Diagnostic test accuracy of an automated device for the MALDI target preparation for microbial identification.

The objective of this study was to evaluate the performance of the Copan Colibrí™ against the manual preparation of the MALDI targets. We analyzed 416 (31 different species) non-duplicate strains covering the most important species identified in clinical routine. We also assessed the intra-strain repeatability between the comparable methods. We then analyzed the performance of this new method after implementation in routine on 12,253 aerobic bacterial isolates and yeasts, encompassing a total of 42 different species. Among the 416 strains analyzed, 6.3% (26/416) and 10.8% (45/416) had a score value < 2 when processed by the Colibri™ and manual method, respectively. Only 5.9% (9/152) of the Gram positive rods and cocci had a score values < 2 by the Colibri™ versus 20.4% (31/152) by the manual method. We confirmed that this relative superiority observed for the Colibri™ was due primarily in the use of the formic acid protocol. For the Gram-negative bacteria, the results of both methods were comparable; 6.6% (17/256) and 4.7% (12/256) had a score value < 2 by the Colibri™ and the manual method, respectively. After implementation in routine, the results according to the Biotyper score cut-off values were distributed as follows: < 1.70: 2.5% (304/12,253), 1.70-1.79: 1.9% (227/12,253), 1.80-1.89: 3.1% (377/12,253), 1.90-1.99: 6.7% (825/12,253), and ≥ 2: 85.9% (10,520/12,253). The Colibrí™ coupled to MALDI-TOF/MS revealed good performances and higher intra-strain repeatability as compared to the manual preparation of the MALDI targets.

Fully Automated EUCAST Rapid Antimicrobial Susceptibility Testing (RAST) from Positive Blood Cultures: Diagnostic Accuracy and Implementation.

The objective of this study was to evaluate the accuracy and robustness of a fully automated EUCAST RAST (rapid antimicrobial susceptibility test) directly from positive blood culture and to appreciate its implementation constraints. This study was conducted in two phases: (i) spiked blood culture bottles (BCs) using 779 non-duplicate clinical isolates and (ii) a prospective clinical trial including 534 positive BCs sequentially processed in routine at the Bacteriology Laboratory of Geneva University Hospitals. The RAST results were assessed against EUCAST standardized disk diffusion testing results. Our first finding was that the results of the spiked BCs precisely predicted the clinical trial results. The overall categorical agreements for all species analyzed were greater than 95% at the different time points. RAST for Pseudomonas aeruginosa, however, raised several challenges. The categorical agreement for imipenem was lower than 95% at 6 h and was not improved with longer incubation times. Additionally, piperacillin-tazobactam, ceftazidime, and cefepime cannot be released at 6 h due to suboptimal performances, but the categorical agreement substantially improved at 8 h. Our results establish that the performance of fully automated EUCAST RAST directly from positive blood culture bottles is consistently robust, even for the detection of extended-spectrum ß-lactamase (ESBL), carbapenemase-producing bacteria, and methicillin-resistant Staphylococcus aureus (MRSA). The automation markedly enhanced the percentage of readable inhibition zones and reduced the percentage of isolates categorized in the area of technical uncertainty (ATU). In summary, a fully automated EUCAST RAST can substantially improve laboratory workflow by reducing hands-on time and removing the strong constraints linked to manual read-outs at precisely defined times.

Performances of automated digital imaging of Gram-stained slides with on-screen reading against manual microscopy.

The objective of this study was to evaluate the performances of the automated digital imaging of Gram-stained slides against manual microscopy. Four hundred forty-three identified Gram-stained slides were included in this study. When both methods agreed, we considered the results as correct, and no further examination was carried out. Whenever the methods gave discrepant results, we reviewed the digital images and the glass slides by manual microscopy to avoid incorrectly read smears. The final result was a consensus of multiple independent reader interpretations. Among the 443 slides analyzed in this study, 101 (22.8%) showed discrepant results between the compared methods. The rates of discrepant results according to the specimen types were 5.7% (9/157) for positive blood cultures, 42% (60/142) for respiratory tract specimens, and 22% (32/144) for sterile site specimens. After a subsequent review of the discrepant slides, the final rate of discrepancies dropped to 7.0% (31/443). The overall agreement between the compared methods and the culture results reached 78% (345/443) and 79% (349/443) for manual microscopy and automated digital imaging, respectively. According to culture results, the specificity for automated digital imaging and manual microscopy were 90.8% and 87.7% respectively. In contrast, sensitivity was 84.1% for the two compared methods. The discrepant results were mostly encountered with microorganism morphologies of rare occurrence. The results reported in this study emphasize that on-screen reading is challenging, since the recognition of morphologies on-screen can appear different as compared to routine manual microscopy. Monitoring of Gram stain errors, which is facilitated by automated digital imaging, remains crucial for the quality control of reported Gram stain results.

