Christophe Ginevra

Rapid identification of Legionella species by mass spectrometry

Legionella species are facultative, intracellular bacteria that infect macrophages and protozoa, with the latter acting as transmission vectors to humans. These fastidious bacteria mostly cause pulmonary tract infections and are routinely identified by various molecular methods, mainly PCR targeting the mip gene and sequencing, which are expensive and time-consuming. Recently, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has emerged as a rapid and inexpensive method for identification of bacterial species. This study evaluated the use of MALDI-TOF-MS for rapid species and serogroup identification of 21 Legionella species recognized as human pathogens. To this end, a reference MS database was developed including 59 Legionella type strains, and a blind test was performed using 237 strains from various species. Two hundred and twenty-three of the 237 strains (94.1 %) were correctly identified at the species level, although ten (4.2 %) were identified with a score lower than 2.0. Fourteen strains (5.9 %) from eight species were misidentified at the species level, including seven (3.0 %) with a significant score, suggesting an intraspecific variability of protein profiles within some species. MALDI-TOF-MS was reproducible but could not identify Legionella strains at the serogroup level. When compared with mip gene sequencing, MALDI-TOF-MS exhibited a sensitivity of 99.2 and 89.9 % for the identification of Legionella strains at the genus and species level, respectively. This study demonstrated that MALDI-TOF-MS is a reliable tool for the rapid identification of Legionella strains at the species level.

Evaluation of a Nested-PCR-Derived Sequence-Based Typing Method Applied Directly to Respiratory Samples from Patients with Legionnaires' Disease

ABSTRACT Sequence-based typing (SBT) is a powerful method based on the sequencing of seven genes of Legionella pneumophila isolates. SBT performed directly on clinical samples has been used only in a limited number of cases. In our study, its efficiency was tested with 63 legionellosis respiratory samples. Sixty-three clinical samples, which included 23 samples from sporadic cases and 40 collected during four French outbreaks, confirmed by culture or urinary antigen testing and all positive by L. pneumophila quantitative PCR were subtyped by SBT according to the European Working Group for Legionella Infections standard scheme. Only 28.6% of the samples provided nucleotide sequences by SBT. Nested-PCR-based SBT (NPSBT) applied to the same respiratory samples was thus evaluated with new PCR primers surrounding the first set of primers used for the SBT. Sequencing results were obtained with 90.5% of the samples. Complete allelic profiles (seven genes sequenced) were obtained for 3.2% versus 53.9% of the samples by SBT and NPSBT, respectively. More importantly, of the 28 culture-negative samples, only 4 did not give any sequencing results. Taken together, NPSBT applied directly to clinical specimens significantly improved epidemiological typing compared to the initial SBT, in particular when no isolates are available.

Identification of legionella in clinical samples.

Currently, several methods are used for the detection of Legionella in clinical samples, and these methods constitute part of the criteria for defining legionellosis cases. Urinary antigen detection is the first-line diagnostic test, although this test is limited to L. pneumophila serogroup 1 (Lp1) (Helbig et al., J Clin Microbiol 41:838-840, 2003). The use of molecular techniques can improve Legionaires disease (LD) diagnosis by detecting other serogroups and species (Diederen et al., J Clin Microbiol 46:671-677, 2008). The isolation of Legionella strains from pulmonary samples by axenic culture is still required to perform further epidemiological investigations (Blyth et al., N S W Public Health Bull 20:157-161, 2009; Fields et al., Clin Microbiol Rev 15:506-526, 2002) but demonstrates various sensitivities. Amoebic coculture has been described as a method to recover Legionella from clinical culture-negative specimens (La Scola et al., J Clin Microbiol 39:365-366, 2001; Rowbotham, J Clin Pathol 36:978-986, 1983) and can be proposed for optimizing Legionella strain isolation from samples contaminated by oropharyngeal flora. Identification of Legionella isolates is based on serological characterization, genotypic methods (with sequencing of the mip gene as the standard method) and, more recently, the Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method.This chapter is limited to the identification of Legionella in clinical samples; antibody detection in human serum will not be discussed.

Characterization of a Novel Composite Staphylococcal Cassette Chromosome mec (SCCmec-SCCcad/ars/cop) in the Neonatal Sepsis-Associated Staphylococcus capitis Pulsotype NRCS-A

ABSTRACT Multiresistant Staphylococcus capitis pulsotype NRCS-A has been reported to be a major pathogen causing nosocomial bacteremia in preterm infants. We report that the NRCS-A strain CR01 harbors a novel 60.9-kb composite staphylococcal cassette chromosome mec (SCCmec) element, composed of an SCCmec with strong homologies to Staphylococcus aureus ST398 SCCmec and of an SCCcad/ars/cop harboring resistance genes for cadmium, arsenic, and copper. Whole-genome-based comparisons of published S. capitis strains suggest that strain CR01 acquired the two elements independently.

