The respiratory and fecal microbiota of beef calves from birth to weaning.

The development and growth of animals coincide with the establishment and maturation of their microbiotas. To evaluate the respiratory and fecal microbiotas of beef calves from birth to weaning, a total of 30 pregnant cows, and their calves at birth, were enrolled in this study. Deep nasal swabs and feces were collected from calves longitudinally, starting on the day of birth and ending on the day of weaning. Nasopharyngeal, vaginal, and fecal samples were also collected from cows, and the microbiotas of all samples were analyzed. The fecal microbiota of calves was enriched with Lactobacillus during the first 8 weeks of life, before being displaced by genera associated with fiber digestion, and then increasing in diversity across time. In contrast, the diversity of calf respiratory microbiota generally decreased with age. At birth, the calf and cow nasal microbiotas were highly similar, indicating colonization from dam contact. This was supported by microbial source-tracking analysis. The structure of the calf nasal microbiota remained similar to that of the cows, until weaning, when it diverged. The changes were driven by a decrease in Lactobacillus and an increase in genera typically associated with bovine respiratory disease, including Mannheimia, Pasteurella, and Mycoplasma. These three genera colonized calves early in life, though Mannheimia was initially transferred from the cow reproductive tract. Path analysis was used to model the interrelationships of calf respiratory and fecal microbiotas. It was observed that respiratory Lactobacillus and fecal Oscillospiraceae UCG-005 negatively affected the abundance of Mannheimia or Pasteurella.IMPORTANCEIn beef cattle production, bovine respiratory disease (BRD) accounts for most of the feedlot morbidities and mortalities. Metaphylaxis is a common management tool to mitigate BRD, however its use has led to increased antimicrobial resistance. Novel methods to mitigate BRD are needed, including microbiota-based strategies. However, information on the respiratory bacteria of beef calves prior to weaning was limited. In this study, it was shown that the microbiota of cows influenced the initial composition of both respiratory and fecal microbiotas in calves. While colonization of the respiratory tract of calves by BRD-associated genera occurred early in life, their relative abundances increased at weaning, and were negatively correlated with respiratory and gut bacteria. Thus, microbiotas of both the respiratory and gastrointestinal tracts have important roles in antagonism of respiratory pathogens and are potential targets for enhancing calf respiratory health. Modulation may be most beneficial, if done prior to weaning, before opportunistic pathogens establish colonization.

Prevalence and molecular epidemiology of carbapenemase-producing Enterobacterales isolated from dog and cat faeces submitted to veterinary laboratories in the USA.

AIMS: To estimate the prevalence of carbapenemase-producing Enterobacterales (CPE) carriage among pets using faecal specimens submitted to veterinary diagnostic laboratories throughout the US. A secondary aim was to employ whole-genome sequencing (WGS) to characterize isolates of CPE from companion animals and compare them to publicly available CPE genomes. METHODS AND RESULTS: To estimate the prevalence of CPE in companion animals in the USA, a multicenter surveillance study including 8 different veterinary diagnostic laboratories from across the USA was conducted. Briefly, remnant faecal specimens from dogs and cats were screened using two selective agar plates (CHROMID Carba and MacConkey with 1 mg/L cefotaxime and 0.125 mg/L meropenem) and presumptive CPE isolates screened by the modified carbapenemase inactivation method for carbapenemase production. A total of 2393 specimens were screened and yielded 196 isolates for carbapenemase screening. A total of 5 isolates from 4 dogs and 1 cat at 3 different veterinary diagnostic laboratories were confirmed to produce a carbapenemase (0.21%). Whole-genome sequencing (WGS) revealed two E. coli (ST167) isolates that both produced an NDM-5 carbapenemase, two Enterobacter hormaechei (ST171) isolates that produced an NDM-5 carbapenemase and a KPC-4 carbapenemase respectively and one Klebsiella oxytoca (ST199) that produced an Oxa-48-type carbapenemase. Both E. coli isolates were found to be within at least 22 SNPs of previously characterized canine and human CPE isolates. CONCLUSIONS: This study demonstrates that the prevalence of CPE among companion animals is relatively low (0.21%) but that given the genetic relatedness of animal isolates to human isolates, additional surveillance is needed.

Genomic characterization of antimicrobial resistance in 61 aquatic bacterial isolates.

