Enterococci as a One Health indicator of antimicrobial resistance.

The rapid increase of antimicrobial-resistant bacteria in humans and livestock is concerning. Antimicrobials are essential for the treatment of disease in modern day medicine, and their misuse in humans and food animals has contributed to an increase in the prevalence of antimicrobial-resistant bacteria. Globally, antimicrobial resistance is recognized as a One Health problem affecting humans, animals, and environment. Enterococcal species are Gram-positive bacteria that are widely distributed in nature. Their occurrence, prevalence, and persistence across the One Health continuum make them an ideal candidate to study antimicrobial resistance from a One Health perspective. The objective of this review was to summarize the role of enterococci as an indicator of antimicrobial resistance across One Health sectors. We also briefly address the prevalence of enterococci in human, animal, and environmental settings. In addition, a 16S RNA gene-based phylogenetic tree was constructed to visualize the evolutionary relationship among enterococcal species and whether they segregate based on host environment. We also review the genomic basis of antimicrobial resistance in enterococcal species across the One Health continuum.

Application of Four Genotyping Methods to Mycoplasma bovis Isolates Derived from Western Canadian Feedlot Cattle.

Mycoplasma bovis is a significant pathogen of feedlot cattle, responsible for chronic pneumonia and polyarthritis syndrome (CPPS). M. bovis isolates (n = 129) were used to compare four methods of phylogenetic analysis and to determine if the isolates' genotypes were associated with phenotypes. Metadata included the health status of the animal from which an isolate was derived (healthy, diseased, or dead), anatomical location (nasopharynx, lung, or joint), feedlot, and production year (2006 to 2018). Four in silico phylogenetic typing methods were used: multilocus sequence typing (MLST), core genome MLST (cgMLST), core genome single nucleotide variant (cgSNV) analysis, and whole-genome SNV (wgSNV) analysis. Using Simpson's diversity index (D) as a proxy for resolution, MLST had the lowest resolution (D = 0.932); cgSNV (D = 0.984) and cgMLST (D = 0.987) generated comparable results; and wgSNV (D = 1.000) provided the highest resolution. Visual inspection of the minimum spanning trees found that the memberships of the clonal complexes and clades had similar structural appearances. Although MLST had the lowest resolution, this methodology was intuitive and easy to apply, and the PubMLST database facilitates the comparison of sequence types across studies. The cg methods had higher resolution than MLST, and the graphical interface software was user-friendly for nonbioinformaticians, but the proprietary software is relatively expensive. The wgSNV approach was the most robust for processing poor-quality sequence data while offering the highest resolution; however, application of its software requires specialized training. None of the four methods could associate genotypes with phenotypes.

Resistance determinants and their genetic context in enterobacteria from a longitudinal study of pigs reared under various husbandry conditions.

Pigs are major reservoirs of resistant Enterobacteriaceae that can reach humans through consumption of contaminated meat or vegetables grown in manure-fertilized soil. Samples were collected from sows during lactation and their piglets at five time points spanning the production cycle. Cefotaxime-resistant bacteria were quantified and isolated from feed, feces, manures and carcasses of pigs reared with penicillin-using or antibiotic-free husbandries. The isolates were characterized by antibiotic susceptibility testing, whole genome sequencing and conjugation assays. The extended spectrum ß-lactamase (ESBL) phenotype was more frequent in isolates originating from antibiotic-free animals, while the bacteria isolated from penicillin-using animals were on average resistant to a greater number of antibiotics. The ESBL-encoding genes identified were bla CTX-M-1, bla CTX-M-15 and bla CMY-2 and they co-localised on plasmids with various genes encoding resistance to ß-lactams, co-trimoxazole, phenicols and tetracycline, all antibiotics used in pig production. Groups of genes conferring the observed resistance and the mobile elements disseminating multidrug resistance were determined. The observed resistance to ß-lactams was mainly due to the complementary actions of penicillin-binding proteins, an efflux pump and ß-lactamases. Most resistance determinants were shared by animals raised with or without antimicrobials. This suggests a key contribution of indigenous enterobacteria maternally transmitted along the sow lineage, regardless of antimicrobial use. It is unclear if the antimicrobial resistance observed in the enterobacteria populations of the commercial pig herds studied were present before the use of antibiotics, or the extent to which historical antimicrobial use exerted a selective pressure defining the resistant bacterial populations in farms using penicillin prophylaxis.Importance: Antimicrobial resistance is a global threat that needs to be fought on numerous fronts along the One Health continuum. Vast quantities of antimicrobials are used in agriculture to ensure animal welfare and productivity, and are arguably a driving force for the persistence of environmental and food-borne resistant bacteria. This study evaluated the impact of conventional, organic and other antibiotic-free husbandry practices on the frequency and nature of antimicrobial resistance genes and multidrug resistant enterobacteria. It provides knowledge about the relative contribution of specific resistance determinants to observed antibiotic resistance. It also showed the clear co-selection of genes coding for extended-spectrum beta-lactamases and genes coding for the resistance to antibiotics commonly used for prophylaxis or in curative treatments in pig operations.

