Proficiency of PCR in hospital settings for nonculture diagnosis of invasive meningococcal infections.

BACKGROUND: Meningococcal meningitis requires rapid diagnosis and immediate management which is enhanced by the use of PCR for the ascertainment of these infections. However, its use is still restricted to reference laboratories. METHODS: We conducted an inter-laboratory study to assess the implementation and the performance of PCR in ten French hospital settings in 2010. RESULTS: Our data are in favour of this implementation. Although good performance was obtained in identifying Neisseria meningitidis positive samples, the main issue was reported in identifying other species (Streptococcus pneumoniae and Haemophilus influenzae) which are also involved in bacterial meningitis cases. CONCLUSIONS: Several recommendations are required and, mainly, PCR should target the major etiological agents (N. meningitidis, S. pneumonia, and H. influenzae) of acute bacterial meningitis. Moreover, PCR should predict the most frequent serogroups of Neisseria meningitidis according to local epidemiology.

Real-time PCR for detection of NDM-1 carbapenemase genes from spiked stool samples.

An in-house quantitative real-time PCR (qPCR) assay using TaqMan chemistry has been developed to detect NDM-1 carbapenemase genes from bacterial isolates and directly from stool samples. The qPCR amplification of bla(NDM-1) DNA was linear over 10 log dilutions (r(2) = 0.99), and the amplification efficiency was 1.03. The qPCR detection limit was reproducibly 1 CFU, or 10 plasmid molecules, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria harboring other ß-lactam resistance genes. Feces spiked with decreasing amounts of enterobacterial isolates producing NDM-1 were spread on ChromID ESBL and on CHROMagar KPC media and were subjected to the qPCR. The limits of carbapenem-resistant bacterial detection from stools was reproducibly 1 × 10(1) to 3 × 10(1) CFU/100 mg feces with ChromID ESBL medium. The CHROMagar KPC culture medium had higher limits of detection (1 × 10(1) to 4 × 10(3) CFU/ml), especially with bacterial isolates having low carbapenem MICs. The limits of detection with the qPCR assay were reproducibly below 1 × 10(1) CFU/100 mg of feces by qPCR assay. Samples spiked with NDM-1-negative bacteria were negative by qPCR. The sensitivity and specificity of the bla(NDM-1) qPCR assay on spiked samples were 100% in both cases. Using an automated DNA extraction system (QIAcube system), the qPCR assay was reproducible. The use of qPCR is likely to shorten the time for bla(NDM-1) detection from 48 h to 4 h and will be a valuable tool for outbreak follow-up in order to rapidly isolate colonized patients and assign them to cohorts.

OXA-163, an OXA-48-related class D ß-lactamase with extended activity toward expanded-spectrum cephalosporins.

Two bla(OXA-48)-like-positive isolates (Klebsiella pneumoniae and Enterobacter cloacae) were recovered in Argentina in 2008 as part of a large-scale survey focused on multidrug resistance in Enterobacteriaceae. In both cases, sequencing identified ß-lactamase OXA-163, differing from OXA-48 by a single amino substitution and a 4-amino-acid deletion. OXA-163 hydrolyzed penicillins, ceftazidime, and cefotaxime, whereas OXA-48 did not. However, OXA-163 had a much lower ability to hydrolyze carbapenems than OXA-48, therefore barely being considered a carbapenemase. In both isolates, the bla(OXA-163) gene was located on plasmids that differed in structure and size. However, a detailed genetic analysis revealed a similar genetic context in those isolates, with the bla(OXA-163) gene being bracketed by novel transposase genes, making this genetic environment different from that reported for the bla(OXA-48) gene. This study identified the first class D ß-lactamase compromising both extended-spectrum cephalosporin and carbapenem activities.

How to detect NDM-1 producers.

Enterobacterial isolates expressing the carbapenemase NDM-1 are emerging worldwide. Twenty-seven NDM-1-positive isolates of worldwide origin were included in this study to identify these strains as not only pathogens but also colonizers of normal flora for infection control screening. Although susceptibility to carbapenems varied, a combined test (IMP/IMP + EDTA), the Etest MBL, and automated susceptibility testing by Vitek2 (bioMérieux) identified those NDM-1 producers as verified by PCR using specific primers. Screening for carriers of NDM-1 producers may be based on media such as the ChromID ESBL culture medium routinely used to screen for extended-spectrum ß-lactamase producers, which gives excellent detection levels with low limits of detection ranging from 8 × 10(0) to 5 × 10(2) CFU/ml. The CHROMagar KPC culture medium had higher limits of detection (1 × 10(1) to 5 × 10(5) CFU/ml) and may be proposed for the follow-up of outbreaks of infections with NDM-1 producers. Colonies growing on these screening media can be verified as NDM-1 producers with molecular methods as described herein.

Use of ChromID extended-spectrum beta-lactamase medium for detecting carbapenemase-producing Enterobacteriaceae.

ChromID extended-spectrum beta-lactamase (ESBL) culture medium is routinely used for screening ESBL producers. This medium was tested for detecting carbapenemase-producing Enterobacteriaceae isolates from a collection of reference strains and compared to the CHROMagar KPC culture medium previously evaluated for detecting KPC-producing isolates. Producers of IMP-, VIM-, and KPC-type carbapenemases with high levels of resistance to cephalosporins and to carbapenems were detected at 1x10(1) CFU/ml. The OXA-48 producers were not detected on ChromID ESBL medium unless coexpressing ESBLs, whereas carbapenemase-producing isolates with MICs of <4 microg/ml were not detected on CHROMagar KPC medium.