Antimicrobial resistance (AMR) in pathogens important to aquatic animal health is of increasing concern but vastly understudied. Antimicrobial therapy is used to both treat and prevent bacterial disease in fish and is critical for a viable aquaculture industry and for maintenance of wild fish populations. Unfortunately, phenotypic antimicrobial susceptibility testing is technically difficult for bacteria recovered from aquatic animal hosts resulting in challenges in resistance monitoring using traditional methods. Whole-genome sequencing provides an appealing methodology for investigation of putative resistance. As part of the ongoing efforts of the FDA CVM Vet-LIRN to monitor AMR, source laboratories cultured and preliminarily identified pathogenic bacteria isolated from various fish species collected in 2019 from across the United States. Sixty-one bacterial isolates were evaluated using whole-genome sequencing. We present here the assembled draft genomes, AMR genes, predicted resistance phenotypes, and virulence factors of the 61 isolates and discuss concurrence of the identifications made by source laboratories using matrix-assisted laser desorption/time-of-flight mass spectrometry.

Mucosal Immunization with Spore-Based Vaccines against Mannheimia haemolytica Enhances Antigen-Specific Immunity.

BACKGROUND: Mannheimia haemolytica is a bovine respiratory pathogen commonly associated with bacterial bronchopneumonia. Current vaccine strategies have shown variable efficacy in feedlot cattle, and therefore novel vaccines are needed. Bacillus subtilis spores have been investigated as a mucosal vaccine platform, due to their ability to bind and present antigens to the mucosa and act as an adjuvant. The aim of this study was to develop two spore-based mucosal vaccines targeting M. haemolytica and evaluate their immunogenicity in mice. METHODS: Two antigen constructs composed of cholera toxin B subunit, M. haemolytica leukotoxin, and either the M. haemolytica outer membrane protein PlpE (MhCP1) or GS60 (MhCP2) were synthesized, purified and then bound to spores as vaccines. In two separate mice trials, the spore-bound vaccines (Spore-MhCP1 and Spore-MhCP2) were administered to mice through intranasal and intragastric routes, while free antigens were administered intranasally and intramuscularly. Unbound spores were also evaluated intranasally. Antigen-specific serum IgG and mucosal IgA from bronchoalveolar lavage, feces, and saliva were measured after vaccination. Mice sera from all treatment groups were assessed for their bactericidal activity against M. haemolytica. RESULTS: In both mice experiments, intramuscular immunization induced the strongest serum IgG antibody response. However, the intranasal administration of Spore-MhCP1 and Spore-MhCP2 elicited the greatest secretory IgA-specific response against leukotoxin, PlpE, and GS60 in bronchoalveolar lavage, saliva, and feces (p < 0.05). Compared to the intranasal administration of free antigen, spore-bound antigen groups showed greater bactericidal activity against M. haemolytica (p < 0.05). CONCLUSIONS: Since intranasally delivered Spore-MhCP1 and Spore-MhCP2 elicited both systemic and mucosal immune responses in mice, these vaccines may have potential to mitigate lung infection in cattle by restricting M. haemolytica colonization and proliferation in the respiratory tract. The efficacy of these mucosal spore-based vaccines merits further assessment against M. haemolytica in cattle.

In Silico Analysis of Shiga Toxin-Producing Escherichia coli O157:H7 Strains from Presumptive Super- and Low-Shedder Cattle.

Cattle are the primary reservoir for STEC O157, with some shedding >104 CFU/g in feces, a phenomenon known as super-shedding (SS). The mechanism(s) responsible for SS are not understood but have been attributed to the environment, host, and pathogen. This study aimed to compare genetic characteristics of STEC O157 strains from cattle in the same commercial feedlot pens with SS or low-shedding (LS) status. Strains from SS (n = 35) and LS (n = 28) collected from 11 pens in three feedlots were analyzed for virulence genes, Shiga toxin-carrying bacteriophage insertion sites, and phylogenetic relationships. In silico analysis showed limited variation regarding virulence gene profiles. Stx-encoding prophage insertion sites mrlA and wrbA for stx1a and stx2a, respectively, were all occupied, but two isolates had fragments of the stx-carrying phage in mrlA and wrbA loci without stx1a and stx2a. All strains screened for lineage-specific polymorphism assay (LSPA-6) were 111111, lineage I. Of the isolates, 61 and 2 were clades 1 and 8, respectively. Phylogenetic analysis revealed that pens with more than one SS had multiple distantly related clusters of SS and LS isolates. Although virulence genes and lineage were largely similar within and across feedlots, multiple genetic origins of strains within a single feedlot pen illustrate challenges for on-farm control of STEC.

