Agnès Ferroni

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.

Rapid Identification of Staphylococci Isolated in Clinical Microbiology Laboratories by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

ABSTRACT Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) of intact bacteria yields a reproducible spectrum depending upon growth conditions, strain, or species. Using whole viable bacteria we describe here the application of MALDI-TOF-MS to the identification of coagulase-negative staphylococci (CoNS). Our aim was, once a bacterium has been recognized as Micrococcaceae, to identify peaks in the spectrum that can be used to identify the species or subspecies. MALDI-TOF-MS was performed using bacteria obtained from one isolated colony. One reference strain for each of the 23 clinically relevant species or subspecies of Micrococcaceae was selected. For each reference strain, the MALDI-TOF-MS profile of 10 colonies obtained from 10 different passages was analyzed. For each strain, only peaks that were conserved in the spectra of all 10 isolated colonies and with a relative intensity above 0.1 were retained, thus leading to a set of 3 to 14 selected peaks per strain. The MALDI-TOF-MS profile of 196 tested strains was then compared with that of the set of selected peaks of each of the 23 reference strains. In all cases the best hit was with the set of peaks of the reference strain belonging to the same species as that of the tested strain, thus demonstrating that the 23 sets of selected peaks can be used as a database for the rapid species identification of CoNS. Similar results were obtained using four different growth conditions. Extending this strategy to other groups of relevant pathogenic bacteria will allow rapid bacterial identification.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Nonfermenting Gram-Negative Bacilli Isolated from Cystic Fibrosis Patients

ABSTRACT The identification of nonfermenting gram-negative bacilli isolated from cystic fibrosis (CF) patients is usually achieved by using phenotype-based techniques and eventually molecular tools. These techniques remain time-consuming, expensive, and technically demanding. We used a method based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) for the identification of these bacteria. A set of reference strains belonging to 58 species of clinically relevant nonfermenting gram-negative bacilli was used. To identify peaks discriminating between these various species, the profile of 10 isolated colonies obtained from 10 different passages was analyzed for each referenced strain. Conserved peaks with a relative intensity greater than 0.1 were retained. The spectra of 559 clinical isolates were then compared to that of each of the 58 reference strains as follows: 400 Pseudomonas aeruginosa, 54 Achromobacter xylosoxidans, 32 Stenotrophomonas maltophilia, 52 Burkholderia cepacia complex (BCC), 1 Burkholderia gladioli, 14 Ralstonia mannitolilytica, 2 Ralstonia pickettii, 1 Bordetella hinzii, 1 Inquilinus limosus, 1 Cupriavidus respiraculi, and 1 Burkholderia thailandensis. Using this database, 549 strains were correctly identified. Nine BCC strains and one R. mannnitolilytica strain were identified as belonging to the appropriate genus but not the correct species. We subsequently engineered BCC- and Ralstonia-specific databases using additional reference strains. Using these databases, correct identification for these species increased from 83 to 98% and from 94 to 100% of cases, respectively. Altogether, these data demonstrate that, in CF patients, MALDI-TOF-MS is a powerful tool for rapid identification of nonfermenting gram-negative bacilli.

Outbreak of nosocomial urinary tract infections due to Pseudomonas aeruginosa in a paediatric surgical unit associated with tap-water contamination

An outbreak of 14 cases of urinary tract infections by Pseudomonas aeruginosa, including six symptomatic infections, occurred from September to November 1994 in a paediatric surgical unit. During the outbreak, urine samples from patients and multiple samples from the environment of patients were tested for the presence of P. aeruginosa. Bacterial isolates were studied by pulsed-field gel electrophoresis. Genotypic analysis showed that most of the isolates from children were different. Multiple P. aeruginosa isolates were also found in the tap water, as the only putative source of contamination. Two of these isolates were identified in two infected patients, indicating possible direct contamination of patients via tap water and this was related to the distal colonization of faucets. Bacteria were eradicated from tap water by replacement of taps. The cluster of cases of P. aeruginosa urinary infection was, therefore, related to multiple contaminations through tap water. These results illustrate an unexpected risk of nosocomial infection and emphasizes the importance of checking tap water to prevent bacterial contamination through handwashing in contaminated water.

