Development of a Multiplex Real-Time PCR Assay for Predicting Macrolide and Tetracycline Resistance Associated with Bacterial Pathogens of Bovine Respiratory Disease

Dutta, Enakshy; Loy, John Dustin; Deal, Caitlyn; Wynn, Emily L.; Clawson, Marion; Clarke, Jennifer; Wang, Bing

doi:10.3390/pathogens10010064

Cited by 10 publications

(6 citation statements)

References 29 publications

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“…Several studies have shown genotype–phenotype concordance rates to vary across antimicrobial drugs, BRD bacteria, genes of interest, testing methods used, history of antimicrobial exposure, animal sampling time point (e.g. time of feedlot arrival, revaccination, or time of BRD diagnosis), and sampling location within the respiratory tract [ 13 , 14 , 60 , 61 ]. Some general reasons for genotype–phenotype discordance include alternative mechanisms of phenotypic resistance, potential presence of ARGs not yet identified [ 14 ], the potential for resistance genes to be present but inactive [ 60 ], and availability of approved breakpoints for each drug-bacterium-host combination [ 62 ].…”

Section: Discussionmentioning

confidence: 99%

Evaluating the potential of third generation metagenomic sequencing for the detection of BRD pathogens and genetic determinants of antimicrobial resistance in chronically ill feedlot cattle

et al. 2022

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Background Bovine respiratory disease (BRD) is an important cause of morbidity and mortality and is responsible for most of the injectable antimicrobial use in the feedlot industry. Traditional bacterial culture can be used to diagnose BRD by confirming the presence of causative pathogens and to support antimicrobial selection. However, given that bacterial culture takes up to a week and early intervention is critical for treatment success, culture has limited utility for informing rapid therapeutic decision-making. In contrast, metagenomic sequencing has the potential to quickly resolve all nucleic acid in a sample, including pathogen biomarkers and antimicrobial resistance genes. In particular, third-generation Oxford Nanopore Technology sequencing platforms provide long reads and access to raw sequencing data in real-time as it is produced, thereby reducing the time from sample collection to diagnostic answer. The purpose of this study was to compare the performance of nanopore metagenomic sequencing to traditional culture and sensitivity methods as applied to nasopharyngeal samples from segregated groups of chronically ill feedlot cattle, previously treated with antimicrobials for nonresponsive pneumonia or lameness. Results BRD pathogens were isolated from most samples and a variety of different resistance profiles were observed across isolates. The sequencing data indicated the samples were dominated by Moraxella bovoculi, Mannheimia haemolytica, Mycoplasma dispar, and Pasteurella multocida, and included a wide range of antimicrobial resistance genes (ARGs), encoding resistance for up to seven classes of antimicrobials. Genes conferring resistance to beta-lactams were the most commonly detected, while the tetH gene was detected in the most samples overall. Metagenomic sequencing detected the BRD pathogens of interest more often than did culture, but there was limited concordance between phenotypic resistance to antimicrobials and the presence of relevant ARGs. Conclusions Metagenomic sequencing can reduce the time from sampling to results, detect pathogens missed by bacterial culture, and identify genetically encoded determinants of resistance. Increasing sequencing coverage of target organisms will be an essential component of improving the reliability of this technology, such that it can be better used for the surveillance of pathogens of interest, genetic determinants of resistance, and to inform diagnostic decisions.

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Section: Discussionmentioning

confidence: 99%

Evaluating the potential of third generation metagenomic sequencing for the detection of BRD pathogens and genetic determinants of antimicrobial resistance in chronically ill feedlot cattle

et al. 2022

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“…This isolate had previously been determined by Sanger sequencing to contain eight AMR genes associated with ICE Pmu1 , namely aphA1 , sul2 , tetR(H) , floR , erm(42) , bla OXA2 , msr(E) , and mph(E) , with four of these genes also associated with ICE Mh1 ; all eight genes had been detected by a mqPCR assay (described below) that had been co-developed by one of the authors (J.D.L.) [ 27 ], hence M. haemolytica strain 2308 was validated for use as positive control for this mqPCR assay. E. coli strain 25922 (American Type Culture Collection) is a laboratory strain that does not possess these genes, and was used as a negative control for the mqPCR assay (described below).…”

Section: Methodsmentioning

confidence: 99%

“…The mqPCR assay and targets were in part based on previous work with applications to BRD pathogens [ 27 ]. Four-plex mqPCR (25-µL reaction) assays included targets, reagents, and primers as described in Table S4 .…”

Section: Methodsmentioning

confidence: 99%

“…A 2.0 µL aliquot of this DNA template was used in the 25-µL PCR reaction. Individual 25-µL reaction PCR assays were conducted using primer pairs as shown in Table S5 [ 27 , 30 , 31 ]. The PCR reaction consisted of 2.5 µL 10X ThermoPol Reaction Buffer, 1.0 μL of each forward and reverse primer (10 μM each, 2 μL total), 0.5 µL dNTP mix (10 mM each dNTP), 0.25 µL NEB Taq DNA Polymerase (New England Biolabs, Ipswich, MA, USA), 17.75 µL UltraPure Water, and 2.0 µL (5 ng/μL) of template DNA.…”

