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.

Keywords: Mannheimia; Pasteurella; antibiotic resistance; genomics; veterinary macrolides.

Figure 1

Structure of the 75.6 kb ICE within the chromosome of P. multocida strain 3358 is depicted by the blue region, with the extent of the two accessory regions indicated in pale red. The size marker dots are spaced at 500 bp. The int1, int2, ICE-rel1, and parB genes in the core regions are shown in green, other essential core genes in yellow, the remaining core genes in black, and antibiotic resistance genes in the accessory regions in red. Genes are annotated according to their known functions or by using the NCBI notation (Supplementary Table S1). All numbered genes are prefixed with pmu3358_ as shown for the first and last genes in this figure. The hybridization sites for the primers used in the multiplex PCR assays are indicated by the arrow heads below the respective genes.

Figure 2

Content of ICEs from the Pasteurellaceae strains determined by whole genome sequencing. The previously reported ICEPmu1 of P. multocida strain 36950 (Michael et al., 2012b) and ICEMh1 from M. haemolytica strain 42548 (Eidam et al., 2015) are included for comparison. The ICEs are annotated here according to the strains containing them, and their sizes can be read from the kilobase pair (kb) scale at the bottom. Color coding of the genes as in Figure 1 with the core genes targeted in the multiplex assay (green), other essential core genes (yellow), antibiotic resistance genes (red) plus additional open reading frames (black). The chromosomal site of ICE insertion left a disrupted tRNA^Leu gene at both ends of the sequence, which is replaced by in all cases by an intact copy of the tRNA^Leu gene located after parA (far right, in this sequence orientation). The multiplex assay was design to give a positive signal for ICE-rel1 but not for the ICE-rel2 gene (green/black stripes) that is evident in some M. haemolytica ICEs. The sensitive strain Pmu4407 contains no ICE genes. Strain Mh11935 also completely lacks resistance genes but contains numerous ICE core region genes, although key genes for ICE transfer and maintenance are missing. The ICE-rel1 gene in ICEMh11935 is truncated and presumably inactive, despite giving a positive multiplex PCR signal. The parB gene of ICEMh11935 is also truncated, and lacks one of the priming regions needed for a PCR signal. All of the four targeted genes int1, int2, ICE-rel1, and parB were 100% conserved in ICEPmu3358, ICEPmu3361, ICEPmu12591, and ICEPmu12601 of the P. multocida strains. Accession codes for the whole genome sequences are listed in Supplementary Table S1.

Figure 3

Identification of ICE-related Pasteurellaceae genes using multiplex PCR. The gels show the analyses of 75 field isolates, 43 of which were P. multocida (identified by the 600 bp band that is specific for Pasteurella spp.) and 32 were M. haemolytica (band at 720 bp and lack of 600 bp band). The four ICE-related PCR products correspond to parB (503 bp), the ICE-specific relaxase gene (437 bp), and the longer and shorter versions of the integrase genes int1 (301 bp) and int2 (215 bp). In strains that lacked all these ICE genes (e.g., Pmu13083) a faint artifact band of 250 bp was sometimes apparent. This band was no longer evident after raising the primer annealing temperature 3°C above the optimal hybridization temperature for the canonical sites. The control lane (-DNA) shows a reaction without template DNA. The bands in the GeneRuler ladder on the left are in 100-bp steps.

Figure 4

Relative numbers of the P. multocida (blue) and M. haemolytica strains (green) containing the ICE core genes screened in the multiplex PCR assay. Twenty-four of the 43 P. multocida isolates gave a positive signal with each of the int1, int2, ICE-rel1, and parB primer combinations; and a similar proportion of M. haemolytica strains (17 of 32) was also shown to contain these four ICE genes. The remainder of the P. multocida strains tested lacked all of the int1, int2, ICE-rel1, and parB genes, but no P. multocida strain was found to harbor a truncated, nonfunctional ICE structure. In contrast, 14 M. haemolytica strains possessed degenerate ICE sequences that would presumably not be capable of promoting intercellular transfer.