Characterization of the hoof bacterial communities in feedlot cattle affected with digital dermatitis, foot rot or both using a surface swab technique.

BACKGROUND: Lameness is defined as altered or abnormal gait due to dysfunction of the locomotor system, and is a health issue of feedlot cattle, having major economic, labour, and welfare implications. Digital dermatitis (DD-a lesion of the plantar surface of the foot) and foot rot (FR-affects the interdigital cleft) are common infectious causes of lameness in feedlots. These hoof lesions can occur alone or in combination (DD + FR) in the same hoof. A total of 208 hoof swabs were collected from three commercial feedlots located in southern Alberta. Every lesion sample was matched with a corresponding control skin sample taken from a healthy contralateral foot. Control skin samples were also collected from cattle with no lesion on any feet. Bacterial communities of three types of hoof lesions (DD, DD + FR, FR) and healthy skin were profiled using 16S amplicon sequencing. RESULTS: Alpha diversity analysis revealed a lower bacterial diversity on DD and FR lesions compared to control skin. Beta diversity analysis showed that bacterial communities of DD, FR, and DD + FR lesions were distinct from those of the control skin. While the impact of feedlot was minimal, lesion type contributed to 22% of the variation observed among bacterial communities (PERMANOVA-R = 0.22, P < 0.01). Compared to the corresponding control skin, there were 11, 12, and 3 differentially abundant (DA) bacterial genera in DD, DD + FR, and FR lesions, respectively. CONCLUSIONS: The bacterial community description of a DD + FR lesion is a novel finding. Not only did lesions lead to altered bacterial communities when compared to healthy skin, but the composition of those communities also differed depending on the hoof lesion. The 16S amplicon sequencing of surface swabs has significant value as a research tool in separating different hoof lesions and can provide additional insights to the polybacterial etiology of DD and FR in feedlot cattle.

Development of a plant-based oral vaccine candidate against the bovine respiratory pathogen Mannheimia haemolytica.

Bovine respiratory disease (BRD) affects feedlot cattle across North America, resulting in economic losses due to animal treatment and reduced performance. In an effort to develop a vaccine candidate targeting a primary bacterial agent contributing to BRD, we produced a tripartite antigen consisting of segments of the virulence factor Leukotoxin A (LktA) and lipoprotein PlpE from Mannheimia haemolytica, fused to a cholera toxin mucosal adjuvant (CTB). This recombinant subunit vaccine candidate was expressed in the leaves of Nicotiana benthamiana plants, with accumulation tested in five subcellular compartments. The recombinant protein was found to accumulate highest in the endoplasmic reticulum, but targeting to the chloroplast was employed for scaling up production due the absence of post-translational modification while still producing feasible levels. Leaves were freeze dried, then orally administered to mice to determine its immunogenicity. Sera from mice immunized with leaf tissue expressing the recombinant antigen contained IgG antibodies, specifically recognizing both LktA and PlpE. These mice also had a mucosal immune response to the CTB+LktA+PlpE protein as measured by the presence of LktA- and PlpE-specific IgA antibodies in lung and fecal material. Moreover, the antigen remained stable at room temperature with limited deterioration for up to one year when stored as lyophilized plant material. This study demonstrated that a recombinant antigen expressed in plant tissue elicited both humoral and mucosal immune responses when fed to mice, and warrants evaluation in cattle.

The Isolation and Characterization of Bacteriophages Infecting Avian Pathogenic Escherichia coli O1, O2 and O78 Strains.

