Gilles Zambardi

Comparative Evaluation of a Prototype Chromogenic Medium (ChromID CARBA) for Detecting Carbapenemase-Producing Enterobacteriaceae in Surveillance Rectal Swabs

ABSTRACT Carbapenemase-producing Enterobacteriaceae (CPE) are an increasing problem worldwide, and rectal swab surveillance is recommended as a component of infection control programs. The performance of a prototype chromogenic medium (chromID CARBA) was evaluated and compared with media tested by four other screening methods: (i) overnight selective enrichment in 5 ml tryptic soy broth with a 10-μg ertapenem disk followed by plating onto MacConkey agar (CDC-TS), (ii) short selective enrichment in 9 ml brain heart infusion broth with a 10-μg ertapenem disk followed by plating onto chromID ESBL medium (ESBL-BH), (iii) direct plating onto chromID ESBL, and (iv) direct plating onto MacConkey agar supplemented with meropenem (1 μg/ml) (MCM). The screening methods were applied to detect CPE in 200 rectal swab specimens taken from different hospitalized patients. Identification and antimicrobial susceptibility were performed by the Vitek 2 system. Carbapenem MICs were checked by Etest. Carbapenemase production was confirmed using the modified Hodge test, combined-disk tests, and PCR assays. In total, 133 presumptive CPE strains were detected. Phenotypic and genotypic assays confirmed 92 strains to be CPE (56 KPC-positive Klebsiella pneumoniae, 29 VIM-positive K. pneumoniae, and 7 KPC-positive Enterobacter aerogenes strains) recovered from 73 patients, while the remaining 41 strains were confirmed to be CPE negative (19 ESBL producers and 22 nonfermenters). chromID CARBA, ESBL-BH, and chromID ESBL exhibited the highest sensitivity (92.4%), followed by CDC-TS and MCM (89.1%) (P = 0.631). The specificity was greater for chromID CARBA (96.9%) and ESBL-BH (93.2%) than for CDC-TS (86.4%), MCM (85.2%), and chromID ESBL (84.7%) (P = 0.014). In conclusion, chromID CARBA was found to be a rapid and accurate culture screening method for active CPE surveillance.

Clinical impact of rapid oxacillin susceptibility testing using a PCR assay in Staphylococcus aureus bactaeremia

OBJECTIVES The aim of this work was to establish the clinical impact of rapid oxacillin susceptibility testing in nosocomial Staphylococcus aureus bacteraemia. METHODS This study was performed in 145 critically ill patients infected by S. aureus. Patients were randomly assigned to one of two groups: patients for whom susceptibility testing was performed using a rapid same day multiplex PCR assay for detection of the staphylococcal mecA (mean delay of response: 6 h) and those for whom testing was accomplished using traditional overnight techniques (21 h). RESULTS The results of this study showed no significant difference between the two groups in terms of age, Simplified Acute Physiologic Score, severity of infection, severity of underlying disease and clinical outcome (control vs. PCR): unfavourable outcome of infection, 12.32 vs. 12.5%; 95% CI for the difference = -11.49 to 11.09 (P = 0.975); unfavourable general outcome, 16.43 vs. 20.83%; 95% CI for the difference = -17.35 to 8.50 (P = 0.497). For the oxacillin-susceptible S. aureus bactaeraemia, results were: unfavourable outcome of infection = 13.04 vs. 11.11%; 95% CI for the difference = -11.38 to 16.18 (P = 0.767); unfavourable general outcome = 13.04 vs. 20.37%; 95% CI for the difference = -22.12 to 8.07 (P = 0.331). CONCLUSION This study seemed to demonstrate that rapid oxacillin susceptibility testing using a PCR assay did not have a major impact on the care and outcome of patients with S. aureus bactaeremia.

Phylogenetic Distribution of CRISPR-Cas Systems in Antibiotic-Resistant Pseudomonas aeruginosa

ABSTRACT Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates. IMPORTANCE P. aeruginosa is both an antibiotic-refractory pathogen and an important model system for type I CRISPR-Cas bacterial immune systems. By combining the genome sequences of 672 newly and previously sequenced genomes, we were able to provide a global view of the phylogenetic distribution, conservation, and potential targets of these systems. This analysis identified a new and putatively mobile P. aeruginosa CRISPR-Cas subtype, characterized the diverse distribution of known CRISPR-inhibiting genes, and provided a potential new use for CRISPR spacer libraries in accessory genome analysis. Our data demonstrated the importance of CRISPR-Cas systems in modulating the accessory genomes of globally distributed strains while also providing substantial data for subsequent genomic and experimental studies in multiple fields. Understanding why certain genotypes of P. aeruginosa are clinically prevalent and adept at horizontally acquiring virulence and antibiotic resistance elements is of major clinical and economic importance. P. aeruginosa is both an antibiotic-refractory pathogen and an important model system for type I CRISPR-Cas bacterial immune systems. By combining the genome sequences of 672 newly and previously sequenced genomes, we were able to provide a global view of the phylogenetic distribution, conservation, and potential targets of these systems. This analysis identified a new and putatively mobile P. aeruginosa CRISPR-Cas subtype, characterized the diverse distribution of known CRISPR-inhibiting genes, and provided a potential new use for CRISPR spacer libraries in accessory genome analysis. Our data demonstrated the importance of CRISPR-Cas systems in modulating the accessory genomes of globally distributed strains while also providing substantial data for subsequent genomic and experimental studies in multiple fields. Understanding why certain genotypes of P. aeruginosa are clinically prevalent and adept at horizontally acquiring virulence and antibiotic resistance elements is of major clinical and economic importance.

