Integrative and Conjugative Elements (ICEs) in Pasteurellaceae Species and Their Detection by Multiplex PCR.
ABSTRACT: Strains of the Pasteurellaceae bacteria Pasteurella multocida and Mannheimia haemolytica are major etiological agents of bovine respiratory disease (BRD). Treatment of BRD with antimicrobials is becoming more challenging due to the increasing occurrence of resistance in infecting strains. In Pasteurellaceae strains exhibiting resistance to multiple antimicrobials including aminoglycosides, beta-lactams, macrolides and sulfonamides, the resistance determinants are often chromosomally encoded within integrative and conjugative elements (ICEs). To gain a more comprehensive picture of ICE structures, we sequenced the genomes of six strains of P. multocida and four strains of M. haemolytica; all strains were independent isolates and eight of them were multiple-resistant. ICE sequences varied in size from 49 to 79 kb, and were comprised of an array of conserved genes within a core region and varieties of resistance genes within accessory regions. These latter regions mainly account for the variation in the overall ICE sizes. From the sequence data, we developed a multiplex PCR assay targeting four conserved core genes required for integration and maintenance of ICE structures. Application of this assay on 75 isolates of P. multocida and M. haemolytica reveals how the presence and structures of ICEs are related to their antibiotic resistance phenotypes. The assay is also applicable to other members of the Pasteurellaceae family including Histophilus somni and indicates how clustering and dissemination of the resistance genes came about.
Project description:Rapid and accurate diagnosis of bovine respiratory disease (BRD) presents a substantial challenge to the North American cattle industry. Here we utilize recombinase polymerase amplification (RPA), a fast and sensitive isothermal DNA-based technology for the detection of four BRD pathogens (Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycoplasma bovis), genes coding antimicrobial resistance (AMR) and integrative conjugative elements (ICE) which can harbor AMR genes. Eleven RPA assays were designed and validated including: a) one conventional species-specific multiplex assay targeting the 4 BRD pathogens, b) two species-specific real-time multiplex RPA assays targeting M. haemolytica/M. bovis and P. multocida/H. somni, respectively with a novel competitive internal amplification control, c) seven conventional assays targeting AMR genes (tetH, tetR, msrE, mphE, sul2, floR, erm42), and d) one real-time assay targeting ICE. Each real-time RPA assay was tested on 100 deep nasopharyngeal swabs (DNPS) collected from feedlot cattle previously assessed for targets using either culture methods and/or polymerase chain reaction (PCR) verification (TC-PCR). The developed RPA assays enabled sensitive and accurate identification of BRD agents and AMR/ICE genes directly from DNPS, in a shorter period than TC-PCR, showing considerable promise as a tool for point-of-care identification of BRD pathogens and antimicrobial resistance genes.
Project description:Horizontal gene transfer of integrative and conjugative elements (ICE) in bacterial pathogens of the bovine respiratory disease (BRD) complex has emerged as a significant cause of antimicrobial resistance (AMR) and therapeutic failure and mortalities in cattle. The aim of this study was to assess an AMR ICE occurring in Pasteurella multocida from a case of BRD, designated ICEMh1 PM22 for its structure and host genome insertion site, and to identify consequences for host fitness and antimicrobial therapy. The modular structure of ICEMh1-like elements found in several related livestock pathogens was compared to ICEMh1 PM22, and the repertoire of cargo genes in variable ICE modules was functionally categorized. AMR genes were identified as frequent additions to the variable modules of ICEMh1-like elements. Random PCR-based mapping of ICEMh1 PM22-genome junctions in transconjugants provided evidence that ICEMh1 PM22 integrates into the tRNA-leu for the UUG codon, and not into tRNA-leu for other codons. This was separately confirmed in the genomes of ICEMh1-like-harboring livestock pathogens. Bacterial genera harboring receptive tRNA-leuUUG were identified to establish the potential host range of ICEMh1-like elements. ICEMh1 PM22-carrying transconjugants in P. multocida and Mannheimia haemolytica were less fit than isogenic strains without the ICE when grown without antimicrobial selection. This fitness cost was abrogated in the presence of subinhibitory concentrations of antimicrobials. Despite this cost, ICEMh1 PM22 was retained in transconjugants in extended culture. To identify possible therapeutic efficiencies, antimicrobial combinations were screened for synergistic interactions against AMR ICEMh1 PM22-carrying transconjugants. No antimicrobial combination tested exhibited synergistic interactions against AMR P. multocida or M. haemolytica harboring ICEMh1 PM22. In conclusion, this study provided information on the structural variation of ICEMh1-like elements, refined the ICE insertion site and potential host range, and demonstrated the risk and consequences for AMR following horizontal transfer of ICE into BRD pathogens.