The Legionella pneumophila Collagen-Like Protein Mediates Sedimentation, Autoaggregation, and Pathogen-Phagocyte Interactions

ABSTRACT Although only partially understood, multicellular behavior is relatively common in bacterial pathogens. Bacterial aggregates can resist various host defenses and colonize their environment more efficiently than planktonic cells. For the waterborne pathogen Legionella pneumophila, little is known about the roles of autoaggregation or the parameters which allow cell-cell interactions to occur. Here, we determined the endogenous and exogenous factors sufficient to allow autoaggregation to take place in L. pneumophila. We show that isolates from Legionella species which do not produce the Legionella collagen-like protein (Lcl) are deficient in autoaggregation. Targeted deletion of the Lcl-encoding gene (lpg2644) and the addition of Lcl ligands impair the autoaggregation of L. pneumophila. In addition, Lcl-induced autoaggregation requires divalent cations. Escherichia coli producing surface-exposed Lcl is able to autoaggregate and shows increased biofilm production. We also demonstrate that L. pneumophila infection of Acanthamoeba castellanii and Hartmanella vermiformis is potentiated under conditions which promote Lcl dependent autoaggregation. Overall, this study shows that L. pneumophila is capable of autoaggregating in a process that is mediated by Lcl in a divalent-cation-dependent manner. It also reveals that Lcl potentiates the ability of L. pneumophila to come in contact, attach, and infect amoebae.

Seeding and Establishment of Legionella pneumophila in Hospitals: Implications for Genomic Investigations of Nosocomial Legionnaires’ Disease

Summary Whole-genome sequencing can be used to support or refute suspected links between hospital water systems and Legionnaires’ disease cases. However, caveats regarding the interpretation of genomic data from Legionella pneumophila are described that should be considered in future investigations.

Evaluation of BMPA, MWY, GVPC and BCYE media for the isolation of Legionella species from respiratory samples☆

Culture media performance is a critical factor in the isolation of Legionellae from respiratory samples. We showed that BMPA and MWY media yielded significantly higher isolation rates than GVPC and BCYE media in regard to performance with samples that harbored low Legionella inocula and high contamination levels.

Contribution of Amoebic Coculture to Recovery of Legionella Isolates from Respiratory Samples: Prospective Analysis over a Period of 32 Months

ABSTRACT We evaluated the contribution of amoebic coculture to the recovery of Legionella spp. from 379 respiratory samples. The sensitivity of axenic culture was 42.1%. The combination of axenic culture with amoebic coculture increased the Legionella isolation rate to 47.1%. Amoebic coculture was particularly efficient in isolating Legionella spp. from respiratory samples contaminated with oropharyngeal flora.

Validation of a Microbead-Based Format for Spoligotyping of Legionella pneumophila

ABSTRACT A 42-plex clustered regularly interspaced short palindromic repeat (CRISPR)-based typing technique (spoligotyping) was recently developed at the French National Reference Center for Legionella. It allows the subtyping of the Legionella pneumophila sequence type 1/Paris pulsotype. In this report, we present the transfer of the membrane-based spoligotyping technique to a microbead-based multiplexed format. This microbead-based high-throughput assay uses devices such as Luminex 200 or the recently launched Magpix system (Luminex Corp., Austin, TX). We designated this new technique LP-SPOL (for L. p neumophila spoligotyping). We used two sets of samples previously subtyped by the membrane-based spoligotyping method to set up and validate the transfer on the two microbead-based systems. The first set of isolates (n = 56) represented the whole diversity of the CRISPR patterns known to date. These isolates were used for transfer setup (determination of spacer cutoffs for both devices). The second set of isolates (n = 245) was used to validate the transfer to the two microbead-based systems. The results obtained by the Luminex 200 system were 100% concordant with those obtained by the Magpix system for the 2 sets of isolates. In total, 10 discrepant results were observed when comparing the membrane-based method to the microbead-based method. These discrepancies were further resolved by repeating either the membrane-based or the microbead-based assay. This new assay is expected to play an emerging role for surveillance of L. pneumophila, starting with one of the most frequent genotypes, the sequence type 1/Paris pulsotype. However, the generalization of this typing method to all L. pneumophila strains is not feasible, since not all L. pneumophila strains contain CRISPRs.

Legionella pneumophila LPS to evaluate urinary antigen tests

Three urinary antigen tests were compared using purified Legionella pneumophila (Lp) LPS. For Lp serogroup1, Sofia®FIA and Binax®EIA limits of detection (LOD) were similar; that of BinaxNOW® lower. For all tests the LOD was higher with LPS from non-Pontiac compared to Pontiac-strains. The LOD was variable for other Lp serogroups.