Emmanuelle Bille

Real-Time Identification of Bacteria and Candida Species in Positive Blood Culture Broths by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

ABSTRACT Delays in the identification of microorganisms are a barrier to the establishment of adequate empirical antibiotic therapy of bacteremia. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) allows the identification of microorganisms directly from colonies within minutes. In this study, we have adapted and tested this technology for use with blood culture broths, thus allowing identification in less than 30 min once the blood culture is detected as positive. Our method is based on the selective recovery of bacteria by adding a detergent that solubilizes blood cells but not microbial membranes. Microorganisms are then extracted by centrifugation and analyzed by MALDI-TOF-MS. This strategy was first tested by inoculating various bacterial and fungal species into negative blood culture bottles. We then tested positive patient blood or fluid samples grown in blood culture bottles, and the results obtained by MALDI-TOF-MS were compared with those obtained using conventional strategies. Three hundred twelve spiked bottles and 434 positive cultures from patients were analyzed. Among monomicrobial fluids, MALDI-TOF-MS allowed a reliable identification at the species, group, and genus/family level in 91%, 5%, and 2% of cases, respectively, in 20 min. In only 2% of these samples, MALDI-TOF MS did not yield any result. When blood cultures were multibacterial, identification was improved by using specific databases based on the Gram staining results. MALDI-TOF-MS is currently the fastest technique to accurately identify microorganisms grown in positive blood culture broths.

MALDI-TOF mass spectrometry tools for bacterial identification in clinical microbiology laboratory.

Since the early 1980s, mass spectrometry has emerged as a particularly powerful tool for analysis and characterization of proteins in research. Recently, bacteriologists have focused their attention on the use of mass spectrometry (MS) for bacterial identification, especially Matrix Assisted Laser Desorption Ionization Time-Of-Flight (MALDI-TOF). Moreover, recent publications have evaluated MALDI-TOF in microbiology laboratory for routine use. MALDI-TOF-MS is a rapid, precise, and cost-effective method for identification of intact bacteria, compared to conventional phenotypic techniques or molecular biology. Furthermore, it allows identification of bacteria directly from clinical samples (blood cultures for example). The goal of this review was to update recent data concerning routine identification of microorganisms by MALDI-TOF in the clinical microbiology laboratory.

A chromosomally integrated bacteriophage in invasive meningococci

Cerebrospinal meningitis is a feared disease that can cause the death of a previously healthy individual within hours. Paradoxically, the causative agent, Neisseria meningitidis, is a common inhabitant of the human nasopharynx, and as such, may be considered a normal, commensal organism. Only in a small proportion of colonized people do the bacteria invade the bloodstream, from where they can cross the blood–brain barrier to cause meningitis. Furthermore, most meningococcal disease is caused by bacteria belonging to only a few of the phylogenetic groups among the large number that constitute the population structure of this genetically variable organism. However, the genetic basis for the differences in pathogenic potential remains elusive. By performing whole genome comparisons of a large collection of meningococcal isolates of defined pathogenic potential we brought to light a meningococcal prophage present in disease-causing bacteria. The phage, of the filamentous family, excises from the chromosome and is secreted from the bacteria via the type IV pilin secretin. Therefore, this element, by spreading among the population, may promote the development of new epidemic clones of N. meningitidis that are capable of breaking the normal commensal relationship with humans and causing invasive disease.

Type IV pilus retraction in pathogenic Neisseria is regulated by the PilC proteins

Pathogenic Neisseria express type IV pili (tfp), which have been shown to play a central role in the interactions of bacteria with their environment. The regulation of piliation thus constitutes a central element in bacterial life cycle. The PilC proteins are outer membrane‐associated proteins that have a key role in tfp biogenesis since PilC‐null mutants appear defective for fibre expression. Moreover, tfp are also subjected to retraction, which is under the control of the PilT nucleotide‐binding protein. In this work, we bring evidence that fibre retraction involves the translocation of pilin subunits to the cytoplasmic membrane. Furthermore, by engineering meningococcal strains that harbour inducible pilC genes, and with the use of meningococcus–cell interaction as a model for the sequential observation of fibre expression and retraction, we show that the PilC proteins regulate PilT‐mediated fibre retraction.