Degradation of antimicrobial resistance genes within stockpiled beef cattle feedlot manure.

Degradation of antimicrobial resistance genes (ARG) in manure from beef cattle administered (kg-1 feed) 44 mg of chlortetracycline (CTC), 44 mg of chlortetracycline plus sulfamethazine (CTCSMZ), 11 mg of tylosin (TYL), or no antimicrobials (Control) was examined. Manure was stockpiled and quantitative PCR (qPCR) was used to assess tetracycline [tet(C), (L), (M), (W)], erythromycin [erm(A), (B), (F), (X)], and sulfamethazine [sul(1), (2)] ARG and 16S rDNA. After 102 d, copies of all ARG decreased by 0.3 to 1.5 log10 copies (g dry matter)-1. Temperature in the interior of piles averaged ≥ 55 °C for 10 d, except for CTCSMZ, but did not reach 55 °C at pile exteriors. Compared to Control, CTCSMZ increased (P < 0.05) tet(C), tet(M), tet(W), sul(1), and sul(2) in stockpiled manure. Copies of 16S rDNA remained higher (P < 0.05) in CTCSMZ than Control for the first 26 d. Levels of most ARG did not differ between the interior and exterior of stockpiles. Our results suggest that stockpiled manure would still introduce ARG to land upon manure application, but at levels lower than if manure was applied fresh.

Comparative genomics of multidrug-resistant Enterococcus spp. isolated from wastewater treatment plants.

BACKGROUND: Wastewater treatment plants (WWTPs) are considered hotspots for the environmental dissemination of antimicrobial resistance (AMR) determinants. Vancomycin-Resistant Enterococcus (VRE) are candidates for gauging the degree of AMR bacteria in wastewater. Enterococcus faecalis and Enterococcus faecium are recognized indicators of fecal contamination in water. Comparative genomics of enterococci isolated from conventional activated sludge (CAS) and biological aerated filter (BAF) WWTPs was conducted. RESULTS: VRE isolates, including E. faecalis (n = 24), E. faecium (n = 11), E. casseliflavus (n = 2) and E. gallinarum (n = 2) were selected for sequencing based on WWTP source, species and AMR phenotype. The pangenomes of E. faecium and E. faecalis were both open. The genomic fraction related to the mobilome was positively correlated with genome size in E. faecium (p < 0.001) and E. faecalis (p < 0.001) and with the number of AMR genes in E. faecium (p = 0.005). Genes conferring vancomycin resistance, including vanA and vanM (E. faecium), vanG (E. faecalis), and vanC (E. casseliflavus/E. gallinarum), were detected in 20 genomes. The most prominent functional AMR genes were efflux pumps and transporters. A minimum of 16, 6, 5 and 3 virulence genes were detected in E. faecium, E. faecalis, E. casseliflavus and E. gallinarum, respectively. Virulence genes were more common in E. faecalis and E. faecium, than E. casseliflavus and E. gallinarum. A number of mobile genetic elements were shared among species. Functional CRISPR/Cas arrays were detected in 13 E. faecalis genomes, with all but one also containing a prophage. The lack of a functional CRISPR/Cas arrays was associated with multi-drug resistance in E. faecium. Phylogenetic analysis demonstrated differential clustering of isolates based on original source but not WWTP. Genes related to phage and CRISPR/Cas arrays could potentially serve as environmental biomarkers. CONCLUSIONS: There was no discernible difference between enterococcal genomes from the CAS and BAF WWTPs. E. faecalis and E. faecium have smaller genomes and harbor more virulence, AMR, and mobile genetic elements than other Enterococcus spp.

Comparative diversity of microbiomes and Resistomes in beef feedlots, downstream environments and urban sewage influent.

