Dataset Information

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.

SUBMITTER: Beker M

PROVIDER: S-EPMC6028734 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Integrative and Conjugative Elements (ICEs) in Pasteurellaceae Species and Their Detection by Multiplex PCR.

Beker Michal M Rose Simon S Lykkebo Claus A CA Douthwaite Stephen S

Frontiers in microbiology 20180626

Strains of the Pasteurellaceae bacteria <i>Pasteurella multocida</i> and <i>Mannheimia haemolytica</i> 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 chromosomall ...[more]

PMID: 29997583

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Project description:BackgroundVibrio alginolyticus is ubiquitous in marine and estuarine environments. In 2012-2013, SXT/R391-like integrative conjugative elements (ICEs) in environmental V. alginolyticus strains were discovered and found to occur in 8.9 % of 192 V. alginolyticus strains, which suggests that V. alginolyticus may be a natural pool possessing resourceful ICEs. However, complete ICE sequences originating from this bacterium have not been reported, which represents a significant barrier to characterizing the ICEs of this bacterium and exploring their relationships with other ICEs. In the present study, we acquired six ICE sequences from five V. alginolyticus strains and performed a comparative analysis of these ICE genomes.ResultsA sequence analysis showed that there were only 14 variable bases dispersed between ICEValE0601 and ICEValHN492. ICEValE0601 and ICEValHN492 were treated as the same ICE. ICEValA056-1, ICEValE0601 and ICEValHN492 integrate into the 5' end of the host's prfC gene, and their Int and Xis share at least 97 % identity with their counterparts from SXT. ICEValE0601 or ICEValHN492 contain 50 of 52 conserved core genes in the SXT/R391 ICEs (not s025 or s026). ICEValA056-2, ICEValHN396 and ICEValHN437 have a different tRNA-ser integration site and a distinct int/xis module; however, the remaining backbone genes are highly similar to their counterparts in SXT/R391 ICEs. DNA sequences inserted into hotspot and variable regions of the ICEs are of various sizes. The variable genes of six ICEs encode a large array of functions to bestow various adaptive abilities upon their hosts, and only ICEValA056-1 contains drug-resistant genes. Many variable genes have orthologous and functionally related genes to those found in SXT/R391 ICEs, such as genes coding for a toxin-antitoxin system, a restriction-modification system, helicases and endonucleases. Six ICEs also contain a large number of unique genes or gene clusters that were not found in other ICEs. Six ICEs harbor more abundant transposase genes compared with other parts of their host genomes. A phylogenetic analysis indicated that transposase genes in these ICEs are highly diverse.ConclusionsICEValA056-1, ICEValE0601 and ICEValHN492 are typical members of the SXT/R391 family. ICEValA056-2, ICEValHN396 and ICEValHN437 form a new atypical group belonging to the SXT/R391 family. In addition to the many genes found to be present in other ICEs, six ICEs contain a large number of unique genes or gene clusters that were not found in other ICEs. ICEs may serve as a carrier for transposable genetic elements (TEs) and largely facilitate the dissemination of TEs.

Project description:Integrative and Conjugative Elements (ICEs) of the SXT/R391 family disseminate multidrug resistance among pathogenic Gammaproteobacteria such as Vibrio cholerae. SXT/R391 ICEs are mobile genetic elements that reside in the chromosome of their host and eventually self-transfer to other bacteria by conjugation. Conjugative transfer of SXT/R391 ICEs involves a transient extrachromosomal circular plasmid-like form that is thought to be the substrate for single-stranded DNA translocation to the recipient cell through the mating pore. This plasmid-like form is thought to be non-replicative and is consequently expected to be highly unstable. We report here that the ICE R391 of Providencia rettgeri is impervious to loss upon cell division. We have investigated the genetic determinants contributing to R391 stability. First, we found that a hipAB-like toxin/antitoxin system improves R391 stability as its deletion resulted in a tenfold increase of R391 loss. Because hipAB is not a conserved feature of SXT/R391 ICEs, we sought for alternative and conserved stabilization mechanisms. We found that conjugation itself does not stabilize R391 as deletion of traG, which abolishes conjugative transfer, did not influence the frequency of loss. However, deletion of either the relaxase-encoding gene traI or the origin of transfer (oriT) led to a dramatic increase of R391 loss correlated with a copy number decrease of its plasmid-like form. This observation suggests that replication initiated at oriT by TraI is essential not only for conjugative transfer but also for stabilization of SXT/R391 ICEs. Finally, we uncovered srpMRC, a conserved locus coding for two proteins distantly related to the type II (actin-type ATPase) parMRC partitioning system of plasmid R1. R391 and plasmid stabilization assays demonstrate that srpMRC is active and contributes to reducing R391 loss. While partitioning systems usually stabilizes low-copy plasmids, srpMRC is the first to be reported that stabilizes a family of ICEs.

