing reliance on in-feed antibiotics.

Bovine Respiratory Disease: Conventional to Culture-Independent Approaches to Studying Antimicrobial Resistance in North America.

Numerous antimicrobial resistance (AMR) surveillance studies have been conducted in North American feedlot cattle to investigate the major bacterial pathogens of the bovine respiratory disease (BRD) complex, specifically: Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. While most bacterial isolates recovered from healthy cattle are susceptible to a repertoire of antimicrobials, multidrug resistance is common in isolates recovered from cattle suffering from BRD. Integrative and conjugative elements (ICE) have gained increasing notoriety in BRD-Pasteurellaceae as they appear to play a key role in the concentration and dissemination of antimicrobial resistant genes. Likewise, low macrolide susceptibility has been described in feedlot isolates of M. bovis. Horizontal gene transfer has also been implicated in the spread of AMR within mycoplasmas, and in-vitro experiments have shown that exposure to antimicrobials can generate high levels of resistance in mycoplasmas via a single conjugative event. Consequently, antimicrobial use (AMU) could be accelerating AMR horizontal transfer within all members of the bacterial BRD complex. While metagenomics has been applied to the study of AMR in the microbiota of the respiratory tract, the potential role of the respiratory tract microbiome as an AMR reservoir remains uncertain. Current and prospective molecular tools to survey and characterize AMR need to be adapted as point-of-care technologies to enhance prudent AMU in the beef industry.

Prevalence, Risk Factors, and Antimicrobial Resistance Profile of Respiratory Pathogens Isolated From Suckling Beef Calves to Reprocessing at the Feedlot: A Longitudinal Study.

Here, we investigated the prevalence and risk factors for the presence of Histophilus somni, Mannheimia haemolytica, Mycoplasma bovis, and Pasteurella multocida in the respiratory tract of calves from the spring processing to the reprocessing at feedlots. Additionally, we characterized, phenotypically and genotypically, the antimicrobial resistance (AMR) profile of the four species. Calves from 22 cow-calf operations were enrolled in the study (n = 30 calves per operation) and sampled by deep nasopharyngeal swabs at three time points: spring processing, weaning, or induction into feedlots, and at reprocessing at the feedlot. Isolates were tested for susceptibility using the minimum inhibitory concentration (MIC) test against commonly administered antimicrobials. Additionally, a subset of isolates underwent whole-genome sequencing to infer presence of AMR genes and resistance determinants. Among studied pathogens, P. multocida was the most prevalent species, regardless of time point, followed by M. haemolytica, M. bovis, and H. somni. For M. bovis, a sharp increase in prevalence was detected at the reprocessing sampling, whereas for P. multocida, an increase in prevalence was observed at the weaning/induction sampling. Comingling and co-location of feedlots were not associated with prevalence of any respiratory pathogen. In terms of AMR, resistance against macrolides was prevalent in M. bovis, with most isolates resistant against tildipirosin, tilmicosin, and tylosin. In general, there was limited evidence to support an increase in resistance rates of respiratory bacteria from the spring processing to reprocessing at feedlots, with the exception of florfenicol resistance in M. bovis, which increased at reprocessing. Metaphylactic administration of tetracyclines at feedlot induction was not associated with the MIC of tetracyclines in any respiratory bacteria. Conversely, there were clear associations between the parenteral use of macrolides as metaphylaxis at the feedlot induction, and increased MIC against macrolides in P. multocida, M. haemolytica, and H. somni. Overall, the AMR phenotypes were corroborated by presence of AMR genes. We hypothesize that the administration of macrolides such as tulathromycin at feedlot induction contributes to historical changes in macrolides MIC data of respiratory bacteria of beef cattle.

Prevalence and Risk Factors Associated With Antimicrobial Resistance in Bacteria Related to Bovine Respiratory Disease-A Broad Cross-Sectional Study of Beef Cattle at Entry Into Canadian Feedlots.

A broad, cross-sectional study of beef cattle at entry into Canadian feedlots investigated the prevalence and epidemiology of antimicrobial resistance (AMR) in Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis, bacterial members of the bovine respiratory disease (BRD) complex. Upon feedlot arrival and before antimicrobials were administered at the feedlot, deep nasopharyngeal swabs were collected from 2,824 feedlot cattle in southern and central Alberta, Canada. Data on the date of feedlot arrival, cattle type (beef, dairy), sex (heifer, bull, steer), weight (kg), age class (calf, yearling), source (ranch direct, auction barn, backgrounding operations), risk of developing BRD (high, low), and weather conditions at arrival (temperature, precipitation, and estimated wind speed) were obtained. Mannheimia haemolytica, P. multocida, and H. somni isolates with multidrug-resistant (MDR) profiles associated with the presence of integrative and conjugative elements were isolated more often from dairy-type than from beef-type cattle. Our results showed that beef-type cattle from backgrounding operations presented higher odds of AMR bacteria as compared to auction-derived calves. Oxytetracycline resistance was the most frequently observed resistance across all Pasteurellaceae species and cattle types. Mycoplasma bovis exhibited high macrolide minimum inhibitory concentrations in both cattle types. Whether these MDR isolates establish and persist within the feedlot environment, requires further evaluation.