Avian pathogenic Escherichia coli (APEC), such as O1, O2 and O78, are important serogroups relating to chicken health, being responsible for colibacillosis. In this study, we isolated and characterized bacteriophages (phages) from hen feces and human sewage in Alberta with the potential for controlling colibacillosis in laying hens. The lytic profile, host range, pH tolerance and morphology of seven APEC-infecting phages (ASO1A, ASO1B, ASO2A, ASO78A, ASO2B, AVIO78A and ASO78B) were assessed using a microplate phage virulence assay and transmission electron microscopy (TEM). The potential safety of phages at the genome level was predicted using AMRFinderPlus and the Virulence Factor Database. Finally, phage genera and genetic relatedness with other known phages from the NCBI GenBank database were inferred using the virus intergenomic distance calculator and single gene-based phylogenetic trees. The seven APEC-infecting phages preferentially lysed APEC strains in this study, with ECL21443 (O2) being the most susceptible to phages (n = 5). ASO78A had the broadest host range, lysing all tested strains (n = 5) except ECL20885 (O1). Phages were viable at a pH of 2.5 or 3.5-9.0 after 4 h of incubation. Based on TEM, phages were classed as myovirus, siphovirus and podovirus. No genes associated with virulence, antimicrobial resistance or lysogeny were detected in phage genomes. Comparative genomic analysis placed six of the seven phages in five genera: Felixounavirus (ASO1A and ASO1B), Phapecoctavirus (ASO2A), Tequatrovirus (ASO78A), Kayfunavirus (ASO2B) and Sashavirus (AVIO78A). Based on the nucleotide intergenomic similarity (<70%), phage ASO78B was not assigned a genus in the siphovirus and could represent a new genus in class Caudoviricetes. The tail fiber protein phylogeny revealed variations within APEC-infecting phages and closely related phages. Diverse APEC-infecting phages harbored in the environment demonstrate the potential to control colibacillosis in poultry.

Auction market placement and a rest stop during transportation affect the respiratory bacterial microbiota of beef cattle.

Background: Bovine respiratory disease (BRD) is a significant health problem in beef cattle production, resulting in considerable economic losses due to mortalities, cost of treatment, and reduced feed efficiency. The onset of BRD is multifactorial, with numerous stressors being implicated, including transportation from farms to feedlots. In relation to animal welfare, regulations or practices may require mandatory rest times during transportation. Despite this, there is limited information on how transportation and rest stops affect the respiratory microbiota. Results: This study evaluated the effect of cattle source (ranch-direct or auction market-derived) and rest stop duration (0 or 8 h of rest) on the upper respiratory tract microbiota and its relationship to stress response indicators (blood cortisol and haptoglobin) of recently weaned cattle transported for 36 h. The community structure of bacteria was altered by feedlot placement. When cattle were off-loaded for a rest, several key bacterial genera associated with BRD (Mannheimia, Histophilus, Pasteurella) were increased for most sampling times after feedlot placement for the ranch-direct cattle group, compared to animals given no rest stop. Similarly, more sampling time points had elevated levels of BRD-associated genera when auction market cattle were compared to ranch-direct. When evaluated across time and treatments several genera including Mannheimia, Moraxella, Streptococcus and Corynebacterium were positively correlated with blood cortisol concentrations. Conclusion: This is the first study to assess the effect of rest during transportation and cattle source on the respiratory microbiota in weaned beef calves. The results suggest that rest stops and auction market placement may be risk factors for BRD, based solely on increased abundance of BRD-associated genera in the upper respiratory tract. However, it was not possible to link these microbiota to disease outcome, due to low incidence of BRD in the study populations. Larger scale studies are needed to further define how transportation variables impact cattle health.

Prevalence and antimicrobial susceptibility of Mycoplasma bovis from the upper and lower respiratory tracts of healthy feedlot cattle and those diagnosed with bovine respiratory disease.

Mycoplasma bovis is an important respiratory pathogen of cattle. In this study, the prevalence and antimicrobial susceptibility of M. bovis were evaluated from two Cohorts of feedlot cattle spanning an 8-year period. In the first study conducted in 2008-2009, nasopharyngeal swabs from cattle sampled at feedlot entry and after 60 days on feed were collected (Cohort 1). In a second study conducted in 2015-2016, nasopharyngeal and trans-tracheal samples were collected from cattle diagnosed with bovine respiratory disease (BRD) and matching healthy controls (Cohort 2). For Cohort 1, the prevalence of M. bovis was lower in cattle at entry compared to when the same individuals were sampled ≥60 days later (P < 0.05). For Cohort 2, the prevalence of M. bovis was greater in both nasopharyngeal and tracheal samples from cattle diagnosed with BRD, compared to controls (P < 0.05). In both Cohorts, almost all isolates were resistant to tilmicosin. Compared to M. bovis from Cohort 1, isolates of Cohort 2 exhibited increased resistance to clindamycin, enrofloxacin, florfenicol, tylosin, and tulathromycin, with the latter showing resistance levels >90 %. These data suggest that antimicrobials used to prevent and treat BRD selected for resistance in M. bovis over the 8-year period. For macrolides, cross-resistance occurred and M. bovis can retain resistance even when antimicrobial selection pressure is removed. Within 9 years of commercial availability of tulathromycin, the majority of M. bovis displayed resistance. Therefore, longitudinal evaluation of resistance in respiratory pathogens is important to ensure efficacious treatment of BRD.

