Bernard La Scola

Patients admitted to intensive care units are frequently exposed to pathogenic microorganisms present in their environment. Exposure to these microbes may lead to the development of hospital-acquired infections that complicate the illness and may be fatal. Amoeba-associated microorganisms (AAMs) are frequently isolated from hospital water networks and are reported to be associated to cases of community and hospital-acquired pneumonia. We used a multiplexed immunofluorescence assay to test for the presence of antibodies against AAMs in sera of intensive care unit (ICU) pneumonia patients and compared to patients at the admission to the ICU (controls). Our results show that some AAMs may be more frequently detected in patients who had hospital-acquired pneumonia than in controls, whereas other AAMs are ubiquitously detected. However, ICU patients seem to exhibit increasing immune response to AAMs when the ICU stay is prolonged. Moreover, concomitant antibodies responses against seven different microorganisms (5 Rhizobiales, Balneatrix alpica, and Mimivirus) were observed in the serum of patients that had a prolonged ICU stay. Our work partially confirms the results of previous studies, which show that ICU patients would be exposed to water amoeba-associated microorganisms, and provides information about the magnitude of AAM infection in ICU patients, especially patients that have a prolonged ICU stay. However, the incidence of this exposure on the development of pneumonia remains to assess.

Two isolates of intra-amoeba-growing bacteria, LegA^T ( = DSM 24804^T = CSUR P146^T) and LegM^T ( = DSM 24805^T = CSUR P145^T), were characterized on the basis of microscopic appearance, staining characteristics, axenic growth at different temperatures and the sequences of the mip, rpoB, 16S rRNA and rnpb genes, as well as the 23S–5S region. Phylogenetic analysis showed that these two isolates lay within the radius of the family Legionellaceae . Furthermore, the analysis of these genes yielded congruent data that indicated that, although strain LegM^T clusters specifically with Legionella feeleii ATCC 35072^T and LegA^T clusters with Legionella nautarum ATCC 49596^T, the divergence observed between these species was greater than that observed between other members of the family. Taken together, these results support the proposal that these two isolates represent novel members of the genus Legionella , and we propose to name them Legionella tunisiensis sp. nov. for LegM^T ( = DSM 24805^T = CSUR P145^T) and Legionella massiliensis sp. nov. for LegA^T ( = DSM 24804^T = CSUR P146^T).

Despite the considerable number of studies reported to date, the causative agents of pneumonia are not completely identified. We comprehensively applied modern and traditional laboratory diagnostic techniques to identify microbiota in patients who were admitted to or developed pneumonia in intensive care units (ICUs). During a three-year period, we tested the bronchoalveolar lavage (BAL) of patients with ventilator-associated pneumonia, community-acquired pneumonia, non-ventilator ICU pneumonia and aspiration pneumonia, and compared the results with those from patients without pneumonia (controls). Samples were tested by amplification of 16S rDNA, 18S rDNA genes followed by cloning and sequencing and by PCR to target specific pathogens. We also included culture, amoeba co-culture, detection of antibodies to selected agents and urinary antigen tests. Based on molecular testing, we identified a wide repertoire of 160 bacterial species of which 73 have not been previously reported in pneumonia. Moreover, we found 37 putative new bacterial phylotypes with a 16S rDNA gene divergence ≥98% from known phylotypes. We also identified 24 fungal species of which 6 have not been previously reported in pneumonia and 7 viruses. Patients can present up to 16 different microorganisms in a single BAL (mean ± SD; 3.77±2.93). Some pathogens considered to be typical for ICU pneumonia such as Pseudomonas aeruginosa and Streptococcus species can be detected as commonly in controls as in pneumonia patients which strikingly highlights the existence of a core pulmonary microbiota. Differences in the microbiota of different forms of pneumonia were documented.

The analysis of three water samples from two cooling towers and one river allowed us to isolate three strains of a novel species of the classAlphaproteobacteriawhich is phylogenetically related to uncultured alphaproteobacteria. Based upon 16S rRNA gene sequence analysis and phenotypic characterization, we propose to name this novel speciesReyranella massiliensisgen. nov., sp. nov., type strain 521^T( = CSUR P115^T = DSM 23428^T). The most closely related cultivable micro-organism to this novel bacterium is a member of the genusMagnetospirillum.

MALDI-TOF mass spectrometry (MS) on intact cells is at the forefront of a new revolution in the routine identification of microorganisms in clinical microbiology laboratories. In addition to its high accuracy for identification, it provides results in just a few minutes. As correct and timely diagnoses are pivotal for the management of bloodstream infections, MALDI-TOF MS has the potential to replace or at least complement other methods for microorganism identification in bloodstream infections. With the improvement of databases and software, this method has an accurate identification rate of nearly 100% on nearly all agar-grown microorganisms and more than 80% on positive blood culture broth tests. Even if it is highly efficient now, after a decade of developments, the technique is currently still under improvement. It lacks standardization and exhaustive databases or more efficient software that are currently under development. Even now, both the technology itself and the reagents are improving significantly. Thus, it is likely that identifications will become more accurate in the future, especially on more complex fluids, such as blood culture broth.

Streptobacillus moniliformis is a fastidious growing Gram-negative bacillus responsible of rat-bite fever. We describe here the first report of this disease in la Réunion and the first isolation using shell vial cell culture from a blood culture bottle with a bacterium suspected to be dead.

With long delays observed between sampling and availability of results, the usefulness of blood cultures in the context of emergency infectious diseases has recently been questioned. Among methods that allow quicker bacterial identification from growing colonies, matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry was demonstrated to accurately identify bacteria routinely isolated in a clinical biology laboratory. In order to speed up the identification process, in the present work we attempted bacterial identification directly from blood culture bottles detected positive by the automate. We prospectively analysed routine MALDI-TOF identification of bacteria detected in blood culture by two different protocols involving successive centrifugations and then lysis by trifluoroacetic acid or formic acid. Of the 562 blood culture broths detected as positive by the automate and containing one bacterial species, 370 (66%) were correctly identified. Changing the protocol from trifluoroacetic acid to formic acid improved identification of Staphylococci, and overall correct identification increased from 59% to 76%. Lack of identification was observed mostly with viridans streptococci, and only one false positive was observed. In the 22 positive blood culture broths that contained two or more different species, only one of the species was identified in 18 samples, no species were identified in two samples and false species identifications were obtained in two cases. The positive predictive value of bacterial identification using this procedure was 99.2%. MALDI-TOF MS is an efficient method for direct routine identification of bacterial isolates in blood culture, with the exception of polymicrobial samples and viridans streptococci. It may replace routine identification performed on colonies, provided improvement for the specificity of blood culture broths growing viridans streptococci is obtained in the near future.