Investigation of Macrolide Resistance Genotypes in Mycoplasma bovis Isolates from Canadian Feedlot Cattle.

Mycoplasma bovis is associated with bovine respiratory disease (BRD) and chronic pneumonia and polyarthritis syndrome (CPPS) in feedlot cattle. No efficacious vaccines for M. bovis exist; hence, macrolides are commonly used to control mycoplasmosis. Whole genome sequences of 126 M. bovis isolates, derived from 96 feedlot cattle over 12 production years, were determined. Antimicrobial susceptibility testing (AST) of five macrolides (gamithromycin, tildipirosin, tilmicosin, tulathromycin, tylosin) was conducted using a microbroth dilution method. The AST phenotypes were compared to the genotypes generated for 23S rRNA and the L4 and L22 ribosomal proteins. Mutations in domains II (nucleotide 748; E. coli numbering) and V (nucleotide 2059 and 2060) of the 23S rRNA (rrl) gene alleles were associated with resistance. All isolates with a single mutation at Δ748 were susceptible to tulathromycin, but resistant to tilmicosin and tildipirosin. Isolates with mutations in both domain II and V (Δ748Δ2059 or Δ748Δ2060) were resistant to all five macrolides. However, >99% of isolates were resistant to tildipirosin and tilmicosin, regardless of the number and positions of the mutations. Isolates with a Δ748 mutation in the 23S rRNA gene and mutations in L4 and L22 were resistant to all macrolides except for tulathromycin.

Antimicrobial resistance among canine enteric Escherichia coli isolates and prevalence of attaching-effacing and extraintestinal pathogenic virulence factors in Spain.

The aim of this study was to estimate the prevalence of antimicrobial resistance (AMR) in Escherichia coli from a dog population in Spain and assess specific virulence factors. Susceptibility to 22 antimicrobials was tested along with the production of extended-spectrum ß-lactamases (ESBLs) and AmpC in faecal isolates from 100 dogs. Virulence-related genes associated with attaching and effacing E. coli (eae, Stx1, Stx2) and extraintestinal pathogenic E. coli - ExPEC - (papC, hlyA and cnf1) were detected by PCR. At least one kind of AMR was observed in 73% of the isolates. The highest prevalences corresponded to penicillin (45%), aminoglycoside (40%) and non-extended spectrum cephalosporin (39%) classes. Multidrug resistance (MDR) was observed in 53.4% of the resistant isolates. No resistance to colistin was found. Production of ESBL/AmpC enzymes was detected in 5% of E. coli. Shiga toxin-producing E. coli were not observed, enteropathogenic E. coli were identified in only 12% of them, and ExPEC were found in 25%. Dog faeces can be a source of E. coli strains potentially presenting a threat to humans through their virulence factors or AMR. The non-hygienic keeping of animals may increase the risk of colonisation of such pathogens in humans.

Antimicrobial Sensitivity Testing of Mycoplasma bovis Isolates Derived from Western Canadian Feedlot Cattle.

Mycoplasma bovis is particularly adept at evading the immune system, resulting in chronic infections of the lungs and joints of feedlot cattle. The chronicity of the lesions results in prolonged antimicrobial therapy, possibly exacerbating antimicrobial resistance. This cross-sectional study generated in vitro antimicrobial susceptibility testing (AST) data on 211 M. bovis isolates recovered from 159 healthy, diseased, and dead cattle, spanning the period of 2006-2018. Nine antimicrobials commonly administered to western Canadian feedlot cattle were assessed. The data were analyzed with non-parametric statistical tests with a level of significance of p < 0.05 (two-tailed). Minimum inhibitory concentration (MIC) values tended to increase between the isolates from healthy versus dead cattle and over time (2006-2018). Isolates from dead versus healthy cattle were more likely to be resistant to tulathromycin, gamithromycin, tylosin and enrofloxacin. There was no difference in the distributions of the MICs generated from the isolates recovered from the lungs and joints (p ≥ 0.124) and the lungs and deep nasal passages (p ≥ 0.157) of the same animals.

A direct qPCR screening approach to improve the efficiency of Mycoplasma bovis isolation in the frame of a broad surveillance study.