Development of a spore-based mucosal vaccine against the bovine respiratory pathogen Mannheimia haemolytica.

Bovine respiratory disease (BRD) is a significant health issue in the North American feedlot industry, causing substantial financial losses due to morbidity and mortality. A lack of effective vaccines against BRD pathogens has resulted in antibiotics primarily being used for BRD prevention. The aim of this study was to develop a mucosal vaccine against the BRD pathogen, Mannheimia haemolytica, using Bacillus subtilis spores as an adjuvant. A chimeric protein (MhCP) containing a tandem repeat of neutralizing epitopes from M. haemolytica leukotoxin A (NLKT) and outer membrane protein PlpE was expressed to produce antigen for adsorption to B. subtilis spores. Adsorption was optimized by comparing varying amounts of antigen and spores, as well as different buffer pH and reaction temperatures. Using the optimal adsorption parameters, spore-bound antigen (Spore-MhCP) was prepared and administered to mice via two mucosal routes (intranasal and intragastric), while intramuscular administration of free MhCP and unvaccinated mice were used as positive and negative control treatments, respectively. Intramuscular administration of MhCP elicited the strongest serum IgG response. However, intranasal immunization of Spore-MhCP generated the best secretory IgA-specific response against both PlpE and NLKT in all samples evaluated (bronchoalveolar lavage, saliva, and feces). Since proliferation of M. haemolytica in the respiratory tract is a prerequisite to lung infection, this spore-based vaccine may offer protection in cattle by limiting colonization and subsequent infection, and Spore-MhCP warrants further evaluation in cattle as a mucosal vaccine against M. haemolytica.

Bacteriophages for the Targeted Control of Foodborne Pathogens.

Foodborne illness is exacerbated by novel and emerging pathotypes, persistent contamination, antimicrobial resistance, an ever-changing environment, and the complexity of food production systems. Sporadic and outbreak events of common foodborne pathogens like Shiga toxigenic E. coli (STEC), Salmonella, Campylobacter, and Listeria monocytogenes are increasingly identified. Methods of controlling human infections linked with food products are essential to improve food safety and public health and to avoid economic losses associated with contaminated food product recalls and litigations. Bacteriophages (phages) are an attractive additional weapon in the ongoing search for preventative measures to improve food safety and public health. However, like all other antimicrobial interventions that are being employed in food production systems, phages are not a panacea to all food safety challenges. Therefore, while phage-based biocontrol can be promising in combating foodborne pathogens, their antibacterial spectrum is generally narrower than most antibiotics. The emergence of phage-insensitive single-cell variants and the formulation of effective cocktails are some of the challenges faced by phage-based biocontrol methods. This review examines phage-based applications at critical control points in food production systems with an emphasis on when and where they can be successfully applied at production and processing levels. Shortcomings associated with phage-based control measures are outlined together with strategies that can be applied to improve phage utility for current and future applications in food safety.

A Single Intranasal Dose of Bacterial Therapeutics to Calves Confers Longitudinal Modulation of the Nasopharyngeal Microbiota: a Pilot Study.

To address the emergence of antimicrobial-resistant pathogens in livestock, microbiome-based strategies are increasingly being sought to reduce antimicrobial use. Here, we describe the effects of intranasal application of bacterial therapeutics (BTs) on the bovine respiratory microbiota and used structural equation modeling to investigate the causal networks after BT application. Beef cattle received (i) an intranasal cocktail of previously characterized BT strains, (ii) an injection of metaphylactic antimicrobial (tulathromycin), or (iii) intranasal saline. Despite being transient colonizers, inoculated BT strains induced longitudinal modulation of the nasopharyngeal bacterial microbiota while showing no adverse effect on animal health. The BT-mediated changes in bacteria included reduced diversity and richness and strengthened cooperative and competitive interactions. In contrast, tulathromycin increased bacterial diversity and antibiotic resistance and disrupted bacterial interactions. Overall, a single intranasal dose of BTs can modulate the bovine respiratory microbiota, highlighting that microbiome-based strategies have potential in being utilized to mitigate bovine respiratory disease in feedlot cattle. IMPORTANCE Bovine respiratory disease (BRD) remains the most significant health challenge affecting the North American beef cattle industry and results in $3 billion in economic losses yearly. Current BRD control strategies mainly rely on antibiotics, with metaphylaxis commonly employed to mitigate BRD incidence in commercial feedlots. However, the emergence of multidrug-resistant BRD pathogens threatens to reduce the efficacy of antimicrobials. Here, we investigated the potential use of novel bacterial therapeutics (BTs) to modulate the nasopharyngeal microbiota in beef calves, which are commonly administered metaphylactic antibiotics to mitigate BRD when sourced from auction markets. By direct comparison of the BTs with an antibiotic commonly used for BRD metaphylaxis in feedlots, this study conveyed the potential use of the BTs to modulate respiratory microbiome and thereby improve resistance against BRD in feedlot cattle.

