Characterization of putative virulence genes on the related RepFIB plasmids harbored by Cronobacter spp

Abstract

Cronobacter spp. are emerging neonatal pathogens that cause meningitis, sepsis, and necrotizing enterocolitis. The genus Chronobacter consists of six species: C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, and Cronobacter genomospecies group 1. Whole-genome sequencing of C. sakazakii BAA-894 and C. turicensis z3032 revealed that they harbor similarly sized plasmids identified as pESA3 (131 kb) and pCTU1 (138 kb), respectively. In silico analysis showed that both plasmids encode a single RepFIB-like origin of replication gene, repA, as well as two iron acquisition systems (eitCBAD and iucABCD/iutA). In a chrome azurol S agar diffusion assay, it was demonstrated that siderophore activity was associated with the presence of pESA3 or pCTU1. Additionally, pESA3 contains a cpa (Cronobacter plasminogen activator) gene and a 17-kb type 6 secretion system (T6SS) locus, while pCTU1 contains a 27-kb region encoding a filamentous hemagglutinin gene (fhaB), its specifc transporter gene (fhaC), and associated putative adhesins (FHA locus), suggesting that these are virulence plasmids. In a repA-targeted PCR assay, 97% of 229 Cronobacter species isolates were found to possess a homologous RepFIB plasmid. All repA PCR-positive strains were also positive for the eitCBAD and iucABCD/iutA iron acquisition systems. However, the presence of cpa, T6SS, and FHA loci depended on species, demonstrating a strong correlation with the presence of virulence traits, plasmid type, and species. These results support the hypothesis that these plasmids have evolved from a single archetypical plasmid backbone through the cointegration, or deletion, of specific virulence traits in each species.

Fig. 1.

Sequence alignment of pESA3 and pCTU1, produced with the Artemis Comparison Tool (ACT). A schematic of each plasmid is shown above (or below) its corresponding ruler. The sequence of pESA3 was modified by rejoining the repA gene at the 3′ end, which is split in the GenBank sequence. The G+C content (as a percentage) is shown between the plasmid ORF schematic and the ACT homology output; pESA3 and pCTU1 have a mean G+C contents of 56.85% and 56.05%, respectively. Select genes or loci are shown in color as follows: eit (red), iuc (orange), parAB and repA (purple), integrase (black) and associated genes (white), cpa (teal), T6SS (blue), and FHA (brown). In the middle section, the red color indicates significant nucleotide homology, as determined by BlastN analysis, between pESA3 and pCTU1, and the location on each plasmid, for example eit, iuc, parAB, and repA. White indicates regions or loci present on one plasmid and absent on the other, e.g., cpa, T6SS, and FHA.

Fig. 2.

Phylogenetic cluster analysis of repA. The evolutionary history was inferred using the neighbor-joining method (64). The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches (64). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method (65), and data shown are the number of base substitutions per site. Codon positions included were the first, second, and third and noncoding positions. All positions containing gaps and missing data were eliminated from the data set (complete deletion option). There were a total of 780 positions in the final data set. Phylogenetic analyses were conducted by using MEGA4 (65).

Fig. 3.

cpa locus of pESA3. The schematic shows the pESA3 cpa locus, 1,427 bp, collapsed region (dashed lines) in pCTU1 (37 bp), and conserved upstream and downstream flanking genes rimL (COG1670; acetyltransferase of the type rimL N-acetylase of ribosomal proteins), MFS-1, the major facilitator superfamily transporter (putative antibiotic resistance drug efflux pump); cpmJK, homologous (41% amino acid identity) to proteins of Photorhabdus luminescens subsp. laumondii TTO1 involved in carbapenem resistance; TR, transcriptional regulator; gloA, gene with the functional domain of lactoylglutathione lyase (also known as glyoxalase I), bleomycin resistance protein, or dioxygenase; and PCR primers (arrows with superscript numbers) used for plasmidotype screening. Primer 1, Δcpafw; primer 2, Δcparv; primer 3, cpafw; primer 4, cparv.

Fig. 4.

The T6SS cluster of pESA3, 16,937 bp long, compared to the collapsed region (dashed lines) on pCTU1 (32 bp), including conserved upstream and downstream flanking genes and PCR primers (arrows with superscript numbers) used for plasmidotype screening. T6SS cluster genes (diagonal shading) are identified by their COG numbers, unless otherwise specified. DUF2931, putative lipoprotein. Flanking genes were identified by closest or most probable BlastX homology. arsCBR, arsenic resistance gene operon; dapA, homologue of dihydrodipicolinate synthetase; PRK11272, drug/metabolite transporter; gntR, putative transcriptional regulator with DNA-binding and aminotransferase domain; dsbGD, disulfide bond formation/isomerization genes; scsA, homologue of suppressor of copper sensitivity from Serratia odorifera DSM 4582; ynaJ, putative inner membrane protein; SMI1_KNR4, regulator of 1,3-β-glucan synthase activity; ymjA, conserved hypothetical protein of Enterobacteriaceae; eitABCD, iron(III) siderophore operon. PCR primers (arrows with superscript numbers) used for plasmidotype screening were as follows: primer 1, Δt6ssfw; primer 2, Δt6ssrv; primer 3, t6ssrv; primer 4, vgrGfw; primer 5, vgrGrv; primer 6, t6ssfw; primer 7, t6ssrv3.

Fig. 5.

FHA locus of pCTU1 (27,421 bp), compared to the collapsed region (dashed lines) of pESA3 (266 bp), including conserved upstream and downstream flanking genes. The FHA cluster includes a homologue of fhaC, two partner secretion proteins, and fhaB, the filamentous hemagglutinin precursor (diagonal gray lines); accessory adhesins (fha1 to -5; black) and accessory outer membrane protein homologues (FHA OMP; vertical gray lines) are also shown. Flanking genes include parAB, chromosome/plasmid partitioning proteins; repA, plasmid replication protein; HP_PGM, His residue phosphotase/phosphoglycerate mutase; COG4705, conserved membrane-anchored protein; rimL, COG1670 acetyltransferase of the type rimL N-acetylase of ribosomal proteins; MFS_1, major facilitator superfamily transporter. Genes encoding hypothetical proteins are indicated by white arrows. PCR primers (indicated by arrows with superscript numbers) used for plasmidotype screening were as follows: primer 1, Δfhafw; primer 2, Δfharv; primer 3, fhafw; primer 4, fharv.

Fig. 6.

Hierarchical clustering of PCR prevalence results for 8 plasmids traits screened from among 224 Cronobacter species isolates. A positive PCR is indicated by a score of 1, and a negative reaction is indicated by a 0. A PCR-positive result of at least one of the targets of the T6SS was considered positive for the group as a whole. Cluster analysis was performed using the unweighted pair group method with arithmetic mean (UPGMA) found in the Bionumerics software suite of programs (Applied Maths, Inc., Austin, TX). The percentage of strains positive for each subgroup was rounded to the nearest whole number.