BACKGROUND: Comparative knowledge of microbiomes and resistomes across environmental interfaces between animal production systems and urban settings is lacking. In this study, we executed a comparative analysis of the microbiota and resistomes of metagenomes from cattle feces, catch basin water, manured agricultural soil and urban sewage. RESULTS: Metagenomic DNA from composite fecal samples (FC; n = 12) collected from penned cattle at four feedlots in Alberta, Canada, along with water from adjacent catchment basins (CB; n = 13), soil (n = 4) from fields in the vicinity of one of the feedlots and urban sewage influent (SI; n = 6) from two municipalities were subjected to Illumina HiSeq2000 sequencing. Firmicutes exhibited the highest prevalence (40%) in FC, whereas Proteobacteria were most abundant in CB (64%), soil (60%) and SI (83%). Among sample types, SI had the highest diversity of antimicrobial resistance (AMR), and metal and biocide resistance (MBR) classes (13 & 15) followed by FC (10 & 8), CB (8 & 4), and soil (6 & 1). The highest antimicrobial resistant (AMR) gene (ARG) abundance was harboured by FC, whereas soil samples had a very small, but unique resistome which did not overlap with FC & CB resistomes. In the beef production system, tetracycline resistance predominated followed by macrolide resistance. The SI resistome harboured ß-lactam, macrolide, tetracycline, aminoglycoside, fluoroquinolone and fosfomycin resistance determinants. Metal and biocide resistance accounted for 26% of the SI resistome with a predominance of mercury resistance. CONCLUSIONS: This study demonstrates an increasing divergence in the nature of the microbiome and resistome as the distance from the feedlot increases. Consistent with antimicrobial use, tetracycline and macrolide resistance genes were predominant in the beef production system. One of the feedlots contributed both conventional (raised with antibiotics) and natural (raised without antibiotics) pens samples. Although natural pen samples exhibited a microbiota composition that was similar to samples from conventional pens, their resistome was less complex. Similarly, the SI resistome was indicative of drug classes used in humans and the greater abundance of mercury resistance may be associated with contamination of municipal water with household and industrial products.

Lower Respiratory Tract Microbiome and Resistome of Bovine Respiratory Disease Mortalities.

Bovine respiratory disease (BRD) continues to be a serious health problem in beef cattle production. A multifactorial condition, BRD encompasses several types of pneumonia that are associated with multiple viral and bacterial agents. Comprehensive identification of microbes associated with BRD fatalities could enhance our understanding of the range of pathogens that contribute to the disease and identify new therapeutic targets. This study used metagenomic analysis to describe the lower respiratory tract microbiome and resistome of 15 feedlot cattle BRD and 3 non-BRD mortalities along with any affiliated integrative and conjugative elements (ICEs). Known bacterial pathogens associated with BRD, including Histophilus somni, Mannheimia haemolytica, and Mycoplasma bovis, were relatively abundant (> 5%) in most, but not all samples. Other relatively abundant genera (> 1%) included Acinetobacter, Bacillus, Bacteroides, Clostridium, Enterococcus, and Pseudomonas. Antimicrobial resistance genes (ARGs) comprised up to 0.5% of sequences and many of these genes were associated with ICEs previously described within the Pasteurellaceae family. A total of 20 putative ICEs were detected among 16 samples. These results document the wide diversity of microorganisms in the lower respiratory tract of cattle that have succumbed to BRD. The data also strongly suggest that antimicrobial-resistant Pasteurellaceae strains are prevalent in BRD cases in Alberta and that the resistance observed is associated with ICEs. The presence of ICEs harboring a wide array of ARGs holds significant consequence for the effectiveness of drug therapies for the control of BRD in beef cattle.

Plasmid Distribution among Escherichia coli from Livestock and Associated Wastewater: Unraveling Factors That Shape the Presence of Genes Conferring Third-Generation Cephalosporin Resistance.