Project description:The objectives of this study were to determine antimicrobial resistance and metal tolerance, and identify associated genes and mobile genetic elements in clinical strains of Histophilus somni isolated from feedlot cattle in Alberta during years 2012-2016 (contemporary isolates, n = 63) and years 1980-1990 (historical isolates, n = 31). Comparison of antimicrobial resistance (AMR) showed a significant increase in resistance among contemporary isolates compared to historical isolates (P < 0.001). Tolerance to copper (Cu) and zinc (Zn) concentrations above 1 mM was observed in 68 and 52% of the contemporary isolates, respectively. The tet(H) gene associated with oxytetracycline resistance and multicopper oxidase (mco) and cation efflux (czcD) genes associated with Cu and Zn tolerance were identified. An integrative conjugative element; ICEHs1, was identified in whole genome sequences of strains resistant to oxytetracycline, which had Cu and Zn minimum inhibitory concentrations (MIC) >1 mM. The length of ICEHs1 was 64,932 bp and it contained 83 genes, including tetracycline resistance gene tetH, a multidrug efflux pump gene ebrB, and metal tolerance genes mco, czcD, and acr3. Comparative genomics of ICEs revealed that ICEHs1 shares high homology with previously described ICEs of Histophilus somni, Pasteurella multocida, and Mannheimia haemolytica. The ICEHs1 is an active element capable of intra- and inter-genus transfer as demonstrated by successful transfer to H. somni and P. multocida recipients. All isolates carrying ICEHs1 were resistant to tetracycline, a commonly used antibiotic in feedlots, and had Cu and Zn MIC higher than 1 mM. Since Cu and Zn are routinely used in feedlots, there is the possibility of co-selection of AMR in H. somni due to selection pressure created by Cu and Zn. Based on results of in-vitro conjugation experiments, ICEHs1 mediated transmission of antimicrobial and metal resistance genes is possible between BRD pathogens in the respiratory tract, potentially undermining treatment options available for histophilosis and BRD.
Project description:The nasopharyngeal (NP) microbiota is important in defining respiratory health in feedlot cattle, with certain NP commensals potentially protecting against bovine respiratory disease (BRD) pathogens. In the present study, we evaluated longitudinal changes in the NP microbiota with a focus on lactic acid-producing bacteria (LAB) and their linkage with BRD-associated bacteria in steers (n = 13) that were first transported to an auction market, and then to a feedlot. Deep nasopharyngeal swabs were collected at the farm before transportation to the auction market (day 0), at feedlot placement (day 2), and 5 (day 7) and 12 (day 14) days after feedlot placement. Swabs were processed for the assessment of the NP microbiota using 16S rRNA gene sequencing, and for the detection of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni by culturing. Possible associations among the top 15 most relatively abundant bacterial genera were predicted using a stepwise-selected generalized linear mixed model. Correlations between LAB and BRD-associated Pasteurellaceae families were also assessed. In addition, antimicrobial activity of selected LAB isolates against M. haemolytica was evaluated in vitro. A noticeable shift was observed in the NP microbial community structure, and in the relative abundance of LAB families as a result of auction market exposure, transport and feedlot placement. Varying degrees of positive or negative associations between the 15 most relatively abundant genera were observed. Many of the LAB families were inversely correlated with the BRD-associated Pasteurellaceae family as the cattle were transported to the auction market and then to the feedlot. Nearly all steers were culture-negative for M. haemolytica and H. somni, and P. multocida became less prevalent after feedlot placement. Isolates from the Lactobacillaceae, Streptococcaceae, and Enterococcaceae families inhibited the growth of M. haemolytica. The results of this study indicated that the NP microbiota became more diverse with an increase in microbial richness following transport to an auction market and feedlot. This study provides evidence of potential cooperation and exclusion taking place in the respiratory microbial community of cattle which may be useful for developing microbial-based strategies to mitigate BRD.