The TIR Homologue Lies near Resistance Genes in Staphylococcus aureus, Coupling Modulation of Virulence and Antimicrobial Susceptibility.

Toll/interleukin-1 receptor (TIR) domains in Toll-like receptors are essential for initiating and propagating the eukaryotic innate immune signaling cascade. Here, we investigate TirS, a Staphylococcus aureus TIR mimic that is part of a novel bacterial invasion mechanism. Its ectopic expression in eukaryotic cells inhibited TLR signaling, downregulating the NF-kB pathway through inhibition of TLR2, TLR4, TLR5, and TLR9. Skin lesions induced by the S. aureus knockout tirS mutant increased in a mouse model compared with wild-type and restored strains even though the tirS-mutant and wild-type strains did not differ in bacterial load. TirS also was associated with lower neutrophil and macrophage activity, confirming a central role in virulence attenuation through local inflammatory responses. TirS invariably localizes within the staphylococcal chromosomal cassettes (SCC) containing the fusC gene for fusidic acid resistance but not always carrying the mecA gene. Of note, sub-inhibitory concentration of fusidic acid increased tirS expression. Epidemiological studies identified no link between this effector and clinical presentation but showed a selective advantage with a SCCmec element with SCC fusC/tirS. Thus, two key traits determining the success and spread of bacterial infections are linked.

Correction: The TIR Homologue Lies near Resistance Genes in Staphylococcus aureus, Coupling Modulation of Virulence and Antimicrobial Susceptibility.

[This corrects the article DOI: 10.1371/journal.ppat.1006092.].

A comparison of Sensititre™ Anaerobe MIC plate with ATB ANA® test for the routine susceptibility testing of common anaerobe pathogens.

The accuracy of the Thermo Scientific™ Sensititre™ Anaerobe MIC plate was assessed against the ATB ANA® test (bioMérieux) on 56 clinically relevant anaerobic strains collected at Geneva University Hospitals. The overall categorical agreement between both methods reached 95%. The Sensititre™ Anaerobe MIC plate had excellent accuracy for most antibiotics tested. When the Sensititre™ Anaerobe MIC plate disagreed with ATB ANA® test, the gradient strip method resolved the antimicrobial susceptibility categories of all the antibiotics tested, except for piperacillin, piperacillin-tazobactam, and penicillin, in favor of the Sensititre™ Anaerobe MIC plate (58% [21 out of 36]). Several very major errors were observed for piperacillin (12.5% [7 out of 56]), piperacillin-tazobactam (12.5% [7 out of 56]), and penicillin (2% [1 out of 56]). The gradient strip method revealed that the categorical differences for piperacillin, piperacillin-tazobactam, and penicillin were at least partly explained by heterogeneity in resistance expression. The Sensititre™ Anaerobe MIC plate offers therefore a useful alternative to the ATB ANA® test for the routine antimicrobial susceptibility testing of anaerobes in clinical microbiology laboratories.

Molecular characterization of fluoroquinolones, macrolides, and imipenem resistance in Haemophilus influenzae: analysis of the mutations in QRDRs and assessment of the extent of the AcrAB-TolC-mediated resistance.

The aims of the present study were to characterize the mechanisms of resistance to fluoroquinolones, macrolides, and imipenem in Haemophilus influenzae, to assess the extent of the AcrAB-TolC-mediated resistance, and to define a core genome multilocus sequence typing (cgMLST) scheme for H. influenzae by using whole-genome sequencing. Four amino acid substitutions in GyrA (at Ser84 and Asp88), ParC (at Ser84), and ParE (at Asp420) were found to be closely associated to the MICs. We did not find any amino acid substitution surrounding the three highly conserved amino acid motifs in PBP3 related to imipenem resistance. All the isolates possessed the ermB gene. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreased the MIC of imipenem by twofold for FQR-6 and fourfold for GE47 and GE88 strains. For erythromycin, the MICs were decreased by twofold. We found that the six FQR isolates were clustered in two groups. The number of different loci within FQR-1_FQR-3_FQR-5 cluster was 6, while FQR-2 and FQR-4 differed for 21 loci. FQR-1_FQR-3_FQR-5 and FQR-2_FQR-4 clusters were distant among each other and compared to 19 genomes downloaded from NCBI, to 8 strains heteroresistant to imipenem, and to 4 strains monoresistant to ciprofloxacin isolated in Denmark. We confirmed that specific amino acid substitutions in GyrA, ParC, and ParE are implicated in quinolone resistance. Additionally, the degree of resistance is related to the number of these amino acid substitutions. We provide robust evidence that drug efflux is one of the substantial mechanisms of imipenem and erythromycin resistance in H. influenzae.