MALDI-TOF MS Andromas strategy for the routine identification of bacteria, mycobacteria, yeasts, Aspergillus spp. and positive blood cultures

All organisms usually isolated in our laboratory are now routinely identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using the Andromas software. The aim of this study was to describe the use of this strategy in a routine clinical microbiology laboratory. The microorganisms identified included bacteria, mycobacteria, yeasts and Aspergillus spp. isolated on solid media or extracted directly from blood cultures. MALDI-TOF MS was performed on 2665 bacteria isolated on solid media, corresponding to all bacteria isolated during this period except Escherichia coli grown on chromogenic media. All acquisitions were performed without extraction. After a single acquisition, 93.1% of bacteria grown on solid media were correctly identified. When the first acquisition was not contributory, a second acquisition was performed either the same day or the next day. After two acquisitions, the rate of bacteria identified increased to 99.2%. The failures reported on 21 strains were due to an unknown profile attributed to new species (9) or an insufficient quality of the spectrum (12). MALDI-TOF MS has been applied to 162 positive blood cultures. The identification rate was 91.4%. All mycobacteria isolated during this period (22) were correctly identified by MALDI-TOF MS without any extraction. For 96.3% and 92.2% of yeasts and Aspergillus spp., respectively, the identification was obtained with a single acquisition. After a second acquisition, the overall identification rate was 98.8% for yeasts (160/162) and 98.4% (63/64) for Aspergillus spp. In conclusion, the MALDI-TOF MS strategy used in this work allows a rapid and efficient identification of all microorganisms isolated routinely.

Evaluation of the Andromas Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Aerobically Growing Gram-Positive Bacilli

ABSTRACT Matrix-associated laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is a rapid and simple microbial identification method. Previous reports using the Biotyper system suggested that this technique requires a preliminary extraction step to identify Gram-positive rods (GPRs), a technical issue that may limit the routine use of this technique to identify pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and 72 species by the direct colony method. This bacterial collection included 40 C. diphtheriae, 13 C. pseudotuberculosis, 19 C. ulcerans, and 270 other Corynebacterium isolates, 32 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia, 75 Actinomyces, 18 Actinobaculum, 11 Propionibacterium acnes, 18 Propionibacterium avidum, 30 Lactobacillus, 21 Bacillus, 2 Rhodococcus equi, 2 Erysipelothrix rhusiopathiae, and 38 other GPR isolates, all identified by reference techniques. Totals of 98.5% and 1.2% of non-Listeria GPR isolates were identified to the species or genus level, respectively. Except for L. grayi isolates that were identified to the species level, all other Listeria isolates were identified to the genus level because of highly similar spectra. These data demonstrate that rapid identification of pathogenic GPRs can be obtained without an extraction step by MALDI-TOF mass spectrometry.

Association of a Bacteriophage with Meningococcal Disease in Young Adults

Despite being the agent of life-threatening meningitis, Neisseria meningitidis is usually carried asymptomatically in the nasopharynx of humans and only occasionally causes disease. The genetic bases for virulence have not been entirely elucidated and the search for new virulence factors in this species is hampered by the lack of an animal model representative of the human disease. As an alternative strategy we employ a molecular epidemiological approach to establish a statistical association of a candidate virulence gene with disease in the human population. We examine the distribution of a previously-identified genetic element, a temperate bacteriophage, in 1288 meningococci isolated from cases of disease and asymptomatic carriage. The phage was over-represented in disease isolates from young adults indicating that it may contribute to invasive disease in this age group. Further statistical analysis indicated that between 20% and 45% of the pathogenic potential of the five most common disease-causing meningococcal groups was linked to the presence of the phage. In the absence of an animal model of human disease, this molecular epidemiological approach permitted the estimation of the influence of the candidate virulence factor. Such an approach is particularly valuable in the investigation of exclusively human diseases.

