Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of ≥97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of ≥99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter andPantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides unambiguous data even for rare isolates, which are reproducible in and between laboratories. The increase in accurate new 16S rDNA sequences and the development of alternative genes for molecular identification of certain taxa should further improve the usefulness of molecular identification of bacteria.

Phylogenetic analyses ofThe phylogenetic relationships of the type strains of 9 Klebsiella species and 20 species from 11 genera of the family Enterobacteriaceae were investigated by performing a comparative analysis of the sequences of the 16S rRNA and rpoB genes. The sequence data were phylogenetically analysed by the neighbourjoining and parsimony methods. The phylogenetic inference of the sequence comparison confirmed that the genus Klebsiella is heterogeneous and composed of species which form three clusters that also included members of other genera, including Enterobacter aerogenes, Erwinia clusters I and II and Tatumella. Cluster I contained the type strains of Klebsiella pneumoniae subsp. pneumoniae, Klebsiella pneumoniae subsp. rhinoscleromatis and Klebsiella pneumoniae subsp. ozaenae. Cluster II contained Klebsiella ornithinolytica, Klebsiella planticola, Klebsiella trevisanii and Klebsiella terrigena, organisms characterized by growth at 10 SC and utilization of L-sorbose as carbon source. Cluster III contained Klebsiella oxytoca. The data from the sequence analyses along with previously reported biochemical and DNA-DNA hybridization data support the division of the genus Klebsiella into two genera and one genogroup. The name Raoultella is proposed as a genus name for species of cluster II and emended definitions of Klebsiella species are proposed.

FranceEntembacter aemgenes is among the five most frequently isolated nosocomial pathogens in France, and this bacterium also shows increasing multidrug resistance. In this study, various E. aerogenes strains isolated from hospital units were characterized for their outer-membrane proteins, antibiotic susceptibilities (inhibition diameters and MICs) and resistance mechanisms associated with modification of envelope permeability (porin alteration and active efflux). Diminished outer-membrane permeability due to porin alterations was found in conjunction with the expression of an enzymic barrier in resistant isolates. Interestingly, changes in the functional expression of porins appeared to play a special role in susceptibility to cefepime. An active efflux to quinolones was also identified. Simultaneous changes in envelope permeability, i.e. a porin deficiency (in) and an efflux mechanism (out), were clearly evident in two clinical strains.

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The purpose of this prospective postmortem study was to assess the diagnostic accuracy of bronchoscopic techniques (bronchoalveolar lavage [BAL] and protected specimen brush [PSB]) and nonbronchoscopic techniques (blind bronchial sampling [BBS] and mini-BAL) in the diagnosis of ventilator-associated pneumonia (VAP). The results of each technique were compared with histology and culture of lung tissue specimens obtained by surgical pneumonectomies in 38 patients who died after at least 72 h of mechanical ventilation. Histology was positive for VAP in 18 patients and negative in 20 patients. There were 12 definite VAP (positive histology and positive lung cultures) and 6 histologic VAP (positive histology and negative cultures). Clinical pulmonary infection score (CPIS) at a threshold of 6 achieved a sensitivity of 72% and a specificity of 85%. When the CPIS was combined with the logarithmic concentration of the predominant microorganism obtained from the BBS sample culture, specificity was increased to 95%, for a threshold of 10. Using 10(3) cfu/ml as the threshold of positivity for cultures obtained with PSB and mini-BAL samples and 10(4) cfu/ml for cultures obtained with BBS and BAL, the respective sensitivities of these techniques for definite VAP were 42, 67, 83, and 58%. The sensitivity of BBS was significantly higher than that of PSB (p < 0.05). The area under the receiver operator characteristic curve was significantly greater for BBS than PSB (p < 0.05). Given that it is more sensitive and noninvasive, BBS is preferable to PSB for the diagnosis of VAP.

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With the increased number of resistant Acinetobacter baumannii strains, it is urgently required to decipher the molecular bases of outer membrane permeability. The analyses of the outer membrane from different A. baumannii strains indicated a modification in the expression of two proteins of 29 and 43 kDa, respectively. By electrophoresis and MALDI-MS analyses, the 43 kDa OMP was identified as a protein belonging to the OprD family, a basic amino acid and imipenem porin.

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