Multiplex PCR for detection of acquired carbapenemase genes

Summary: The spread of Enterobacteriaceae, primarily Klebsiella pneumoniae, producing KPC, VIM, IMP, and NDM carbapenemases, is causing an unprecedented public health crisis. Carbapenemase-producing enterobacteria (CPE) infect mainly hospitalized patients but also have been spreading in long-term care facilities. Given their multidrug resistance, therapeutic options are limited and, as discussed here, should be reevaluated and optimized. Based on susceptibility data, colistin and tigecycline are commonly used to treat CPE infections. Nevertheless, a review of the literature revealed high failure rates in cases of monotherapy with these drugs, whilst monotherapy with either a carbapenem or an aminoglycoside appeared to be more effective. Combination therapies not including carbapenems were comparable to aminoglycoside and carbapenem monotherapies. Higher success rates have been achieved with carbapenem-containing combinations. Pharmacodynamic simulations and experimental infections indicate that modification of the current patterns of carbapenem use against CPE warrants further attention. Epidemiological data, though fragmentary in many countries, indicate CPE foci and transmission routes, to some extent, whilst also underlining the lack of international collaborative systems that could react promptly and effectively. Fortunately, there are sound studies showing successful containment of CPE by bundles of measures, among which the most important are active surveillance cultures, separation of carriers, and assignment of dedicated nursing staff.

Carbapenemases in Klebsiella pneumoniae and Other Enterobacteriaceae: an Evolving Crisis of Global Dimensions

Multilocus sequence typing reveals that many bacterial species have a clonal structure and that some clones are widespread. This underlying phylogeny was not revealed by pulsed-field gel electrophoresis, a method better suited to short-term outbreak investigation. Some global clones are multiresistant and it is easy to assume that these have disseminated from single foci. Such conclusions need caution, however, unless there is a clear epidemiological trail, as with KPC carbapenemase-positive Klebsiella pneumoniae ST258 from Greece to northwest Europe. Elsewhere, established clones may have repeatedly and independently acquired resistance. Thus, the global ST131 Escherichia coli clone most often has CTX-M-15 extended-spectrum β-lactamase (ESBL), but also occurs without ESBLs and as a host of many other ESBL types. We explore this interaction of clone and resistance for E. coli, K. pneumoniae, Acinetobacter baumannii- a species where three global lineages dominate--and Pseudomonas aeruginosa, which shows clonal diversity, but includes the relatively 'tight' serotype O12/Burst Group 4 cluster that has proved adept at acquiring resistances--from PSE-1 to VIM-1 β-lactamases--for over 20 years. In summary, 'high-risk clones' play a major role in the spread of resistance, with the risk lying in their tenacity--deriving from poorly understood survival traits--and a flexible ability to accumulate and switch resistance, rather than to constant resistance batteries.

Multiresistant Gram-negative bacteria: the role of high-risk clones in the dissemination of antibiotic resistance

Objectives To develop a single multiplex real-time PCR assay to detect six different genetic types of carbapenemases already identified in Enterobacteriaceae (KPC, GES, NDM, IMP, VIM and OXA-48). Methods A total of 58 bacterial isolates were tested. Thirty were previously characterized as resistant to carbapenems and documented by PCR and sequencing analysis to carry the following genes: bla(KPC) type, bla(GES) type, bla(IMP) type, bla(VIM) type, bla(OXA-48) and bla(NDM-1). These positive strains included 21 Enterobacteriaceae, 1 Acinetobacter baumannii and 8 Pseudomonas aeruginosa isolates. The remaining 28 isolates previously tested susceptible to carbapenems and were negative for these genes. Bacterial DNA was extracted using the easyMag extractor (bioMerieux, France). The real-time PCR was performed using the Rotor-Gene 6000 instrument (Corbett Life Science, Australia) and specific primers for each carbapenemase target were designed using the DNAStar software (Madison, WI, USA). Results Each one of the six carbapenemase genes tested presented a different melting curve after PCR amplification. The melting temperature (T(m)) analysis of the amplicons identified was as follows: bla(IMP) type (T(m) 80.1°C), bla(OXA-48) (T(m) 81.6°C), bla(NDM-1) (T(m) 84°C), bla(GES) type (T(m) 88.6°C), bla(VIM) type (T(m) 90.3°C) and bla(KPC) type (T(m) 91.6°C). No amplification was detected among the negative samples. The results showed 100% concordance with the genotypes previously identified. Conclusions The new assay was able to detect the presence of six different carbapenemase gene types in a single 3 h PCR.

/pdf/rapid-detection-of-carbapenemase-genes-by-multiplex-real-74ng2k0k7y.pdf

Rapid detection of carbapenemase genes by multiplex real-time PCR

Nosocomial bloodstream infections (nBSIs) are an important cause of morbidity and mortality. Data from a nationwide, concurrent surveillance study, Brazilian SCOPE (Surveillance and Control of Pathogens of Epidemiological Importance), were used to examine the epidemiology and microbiology of nBSIs at 16 Brazilian hospitals. In our study 2,563 patients with nBSIs were included from 12 June 2007 to 31 March 2010. Ninety-five percent of BSIs were monomicrobial. Gram-negative organisms caused 58.5% of these BSIs, Gram-positive organisms caused 35.4%, and fungi caused 6.1%. The most common pathogens (monomicrobial) were Staphylococcus aureus (14.0%), coagulase-negative staphylococci (CoNS) (12.6%), Klebsiella spp. (12.0%), and Acinetobacter spp. (11.4%). The crude mortality was 40.0%. Forty-nine percent of nBSIs occurred in the intensive-care unit (ICU). The most frequent underlying conditions were malignancy, in 622 patients (24.3%). Among the potential factors predisposing patients to BSI, central venous catheters were the most frequent (70.3%). Methicillin resistance was detected in 157 S. aureus isolates (43.7%). Of the Klebsiella sp. isolates, 54.9% were resistant to third-generation cephalosporins. Of the Acinetobacter spp. and Pseudomonas aeruginosa isolates, 55.9% and 36.8%, respectively, were resistant to imipenem. In our multicenter study, we found high crude mortality and a high proportion of nBSIs due to antibiotic-resistant organisms.

Nosocomial bloodstream infections in Brazilian Hospitals: Analysis of 2,563 cases from a prospective nationwide surveillance study

Metallo-β-lactamase enzymes (MβL) are encoded by transferable genes, which appear to spread rapidly among gram-negative bacteria The objective of this study was to develop a multiplex real-time PCR assay followed by a melt curve step for rapid detection and identification of genes encoding MβL-type enzymes based on the amplicon melting peak The reference sequences of all genes encoding IMP and VIM types, SPM-1, GIM-1, and SIM-1 were downloaded from GenBank, and primers were designed to obtain amplicons showing different sizes and melting peak temperatures (Tm) The real-time PCR assay was able to detect all MβL-harboring clinical isolates, and the Tm-assigned genotypes were 100% coincident with previous sequencing results This assay could be suitable for identification of MβL-producing gram-negative bacteria by molecular diagnostic laboratories

Katia A. Kiyota

Papers

Rapid Detection and Identification of Metallo-β-Lactamase-Encoding Genes by Multiplex Real-Time PCR Assay and Melt Curve Analysis