Spread of OXA-48-encoding plasmid in Turkey and beyond.

Eighteen carbapenem-resistant, OXA-48-positive enterobacterial isolates recovered from Turkey, Lebanon, Egypt, France, and Belgium were analyzed. In most isolates, similar 70-kb plasmids carrying the carbapenemase gene bla(OXA-48) were identified. That gene was located within either transposon Tn1999 or transposon Tn1999.2, which was always inserted within the same gene. This work highlights the current plasmid-mediated dissemination of the OXA-48 carbapenemase worldwide.

Integron mobilization unit as a source of mobility of antibiotic resistance genes.

Antibiotic resistance genes are spread mostly through plasmids, integrons (as a form of gene cassettes), and transposons in gram-negative bacteria. We describe here a novel genetic structure, named the integron mobilization unit (IMU), that has characteristics similar to those of miniature inverted transposable elements (MITEs). Two IMUs (288 bp each) were identified from a carbapenem-resistant Enterobacter cloacae isolate that formed a composite structure encompassing a defective class 1 integron containing the carbapenem resistance gene bla(GES-5). This beta-lactamase gene was located on a 7-kb IncQ-type plasmid named pCHE-A, which was sequenced completely. The plasmid pCHE-A was not self conjugative but was mobilizable, and it was successfully transferred from E. cloacae to Pseudomonas aeruginosa. The in silico analysis of the extremities of the IMU elements identified similarities with those of insertion sequence ISSod9 from Shewanella oneidensis MR-1. The mobilization of the IMU composite structure was accomplished by using the transposase activity of ISSod9 that was provided in trans. This is the first identification of MITE-type structures as a source of gene mobilization, implicating here a clinically relevant antibiotic resistance gene.

Outbreak of CTX-M-15-producing Klebsiella pneumoniae in the intensive care unit of a French hospital.

The CTX-M-15 extended spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae isolates were identified in 36 patients hospitalized from December 2006 to September 2007 in the medical intensive care unit (ICU) of the Bicêtre hospital, South Paris, France. The incidence of colonization and/or infection was 4.8%. Eighty-nine percent of the ESBL-producing K. pneumoniae isolates were acquired in the ICU, and only 8.3% of the patients were infected. Pulsed field gel electrophoresis (PFGE) analysis of the isolates showed that 32 isolates were clonally related and contained a 160-kb plasmid carrying the bla(CTX-M-15), bla(OXA-1), bla(TEM-1), and aac6'-Ib-cr genes. CTX-M-15-producing Escherichia coli isolates collected in the ward during the same period of time contained distinct plasmids and were not clonally related. This study highlights the possible occurrence of outbreaks due to CTX-M-producing K. pneumoniae within hospital settings, whereas CTX-Ms are mostly reported in E. coli in community-acquired infections.

Spread of OXA-48-positive carbapenem-resistant Klebsiella pneumoniae isolates in Istanbul, Turkey.

The first outbreak of carbapenem-resistant Klebsiella pneumoniae isolates producing the plasmid-encoded carbapenem-hydrolyzing oxacillinase OXA-48 is reported. The 39 isolates belonged to two different clones and were collected at the University Hospital of Istanbul, Turkey, from May 2006 to February 2007, and they coproduced various beta-lactamases (SHV-12, OXA-9, and TEM-1 for clone A and CTX-M-15, TEM-1, and OXA-1 for clone B).

Performance of chromID ESBL, a chromogenic medium for detection of Enterobacteriaceae producing extended-spectrum beta-lactamases.

The chromogenic agar medium chromID ESBL (bioMérieux) was compared with BLSE agar medium (AES) for selective isolation and presumptive identification of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae from clinical samples. A total of 765 samples (468 rectal swabs, 255 urine samples and 42 pulmonary aspirations) obtained from 547 patients was processed. All bacterial strains isolated on either medium were further characterized using biochemical tests, and ESBL producers were confirmed by synergy testing. Genetic characterization of ESBL genes was determined by PCR. A total of 33 ESBL-producing Enterobacteriaceae strains [Escherichia coli (n=16), Klebsiella pneumoniae (n=8), Enterobacter spp. (n=3), Citrobacter spp. (n=5) and Proteus mirabilis (n=1)] was recovered. The sensitivity after 24 h incubation was 88 % for chromID ESBL and 85 % for BLSE agar. At 48 h, the sensitivity of chromID ESBL increased to 94 % and was higher than that obtained with BLSE agar. The positive predictive value at 24 h for chromID ESBL was 38.7 % [95 % confidence interval (95 % CI) 28.3 -50.2 %)], which was significantly higher than that for BLSE agar [15.4 %, 95 % CI 10.1 -21.5 %]. On both media, false-positive results were mostly due to Pseudomonas aeruginosa and to Enterobacteriaceae overproducing chromosomal cephalosporinase (Enterobacter spp.) or a chromosomal penicillinase (Klebsiella oxytoca). This study showed that chromID ESBL, a ready-to-use chromogenic selective medium, is sensitive and specific for rapid, presumptive identification of ESBL-producing Enterobacteriaceae. Its chromogenic properties and its selectivity are particularly useful in specimens containing resident associated flora.