Staphylococcus epidermidis SrrAB regulates bacterial growth and biofilm formation differently under oxic and microaerobic conditions.

SrrAB expression in Staphylococcus epidermidis strain 1457 (SE1457) was upregulated during a shift from oxic to microaerobic conditions. An srrA deletion (ΔsrrA) mutant was constructed for studying the regulatory function of SrrAB. The deletion resulted in retarded growth and abolished biofilm formation both in vitro and in vivo and under both oxic and microaerobic conditions. Associated with the reduced biofilm formation, the ΔsrrA mutant produced much less polysaccharide intercellular adhesion (PIA) and showed decreased initial adherence capacity. Microarray analysis showed that the srrA mutation affected transcription of 230 genes under microaerobic conditions, and 51 genes under oxic conditions. Quantitative real-time PCR confirmed this observation and showed downregulation of genes involved in maintaining the electron transport chain by supporting cytochrome and quinol-oxidase assembly (e.g., qoxB and ctaA) and in anaerobic metabolism (e.g., pflBA and nrdD). In the ΔsrrA mutant, the expression of the biofilm formation-related gene icaR was upregulated under oxic conditions and downregulated under microaerobic conditions, whereas icaA was downregulated under both conditions. An electrophoretic mobility shift assay further revealed that phosphorylated SrrA bound to the promoter regions of icaR, icaA, qoxB, and pflBA, as well as its own promoter region. These findings demonstrate that in S. epidermidis SrrAB is an autoregulator and regulates biofilm formation in an ica-dependent manner. Under oxic conditions, SrrAB modulates electron transport chain activity by positively regulating qoxBACD transcription. Under microaerobic conditions, it regulates fermentation processes and DNA synthesis by modulating the expression of both the pfl operon and nrdDG.

Modelling antibiotic and cytotoxic isoquinoline effects in Staphylococcus aureus, Staphylococcus epidermidis and mammalian cells.

Isoquinolines (IQs) are natural substances with an antibiotic potential we aim to optimize. Specifically, IQ-238 is a synthetic analog of the novel-type N,C-coupled naphthylisoquinoline (NIQ) alkaloid ancisheynine. Recently, we developed and tested other IQs such as IQ-143. By utilizing genome-wide gene expression data, metabolic network modelling and Voronoi tessalation based data analysis - as well as cytotoxicity measurements, chemical properties calculations and principal component analysis of the NIQs - we show that IQ-238 has strong antibiotic potential for staphylococci and low cytotoxicity against murine or human cells. Compared to IQ-143, systemic effects are less pronounced. Most enzyme activity changes due to IQ-238 are located in the carbohydrate metabolism. Validation includes metabolite measurements on biological replicates. IQ-238 delineates key properties and a chemical space for a good therapeutic window. The combination of analysis methods allows suggestions for further lead development and yields an in-depth look at staphylococcal adaptation and network changes after antibiosis. Results are compared to eukaryotic host cells.

Draft Genome Sequence of a Mixed-Serogroup W/Y Invasive Neisseria meningitidis Strain.

We report the genome of a Neisseria meningitidis strain (GE-156) that was isolated in Switzerland from a patient diagnosed with bacteremia. The strain belongs to a rare mixed serogroup W/Y and sequence type 11847 (clonal complex 167), as revealed by both routine laboratory examination and genomic sequencing.

Invasive Mold Infections in Allogeneic Hematopoietic Cell Transplant Recipients in 2020: Have We Made Enough Progress?

