Dataset Information

Novel Quantitative PCR for Rhodococcus equi and Macrolide Resistance Detection in Equine Respiratory Samples.

ABSTRACT: R. equi is an important veterinary pathogen that takes the lives of many foals every year. With the emergence and spread of MDR R. equi to current antimicrobial treatment, new tools that can provide a fast and accurate diagnosis of the disease and antimicrobial resistance profile are needed. Here, we have developed and analytically validated a multiplex qPCR for the simultaneous detection of R. equi and related macrolide resistance genes in equine respiratory samples. The three sets of oligos designed in this study to identify R. equi housekeeping gene choE and macrolide resistance genes erm(46) and erm(51) showed high analytic sensitivity with a limit of detection (LOD) individually and in combination below 12 complete genome copies per PCR reaction, and an amplification efficiency between 90% and 147%. Additionally, our multiplex qPCR shows high specificity in in-silico analysis. Furthermore, it did not present any cross-reaction with normal flora from the equine respiratory tract, nor commonly encountered respiratory pathogens in horses or other genetically close organisms. Our new quantitative PCR is a trustable tool that will improve the speed of R. equi infection diagnosis, as well as helping in treatment selection.

SUBMITTER: Narvaez SA

PROVIDER: S-EPMC9099730 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Novel Quantitative PCR for Rhodococcus equi and Macrolide Resistance Detection in Equine Respiratory Samples.

Narváez Sonsiray Álvarez SÁ Fernández Ingrid I Patel Nikita V NV Sánchez Susan S

Animals : an open access journal from MDPI 20220503 9

R. equi is an important veterinary pathogen that takes the lives of many foals every year. With the emergence and spread of MDR R. equi to current antimicrobial treatment, new tools that can provide a fast and accurate diagnosis of the disease and antimicrobial resistance profile are needed. Here, we have developed and analytically validated a multiplex qPCR for the simultaneous detection of R. equi and related macrolide resistance genes in equine respiratory samples. The th ...[more]

PMID: 35565598

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Project description:Conjugation is one of the main mechanisms involved in the spread and maintenance of antibiotic resistance in bacterial populations. We recently showed that the emerging macrolide resistance in the soilborne equine and zoonotic pathogen Rhodococcus equi is conferred by the erm(46) gene carried on the 87-kb conjugative plasmid pRErm46. Here, we investigated the conjugal transferability of pRErm46 to 14 representative bacteria likely encountered by R. equi in the environmental habitat. In vitro mating experiments demonstrated conjugation to different members of the genus Rhodococcus as well as to Nocardia and Arthrobacter spp. at frequencies ranging from ∼10-2 to 10-6 pRErm46 transfer was also observed in mating experiments in soil and horse manure, albeit at a low frequency and after prolonged incubation at 22 to 30°C (environmental temperatures), not 37°C. All transconjugants were able to transfer pRErm46 back to R. equi Conjugation could not be detected with Mycobacterium or Corynebacterium spp. or several members of the more distant phylum Firmicutes such as Enterococcus, Streptococcus, or Staphylococcus Thus, the pRErm46 host range appears to span several actinobacterial orders with certain host restriction within the Corynebacteriales All bacterial species that acquired pRErm46 expressed increased macrolide resistance with no significant deleterious impact on fitness, except in the case of Rhodococcus rhodnii Our results indicate that actinobacterial members of the environmental microbiota can both acquire and transmit the R. equi pRErm46 plasmid and thus potentially contribute to the maintenance and spread of erm(46)-mediated macrolide resistance in equine farms.IMPORTANCE This study demonstrates the efficient horizontal transfer of the Rhodococcus equi conjugative plasmid pRErm46, recently identified as the cause of the emerging macrolide resistance among equine isolates of this pathogen, to and from different environmental Actinobacteria, including a variety of rhodococci as well as Nocardia and Arthrobacter spp. The reported data support the notion that environmental microbiotas may act as reservoirs for the endemic maintenance of antimicrobial resistance in an antibiotic pressurized farm habitat.