A key concern with agricultural wastewater storage ponds is that they may provide an environment conducive for horizontal exchange of antibiotic resistance genes (ARGs), thereby facilitating the emergence of antibiotic resistant pathogens. Central to this exchange are mobile genetic elements like plasmids; yet, the factors shaping their presence in agricultural environments remain poorly understood. Here, using Escherichia coli as a model bacterium, we examined genetic backgrounds and plasmid profiles of generic fecal and wastewater isolates and those possessing blaCTX-M and blaCMY-2 genes (which confer resistance to third-generation cephalosporins) to delineate factors shaping the environmental persistence of plasmid-associated ARGs in beef cattle feedlots. The wastewater environment exerted minimal influence on plasmid repertoires, as the number of plasmids and distribution of different incompatibility groups did not differ between generic fecal and wastewater isolates. The blaCTX-M and blaCMY-2 genes were associated with IncF and IncA/C plasmids, respectively, and host isolates possessing these ARGs had fewer plasmids than generic isolates, suggesting ARG-bearing plasmids may associate predominantly with such hosts to compensate for the metabolic burden imposed by these plasmids. Phylogeny also appeared to be a factor for blaCTX-M genes, as their bacterial hosts were restricted to particular genetic lineages, including the environmentally adapted ET-1 clade, as noted previously for these genes. Ultimately, these findings have important implications for evaluating human health risks of agricultural wastewater with respect to environmental persistence of ARGs and may help identify options for improving wastewater treatment.

Neptune: a bioinformatics tool for rapid discovery of genomic variation in bacterial populations.

The ready availability of vast amounts of genomic sequence data has created the need to rethink comparative genomics algorithms using 'big data' approaches. Neptune is an efficient system for rapidly locating differentially abundant genomic content in bacterial populations using an exact k-mer matching strategy, while accommodating k-mer mismatches. Neptune's loci discovery process identifies sequences that are sufficiently common to a group of target sequences and sufficiently absent from non-targets using probabilistic models. Neptune uses parallel computing to efficiently identify and extract these loci from draft genome assemblies without requiring multiple sequence alignments or other computationally expensive comparative sequence analyses. Tests on simulated and real datasets showed that Neptune rapidly identifies regions that are both sensitive and specific. We demonstrate that this system can identify trait-specific loci from different bacterial lineages. Neptune is broadly applicable for comparative bacterial analyses, yet will particularly benefit pathogenomic applications, owing to efficient and sensitive discovery of differentially abundant genomic loci. The software is available for download at: http://github.com/phac-nml/neptune.

Dissipation of antimicrobial resistance genes in compost originating from cattle manure after direct oral administration or post-excretion fortification of antimicrobials.

Dissipation of antimicrobial resistance genes (ARG) during composting of cattle manure generated through fortification versus administration of antimicrobials in feed was compared. Manure was collected from cattle fed diets containing (kg-1) dry matter (DM): (1) 44 mg chlortetracycline (CTC), (2) a mixture of 44 mg each of chlortetracycline and sulfamethazine (CTCSMZ), (3) 11 mg tylosin (TYL) or (4) Control, no antimicrobials. Manures were composted for 30 d with a single mixing after 16 d to generate the second heating cycle. Quantitative PCR (qPCR) was used to measure 16S rDNA and tetracycline (tet), erythromycin (erm) and sulfamethazine (sul) genes. Temperature peaks ranged from 48 to 68°C across treatments in the first composting cycle, but except for the control, did not exceed 55°C in the second cycle. Copy numbers of 16S rDNA decreased (P < 0.05) during composting, but were not altered by antimcrobials. Except tet(L), all ARG decreased by 0.1-1.6 log10 g DM-1 in the first cycle, but some genes (tet[B], tet[L], erm[F], erm[X]) increased (P < 0.05) by 1.0-3.1 log10 g DM-1 in the second. During composting, levels of tet(M) and tet(W) in CTC, erm(A), erm(B) and erm(X) in TYL, and sul(1) in CTCSMZ remained higher (P < 0.05) in fed than fortified treatments. The dissipation of ARG during composting of manure fortified with antimicrobials differs from manure generated by cattle that are administered antimicrobials in feed, and does not always align with the dissipation of antimicrobial residues.

Comparative genomics of Enterococcus spp. isolated from bovine feces.