BACKGROUND: Despite progress in diagnostic, prevention, and treatment strategies, invasive mold infections (IMIs) remain the leading cause of mortality in allogeneic hematopoietic cell transplant (allo-HCT) recipients. METHODS: We describe the incidence, risk factors, and mortality of allo-HCT recipients with proven/probable IMI in a retrospective single-center 10-year (01/01/2010-01/01/2020) cohort study. RESULTS: Among 515 allo-HCT recipients, 48 (9.3%) patients developed 51 proven/probable IMI: invasive aspergillosis (IA; 34/51, 67%), mucormycosis (9/51, 18%), and other molds (8/51, 15%). Overall, 35/51 (68.6%) breakthrough IMIs (bIMIs) were identified: 22/35 (62.8%) IA and 13/35 (37.1%) non-IA IMI. One-year IMI cumulative incidence was 7%: 4.9% and 2.1% for IA and non-IA IMI, respectively. Fourteen (29.2 %), 10 (20.8%), and 24 (50.0%) patients were diagnosed during the first 30, 31-180, and >180 days post-HCT, respectively. Risk factors for IMI included prior allo-HCT (sub hazard ratio [SHR], 4.06; P = .004) and grade ≥2 acute graft-vs-host disease (aGvHD; SHR, 3.52; P < .001). All-cause 1-year mortality was 33% (170/515): 48% (23/48) and 31.5% (147/467) for patients with and without IMI (P = .02). Mortality predictors included disease relapse (hazard ratio [HR], 7.47; P < .001), aGvHD (HR, 1.51; P = .001), CMV serology-positive recipients (HR, 1.47; P = .03), and IMI (HR, 3.94; P < .001). All-cause 12-week mortality for patients with IMI was 35.4% (17/48): 31.3% (10/32) for IA and 43.8% (7/16) for non-IA IMI (log-rank P = .47). At 1 year post-IMI diagnosis, 70.8% (34/48) of the patients were dead. CONCLUSIONS: IA mortality has remained relatively unchanged during the last 2 decades. More than two-thirds of allo-HCT recipients with IMI die by 1 year post-IMI diagnosis. Dedicated intensified research efforts are required to further improve clinical outcomes.

Daptomycin resistance mechanisms in clinically derived Staphylococcus aureus strains assessed by a combined transcriptomics and proteomics approach.

OBJECTIVES: The development of daptomycin resistance in Staphylococcus aureus is associated with clinical treatment failures. The mechanism(s) of such resistance have not been clearly defined. METHODS: We studied an isogenic daptomycin-susceptible (DAP(S)) and daptomycin-resistant (DAP(R)) S. aureus strain pair (616; 701) from a patient with relapsing endocarditis during daptomycin treatment, using comparative transcriptomic and proteomic techniques. RESULTS: Minor differences in the genome content were found between strains by DNA hybridization. Transcriptomic analyses identified a number of genes differentially expressed in important functional categories: cell division; metabolism of bacterial envelopes; and global regulation. Of note, the DAP(R) isolate exhibited reduced expression of the major cell wall autolysis gene coincident with the up-regulation of genes involved in cell wall teichoic acid production. Using quantitative (q)RT-PCR on the gene cadre putatively involved in cationic peptide resistance, we formulated a putative regulatory network compatible with microarray data sets, mainly implicating bacterial envelopes. Of interest, qRT-PCR of this same gene cadre from two distinct isogenic DAP(S)/DAP(R) clinical strain pairs revealed evidence of other strain-dependent networks operative in the DAP(R) phenotype. Comparative proteomics of 616 versus 701 revealed a differential abundance of proteins in various functional categories, including cell wall-associated targets and biofilm formation proteins. Phenotypically, strains 616 and 701 showed major differences in their ability to develop bacterial biofilms in the presence of the antibacterial lipid, oleic acid. CONCLUSIONS: Compatible with previous in vitro observations, in vivo-acquired DAP(R) in S. aureus is a complex, multistep phenomenon involving: (i) strain-dependent phenotypes; (ii) transcriptome adaptation; and (iii) modification of the lipid and protein contents of cellular envelopes.

Role of the SaeRS two-component regulatory system in Staphylococcus epidermidis autolysis and biofilm formation.