Genotypic and Phenotypic Modifications of Neisseria meningitidis after an Accidental Human Passage

A scientist in our laboratory was accidentally infected while working with Z5463, a Neisseria meningitidis serogroup A strain. She developed severe symptoms (fever, meningism, purpuric lesions) that fortunately evolved with antibiotic treatment to complete recovery. Pulse-field gel electrophoresis confirmed that the isolate obtained from the blood culture (Z5463BC) was identical to Z5463, more precisely to a fourth subculture of this strain used the week before the contamination (Z5463PI). In order to get some insights into genomic modifications that can occur in vivo, we sequenced these three isolates. All the strains contained a mutated mutS allele and therefore displayed an hypermutator phenotype, consistent with the high number of mutations (SNP, Single Nucleotide Polymorphism) detected in the three strains. By comparing the number of SNP in all three isolates and knowing the number of passages between Z5463 and Z5463PI, we concluded that around 25 bacterial divisions occurred in the human body. As expected, the in vivo passage is responsible for several modifications of phase variable genes. This genomic study has been completed by transcriptomic and phenotypic studies, showing that the blood strain used a different haemoglobin-linked iron receptor (HpuA/B) than the parental strains (HmbR). Different pilin variants were found after the in vivo passage, which expressed different properties of adhesion. Furthermore the deletion of one gene involved in LOS biosynthesis (lgtB) results in Z5463BC expressing a different LOS than the L9 immunotype of Z2491. The in vivo passage, despite the small numbers of divisions, permits the selection of numerous genomic modifications that may account for the high capacity of the strain to disseminate.

Mechanisms of meningeal invasion by a bacterial extracellular pathogen, the example of Neisseria meningitidis

The blood-cerebrospinal fluid (CSF) barrier physiologically protects the meningeal spaces from bloodborne bacterial pathogens, due to the existence of specialized junctional interendothelial complexes. A few bacterial pathogens are able to reach the subarachnoidal space and cause bacterial meningitis in humans, a rare but dreadful disease. Surprisingly, most of them are extracellular commensals of the nasopharynx (Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae) or of the digestive tract (Escherichia coli and Streptococcus agalactiae). The particular ability of these pathogens to induce meningitis is related to virulence factors that allow them to escape host innate immunity, to multiply within the serum, and to interact closely with the endothelial front line of defense of the blood-CSF barrier. In vitro studies using microvascular brain endothelial cell lines have shown that induced transcytosis may be a common route used by H. influenzae, S. pneumoniae, E. coli and S. agalactiae to reach the CSF. N. meningitidis is a strict human pathogen that interacts very tightly with endothelial cells. Adhesion of the meningococcus is mediated by type IV pili that induce a localized remodeling of the sub cortical cytoskeleton, leading to the formation of endothelial membrane protrusions that anchor bacterial colonies at the endoluminal face of the endothelial cell membrane, allowing a better resistance to blood flow. Recent work has shown that N. meningitidis is also able to recruit the polarity complex Par3/Par6/aPKC that re-routes endothelial cell adhesion molecules of interendothelial junctions, opening a paracellular route for bacteria to cross the endothelial barrier.

Bacteriologic epidemiology and empirical treatment of pediatric complicated appendicitis

Preoperative samples in the context of complicated appendicitis (CA) are rarely collected, and there is no consensus regarding the optimal antibiotic therapy in children. To help optimize empirical preoperative treatment, we studied clinical and bacteriologic data from a prospective cohort of 93 children with CA in a French hospital. All the bacteria isolated from peritoneal fluids were identified, using phenotypic and/or molecular techniques. The most commonly recovered species were Escherichia coli (71%), Streptococcus group milleri (34%), anaerobes (20%), and Pseudomonas aeruginosa (19%). The association piperacillin-tazobactam is an accurate choice of empirical therapy as it is active against 97% of bacteria. A third-generation cephalosporin with metronidazole in association with an aminoglycoside is a good alternative. Although antibiotic use may be considered as an adjunct to surgical intervention of CA, the appropriate use of preoperative antibiotics is essential and must be constantly reevaluated according to the bacterial epidemiology.