Soil antibiotic resistance genes accumulate at different rates over four decades of manure application.

Manure can be a source of antibiotic resistance genes (ARGs) that enter the soil. However, previous studies assessing ARG persistence in soil have generally lacked continuity over sampling times, consistency of location, and assessing the impact of discontinuing manure application. We evaluated both short- and long-term ARG accumulation dynamics in soil with a 40-year known history of manure use. Manure application caused a greater abundance of tetracycline, macrolide, and sulfonamide ARGs in the soil. There was an initial spike in ARG abundance resulting from manure bacteria harboring ARGs being introduced to soil, followed by resident soil bacteria out-competing them, which led to ARG dissipation within a year. However, over four decades, annual manure application caused linear or exponential ARG accumulation, and bacteria associated with ARGs differed compared to those in the short term. Eleven years after discontinuing manure application, most soil ARG levels declined but remained elevated. We systematically explored the historical accumulation of ARGs in manured soil, and provide insight into factors that affect their persistence.

Non-carbapenemase producing carbapenem-resistant Klebsiella pneumoniae isolated from the urinary tract of a dog.

Objective: Carbapenems are broad-spectrum ß-lactams with excellent activity against multidrug-resistant (MDR) Enterobacterales. Unfortunately, resistance to carbapenems within this bacterial family, known as carbapenem-resistant Enterobacterales (CRE), occurs and challenges the ability to treat difficult MDR infections. Although the impact of carbapenem-resistance has been greatest in human medicine, reports in the veterinary literature are increasing especially as national veterinary antimicrobial resistance surveillance programs are now in place. In this brief communication, we report the isolation of a non-carbapenemase-producing, carbapenem-resistant Klebsiella pneumoniae from the urine of a dog, discuss the likely mechanism of resistance, and wider implications. Animal: Canine. Procedure: Whole genome sequencing and phenotypic antimicrobial susceptibility testing was performed on a K. pneumoniae isolated from the urine of a dog. Results: Antimicrobial susceptibility testing identified phenotypic resistance to imipenem and meropenem. Phenotypic detection of carbapenemase production was negative. Whole genome sequencing identified efflux pump genes associated with carbapenem resistance and point mutations in membrane porin genes. No carbapenemase gene was identified. Conclusion: Phenotypic antimicrobial susceptibility testing identified the K. pneumoniae as a non-carbapenemase producing carbapenem-resistant organism with the proposed genotypic mechanism including alteration of efflux pumps and membrane porin activity and/or expression. Clinical significance: Currently, there is limited use of carbapenem antimicrobial drugs in veterinary medicine, and practitioners may be unfamiliar or unaware of this type of resistance, its significance on routine antimicrobial susceptibility test reports, and implications for antimicrobial therapy and public health. Carbapenem-resistant Enterobacterales are infrequently isolated from companion animals; however, due to increasing adoption of advanced medical and surgical interventions, they may become more prevalent.