BACKGROUND: Staphylococcus epidermidis (SE) has emerged as one of the most important causes of nosocomial infections. The SaeRS two-component signal transduction system (TCS) influences virulence and biofilm formation in Staphylococcus aureus. The deletion of saeR in S. epidermidis results in impaired anaerobic growth and decreased nitrate utilization. However, the regulatory function of SaeRS on biofilm formation and autolysis in S. epidermidis remains unclear. RESULTS: The saeRS genes of SE1457 were deleted by homologous recombination. The saeRS deletion mutant, SE1457ΔsaeRS, exhibited increased biofilm formation that was disturbed more severely (a 4-fold reduction) by DNase I treatment compared to SE1457 and the complementation strain SE1457saec. Compared to SE1457 and SE1457saec, SE1457ΔsaeRS showed increased Triton X-100-induced autolysis (approximately 3-fold) and decreased cell viability in planktonic/biofilm states; further, SE1457ΔsaeRS also released more extracellular DNA (eDNA) in the biofilms. Correlated with the increased autolysis phenotype, the transcription of autolysis-related genes, such as atlE and aae, was increased in SE1457ΔsaeRS. Whereas the expression of accumulation-associated protein was up-regulated by 1.8-fold in 1457ΔsaeRS, the expression of an N-acetylglucosaminyl transferase enzyme (encoded by icaA) critical for polysaccharide intercellular adhesin (PIA) synthesis was not affected by the deletion of saeRS. CONCLUSIONS: Deletion of saeRS in S. epidermidis resulted in an increase in biofilm-forming ability, which was associated with increased eDNA release and up-regulated Aap expression. The increased eDNA release from SE1457ΔsaeRS was associated with increased bacterial autolysis and decreased bacterial cell viability in the planktonic/biofilm states.

Impact of oleic acid (cis-9-octadecenoic acid) on bacterial viability and biofilm production in Staphylococcus aureus.

Staphylococcus aureus is responsible for a broad variety of chronic infections. Most S. aureus clinical isolates show the capacity to adhere to abiotic surfaces and to develop biofilms. Because S. aureus growing in a biofilm is highly refractory to treatment, inhibition of biofilm formation represents a major therapeutic objective. We evaluated the effects of oleic acid on primary adhesion and biofilm production in eight genotypically different S. aureus strains as well as in the biofilm-negative Staphylococcus carnosus strain TM300. Oleic acid inhibited primary adhesion but increased biofilm production in every S. aureus strain tested. Staphylococcus aureus strain UAMS-1 was then selected as a model organism for studying the mechanisms triggered by oleic acid on the formation of a biofilm in vitro. Oleic acid inhibited the primary adhesion of UAMS-1 dose dependently with an IC(50) around 0.016%. The adherent bacterial population decreased proportionally with increasing concentrations of oleic acid whereas an opposite effect was observed on the planktonic population. Overall, the total bacterial counts remained stable. Macroscopic detachments and clumps were visible from the adherent bacterial population. In the presence of oleic acid, the expression of sigB, a gene potentially involved in bacterial survival through an effect on fatty acid composition, was not induced. Our results suggest a natural protective effect of oleic acid against primary adhesion.

Transcriptional Modulation of Penicillin-Binding Protein 1b, Outer Membrane Protein P2 and Efflux Pump (AcrAB-TolC) during Heat Stress Is Correlated to Enhanced Bactericidal Action of Imipenem on Non-typeable Haemophilus influenzae.

Objective: The purpose of the present study was to investigate the penicillin binding proteins (PBPs), drug influx and efflux modulations during heat stress and their effects on the bactericidal action of imipenem on non-typeable Haemophilus influenzae (NTHi). Methods: The two NTHi clinical isolates (GE47 and GE88, imipenem MICs by E-test > 32 µg/mL) examined in this study were collected at Geneva University Hospitals. The imipenem killing activity was assessed after incubation of the NTHi strains at either 37 or 42°C for 3 h with increasing concentrations of imipenem. The detection of PBPs was carried out by Bocillin-FL. Global transcriptional changes were monitored by RNA-seq after pre-incubation of bacterial cells at either 37 or 42°C, and the expression levels of relevant target genes were confirmed by qRT-PCR. Results: Quantitation of NTHi viable cells after incubation with 0.25 µg/mL of imipenem for 3 h revealed more than a twofold decrease in GE47 and GE88 viable cells at 42°C as compared to 37°C. Transcriptome analysis showed that under heat stress conditions, there were 141 differentially expressed genes with a | log2(fold change)| > 1, including 67 up-regulated and 74 down-regulated genes. The expression levels of ponB (encoding PBP1b) and acrR (regulator of AcrAB-TolC efflux pump) were significantly increased at 42°C. In contrast, the transcript levels of ompP2 (encoding the outer membrane protein P2) and acrB gene (encoding AcrB) were significantly lower under heat stress condition. Conclusion: This study shows that the transcriptional modulation of ponB, ompP2, acrR, and acrB in the heat stress response is correlated to enhanced antimicrobial effects of imipenem on non-typeable H. influenzae.

Exploring glycopeptide-resistance in Staphylococcus aureus: a combined proteomics and transcriptomics approach for the identification of resistance-related markers.

