Search

ABSTRACTThe recognition of carbapenemase-producing Enterobacteriaceae(CPE) isolates is a major laboratory challenge, and their inappropriate or delayed detection may have negative impacts on patient management and on the implementation of infection control measures. We describe here a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-based method to detect carbapenemase activity in Enterobacteriaceae. After a 20-min incubation of the isolate with 0.5 mg/ml imipenem at 37°C, supernatants were analyzed by MALDI-TOF in order to identify peaks corresponding to imipenem (300 Da) and an imipenem metabolite (254 Da). A total of 223 strains, 77 CPE (OXA-48 variants, KPC, NDM, VIM, IMI, IMP, and NMC-A) and 146 non-CPE (cephalosporinases, extended-spectrum ß-lactamases [ESBLs], and porin defects), were tested and used to calculate a ratio of imipenem hydrolysis: mass spectrometry [MS] ratio = metabolite/(imipenem + metabolite). An MS ratio cutoff was statistically determined to classify strains as carbapenemase producers (MS ratio of =0.82). We validated this method first by testing 30 of our 223 isolates (15 CPE and 15 non-CPE) 10 times to calculate an intraclass correlation coefficient (ICC of 0.98), showing the excellent repeatability of the method. Second, 43 strains (25 CPE and 18 non-CPE) different from the 223 strains used to calculate the ratio cutoff were used as external controls and blind tested. They yielded sensitivity and specificity of 100%. The total cost per test is <0.10 U.S. dollars (USD). This easy-to-perform assay is time-saving, cost-efficient, and highly reliable and might be used in any routine laboratory, given the availability of mass spectrometry, to detect CPE.

ABSTRACTThe Check-MDR CT102 microarray, aimed at identifying bacteria producing extended-spectrum ß-lactamase (ESBL) (SHV, TEM, and CTX-M) and carbapenemase (KPC, OXA-48, VIM, IMP, and NDM-1), was evaluated on a total of 144 Gram-negative strains expressing various ß-lactamases. The sensitivity and specificity were 100% for most tested genes, suggesting that this assay allows accurate identification of common ESBL and carbapenemase producers from bacterial cultures.

ABSTRACTCarbapenem-resistant Acinetobacter baumanniiisolates were obtained from 17 patients between September 2004 and August 2005 at the Academisch Ziekenhuis Vrije Universiteit Brussel, Brussels, Belgium. These multidrug-resistant isolates, which belonged to a single clone, remained susceptible to colistin and tigecycline only and produced the carbapenem-hydrolyzing oxacillinase OXA-58. This study highlights the importance of the intercountry spread of this ß-lactamase-mediated resistance mechanism and its epidemic evolution.

ABSTRACTA pyrosequencing technique was used for identification of extended-spectrum ß-lactamases (ESBLs) of GES type. These ß-lactamases are isolated increasingly emerging in gram-negative bacteria worldwide. This rapid and reliable identification method is interesting, since GES variants, including not only expanded-spectrum cephalosporins but also carbapenems, cephamycins, and monobactams, are the only ESBLs that possess different hydrolysis profiles.

ABSTRACTCarbapenem-resistant Acinetobacter baumanniiisolates were obtained from 24 patients between March and May 2004 at the Centre Hospitalier de Polyne´sie Franc¸aise, Tahiti, French Polynesia. The isolates were multidrug resistant, produced the carbapenemase OXA-23, and belonged to a single clone presenting several subtypes, suggesting an endemic situation. This study further illustrates the global spread of this kind of ß-lactamase-mediated resistance.

ABSTRACTThe spread of the plasmid-mediated carbapenem-hydrolyzing oxacillinase OXA-58 was detected in Acinetobactersp. clinical isolates from southern Europe, the Balkans, and central Turkey. It may contribute significantly to the emergence of carbapenem resistance in Acinetobacterspp., at least in this part of the world.