BACKGROUND: Enterococcus is ubiquitous in nature and is a commensal of both the bovine and human gastrointestinal (GI) tract. It is also associated with clinical infections in humans. Subtherapeutic administration of antibiotics to cattle selects for antibiotic resistant enterococci in the bovine GI tract. Antibiotic resistance genes (ARGs) may be present in enterococci following antibiotic use in cattle. If located on mobile genetic elements (MGEs) their dissemination between Enterococcus species and to pathogenic bacteria may be promoted, reducing the efficacy of antibiotics. RESULTS: We present a comparative genomic analysis of twenty-one Enterococcus spp. isolated from bovine feces including Enterococcus hirae (n = 10), Enterococcus faecium (n = 3), Enterococcus villorum (n = 2), Enterococcus casseliflavus (n = 2), Enterococcus faecalis (n = 1), Enterococcus durans (n = 1), Enterococcus gallinarum (n = 1) and Enterococcus thailandicus (n = 1). The analysis revealed E. faecium and E. faecalis from bovine feces share features with human clinical isolates, including virulence factors. The Tn917 transposon conferring macrolide-lincosamide-streptogramin B resistance was identified in both E. faecium and E. hirae, suggesting dissemination of ARGs on MGEs may occur in the bovine GI tract. An E. faecium isolate was also identified with two integrative conjugative elements (ICEs) belonging to the Tn916 family of ICE, Tn916 and Tn5801, both conferring tetracycline resistance. CONCLUSIONS: This study confirms the presence of enterococci in the bovine GI tract possessing ARGs on MGEs, but the predominant species in cattle, E. hirae is not commonly associated with infections in humans. Analysis using additional complete genomes of E. faecium from the NCBI database demonstrated differential clustering of commensal and clinical isolates, suggesting that these strains may be specifically adapted to their respective environments.

Environmental Growth of Enterococci and Escherichia coli in Feedlot Catch Basins and a Constructed Wetland in the Absence of Fecal Input.

Population structures of fecal indicator bacteria (FIB) isolated from catch basins, a constructed wetland, and feces from a beef cattle feedlot were compared over a two-year period. Enterococcus hirae accounted for 92% of the fecal isolates, whereas secondary environments were characterized by greater relative abundance of environmentally adapted species including Enterococcus casseliflavus. While enterococci densities in the catch basins and wetland were similar under wet and drought conditions, E. hirae predominated during rainy periods, while E. casseliflavus predominated during drought conditions. Environmentally adapted species accounted for almost half of the erythromycin resistant enterococci isolated from the wetland. Densities of Escherichia coli were also comparable during wet versus drought conditions, and the relative abundance of strains from environmentally adapted clades was greater in secondary environments compared to feces. Unlike enterococci, fewer environmentally adapted E. coli strains were isolated on selective media containing ceftriaxone from the wetland compared to feces, suggesting resistance to this antibiotic may not be well maintained in the absence of selective pressure. Overall, these findings suggest that secondary environments select for environmentally adapted FIB. While these species and clades tend to be of limited clinical relevance, they could potentially serve as reservoirs of antimicrobial resistance.

Dissipation of Antimicrobial Resistance Determinants in Composted and Stockpiled Beef Cattle Manure.

Windrow composting or stockpiling reduces the viability of pathogens and antimicrobial residues in manure. However, the impact of these manure management practices on the persistence of genes coding for antimicrobial resistance is less well known. In this study, manure from cattle administered 44 mg of chlortetracycline kg feed (dry wt. basis) (CTC), 44 mg of CTC and 44 mg of sulfamethazine kg feed (CTCSMZ), 11 mg of tylosin kg feed (TYL), and no antimicrobials (control) were composted or stockpiled over 102 d. Temperature remained ≥55°C for 35 d in compost and 2 d in stockpiles. Quantitative PCR was used to measure levels of 16S rRNA genes and tetracycline [(B), (C), (L), (M), (W)], erythromycin [(A), (B), (F), (X)], and sulfamethazine [(1), (2)] resistance determinants. After 102 d, 16S rRNA genes and all resistance determinants declined by 0.5 to 3 log copies per gram dry matter. Copies of 16S rRNA genes were affected ( < 0.05) by antimicrobials with the ranking of control > CTC = TYL > CTCSMZ. Compared with the control, antimicrobials did not increase the abundance of resistance genes in either composted or stockpiled manure, except (M) and (2) in CTCSMZ ( < 0.05). The decline in 16S rRNA genes and resistance determinants was higher ( < 0.05) in composted than in stockpiled manure. We conclude that composting may be more effective than stockpiling in reducing the introduction of antimicrobial resistance genes into the environment before land application of manure.

Pathogens of bovine respiratory disease in North American feedlots conferring multidrug resistance via integrative conjugative elements.