Objectif: Les carbapénèmes sont des ß-lactamines à large spectre avec une excellente activité contre les Enterobacterales multirésistantes (MDR). Malheureusement, la résistance aux carbapénèmes au sein de cette famille bactérienne, connue sous le nom d'Enterobacterales résistantes aux carbapénèmes (CRE), se produit et remet en question la capacité de traiter les infections MDR difficiles. Bien que l'impact de la résistance aux carbapénèmes ait été plus important en médecine humaine, les rapports dans la littérature vétérinaire se multiplient, d'autant plus que des programmes nationaux de surveillance de la résistance aux antimicrobiens vétérinaires sont désormais en place. Dans cette brève communication, nous rapportons l'isolement d'une Klebsiella pneumoniae non-productrice de carbapénémase et résistante aux carbapénèmes à partir de l'urine d'un chien, discutons du mécanisme probable de résistance et des implications plus larges. Animal: Canin. Procédure: Le séquençage du génome entier et les tests de sensibilité phénotypique aux antimicrobiens ont été effectués sur un isolat de K. pneumoniae provenant de l'urine d'un chien. Résultats: Les tests de sensibilité aux antimicrobiens ont identifié une résistance phénotypique à l'imipénème et au méropénème. La détection phénotypique de production de carbapénèmase était négative. Le séquençage du génome entier a identifié des gènes de pompe à efflux associés à la résistance aux carbapénèmes et à des mutations ponctuelles dans les gènes des porines membranaires. Aucun gène de carbapénémase n'a été identifié. Conclusion: Les tests de sensibilité phénotypique aux antimicrobiens ont identifié cet isolat de K. pneumoniae comme un organisme résistant aux carbapénèmes ne produisant pas de carbapénémase avec le mécanisme génotypique proposé, y compris l'altération des pompes à efflux et l'activité et/ou l'expression de porines membranaires. Signification clinique: Actuellement, l'utilisation des médicaments antimicrobiens à base de carbapénème en médecine vétérinaire est limitée, et les praticiens peuvent ne pas être familiers ou ne pas être au fait de ce type de résistance, de son importance dans les rapports de routine sur les tests de sensibilité aux antimicrobiens et de ses implications pour la thérapie antimicrobienne et la santé publique. Les Enterobacterales résistantes aux carbapénèmes sont rarement isolées des animaux de compagnie; cependant, en raison de l'adoption croissante d'interventions médicales et chirurgicales avancées, elles peuvent devenir plus répandues.(Traduit par Dr Serge Messier).

The fish pathogen Flavobacterium columnare represents four distinct species: Flavobacterium columnare, Flavobacterium covae sp. nov., Flavobacterium davisii sp. nov. and Flavobacterium oreochromis sp. nov., and emended description of Flavobacterium columnare.

Flavobacterium columnare is the causative agent of columnaris disease in freshwater fish and four discrete genetic groups exist within the species, suggesting that the species designation requires revision. The present study determined the taxonomic status of the four genetic groups of F. columnare using polyphasic and phylogenomic approaches and included five representative isolates from each genetic group (including type strain ATCC 23463T; genetic group 1). 16S rRNA gene sequence analysis revealed genetic group 2 isolate AL-02-36T, genetic group 3 isolate 90-106T, and genetic group 4 isolate Costa Rica 04-02-TNT shared less than <98.8 % sequence identity to F. columnare ATCC 23463T. Phylogenetic analyses of 16S rRNA and gyrB genes using different methodologies demonstrated the four genetic groups formed well-supported and distinct clades within the genus Flavobacterium. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (GGDC) values between F. columnare ATCC 23463T, genetic group 2 isolate AL-02-36T, genetic group 3 isolate 90-106T, and genetic group 4 isolate Costa Rica 04-02-TNT were less than 90.84% and 42.7%, respectively. Biochemical and physiological characteristics were similar among the four genetic groups; however, quantitative differences in fatty acid profiles were detected and MALDI-TOF analyses demonstrated numerous distinguishing peaks unique to each genetic group. Chemotaxonomic, MALDI-TOF characterization and ANI/GGDC calculations afforded differentiation between the genetic groups, indicating each group is a discrete species. Herein, the names F. covae sp. nov. (AL-02-36T), F. davisii sp. nov. (90-106T), and F. oreochromis sp. nov. (Costa Rica 04-02-TNT) are proposed to represent genetic groups 2, 3, and 4, respectively.

Fluorescence activated cell sorting and fermentation analysis to study rumen microbiome responses to administered live microbials and yeast cell wall derived prebiotics.

Rapid dietary changes, such as switching from high-forage to high-grain diets, can modify the rumen microbiome and initiate gastrointestinal distress, such as bloating. In such cases, feed additives, including prebiotics and live microbials, can be used to mitigate these negative consequences. Bio-Mos® is a carbohydrate-based prebiotic derived from yeast cells that is reported to increase livestock performance. Here, the responses of rumen bacterial cells to Bio-Mos® were quantified, sorted by flow cytometry using fluorescently-labeled yeast mannan, and taxonomically characterized using fluorescence in situ hybridization and 16S rRNA sequencing. Further, to evaluate the effects of bovine-adapted Bacteroides thetaiotaomicron administration as a live microbial with and without Bio-Mos® supplementation, we analyzed microbial fermentation products, changes to carbohydrate profiles, and shifts in microbial composition of an in vitro rumen community. Bio-Mos® was shown to be an effective prebiotic that significantly altered microbial diversity, composition, and fermentation; while addition of B. thetaiotaomicron had no effect on community composition and resulted in fewer significant changes to microbial fermentation. When combined with Bio-Mos®, there were notable, although not significant, changes to major bacterial taxa, along with increased significant changes in fermentation end products. These data suggest a synergistic effect is elicited by combining Bio-Mos® and B. thetaiotaomicron. This protocol provides a new in vitro methodology that could be extended to evaluate prebiotics and probiotics in more complex artificial rumen systems and live animals.

