Population genetics of the nomenspecies Enterobacter cloacae

Abstract

The genetic heterogeneity of the nomenspecies Enterobacter cloacae is well known. Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter dissolvens, Enterobacter hormaechei, Enterobacter kobei, and Enterobacter nimipressuralis are closely related to it and are subsumed in the so-called E. cloacae complex. DNA-DNA hybridization studies performed previously identified at least five DNA-relatedness groups of this complex. In order to analyze the genetic structure and the phylogenetic relationships between the clusters of the nomenspecies E. cloacae, 206 strains collected from 22 hospitals, a veterinarian, and an agricultural center in 11 countries plus all 13 type strains of the genus and reference strain CDC 1347-71(R) were examined with a combination of sequence and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses of the three housekeeping genes hsp60, rpoB, and hemB as well as ampC, the gene of a class C beta-lactamase. Based on the neighbor-joining tree of the hsp60 sequences, 12 genetic clusters (I to XII) and an unstable sequence crowd (xiii) were identified. The robustness of the genetic clusters was confirmed by analyses of rpoB and hemB sequences and ampC PCR-RFLPs. Sequence crowd xiii split into two groups after rpoB analysis. Only three strains formed a cluster with the type strain of E. cloacae, indicating that the minority of isolates identified as E. cloacae truly belong to the species; 13% of strains grouped with other type strains of the genus, suggesting that the phenotypes of these species seem to be more heterogeneous than so far believed. Three clusters represented 70% of strains, but none of them included a type or reference strain. The genetic clustering presented in this study might serve as a framework for future studies dealing with taxonomic, evolutionary, epidemiological, or pathogenetic characteristics of bacteria belonging to the E. cloacae complex.

FIG.1.

Neighbor-joining tree after analyzing 273 nucleotides (91 variable) of the hsp60 gene of 206 study strains and 14 type and reference strains of the genus Enterobacter. The tree was rooted with the hsp60 sequence of E. aerogenes downloaded from GenBank (ENAE, accession number indicated). Type strains are labeled with T, reference strains with R, isolates from plants with a flower symbol, and isolates from animals with a picture of a dog. The animals from which strains were isolated are indicated after strain denominations, as are the abbreviations of the centers (see below) from which the strains originated. If several strains from one center had identical sequences, they were pooled at one spot of the tree, and the number of pooled strains is indicated after the center abbreviation. The scale gives the Jukes-Cantor distance along the branches. Numbers at the nodes of the tree indicate bootstrap values obtained after 100 replicates. A group of strains were considered a genetic cluster (I to XII) if the mean distinctness parameter k was above 2.0 (Table 3); otherwise it was called a sequence crowd (xiii). Cluster and sequence crowd denominations are indicated at the right side of the figure. Species abbreviations: ENAM, E. amnigenus; ENAS, E. asburiae; ENCA, E. cancerogenus; ENCL, E. cloacae; ENCO, E. cowanii; ENDI, E. dissolvens; ENGE, E. gergoviae; ENHO, E. hormaechei; ENIN, E. intermedius; ENKO, E. kobei; ENNI, E. nimipressuralis; ENPY, E. pyrinus; ENSA, E. sakazakii. Center abbreviations: AUIN, Innsbruck, Austria; BEBR, Brussels, Belgium; FRMA, Marseille, France; GEAA, Aachen, Germany; GEBE, Berlin, Germany; GEFB, Freiburg, Germany; GEFR, Frankfurt, Germany; GEGE, Gelsenkirchen, Germany; GEHA, Hannover, Germany; GEHE, Heidelberg, Germany; GEJE, Jena, Germany; GEKI, Kiel, Germany; GEMU, Munich, Germany; GERE, Regensburg, Germany; GETU, Tuebingen, Germany; NOBE, Bergen, Norway; POKR, Cracow, Poland; SPMA, Mallorca, Spain; SVST, Stockholm, Sweden; SVVA, Vaexjoe, Sweden; SWLA, Lausanne, Switzerland; UKLA, Lanarkshire, Scotland, United Kingdom.

FIG.2.

rpoB tree and ampC PCR-RFLP. Left side: Neighbor-joining tree based on the analysis of 1,008 nucleotides (142 nucleotides variable) of the rpoB gene of 78 study strains. The tree was rooted with the rpoB sequence of the E. gergoviae type strain. Bootstrap values after 100 replicates are indicated at the nodes of the tree. Cluster denominations, labeling, and abbreviations correspond to those of Fig. 1. Sequence crowd xiii was split into groups xiiia and xiiib. Right side: For PCR-RFLP analysis. PCR products of partial ampC genes were restricted with HaeIII, electrophoresed in 3% agarose, and strained with ethidium bromide. Resulting clusters were designated a to k.

FIG. 3.

Neighbor-joining tree based on the analysis of 214 nucleotides (68 nucleotides variable) of the hemB gene of 40 study strains. Only the genetic clusters (not the sequence crowd) were considered. The tree was rooted with the hemB sequence of Escherichia coli (GenBank accession number L44595). Cluster denominations, labeling, and abbreviations correspond to those of Fig. 1.