ABSTRACTAlthough enterococci expressing acquired vancomycin resistance phenotype have been reported increasingly worldwide, they have been rarely reported in France. From August to December 2004 we faced an outbreak of vancomycin-resistant Enterococcus faecium(VRE) isolates in the nephrology department at Bice^tre Hospital (K.-Bice^tre, France). The expression of the glycopeptide resistance varied among the 26 VRE isolates, with vancomycin MICs ranging from 12 to >256 µg/ml, whereas teicoplanin MICs ranged from 4 to 48 µg/ml. However, several strains appeared to be susceptible to glycopeptides according to disk diffusion testing and expressed resistance only after subculture with glycopeptides. In addition, a heterogeneous expression of glycopeptide resistance was also observed. This so-called VanD-like phenotype of resistance (low-level resistance to vancomycin and mostly susceptibility to teicoplanin) was surprisingly associated with a vanAgene. Plasmid extraction and mating-out experiments indicated that the vanAgene was located on a 200-kb self-transferable plasmid. Pulsed-field gel electrophoresis identified mostly dissemination of a single clone, whereas diffusion of the VanA-positive plasmid in different genomic backgrounds had also occurred. The vanAgene was part of a vanA-type operon for expression of resistance located on a Tn1546-like transposon. Sequencing of this transposon identified insertion of insertion sequence IS16in the vanYgene that encodes a d,d-carboxypeptidase that might explain in part the peculiar VanD-type phenotype of resistance. This report is the first description of a VRE outbreak in France and underlines the difficulty in detecting this organism due to variability on the expression of the glycopeptide resistance trait, if any.

ABSTRACTSeven NDM-positive Acinetobacter baumanniiisolates of worldwide origin were studied to evaluate the best technique for their identification. Detection of carbapenemase producers based on the measurement of carbapenemase activity by UV spectrophotometry (as for A. baumanniistrains producing other types of carbapenemase), or by the modified Hodge test, failed. Inhibition activity using EDTA was a sensitive technique but lacked specificity compared to molecular techniques, which remain the gold standard.

ABSTRACTVancomycin-resistant enterococci (VRE) are becoming widespread worldwide, and the rapid identification of VRE carriers from surveillance cultures is crucial for the efficient control of their spread. We assessed a new selective chromogenic medium, chromID VRE (bioMe´rieux, France), that enhanced the isolation and presumptive identification of VRE directly from rectal swabs and reduced unnecessary confirmatory and time-consuming tests.

ABSTRACTAs carbapenemase-producing Gram-negative bacilli (CP-GNB) (Enterobacteriaceae, Pseudomonadaceae, and Acinetobacterspp.) are becoming a major public health issue, there is an urgent need for accurate and fast diagnostic tests. The Amplidiag CarbaR+VRE assay is a multiplex nucleic acid-based in vitrodiagnostic test intended for the detection of CP-GNB and vancomycin-resistant enterococci (VRE) from cultured colonies. We have evaluated its ability to detect carbapenemase genes in 100 well-characterized GNB and in 200 consecutive enterobacterial isolates with reduced susceptibility to carbapenems that were referred to the French National Reference Center for carbapenem resistance. The assay has been validated on purified DNA but also directly on colonies. The Amplidiag CarbaR+VRE assay could detect all KPC, NDM, VIM, IMP, and OXA-48-like variants tested and all acquired carbapenem-hydrolyzing oxacillinases from Acinetobacter baumannii(OXA-23, OXA-24/-40, and OXA-58) as well as the overexpressed chromosomally encoded OXA-51-like ß-lactamase associated with an upstream inserted ISAba1. However, as claimed by the manufacturer, other carbapenemases such as GES-like carbapenemases (GES-2, GES-5, and GES-14), GIM-1, AIM-1, SPM-1, DIM-1, OXA-198 in Pseudomonas aeruginosa, or OXA-143-like in A. baumanniiwere not detected. Amplidiag CarbaR+VRE's performance values were high (100% sensitivity and 99% specificity) as it could detect the five major carbapenemases—NDM, VIM, IMP, KPC, and OXA-48—as well as OXA-type carbapenemases from Acinetobacterspp. that are currently emerging also among Proteus mirabilisand other enterobacterial isolates. It can provide a result directly from colonies growing on Mueller-Hinton (MH) agar or on selective screening medium in less than 2 h. Further evaluations are now necessary to determine the performance values directly on rectal swabs.