Project description:The bacterial pathogens Mannheimia haemolytica and Pasteurella multocida are major etiological agents in respiratory tract infections of cattle. Although these infections can generally be successfully treated with veterinary macrolide antibiotics, a few recent isolates have shown resistance to these drugs. Macrolide resistance in members of the family Pasteurellaceae is conferred by combinations of at least three genes: erm(42), which encodes a monomethyltransferase and confers a type I MLS(B) (macrolide, lincosamide, and streptogramin B) phenotype; msr(E), which encodes a macrolide efflux pump; and mph(E), which encodes a macrolide-inactivating phosphotransferase. Here, we describe a multiplex PCR assay that detects the presence of erm(42), msr(E), and mph(E) and differentiates between these genes. In addition, the assay distinguishes P. multocida from M. haemolytica by amplifying distinctive fragments of the 23S rRNA (rrl) genes. One rrl fragment acts as a general indicator of gammaproteobacterial species and confirms whether the PCR assay has functioned as intended on strains that are negative for erm(42), msr(E), and mph(E). The multiplex system has been tested on more than 40 selected isolates of P. multocida and M. haemolytica and correlated with MICs for the veterinary macrolides tulathromycin and tilmicosin, and the newer compounds gamithromycin and tildipirosin. The multiplex PCR system gives a rapid and robustly accurate determination of macrolide resistance genotypes and bacterial genus, matching results from microbiological methods and whole-genome sequencing.
Project description:Although 90% of BRD relapses are reported to receive retreatment with a different class of antimicrobial, studies examining the impact of antimicrobial selection (i.e. bactericidal or bacteriostatic) on retreatment outcomes and the emergence of antimicrobial resistance (AMR) are deficient in the published literature. This survey was conducted to determine the association between antimicrobial class selection for treatment and retreatment of BRD relapses on antimicrobial susceptibility of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni. Pathogens were isolated from samples submitted to the Iowa State University Veterinary Diagnostic Laboratory from January 2013 to December 2015. A total of 781 isolates with corresponding animal case histories, including treatment protocols, were included in the analysis. Original susceptibility testing of these isolates for ceftiofur, danofloxacin, enrofloxacin, florfenicol, oxytetracycline, spectinomycin, tilmicosin, and tulathromycin was performed using Clinical and Laboratory Standards Institute guidelines. Data were analyzed using a Bayesian approach to evaluate whether retreatment with antimicrobials of different mechanistic classes (bactericidal or bacteriostatic) increased the probability of resistant BRD pathogen isolation in calves. The posterior distribution we calculated suggests that an increased number of treatments is associated with a greater probability of isolates resistant to at least one antimicrobial. Furthermore, the frequency of resistant BRD bacterial isolates was greater with retreatment using antimicrobials of different mechanistic classes than retreatment with the same class. Specifically, treatment protocols using a bacteriostatic drug first followed by retreatment with a bactericidal drug were associated with a higher frequency of resistant BRD pathogen isolation. In particular, first treatment with tulathromycin (bacteriostatic) followed by ceftiofur (bactericidal) was associated with the highest probability of resistant M. haemolytica among all antimicrobial combinations. These observations suggest that consideration should be given to antimicrobial pharmacodynamics when selecting drugs for retreatment of BRD. However, prospective studies are needed to determine the clinical relevance to antimicrobial stewardship programs in livestock production systems.
Project description:A novel variant of the AAD(3?) class of aminoglycoside-modifying enzymes was discovered in fatal bovine respiratory disease-associated pathogens Pasteurella multocida and Histophilus somni. The aadA31 gene encodes a spectinomycin/streptomycin adenylyltransferase and was located in a variant of the integrative and conjugative element ICEMh1, a mobile genetic element transmissible among members of the family Pasteurellaceae. The gene was also detected in Mannheimia haemolytica from a case of porcine pneumonia and in Moraxella bovoculi from a case of keratoconjunctivitis. IMPORTANCE Aminoglycosides are important antimicrobials used worldwide for prophylaxis and/or therapy in multiple production animal species. The emergence of new resistance genes jeopardizes current pathogen detection and treatment methods. The risk of resistance gene transfer to other animal and human pathogens is elevated when resistance genes are carried by mobile genetic elements. This study identified a new variant of a spectinomycin/streptomycin resistance gene harbored in a self-transmissible mobile element. The gene was also present in four different bovine pathogen species.