Project description:The self-transferring integrative and conjugative elements (ICEs) are large genomic segments carrying several bacterial adaptive functions including antimicrobial resistance (AMR). SXT/R391 family is one of the ICEs extensively studied in cholera-causing pathogen Vibrio cholerae. The genetic characteristics of ICE-SXT/R391 in V. cholerae are dynamic and region-specific. These ICEs in V. cholerae are strongly correlated with resistance to several antibiotics such as tetracycline, streptomycin and trimethoprim-sulfamethoxazole. We screened V. cholerae O1 strains isolated from cholera patients in Kolkata, India from 2008 to 2015 for antibiotic susceptibility and the presence of ICEs, and subsequently sequenced their conserved genes. Resistance to tetracycline, streptomycin and trimethoprim-sulfamethoxazole was detected in strains isolated during 2008-2010 and 2014-2015. The genes encoding resistance to tetracycline (tetA), trimethoprim-sulfamethoxazole (dfrA1 and sul2), streptomycin (strAB), and chloramphenicol (floR) were detected in the ICEs of these strains. There was a decrease in overall drug resistance in V. cholerae associated with the ICEs in 2011. DNA sequence analysis also showed that AMR in these strains was conferred mainly by two types of ICEs, i.e., ICETET (comprising tetA, strAB, sul2, and dfrA1) and ICEGEN (floR, strAB, sul2, and dfrA1). Based on the genetic structure, Kolkata strains of V. cholerae O1 had distinct genetic traits different from the ICEs reported in other cholera endemic regions. Transfer of AMR was confirmed by conjugation with sodium azide resistant Escherichia coli J53. In addition to the acquired resistance to streptomycin and trimethoprim-sulfamethoxazole, the conjugally transferred (CT) E. coli J53 with ICE showed higher resistance to chloramphenicol and tetracycline than the donor V. cholerae. Pulsed-field gel electrophoresis (PFGE) based clonal analysis revealed that the V. cholerae strains could be grouped based on their ICEs and AMR patterns. Our findings demonstrate the epidemiological importance of ICEs and their role in the emergence of multidrug resistance (MDR) in El Tor vibrios.