Project description:Rhodococcus equi is a leading cause of severe pneumonia in foals. Standard treatment is dual antimicrobial therapy with a macrolide and rifampin, but the emergence of macrolide- and rifampin-resistant R. equi isolates is an increasing problem. The objective of this study was to determine the effect of macrolide and/or rifampin resistance on fitness of R. equi Three unique isogenic sets were created, each consisting of four R. equi strains, as follows: a susceptible parent isolate, strains resistant to macrolides or rifampin, and a dual macrolide- and rifampin-resistant strain. Each isogenic set's bacterial growth curve was generated in enriched medium, minimal medium (MM), and minimal medium without iron (MM-I). Bacterial survival in soil was analyzed over 12 months at -20°C, 4°C, 25°C, and 37°C, and the ability of these strains to retain antimicrobial resistance during sequential subculturing was determined. Insertion of the mobile element conferring macrolide resistance had minimal effect on in vitro growth. However, two of three rpoB mutations conferring rifampin resistance resulted in a decreased growth rate in MM. In soil, macrolide- or rifampin-resistant R. equi strains exhibited limited growth compared to that of the susceptible R. equi isolate at all temperatures except -20°C. During subculturing, macrolide resistance was lost over time, and two of three rpoB mutations reverted to the wild-type form. The growth of rifampin-resistant R. equi colonies is delayed under nutrient restriction. In soil, possession of rifampin or macrolide resistance results in decreased fitness. Both macrolide and rifampin resistance can be lost after repeated subculturing.IMPORTANCE This work advances our understanding of the opportunistic environmental pathogen Rhodococcus equi, a disease agent affecting horses and immunocompromised people. R. equi is one of the most common causes of severe pneumonia in young horses. For decades, the standard treatment for R. equi pneumonia in horses has been dual antimicrobial therapy with a macrolide and rifampin; effective alternatives to this combination are lacking. The World Health Organization classifies these antimicrobial agents as critically important for human medicine. Widespread macrolide and rifampin resistance in R. equi isolates is a major emerging problem for the horse-breeding industry and might also adversely impact human health if resistant strains infect people or transfer resistance mechanisms to other pathogens. This study details the impact of antimicrobial resistance on R. equi fitness, a vital step for understanding the ecology and epidemiology of resistant R. equi isolates, and will support development of novel strategies to combat antimicrobial resistance.

Project description:BackgroundSurveillance of antimicrobial resistance (AMR) among veterinary pathogens is necessary to identify clinically relevant patterns of AMR and to inform antimicrobial use practices. Streptococcus equi subsp. zooepidemicus and Rhodococcus equi are bacterial pathogens of major clinical importance in horses and are frequently implicated in respiratory tract infections. The objectives of this study were to describe antimicrobial resistance patterns and identify predictors of AMR and multidrug resistance (MDR) (resistance to three or more antimicrobial classes) among equine S. zooepidemicus and R. equi isolates.MethodsAntimicrobial susceptibility data from equine specimens submitted to the University of Kentucky Veterinary Diagnostic Laboratory between 2012 and 2017 were used in the study. Temporal trends in AMR and MDR were assessed using the Cochran-Armitage test. Logistic regression was used to identify associations between patient characteristics and the following outcomes: (a) MDR among S. zooepidemicus isolates, and (b) resistance to macrolides and ansamycins (rifampin) among R. equi isolates. Logistic regression was also used to investigate whether resistance of S. zooepidemicus and R. equi isolates to an antimicrobial class could be predicted by resistance to other drug classes.ResultsThe vast majority of S. zooepidemicus (99.6%) and R. equi isolates (83%) were resistant to at least one antimicrobial agent, but no significant temporal trends in AMR were observed. Approximately half (53.3%) of the S. zooepidemicus isolates were multidrug-resistant, and there was a significant (p < 0.001) increasing temporal trend of MDR among S. zooepidemicus isolates. Resistance to penicillin, which is typically recommended for treatment of suspected S. zooepidemicus infections, also increased during the study period, from 3.3% to 9.5%. Among R. equi isolates, 19.2% were resistant to one or more macrolide antibiotics, 24% were resistant to rifampin, and 15.6% were resistant to both macrolide(s) and rifampin. For both organisms, resistance to an antimicrobial class could be predicted based on resistance profiles to other drug classes. For instance, significant (p < 0.01) predictors of β-lactam resistance among S. zooepidemicus isolates included resistance to macrolides (Odds Ratio (OR) = 14.7) and ansamycins (OR = 9.3). Resistance to phenicols (OR = 3.7) and ansamycins (OR = 19.9) were associated with higher odds of macrolide resistance among R. equi isolates.ConclusionsThe increase in MDR among S. zooepidemicus isolates is concerning. The observed levels of resistance to macrolides and rifampin among R. equi are also worrisome given the limited number of antimicrobials available for treatment of this organism. The findings of this study highlight the importance of ongoing surveillance of AMR to guide treatment decisions and directions for future research.

Dataset Information

Novel Quantitative PCR for Rhodococcus equi and Macrolide Resistance Detection in Equine Respiratory Samples.

Publications

Novel Quantitative PCR for <i>Rhodococcus equi</i> and Macrolide Resistance Detection in Equine Respiratory Samples.

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