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.

Antimicrobial Resistance in Members of the Bacterial Bovine Respiratory Disease Complex Isolated from Lung Tissue of Cattle Mortalities Managed with or without the Use of Antimicrobials

“…In Pasteurellaceae species, along with many other genes related to ICE replication, conjugative transfer and chromosomal integration, three genes (also present in ICEPmu1) int2 (Pmu02880 in ICEPmu1), a paralogue of int1 XerD-family tyrosine integrase Pmu02700 [27], rel1, the ICE-relaxase (Pmu02890 in ICEPmu1), and parB (Pmu03590 in ICEPmu1), encoding partitioning protein associated with ICE replication [27] were among the most conserved genes present in the ICE region in sequenced isolates with 100% nucleotide sequence identity across three species. Table 9.…”

A Sensitive and Accurate Recombinase Polymerase Amplification Assay for Detection of the Primary Bacterial Pathogens Causing Bovine Respiratory Disease

“…Furthermore, detection of M. haemolytica was specific to serotypes A1 and A6, those most commonly associated with disease, while excluding all other serotypes, including A2 a common bovine commensal (30,31). Beker et al (13) developed a multiplex PCR assay targeting four conserved core genes required for integration and maintenance of ICE structures within the Pasteurellaceae family and demonstrated relevance of this assay to detecting these elements in P. multocida and M. haemolytica (13). Furthermore, RPA has recently been utilized for detection of P. multocida in cattle (32).…”

“…No BRD pathogens were detected in 9% of ICE-positive DNPS samples. Due to the transmissible nature of ICE, this suggests that BRD pathogens may be transferring ICE to other bacterial species (13,41). A closely related species, Bibersteinia trehalosi, as well as Moraxella and Acinetobacter may also contain ICE (9,31).…”

“…Excluding M. bovis, the genomes of BRD pathogens often contain integrative conjugative elements (ICE), mobile genetic elements that can harbor multiple AMR genes and encode the conjugation machinery required for transfer of ICE between BRD pathogens and to other bacteria (4,9). The resulting multi-drug resistance (MDR) among some BRD pathogens containing ICE presents a significant challenge for the efficacy of antimicrobial therapy as a treatment for BRDClawson et al (12) found that the gene tet(H), which confers tetracycline resistance was present in all AMR M. haemolytica strains isolated from confirmed BRD cases, and was also frequently found in P. multocida (13) and H. somni ICE (14). Furthermore, tet(H) was adjacent to the transposase gene tnpA, a core ICE gene associated with increased minimum inhibitory antimicrobial concentrations in M. haemolytica, H. somni, and P. multocida (15).…”

Intranasal Bacterial Therapeutics Reduce Colonization by the Respiratory Pathogen Mannheimia haemolytica in Dairy Calves

“…Increasing evidence indicates that commensal bacteria in the bovine nasopharynx may prevent the colonization and proliferation of bacterial pathogens through a variety of mechanisms, including direct antagonism (i.e., antimicrobial properties), competition for nutrients and adhesion sites, and host immunomodulatory effects (15)(16)(17)(18). Among commensal bacteria, lactic acid-producing bacteria (LAB), and more specifically Lactobacillus spp., may be important in providing colonization resistance against bacterial respiratory pathogens (19)(20)(21).…”