Culturing Mycoplasma bovis is laborious and unpredictable with most laboratories relying on molecular methods for its detection and identification. However, bacterial culture is still necessary to relate phenotypic characteristics to genotypic traits within and between individual strains. Thus, the main objective of this study was to develop a procedure that saved time and consumables during the culturing of M. bovis within the scope of a broad antimicrobial resistance surveillance project. Deep nasopharyngeal swabs (DNPS) collected from feedlot cattle upon arrival at 10 Southern Alberta feedlots were enriched in broth and an aliquot of the culture was directly used in a M. bovis-specific quantitative PCR (qPCR) assay. Only qPCR-positive cultures were plated onto agar media for the isolation of M. bovis. The detection of M. bovis from broth culture by direct-culture-qPCR proved to be more sensitive (1.61 × 102 CFU/mL) than using a commercial kit (1.61 × 103 CFU/mL) to extract DNA from pure cultures of M. bovis. When isolation of M. bovis from broth-enriched DNPS (n = 208 samples) was used as the gold standard for diagnostics, the qPCR screening approach showed 100% sensitivity, 87.27% specificity, and a kappa index = 0.87 (strong agreement). In contrast, qPCR of DNPS samples (n = 58) exhibited 100% sensitivity, 42.86% specificity, and a kappa index = 0.49 (weak agreement). The qPCR protocol described here together with a high throughput direct-culture-qPCR approach for sample testing made it possible to reduce the labor and cost of M. bovis isolation by eliminating the need to process 97.3% of M. bovis-negative samples. This was possible through the use of qPCR Ct values as a predictive tool of the likelihood of M. bovis isolation. This new procedure could be evaluated for its use in antimicrobial resistance surveillance programs that focus on Mycoplasma species.

Antimicrobial Susceptibilities and Phylogenetic Analyses of Enterococcus hirae Isolated from Broilers with Valvular Endocarditis.

Enterococcus hirae is a zoonotic Enterococcus species that causes opportunistic infections in both humans and animals and can be transmitted by contact with animals or through contaminated food. The aim of this study was to investigate the importance of E. hirae in broilers with endocarditis, as well as the antimicrobial resistance patterns and genetic relatedness of the isolates. A total of 477 three- to five-week-old broilers were studied during five fattening periods on a farm with mortality due to endocarditis. Endocarditis was observed in 27 chickens (5.66%), and samples were taken for pathological, microbiological, and molecular studies. Lesions were mainly found in the right atrioventricular valve and corresponded with a fibrinous endocarditis. Enterococcus hirae was identified in all cases. Pulsed-field gel electrophoresis results showed clonality among some isolates, with one pulsotype harboring 11 isolates that were found throughout the study. Most of the isolates showed multi-drug-resistant phenotypes. These results confirm that E. hirae is a significant cause of endocarditis in broilers, and suggest that broilers may be important carriers of antimicrobial-resistant E. hirae that might enter into the food chain.

Susceptibilidad antimicrobiana y análisis filogenético de Enterococcus hirae aislados de pollos de engorde con endocarditis valvular. Enterococcus hirae es una especie zoonótica de enterococo que provoca infecciones oportunistas en el hombre y en los animales y que puede transmitirse mediante el contacto con animales o a través de alimentos contaminados. El objetivo de este estudio fue la investigación de la importancia de E. hirae en pollos de engorde con endocarditis, así como el estudio de sus patrones de resistencia antimicrobiana y la relación genética entre los aislados. Se estudiaron 477 pollos de engorde de tres a cinco semanas de edad, durante cinco periodos de engorde, en una granja con historial de muertes por endocarditis. Se detectó endocarditis en 27 pollos (5.66%) y se recolectaron muestras para estudios histopatológicos, microbiológicos y moleculares. Las lesiones se observaron principalmente en la válvula atrioventricular derecha, correspondiendo con una endocarditis fibrinosa. En todos los casos se identificó E. hirae. Mediante electroforesis en gel de campo con pulsaciones se detectó clonalidad en algunos aislados, con once aislados agrupados en un pulsotipo, los cuales fueron detectados a lo largo de todo el estudio. La mayoría de los aislados presentaban fenotipos multirresistentes a varios antibióticos. Estos resultados confirman que E. hirae es una causa importante de endocarditis en pollos de engorde y que estos pueden ser portadores importantes de cepas multirresistentes de E. hirae, las cuales podrían entrar en la cadena alimentaria.