Project description:Bovine respiratory diseases (BRD) are widespread in veal calf feedlots. Several pathogens are implicated, both viruses and bacteria, one of which, Mycoplasma bovis, is under-researched. This worldwide-distributed bacterium has been shown to be highly resistant in vitro to the main antimicrobials used to treat BRD. Our objective was to monitor the relative prevalence of M. bovis during BRD episodes, its diversity, and its resistance phenotype in relation to antimicrobial use. For this purpose, a two-year longitudinal follow-up of 25 feedlots was organized and 537 nasal swabs were collected on 358 veal calves at their arrival in the lot, at the BRD peak and 4 weeks after collective antimicrobial treatments. The presence of M. bovis was assessed by real-time PCR and culture. The clones isolated were then subtyped (polC subtyping and PFGE analysis), and their susceptibility to five antimicrobials was determined. The course of the disease and the antimicrobials used had no influence on the genetic diversity of the M. bovis strains: The subtype distribution was the same throughout the BRD episode and similar to that already described in France, with a major narrowly-variable subtype circulating, st2. The same conclusion holds for antimicrobial resistance (AMR) phenotypes: All the clones were already multiresistant to the main antimicrobials used (except for fluoroquinolones) prior to any treatments. By contrast, changes of AMR phenotypes could be suspected for Pasteurellaceae in two cases in relation to the treatments registered.
Project description:We investigated the occurrence of infectious pathogens during an outbreak of bovine respiratory disease (BRD) in a beef cattle feedlot in southern Brazil that has a high risk of developing BRD. Nasopharyngeal swabs were randomly collected from steers ( n = 23) and assessed for the presence of infectious agents of BRD by PCR and/or RT-PCR assays. These included: Histophilus somni, Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, bovine respiratory syncytial virus (BRSV), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV), bovine alphaherpesvirus 1 (BoHV-1), and bovine parainfluenza virus 3 (BPIV-3). Pulmonary sections of one steer that died with clinical BRD were submitted for pathology and molecular testing. The frequencies of the pathogens identified from the nasopharyngeal swabs were: H. somni 39% (9 of 23), BRSV 35% (8 of 23), BCoV 22% (5 of 23), and M. haemolytica 13% (3 of 23). PCR or RT-PCR assays did not identify P. multocida, M. bovis, BoHV-1, BVDV, or BPIV-3 from the nasopharyngeal swabs. Single and concomitant associations of infectious agents of BRD were identified. Fibrinous bronchopneumonia was diagnosed in one steer that died; samples were positive for H. somni and M. haemolytica by PCR. H. somni, BRSV, and BCoV are important disease pathogens of BRD in feedlot cattle in Brazil, but H. somni and BCoV are probably under-reported.
Project description:Over a two-year period, Mannheimia haemolytica (MH; n = 113), Pasteurella multocida (PM; n = 47), Histophilus somni (HS; n = 41) and Mycoplasma bovis (MB; n = 227) were isolated from bovine lung tissue at necropsy from cattle raised conventionally (CON, n = 29 feedlots) or without antimicrobials [natural (NAT), n = 2 feedlots]. Excluding MB, isolates were assayed by PCR to detect the presence of 13 antimicrobial resistance (AMR) genes and five core genes associated with integrative and conjugative elements (ICEs). Antimicrobial susceptibility phenotypes and minimum inhibitory concentrations (MICs, µg/mL) were determined for a subset of isolates (MH, n = 104; PM, n = 45; HS, n = 23; and MB, n = 61) using Sensititre analyses. A subset of isolates (n = 21) was also evaluated by whole-genome sequencing (WGS) based on variation in AMR phenotype. All five ICE core genes were detected in PM and HS by PCR, but only 3/5 were present in MH. Presence of mco and tnpA ICE core genes in MH was associated with higher MICs (p < 0.05) for all tetracyclines, and 2/3 of all macrolides, aminoglycosides and fluoroquinolones evaluated. In contrast, association of ICE core genes with MICs was largely restricted to macrolides for PM and to individual tetracyclines and macrolides for HS. For MH, the average number of AMR genes markedly increased (p < 0.05) in year 2 of the study due to the emergence of a strain that was PCR positive for all 13 PCR-tested AMR genes as well as two additional AMR genes (aadA31 and blaROB-1) detected by WGS. Conventional management of cattle increased (p < 0.05) MICs of tilmicosin and tulathromycin for MH; neomycin and spectinomycin for PM; and gamithromycin and tulathromycin for MB. The average number of PCR-detected AMR genes in PM was also increased (p < 0.05) in CON mortalities. This study demonstrates increased AMR especially to macrolides by bovine respiratory disease organisms in CON as compared to NAT feedlots and a rapid increase in AMR following dissemination of strain(s) carrying ICE-associated multidrug resistance.