Evolution in quantum leaps: multiple combinatorial transfers of HPI and other genetic modules in Enterobacteriaceae

Abstract

Horizontal gene transfer is a key step in the evolution of Enterobacteriaceae. By acquiring virulence determinants of foreign origin, commensals can evolve into pathogens. In Enterobacteriaceae, horizontal transfer of these virulence determinants is largely dependent on transfer by plasmids, phages, genomic islands (GIs) and genomic modules (GMs). The High Pathogenicity Island (HPI) is a GI encoding virulence genes that can be transferred between different Enterobacteriaceae. We investigated the HPI because it was present in an Enterobacter hormaechei outbreak strain (EHOS). Genome sequence analysis showed that the EHOS contained an integration site for mobile elements and harbored two GIs and three putative GMs, including a new variant of the HPI (HPI-ICEEh1). We demonstrate, for the first time, that combinatorial transfers of GIs and GMs between Enterobacter cloacae complex isolates must have occurred. Furthermore, the excision and circularization of several combinations of the GIs and GMs was demonstrated. Because of its flexibility, the multiple integration site of mobile DNA can be considered an integration hotspot (IHS) that increases the genomic plasticity of the bacterium. Multiple combinatorial transfers of diverse combinations of the HPI and other genomic elements among Enterobacteriaceae may accelerate the generation of new pathogenic strains.

Figure 1. Integration hotspot with genomic islands and genetic modules of E. hormaechei 05-545.

Accession no. FN297818. White: chromosomal DNA of E. hormaechei 05-545; red: asn tRNA; turquoise: direct repeat attO; green: genetic island EhGI1; yellow: the conserved region of HPI-ICEEh1encoding the integrase and yersiniabactin production, regulation and uptake; orange: the putative integrative and conjugative element of HPI-ICE-Eh1; blue: genetic module 3 (EhGM3); purple: genetic module 4 (EhGM4); brown: genetic module 5 (EhGM5). In Box 1-5 the red arrows indicate primer positions for the linkage PCR to determine whether genomic islands are located next to each other and in which direction (see Table 3 (and Table S2) for results and Table S1 for primer characteristics). Red numbered boxes indicate positions of the amplified products by PCR for IHS characterization with primers as described in Table S1.

Figure 2. Sequencing approach for the intB gene of all HPI-positive isolates used in this study.

A) Region bp 41,120-45,349 of FN297818 (Box 2 in Figure 1). Amplification primers (black): pre-GI1-F and YbtS-R. Sequence primers red and black for the obtained PCR product: pre-GI1-F, IntBseq2F, IntB-F, YbtS-R, IntB-R, and IntBseq1R. PCR-positive and sequenced isolates: EHOS isolates: 01-083, 01-234, 02-195, 02-203, 02-477, 03-375, 03-525, 03-577, 04-640, R1568, 05-545. Non-EHOS isolates but with the same content in the integration hotspot: 03-273, 05-349. A non-EHOS isolate with another integration hotspot: 05-316. EHOS isolates 05-761, 06-339, H9 were PCR-positive but not sequenced because ten other EHOS isolates were sequenced and did not show polymorphisms. For the other color codes see Figure 1. B) Region bp 383–4423 of AF091251. Amplification primers (black): pre-IntB-F and YbtS-R. Sequence primers red and black for the obtained PCR product: pre-IntB-F, IntBseq2F, IntB-F, YbtS-R, IntB-R, and IntBseq1R. PCR-positive and sequenced isolates: 01-084, 01-306, 02-023, 02-272, 03-018, 03-339, 03-426, 03-595, 03-613, 03-635, 03-642, 03-739, 03-895, 05-189, 05-202, 05-680, 06-316, 10A275, 10E013, 14A001, 18D099, and X2327. From the isolates 03-093, 03-192, 03-414, and R0332 the amplification product was approximately 350 bp smaller than expected. Sequence results indicated that these isolates contained a truncated intB gene. Moreover, bp 124–470 was deleted compared to the wild-type intB gene.

Figure 3. Sequence strategy for the extreme right side of the integration hotspot.

PCR amplification was performed on isolates with EhGI-1 used in this study. Using a PCR amplification reaction with the primers IHS-iutA-F and ORF7-2R, a product was obtained only from the 12 tested EHOS isolates and isolates 03-273 and 05-349. The amplified products were partly sequenced with primers Rev-IntB-GI1-out and For-iutA-tRNA-seq. Accession no. GQ891736-GQ891749.

Figure 4. Phylogenetic tree based on the sequence of the conservative part of the HPI-ICE of 23 Enterobacteriaceae.

The phylogenetic tree is based on the sequences of the conservative part of the HPI-ICE (homologous to bp 42589-72874 of accession no. FN297818) of 23 Enterobacteriaceae clustered with Clonalframe. Numbers indicate confidence values of the branches. The phylogenetic group membership of the E. coli strain is indicated between brackets.

Figure 5. Phylogenetic tree based on 58 intB sequences.

The phylogenetic tree is based on a neighbor-joining algorithm and 10,000 bootstrap iterations with Mega4.0 on intB sequences, showing the relationships among the 58 intB sequences included in this study. The scale bar represents a 1% difference in nucleotide sequence. Red: ECC isolates with identical intB sequences and otherwise genotyped as the EHOS. The tree is divided into three separate branches with high bootstrap values except for Klebsiella pneumoniae ICEkp1, which demonstrated low bootstrap values with other isolates, suggesting it is a separate cluster.

Figure 6. Phylogenetic tree based on 96 792-bp fragment of intB sequences.

Phylogenetic tree based on a neighbor-joining algorithm and 10,000 bootstrap iterations with Mega4.0 for a 792-bp fragment of the intB gene that shows the relationships among the 96 intB sequences included in this study. The scale bar represents a 1% difference in nucleotide sequence. Each genus is depicted with a different color. The tree is divided into three separate braches with high bootstrap values except for Klebsiella pneumoniae ICEkp1 and E. coli 3172/97, which demonstrated low bootstrap values with the other isolates, suggesting separate clusters or recombination of the gene. Cluster 1 contains 68 isolates of nine different species; cluster 2 contains two Y. enterocolitica species; and cluster 3 contains five E. coli strains, seven ECC strains, eleven EHOS strains and one K. pneumoniae strain, in which two ECCs, all EHOSs and E. coli ECOR31 are identical.