Incidence and characterization of Clostridium difficile in a secondary care hospital in Spain

Introduction: Clostridium difficile (C. difficile) is a major nosocomial infectious agent in hospitals. Previous studies have addressed the high proportion of infection episodes that are overlooked in health care facilities. Objective: the main aim of this study was to characterize C. difficile clinical cases that occurred in a secondary care hospital during a five-month period. Material and methods: for this purpose, a total of 137 stool samples from the same number of patients with diarrhea were analyzed for the presence of C. difficile by culture techniques. An enzyme immunoassay (EIA) test for the detection of C. difficile and its toxins was also used in 50 cases (36.5%) for diagnostic purposes. Results: a total of 14 (10.2%) C. difficile isolates were obtained, of which nine (64.3%) were toxigenic. A mean incidence of 3.2 episodes of C. difficile infections (CDI) per 10,000 patients-days was estimated for the study period. Around 56% of the CDI cases were determined as hospital-acquired, whereas 44% originated in the community. Among these, only two episodes (22.2%) were detected in the hospital by the EIA test, which indicated that the hospital CDI detection protocol needed to be revised. One unusual C. difficile isolate was negative for all toxin genes examined and also for the non-toxigenic strain assay, which highlights the need to perform genome sequencing to study its pathogenicity locus insertion site organization. A stable metronidazole-resistant C. difficile strain and three strains showing multidrug resistance were detected in this study, suggesting that C. difficile antimicrobial susceptibility surveillance programs should be established in this health-care facility

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Serotyping and antimicrobial resistance of Mannheimia haemolytica strains from European cattle with bovine respiratory disease.

The objective of this study was to assess the prevalence of three serotypes, A1, A2, and A6 in 98 M. haemolytica isolates collected from clinical BRD cases in European cattle and assess their antimicrobial resistance profiles. Isolates were characterized by serotyping (plate agglutination and serotype specific PCR) and antimicrobial susceptibility testing. The study identified a predominance of serotypes A1 (59%) and A6 (22%) in European M. haemolytica isolates exhibiting a relatively low level of antimicrobial resistance. A comprehensive understanding of the relative prevalence of different M. haemolytica serotypes in Europe informs a targeted approach for vaccine design against BRD.

Incidence and characterization of Clostridium difficile in a secondary care hospital in Spain.

INTRODUCTION: Clostridium difficile (C. difficile) is a major nosocomial infectious agent in hospitals. Previous studies have addressed the high proportion of infection episodes that are overlooked in health care facilities. OBJECTIVE: the main aim of this study was to characterize C. difficile clinical cases that occurred in a secondary care hospital during a five-month period. MATERIAL AND METHODS: for this purpose, a total of 137 stool samples from the same number of patients with diarrhea were analyzed for the presence of C. difficile by culture techniques. An enzyme immunoassay (EIA) test for the detection of C. difficile and its toxins was also used in 50 cases (36.5%) for diagnostic purposes. RESULTS: a total of 14 (10.2%) C. difficile isolates were obtained, of which nine (64.3%) were toxigenic. A mean incidence of 3.2 episodes of C. difficile infections (CDI) per 10,000 patients-days was estimated for the study period. Around 56% of the CDI cases were determined as hospital-acquired, whereas 44% originated in the community. Among these, only two episodes (22.2%) were detected in the hospital by the EIA test, which indicated that the hospital CDI detection protocol needed to be revised. One unusual C. difficile isolate was negative for all toxin genes examined and also for the non-toxigenic strain assay, which highlights the need to perform genome sequencing to study its pathogenicity locus insertion site organization. A stable metronidazole-resistant C. difficile strain and three strains showing multidrug resistance were detected in this study, suggesting that C. difficile antimicrobial susceptibility surveillance programs should be established in this health-care facility.

Preliminary studies on isolates of Clostridium difficile from dogs and exotic pets.

BACKGROUND: Clostridium difficile infection (CDI) is recognised as an emerging disease in both humans and some animal species. During the past few years, insights into human CDI epidemiology changed and C. difficile is also considered as an emerging community-acquired pathogen. Certain ribotypes (RT) are possibly associated with zoonotic transmission. The objective of this study was to assess the presence of C. difficile in a population of pets and to characterise the isolates. RESULTS: Faecal samples from a total of 90 diarrhoeic dogs and 24 from exotic animal species (both diarrhoeic and non-diarrhoeic) were analysed. Clostridium difficile was isolated from 6 (6.7%) dogs and one reptile sample (4.2%). Four (66.7%) of the six dog strains were capable of producing toxins. Four known different RTs were detected in dogs (010, 014, 123 and 358) and a new one was found in a faecal sample of an exotic animal. This new RT isolate was negative for all toxin genes tested and belonged to sequence type 347 which has been proposed as a Clade-III member. Importantly, two dog strains showed a stable resistance to metronidazole (initial MIC values: 128 and 48 µg/ml). CONCLUSIONS: The results obtained in this study suggest the implementation of antimicrobial susceptibility surveillance programs to assess the prevalence of metronidazole resistance in dogs; molecular studies to elucidate C. difficile metronidazole resistance mechanisms are warranted. Based on the similarity between the ribotypes observed in dogs and those described in humans, the zoonotic transmission should be further explored. Furthermore, exotic animals have shown to harbor uncommon C. difficile strains which require further genomic studies.