Comparison of pathogenic bacteria in the upper and lower respiratory tracts of cattle either directly transported to a feedlot or co-mingled at auction markets prior to feedlot placement.

Introduction: Bacterial bronchopneumonia (BP) has been associated with purchasing cattle through auction markets. However, whether auction markets are a source of BP-associated bacterial pathogens is unknown. This study evaluated prevalence, antimicrobial susceptibility, and genetic relatedness (using pulsed-field gel electrophoresis, PFGE) of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni isolated from cattle either transported to an auction market prior to feedlot placement (AUC), or directly to a feedlot from a farm (RANC). Methods: Two groups of cattle were enrolled (N = 30 per group) from two separate farms with 15 animals from an individual farm designated as AUC or RANC. Deep nasal swab (DNS) and trans-tracheal aspirates (TTA) were collected on day 0 at weaning (T0) and on day 2 at on-arrival processing at the feedlot (T1). The DNS were also collected on day 9 (T2) and day 30 (T3) after arrival at the feedlot. Results and discussion: In both TTA and DNS, prevalence of bacteria did not differ between AUC and RANC groups (P > 0.05). None of the bacteria isolated at T0 were resistant to antimicrobials and diversity of all bacteria was greatest at T0 and T1. In Group 1 cattle, 100% of P. multocida isolated at T2 and T3 were multi-drug resistant. These isolates were highly related (>90%) according to PFGE, with most being clones. Though limited in size, results for animals evaluated in this study suggested that auction markets were not a major source of resistant BP pathogens, however, horizontal transmission of a multi-resistant strain of P. multocida occurred in a feedlot. Spread of resistant P. multocida was likely due to the selective pressures imposed by feedlot antimicrobial use and encoded resistance by the bacteria.

Microbial Variability of Commercial Equine Probiotics.

Probiotics are defined as live microorganisms that confer a health benefit to the host when administered in adequate amounts, therefore the presence of viable microorganisms is essential. Previous studies reported inconsistencies when comparing contents of probiotics marketed for use in animals with label claims. However, to the author's knowledge the variability of the same product between different lots has yet to be evaluated. The objective of this observational study was to evaluate the content and inter-lot variability of commercial equine probiotic products using culture dependent and independent techniques. Eleven probiotics marketed for use in horses were evaluated for microbial content using culture-dependent and independent techniques. The content was then compared to microorganisms listed and quantities on the label of the probiotic. Statistical analyses were performed using a Kruskal-Wallis test. None of the 11 probiotics met their label claim. Nine of the 11 did not have statistically significant inter-lot variability. Several products lacked microorganisms listed on the label based on both culture and polymerase chain reaction (PCR) analyses.

Effect of an In Vitro Proximal Gastrointestinal Tract on Viability of Commercially Available Equine Probiotics.

Probiotics, by definition, are live micro-organisms and should remain viable when they reach the intended site of action which is typically the cecum and/or colon. In humans, probiotics often need enteric protection to survive transit through the proximal gastrointestinal (GI) tract. Typically, equine probiotics do not advertise enteric protection and to the author's knowledge the viability of equine probiotics after exposure to the proximal GI tract has not been evaluated. The objective of this study was to evaluate the effect of an in vitro simulation of the equine proximal GI tract on probiotic viability. We hypothesized that the simulated proximal GI tract would adversely effect microbial viability and that the adverse effects would be partially ameliorated by increasing the gastric pH to 4. A total of 11 products were evaluated of which six had at least one micro-organism that was adversely effected by exposure to the proximal GI tract and four of which had at least one micro-organism that was adversely affected when the gastric pH was increased to 4.0. Results from this study indicate that some micro-organisms in equine probiotics do not appear to be adversely affected by exposure to the equine proximal GI tract.