Microbiological Diagnosis of Empyema in Children: Comparative Evaluations by Culture, Polymerase Chain Reaction, and Pneumococcal Antigen Detection in Pleural Fluids

BACKGROUND Pleural empyema is an increasingly reported complication of pneumonia in children. Microbiological diagnostic tests for empyema by culture frequently have false-negative results due to previous administration of antibiotics. Molecular diagnosis by broad-range 16S ribosomal DNA (rDNA) polymerase chain reaction (PCR) and rapid pneumococcal antigen detection are reliable tools, but their diagnostic value has not been clearly established for pleural fluid samples. Pneumococcal antigen detection has only been validated for urine and cerebrospinal fluid samples. METHODS Over 4 years, pleural fluid specimens were collected from 78 children with pleural empyema. Standard culture, pneumococcal antigen detection by latex agglutination (Pastorex; Bio-Rad) and immunochromatographic testing (Binax NOW Streptococcus pneumoniae), and 16S rDNA PCR were performed on these specimens. Pneumococcal identification by 16S rDNA PCR and sequencing was confirmed by pneumolysin PCR. RESULTS Of the 78 cases of pleural empyema, 60 (77%) were microbiologically documented by culture or 16S rDNA PCR. Of the 40 pneumococcal empyema cases, 17 (43%) were only diagnosed by PCR and 23 with PCR and culture. The sensitivity and specificity of the latex antigen detection (with the use of culture and/or PCR as the test standard) were 90% and 95%, respectively. The immunochromatographic test detected pneumococcal antigens in 3 additional specimens for which latex agglutination results were negative, thereby increasing the sensitivity of antigen detection. CONCLUSIONS Pneumococcal antigen detection in pleural fluid specimens from children provides a rapid and sensitive method of diagnosis of pneumococcal empyema, which can be confirmed by specific pneumolysin PCR when culture results are negative. Broad-range 16S rDNA PCR has value in detecting bacterial agents responsible for culture-negative pleural empyema.

Rapid Identification of Mycobacterial Whole Cells in Solid and Liquid Culture Media by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

ABSTRACT Mycobacterial identification is based on several methods: conventional biochemical tests that require several weeks for accurate identification, and molecular tools that are now routinely used. However, these techniques are expensive and time-consuming. In this study, an alternative method was developed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). This approach allows a characteristic mass spectral fingerprint to be obtained from whole inactivated mycobacterial cells. We engineered a strategy based on specific profiles in order to identify the most clinically relevant species of mycobacteria. To validate the mycobacterial database, a total of 311 strains belonging to 31 distinct species and 4 species complexes grown in Löwenstein-Jensen (LJ) and liquid (mycobacterium growth indicator tube [MGIT]) media were analyzed. No extraction step was required. Correct identifications were obtained for 97% of strains from LJ and 77% from MGIT media. No misidentification was noted. Our results, based on a very simple protocol, suggest that this system may represent a serious alternative for clinical laboratories to identify mycobacterial species.

Mycobacterium abscessus and children with cystic fibrosis.

We prospectively studied 298 patients with cystic fibrosis (mean age 11.3 years; range 2 months to 32 years; sex ratio, 0.47) for nontuberculous mycobacteria in respiratory samples from January 1, 1996, to December 31, 1999. Mycobacterium abscessus was by far the most prevalent nontuberculous mycobacterium: 15 patients (6 male, 9 female; mean age 11.9 years; range 2.5–22 years) had at least one positive sample for this microorganism (versus 6 patients positive for M. avium complex), including 10 with >3 positive samples (versus 3 patients for M. avium complex). The M. abscessus isolates from 14 patients were typed by pulsed-field gel electrophoresis: each of the 14 patients harbored a unique strain, ruling out a common environmental reservoir or person-to-person transmission. Water samples collected in the cystic fibrosis center were negative for M. abscessus. This major mycobacterial pathogen in children and teenagers with cystic fibrosis does not appear to be acquired nosocomially.

Age-Related Prevalence and Distribution of Nontuberculous Mycobacterial Species among Patients with Cystic Fibrosis

ABSTRACT We studied the prevalence and species distribution of nontuberculous mycobacteria (NTM) in relation to age in 385 patients with cystic fibrosis (CF) (mean age ± standard deviation [range], 12.0 ± 6.1 [1 to 24] years; sex ratio, 0.53) attending three Parisian centers. The overall prevalence of NTM in sputum was 8.1% (31 out of 385). The following NTM were isolated (n = 33): Mycobacterium abscessus (n = 13, 39.4%), Mycobacterium avium complex (MAC) (n = 7, 21.2%), Mycobacterium gordonae (n = 6, 18.2%), and other (n = 7, 21.2%). Sixteen patients met the American Thoracic Society microbiological criteria for NTM infection, including 11 patients positive for M. abscessus, 4 for MAC, and 1 for MAC and Mycobacterium kansasii. The overall prevalence of NTM was significantly lower in patients under 15 years old than for patients equal to or more than 15 years old (4.8 versus 14.9%, respectively; P = 0.001). M. abscessus was isolated at all ages, while MAC was not recovered before 15 years (prevalence of 0.0 and 5.2% in patients aged 1 to 14 and 15 to 24, respectively; P = 0.001).

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.