Project description:The discovery of integrative conjugative elements (ICEs) in wall-less mycoplasmas and the demonstration of their role in massive gene flows within and across species have shed new light on the evolution of these minimal bacteria. Of these, the ICE of the ruminant pathogen Mycoplasma agalactiae (ICEA) represents a prototype and belongs to a new clade of the Mutator-like superfamily that has no preferential insertion site and often occurs as multiple chromosomal copies. Here, functional genomics and mating experiments were combined to address ICEA functions and define the minimal ICEA chassis conferring conjugative properties to M. agalactiae Data further indicated a complex interaction among coresident ICEAs, since the minimal ICEA structure was influenced by the occurrence of additional ICEA copies that can trans-complement conjugation-deficient ICEAs. However, this cooperative behavior was limited to the CDS14 surface lipoprotein, which is constitutively expressed by coresident ICEAs, and did not extend to other ICEA proteins, including the cis-acting DDE recombinase and components of the mating channel whose expression was detected only sporadically. Remarkably, conjugation-deficient mutants containing a single ICEA copy knocked out in cds14 can be complemented by neighboring cells expressing CDS14. This result, together with those revealing the conservation of CDS14 functions in closely related species, may suggest a way for mycoplasma ICEs to extend their interaction outside their chromosomal environment. Overall, this report provides a first model of conjugative transfer in mycoplasmas and offers valuable insights into understanding horizontal gene transfer in this highly adaptive and diverse group of minimal bacteria.IMPORTANCE Integrative conjugative elements (ICEs) are self-transmissible mobile genetic elements that are key mediators of horizontal gene flow in bacteria. Recently, a new category of ICEs was identified that confer conjugative properties to mycoplasmas, a highly adaptive and diverse group of wall-less bacteria with reduced genomes. Unlike classical ICEs, these mobile elements have no preferential insertion specificity, and multiple mycoplasma ICE copies can be found randomly integrated into the host chromosome. Here, the prototype ICE of Mycoplasma agalactiae was used to define the minimal conjugative machinery and to propose the first model of ICE transfer in mycoplasmas. This model unveils the complex interactions taking place among coresident ICEs and suggests a way for these elements to extend their influence outside their chromosomal environment. These data pave the way for future studies aiming at deciphering chromosomal transfer, an unconventional mechanism of DNA swapping that has been recently associated with mycoplasma ICEs.

Project description:We sequenced the entire genomes of ten biphenyl/PCB degrading bacterial strains (KF strains) isolated from biphenyl-contaminated soil in Kitakyushu, Japan. All the strains were Gram-negative bacteria belonging to ?- and ?-proteobacteria. Out of the ten strains, nine strains carried a biphenyl catabolic bph gene cluster as integrative conjugative elements (ICEs), and they were classified into four groups based on the structural features of the bph genes. Group I (five strains) possessed bph genes that were very similar to the ones in Pseudomonas furukawaii KF707 (formerly Pseudomonas pseudoalcaligenes KF707), which is one of the best characterized biphenyl-utilizing strains. This group of strains carried salicylate catabolic sal genes that were approximately 6-kb downstream of the bph genes. Group II (two strains) possessed bph and sal genes similar to the ones in KF707, but these strains lacked the bphX region between bphC and bphD, which is involved in the downstream catabolism of biphenyl. These bph-sal clusters in groups I and II were located on an integrative conjugative element that was larger than 110 kb, and they were named ICEbph-sal. Our previous study demonstrated that the ICEbph-sal of Pseudomonas putida KF715 in group II existed both in an integrated form in the chromosome (referred to as ICEbph-salKF715 (integrated)) and in a extrachromosomal circular form (referred to as ICEbph-sal (circular)) (previously called pKF715A, 483 kb) in the stationary culture. The ICEbph-sal was transferred from KF715 into P. putida AC30 and P. putida KT2440 with high frequency, and it was maintained stably as an extrachromosomal circular form. The ICEbph-salKF715 (circular) in these transconjugants was further transferred to P. putida F39/D and then integrated into the chromosome in one or two copies. Meanwhile, group III (one strain) possessed bph genes, but not sal genes. The nucleotide sequences of the bph genes in this group were less conserved compared to the genes of the strains belonging to groups I and II. Currently, there is no evidence to indicate that the bph genes in group III are carried by a mobile element. Group IV (two strains) carried bph genes as ICEs (59-61 kb) that were similar to the genes found in Tn4371 from Cupriavidus oxalacticus A5 and ICEKKS1024677 from the Acidovorax sp. strain KKS102. Our study found that bph gene islands have integrative functions, are transferred among soil bacteria, and are diversified through modification.

Dataset Information

Integrative and Conjugative Elements (ICEs) in Pasteurellaceae Species and Their Detection by Multiplex PCR.

Publications

Integrative and Conjugative Elements (ICEs) in Pasteurellaceae Species and Their Detection by Multiplex PCR.

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