In this study, we determined the prevalence of bovine respiratory disease (BRD)-associated viral and bacterial pathogens in cattle and characterized the genetic profiles, antimicrobial susceptibilities, and nature of antimicrobial resistance determinants in collected bacteria. Nasopharyngeal swab and lung tissue samples from 68 BRD mortalities in Alberta, Canada (n = 42), Texas (n = 6), and Nebraska (n = 20) were screened using PCR for bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus, bovine herpesvirus 1, parainfluenza type 3 virus, Mycoplasma bovis, Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. Excepting bovine herpesvirus 1, all agents were detected. M. haemolytica (91%) and BVDV (69%) were the most prevalent, with cooccurrence in 63% of the cattle. Isolates of M. haemolytica (n = 55), P. multocida (n = 8), and H. somni (n = 10) from lungs were also collected. Among M. haemolytica isolates, a clonal subpopulation (n = 8) was obtained from a Nebraskan feedlot. All three bacterial pathogens exhibited a high rate of antimicrobial resistance, with 45% exhibiting resistance to three or more antimicrobials. M. haemolytica (n = 18), P. multocida (n = 3), and H. somni (n = 3) from Texas and Nebraska possessed integrative conjugative elements (ICE) that conferred resistance for up to seven different antimicrobial classes. ICE were shown to be transferred via conjugation from P. multocida to Escherichia coli and from M. haemolytica and H. somni to P. multocida. ICE-mediated multidrug-resistant profiles of bacterial BRD pathogens could be a major detriment to many of the therapeutic antimicrobial strategies currently used to control BRD.

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.

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.

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.

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.

Sinorhizobium meliloti phospholipase C required for lipid remodeling during phosphorus limitation.

Rhizobia are Gram-negative soil bacteria able to establish nitrogen-fixing root nodules with their respective legume host plants. Besides phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine, rhizobial membranes contain phosphatidylcholine (PC) as a major membrane lipid. Under phosphate-limiting conditions of growth, some bacteria replace their membrane phospholipids with lipids lacking phosphorus. In Sinorhizobium meliloti, these phosphorus-free lipids are sulfoquinovosyl diacylglycerol, ornithine-containing lipid, and diacylglyceryl trimethylhomoserine (DGTS). Pulse-chase experiments suggest that the zwitterionic phospholipids phosphatidylethanolamine and PC act as biosynthetic precursors of DGTS under phosphorus-limiting conditions. A S. meliloti mutant, deficient in the predicted phosphatase SMc00171 was unable to degrade PC or to form DGTS in a similar way as the wild type. Cell-free extracts of Escherichia coli, in which SMc00171 had been expressed, convert PC to phosphocholine and diacylglycerol, showing that SMc00171 functions as a phospholipase C. Diacylglycerol , in turn, is the lipid anchor from which biosynthesis is initiated during the formation of the phosphorus-free membrane lipid DGTS. Inorganic phosphate can be liberated from phosphocholine. These data suggest that, in S. meliloti under phosphate-limiting conditions, membrane phospholipids provide a pool for metabolizable inorganic phosphate, which can be used for the synthesis of other essential phosphorus-containing biomolecules. This is an example of an intracellular phospholipase C in a bacterial system; however, the ability to degrade endogenous preexisting membrane phospholipids as a source of phosphorus may be a general property of Gram-negative soil bacteria.

Evaluation of metagenomic assembly methods for the detection and characterization of antimicrobial resistance determinants and associated mobilizable elements.

Antimicrobial resistance genes (ARGs) can be transferred between members of a bacterial population by mobile genetic elements (MGE). Understanding the risk of these transfer events is important in monitoring and predicting antimicrobial resistance (AMR), especially in the context of a One Health Continuum. However, there is no universally accepted method for detection of ARGs and MGEs, and especially for determining their linkages. This study used publicly available shotgun metagenomic DNA short-read (Illumina, 100 bp paired-end) sequence data from samples across the One Health Continuum (including beef cattle composite feces from feedlots, catch basin water at feedlots, agricultural soil from feedlot manured surrounding fields, and urban/municipal sewage influent from two municipal wastewater treatment plants) to develop a workflow to identify and associate ARGs and MGEs. ARG- and MGE-based targeted-assemblies with available short-read data were unable to meet this analysis goal. In contrast, de novo assembly of contigs provided enough sequence context to associate ARGs and MGEs, without compromising discovery rate. However, to estimate the relative abundance of these elements, unassembled sequence data must still be used.