The sodA gene as a target for phylogenetic dissection of the genus Haemophilus and accurate identification of human clinical isolates.

The genus Haemophilus constitutes a heterogeneous group of Pasteurellaceae species, and conventional identification of isolates other than Haemophilus influenzae and Haemophilus parainfluenzae is often challenging. Here, simple colony-PCR and sequencing assays with the same pair of degenerate primers were used to characterize a 449- to 458-bp fragment (sodA(int)) internal to the sodA gene encoding the manganese-dependent superoxide dismutase in type strains of all 15 Haemophilus species and Actinobacillus actinomycetemcomitans. The topology of a sodA(int)-based phylogenetic tree was in general agreement with that inferred from the analysis of 16S rRNA and other housekeeping gene sequences, but allowed more confident delineation of the main clusters of species. The sodA(int) sequences showed a markedly higher divergence than those of the corresponding 16S rRNA genes, and 38 independent human clinical isolates were identified by comparing their sodA(int) sequence to those of the type species. Except for one Haemophilus aphrophilus strain, all isolates were unambiguously characterized in spite of a high intraspecific sodA(int) sequence diversity. This study provides a comprehensive sequence-based phylogenetic analysis of the entire genus Haemophilus, and confirms that sodA is a potent target for the identification of clinical isolates of Pasteurellaceae. This approach might contribute to the taxonomic reappraisal of this family, and to the development of diagnostic tools.

Rapid and accurate identification of human isolates of Pasteurella and related species by sequencing the sodA gene.

The identification of Pasteurella and related bacteria remains a challenge. Here, a 449- to 473-bp fragment (sodA(int)) internal to the sodA gene, encoding the manganese-dependent superoxide dismutase, was amplified and sequenced with a single pair of degenerate primers from the type strains of Pasteurella (18 strains), Gallibacterium (1 strain), and Mannheimia (5 strains) species. The sodA(int)-based phylogenetic tree was in general agreement with that inferred from the analysis of the corresponding 16S rRNA gene sequences, with members of the Pasteurella sensu stricto cluster (Pasteurella multocida, Pasteurella canis, Pasteurella dagmatis, and Pasteurella stomatis) forming a monophyletic group and Gallibacterium and Mannheimia being independent monophyletic genera. However, the sodA(int) sequences showed a markedly higher divergence than the corresponding 16S rRNA genes, confirming that sodA is a potent target to differentiate related species. Thirty-three independent human clinical isolates phenotypically assigned to 13 Pasteurella species by a reference laboratory were successfully identified by comparing their sodA(int) sequences to those of the type species. In the course of this work, we identified the first Gallibacterium anatis isolate ever reported from a human clinical specimen. The sodA(int) sequences of the clinical isolates displayed less than 2.5% divergence from those of the corresponding type strains, except for the Pasteurella pneumotropica isolates, which were closely related to each other (> 98% sodA(int) sequence identity) but shared only 92% sodA(int) identity with the type strain. The method described here provides a rapid and accurate tool for species identification of Pasteurella isolates when access to a sequencing facility is available.

Changes in staphylococcal cassette chromosome type and antibiotic resistance profile in methicillin-resistant Staphylococcus aureus isolates from a French hospital over an 11 year period.

BACKGROUND: In this study, we investigated the relationship between changes in antibiotic resistance and distribution of staphylococcal cassette chromosome (SCC) types amongst methicillin-resistant Staphylococcus aureus (MRSA) isolates expressing the most frequently encountered profiles of antibiotic resistance over an 11 year period in the University Hospital of Rennes, France. METHODS: Antibiotic susceptibilities were determined by agar diffusion. SCC typing was performed using PCR. PFGE demonstrated that isolates were phylogenetically related. RESULTS: Fourteen profiles of antibiotic resistance were defined among MRSA isolates. For each resistance profile, only one SCC type was associated: four patterns corresponded to SCC type I or IA, nine to SCC type IV or IVA, and none to types II and III. One was not typed. PFGE indicated that isolates with SCC type I or IA belong to a single genetic lineage, which also includes most of the epidemic isolates, which carry SCC type IVA. In contrast to type I or IA, isolates with SCC type IV or IVA were found to be associated with several different PFGE clusters, although not all of these represent epidemic isolates. CONCLUSIONS: During the course of the study, the spectrum of antibiotic resistance in MRSA isolates decreased. This occurred due to the emergence of strains with SCC type IV or IVA, which are susceptible to more antibiotics than type I or IA strains. The greater prevalence of such isolates could not be linked conclusively to the presence of SCC type IV or IVA, or to one particular PFGE cluster.

Nine-year surveillance of methicillin-resistant Staphylococcus aureus in a hospital suggests instability of mecA DNA region in an epidemic strain.

The distributions of the antibiotic resistance patterns in a population of Staphylococcus aureus isolates from a teaching hospital were studied over a 9-year period. The results indicate the existence of successive major epidemic methicillin-resistant strains and the emergence of a methicillin-susceptible strain with an unusual resistance pattern. Our findings suggest that this methicillin-susceptible S. aureus strain could be derived from the dominant gentamicin-susceptible methicillin-resistant S. aureus strain with the loss of a 40-kb DNA fragment.