BACKGROUND: To unravel molecular targets involved in glycopeptide resistance, three isogenic strains of Staphylococcus aureus with different susceptibility levels to vancomycin or teicoplanin were subjected to whole-genome microarray-based transcription and quantitative proteomic profiling. Quantitative proteomics performed on membrane extracts showed exquisite inter-experimental reproducibility permitting the identification and relative quantification of >30% of the predicted S. aureus proteome. RESULTS: In the absence of antibiotic selection pressure, comparison of stable resistant and susceptible strains revealed 94 differentially expressed genes and 178 proteins. As expected, only partial correlation was obtained between transcriptomic and proteomic results during stationary-phase. Application of massively parallel methods identified one third of the complete proteome, a majority of which was only predicted based on genome sequencing, but never identified to date. Several over-expressed genes represent previously reported targets, while series of genes and proteins possibly involved in the glycopeptide resistance mechanism were discovered here, including regulators, global regulator attenuator, hyper-mutability factor or hypothetical proteins. Gene expression of these markers was confirmed in a collection of genetically unrelated strains showing altered susceptibility to glycopeptides. CONCLUSION: Our proteome and transcriptome analyses have been performed during stationary-phase of growth on isogenic strains showing susceptibility or intermediate level of resistance against glycopeptides. Altered susceptibility had emerged spontaneously after infection with a sensitive parental strain, thus not selected in vitro. This combined analysis allows the identification of hundreds of proteins considered, so far as hypothetical protein. In addition, this study provides not only a global picture of transcription and expression adaptations during a complex antibiotic resistance mechanism but also unravels potential drug targets or markers that are constitutively expressed by resistant strains regardless of their genetic background, amenable to be used as diagnostic targets.

Screening for staphylococcal superantigen genes shows no correlation with the presence or the severity of chronic rhinosinusitis and nasal polyposis.

BACKGROUND: Staphylococcus aureus secretes numerous exotoxins which may exhibit superantigenic properties. Whereas the virulence of several of them is well documented, their exact biological effects are not fully understood. Exotoxins may influence the immune and inflammatory state of various organs, including the sinonasal mucosa: their possible involvement in chronic rhinosinusitis has been suggested and is one of the main trends in current research. The aim of this study was to investigate whether the presence of any of the 22 currently known staphylococcal exotoxin genes could be correlated with chronic rhinosinusitis. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a prospective, multi-centred European study, analysing 93 Staphylococcus aureus positive swabs taken from the middle meatus of patients suffering from chronic rhinosinusitis, with or without nasal polyposis, and controls. Strains were systematically tested for the presence of the 22 currently known exotoxin genes and genotyped according to their agr groups. No direct correlation was observed between chronic rhinosinusitis, with or without nasal polyposis, and either agr groups or the presence of the most studied exotoxins genes (egc, sea, seb, pvl, exfoliatins or tsst-1). However, genes for enterotoxins P and Q were frequently observed in nasal polyposis for the first time, but absent in the control group. The number of exotoxin genes detected was not statistically different among the 3 patient groups. CONCLUSIONS/SIGNIFICANCE: Unlike many previous studies have been suggesting, we did not find any evident correlation between staphylococcal exotoxin genes and the presence or severity of chronic rhinosinusitis with or without nasal polyposis.

Development and evaluation of a rapid strategy to determine enterotoxin gene content in Staphylococcus aureus.

Enterotoxins of S. aureus are important molecules displaying superantigenic properties. To date no less than 18 enterotoxins have been identified in S. aureus and their role has been documented in very diverse diseases. Using available nucleotide sequence information, we developed a rapid and automated PCR-based approach to evaluate enterotoxin content in S aureus. We studied a collection of S. aureus strains previously analyzed for enterotoxins gene content and report a perfect correlation between simplex and multiplex PCR assays for the presence of all enterotoxin genes described so far. The determination of enterotoxin content relies on 4 multiplex PCR tubes whose amplification products are resolved by a rapid microcapillary electrophoresis. Automated analysis of the PCR profiles evaluates for the presence of the 18 enterotoxin genes in less than 3 h and at moderate cost. Finally, the use of enterotoxin gene content for genotyping purpose was compared to multi-locus variable number of tandem repeat assay and spa genotyping. Analysis revealed an important homogeneity of the genetic backgrounds for strains harboring the egc cluster as well as a large diversity for strains harboring other enterotoxins but lacking the egc cluster. A combined genotyping method that includes rapid enterotoxin content determination appears informative for various epidemiological survey purposes.