Section: Methodsmentioning

confidence: 99%

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Association of ISVsa3 with Multidrug Resistance in Salmonella enterica Isolates from Cattle (Bos taurus)

et al. 2023

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Salmonella enterica is, globally, an important cause of human illness with beef being a significant attributable source. In the human patient, systemic Salmonella infection requires antibiotic therapy, and when strains are multidrug resistant (MDR), no effective treatment may be available. MDR in bacteria is often associated with the presence of mobile genetic elements (MGE) that mediate horizontal spread of antimicrobial resistance (AMR) genes. In this study, we sought to determine the potential relationship of MDR in bovine Salmonella isolates with MGE. The present study involved 111 bovine Salmonella isolates obtained collectively from specimens derived from healthy cattle or their environments at Midwestern U.S. feedyards (2000–2001, n = 19), or specimens from sick cattle submitted to the Nebraska Veterinary Diagnostic Center (2010–2020, n = 92). Phenotypically, 33/111 isolates (29.7%) were MDR (resistant to ≥3 drug classes). Based on whole-genome sequencing (WGS; n = 41) and PCR (n = 111), a MDR phenotype was strongly associated (OR = 186; p < 0.0001) with carriage of ISVsa3, an IS91-like Family transposase. In all 41 isolates analyzed by WGS ((31 MDR and 10 non-MDR (resistant to 0–2 antibiotic classes)), MDR genes were associated with carriage of ISVsa3, most often on an IncC type plasmid carrying blaCMY-2. The typical arrangement was floR, tet(A), aph(6)-Id, aph(3″)-Ib, and sul2 flanked by ISVsa3. These results suggest that AMR genes in MDR S. enterica isolates of cattle are frequently associated with ISVsa3 and carried on IncC plasmids. Further research is needed to better understand the role of ISVsa3 in dissemination of MDR Salmonella strains.

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“…Due to the multifactorial and polymicrobial nature of BRD, effort has been made to illustrate host factors, management schema, etiological associations, and stressful environmental factors associated with disease development and progression 1 , 2 , 4 . Recent research has been focused on predicting BRD susceptibility and outcomes over time 5 – 8 . Unfortunately, clinical diagnostic and prognostic prediction models remain contested, and mechanistic information regarding host–pathogen interactions and the development of clinical BRD is not fully understood.…”

Section: Introductionmentioning

confidence: 99%

Genes and regulatory mechanisms associated with experimentally-induced bovine respiratory disease identified using supervised machine learning methodology

Scott

Woolums

Swiderski

et al. 2021

Sci Rep

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Bovine respiratory disease (BRD) is a multifactorial disease involving complex host immune interactions shaped by pathogenic agents and environmental factors. Advancements in RNA sequencing and associated analytical methods are improving our understanding of host response related to BRD pathophysiology. Supervised machine learning (ML) approaches present one such method for analyzing new and previously published transcriptome data to identify novel disease-associated genes and mechanisms. Our objective was to apply ML models to lung and immunological tissue datasets acquired from previous clinical BRD experiments to identify genes that classify disease with high accuracy. Raw mRNA sequencing reads from 151 bovine datasets (n = 123 BRD, n = 28 control) were downloaded from NCBI-GEO. Quality filtered reads were assembled in a HISAT2/Stringtie2 pipeline. Raw gene counts for ML analysis were normalized, transformed, and analyzed with MLSeq, utilizing six ML models. Cross-validation parameters (fivefold, repeated 10 times) were applied to 70% of the compiled datasets for ML model training and parameter tuning; optimized ML models were tested with the remaining 30%. Downstream analysis of significant genes identified by the top ML models, based on classification accuracy for each etiological association, was performed within WebGestalt and Reactome (FDR ≤ 0.05). Nearest shrunken centroid and Poisson linear discriminant analysis with power transformation models identified 154 and 195 significant genes for IBR and BRSV, respectively; from these genes, the two ML models discriminated IBR and BRSV with 100% accuracy compared to sham controls. Significant genes classified by the top ML models in IBR (154) and BRSV (195), but not BVDV (74), were related to type I interferon production and IL-8 secretion, specifically in lymphoid tissue and not homogenized lung tissue. Genes identified in Mannheimia haemolytica infections (97) were involved in activating classical and alternative pathways of complement. Novel findings, including expression of genes related to reduced mitochondrial oxygenation and ATP synthesis in consolidated lung tissue, were discovered. Genes identified in each analysis represent distinct genomic events relevant to understanding and predicting clinical BRD. Our analysis demonstrates the utility of ML with published datasets for discovering functional information to support the prediction and understanding of clinical BRD.

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Development of a Multiplex Real-Time PCR Assay for Predicting Macrolide and Tetracycline Resistance Associated with Bacterial Pathogens of Bovine Respiratory Disease

Cited by 10 publications

References 29 publications

Evaluating the potential of third generation metagenomic sequencing for the detection of BRD pathogens and genetic determinants of antimicrobial resistance in chronically ill feedlot cattle

Evaluating the potential of third generation metagenomic sequencing for the detection of BRD pathogens and genetic determinants of antimicrobial resistance in chronically ill feedlot cattle

Association of ISVsa3 with Multidrug Resistance in Salmonella enterica Isolates from Cattle (Bos taurus)

Genes and regulatory mechanisms associated with experimentally-induced bovine respiratory disease identified using supervised machine learning methodology

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