Comparative evaluation of a novel chromogenic medium (chromID OXA-48) for detection of OXA-48 producing Enterobacteriaceae.

Comparative evaluation of the recently developed chromogenic culture medium chromID OXA-48 (bioMérieux) with chromID CARBA (bioMérieux) and SUPERCARBA showed that chromID OXA-48 and SUPERCARBA media have the highest sensitivity for detection of OXA-48 producing Enterobacteriaceae (91% and 93%) comparatively to chromID CARBA (21 %). The chromID OXA-48 has the highest specificity, with 100%, as compared to 53% and 68% for the SUPERCARBA and chromID CARBA media for detecting those OXA-48 producers.

Laboratory diagnosis of oxacillin resistance in Staphylococcus aureus by a multiplex-polymerase chain reaction assay

A polymerase chain reaction (PCR) test was developed in which the mecA gene responsible for the intrinsic resistance to oxacillin in Staphylococcus aureus and the gyrA gene, always present in this species, were amplified in one operation. Among the 468 clinical isolates tested, the results obtained for 454 of the isolates (97%) were consistent with those of MIC determination. Discrepant results were noted for strains with low-level oxacillin resistance (MICs, 4-8 micrograms/ml) and mecA gene negative. For these strains, susceptibility to oxacillin was restored in the presence of a beta-lactamase inhibitor, which suggested a resistance by penicillinase hyperproduction. In contrast, all of the high-level resistant strains (MICs, > 8 micrograms/ml) carried the mecA gene. The presence of this gene has frequently been associated with resistance to gentamicin, tetracycline, erythromycin, lincomycin, and pefloxacin. The PCR assay described in this study can be accomplished with ease and total confidence in the clinical microbiologic laboratory for a rapid and effective establishment of antistaphylococcal chemotherapy.

Rapid Bacterial Identification, Resistance, Virulence and Type Profiling using Selected Reaction Monitoring Mass Spectrometry

Mass spectrometry (MS) in Selected Reaction Monitoring (SRM) mode is proposed for in-depth characterisation of microorganisms in a multiplexed analysis. Within 60–80 minutes, the SRM method performs microbial identification (I), antibiotic-resistance detection (R), virulence assessment (V) and it provides epidemiological typing information (T). This SRM application is illustrated by the analysis of the human pathogen Staphylococcus aureus, demonstrating its promise for rapid characterisation of bacteria from positive blood cultures of sepsis patients.

Evaluation of Etest® strips for detection of KPC and metallo-carbapenemases in Enterobacteriaceae

The performance of Etest KPC and MBL strips (bioMérieux) was evaluated as compared to other phenotypic tests for detecting carbapenemases of the KPC-type and metallo-β-lactamases, respectively, on 133 well-characterized enterobacterial isolates. KPC and meropenem-containing MP/MPI Etest had high sensitivity (>92 %) and specificity (>97 %).

European multicentre evaluation of a commercial system for identification of methicillin-resistant staphylococcus aureus

A commercial system for the rapid detection of methicillin-resistantStaphylococcus aureus, the BBL Crystal MRSA test (C-MRSA ID; Becton Dickinson, USA), was evaluated prospectively and compared with a polymerase chain reaction test for the presence of themecA gene. Ten European centres tested a total of 676 isolates ofStaphylococcus aureus from blood cultures. The system correctly identified 661 (97.8%) isolates within 4 h. All but threemecA gene-negative isolates (99.4% specificity) yielded a negative C-MRSA ID reaction, and 158 of 170mecA gene-positive isolates were accurately detected (92.9% sensitivity). After repeated testing of discrepant results, sensitivity and specificity increased to 99% and 100%, respectively.

MCR: modern colistin resistance

Recently, plasmid-mediated and, therefore, transferable bacterial polymyxin resistance was discovered in strains from both humans and animals. Such a trait may widely spread geographically, while simultaneously crossing microbial species barriers. This may ultimately render the “last resort” polymyxin antibiotics therapeutically useless. Colistin is currently used to treat infections caused by Gram-negative carbapenemase producers and colistin resistance may lead to practical pan-antibiotic resistance. We here analyzed the medical and diagnostic consequences of (emerging) colistin resistance and propose pathways toward adequate diagnostics for timely detection of both asymptomatic carriage and infection. Culture-based testing using chromogenic and selective media for screening clinical (and veterinary) specimens may constitute key tools for that purpose. Relevant molecular tests are also discussed.

Correlation between phenotypic antibiotic susceptibility and the resistome in Pseudomonas aeruginosa

Genetic determinants of antibiotic resistance (AR) have been extensively investigated. High-throughput sequencing allows for the assessment of the relationship between genotype and phenotype. A panel of 672 Pseudomonas aeruginosa strains was analysed, including representatives of globally disseminated multidrug-resistant and extensively drug-resistant clones; genomes and multiple antibiograms were available. This panel was annotated for AR gene presence and polymorphism, defining a resistome in which integrons were included. Integrons were present in >70 distinct cassettes, with In5 being the most prevalent. Some cassettes closely associated with clonal complexes, whereas others spread across the phylogenetic diversity, highlighting the importance of horizontal transfer. A resistome-wide association study (RWAS) was performed for clinically relevant antibiotics by correlating the variability in minimum inhibitory concentration (MIC) values with resistome data. Resistome annotation identified 147 loci associated with AR. These loci consisted mainly of acquired genomic elements and intrinsic genes. The RWAS allowed for correct identification of resistance mechanisms for meropenem, amikacin, levofloxacin and cefepime, and added 46 novel mutations. Among these, 29 were variants of the oprD gene associated with variation in meropenem MIC. Using genomic and MIC data, phenotypic AR was successfully correlated with molecular determinants at the whole-genome sequence level.