Te-Din Huang

Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of Nocardia species.

ABSTRACT The identification of Nocardia species, usually based on biochemical tests together with phenotypic in vitro susceptibility and resistance patterns, is a difficult and lengthy process owing to the slow growth and limited reactivity of these bacteria. In this study, a panel of 153 clinical and reference strains of Nocardia spp., altogether representing 19 different species, were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). As reference methods for species identification, full-length 16S rRNA gene sequencing and phenotypical biochemical and enzymatic tests were used. In a first step, a complementary homemade reference database was established by the analysis of 110 Nocardia isolates (pretreated with 30 min of boiling and extraction) in the MALDI BioTyper software according to the manufacturers recommendations for microflex measurement (Bruker Daltonik GmbH, Leipzig, Germany), generating a dendrogram with species-specific cluster patterns. In a second step, the MALDI BioTyper database and the generated database were challenged with 43 blind-coded clinical isolates of Nocardia spp. Following addition of the homemade database in the BioTyper software, MALDI-TOF MS provided reliable identification to the species level for five species of which more than a single isolate was analyzed. Correct identification was achieved for 38 of the 43 isolates (88%), including 34 strains identified to the species level and 4 strains identified to the genus level according to the manufacturers log score specifications. These data suggest that MALDI-TOF MS has potential for use as a rapid (<1 h) and reliable method for the identification of Nocardia species without any substantial costs for consumables.

Rapid emergence and spread of OXA-48-producing carbapenem-resistant Enterobacteriaceae isolates in Belgian hospitals

During a polymerase chain reaction (PCR)-based surveillance study of β-lactam resistance, 19 OXA-48-positive enterobacterial isolates were detected at nine Belgian hospitals from January 2010 to April 2011. Most cases were presumed to have been locally acquired and were detected in patients who had not travelled abroad. Clonally related outbreaks occurred in two different cities. The majority of isolates co-produced several β-lactamases as well as non-β-lactam resistance genes. This report highlights the rapid emergence and spread of OXA-48-producing Enterobacteriaceae in Belgium.

Plasmid-Encoded Carbapenem-Hydrolyzing {beta}-Lactamase OXA-48 in an imipenem-susceptible Klebsiella pneumoniae strain from Belgium.

Emergence and dissemination of Enterobacteriaceae isolates harboring carbapenemases represent a significant threat to the management of nosocomial infections (6). ...

GES Extended-Spectrum β-Lactamases in Acinetobacter baumannii Isolates in Belgium

ABSTRACT During a PCR-based surveillance study of β-lactam resistance, 125 multidrug-resistant (MDR) Acinetobacter baumannii isolates were obtained from 18 hospitals in Belgium from January 2008 to December 2009. Nine GES-positive A. baumannii isolates were detected at 6 Belgian hospitals. DNA sequencing of the blaGES genes identified GES-11, GES-12, and a novel variant GES-14, which differs from GES-11 by a single amino acid substitution (Gly170Ser). All index isolates were travel associated and originated from patients transferred from Turkey (n = 2), Egypt (n = 2), and Palestinian territories (Gaza) (n = 2). A nosocomial outbreak involving three additional patients occurred in a burn unit at a single hospital. No clonal relatedness could be established between the 6 index isolates by pulsed-field gel electrophoresis (PFGE) analysis. Three different alleles (the plasmid-located blaGES-11 and blaGES-12 and a likely chromosomally located novel variant blaGES-14) were detected as part of a class 1 integron, also including the aac6′Ib and dfrA7 genes. Restriction analysis of plasmids suggests a common origin for the plasmids bearing blaGES-11 and blaGES-12. Cloning of the blaGES genes in Escherichiacoli identified GES-14 as hydrolyzing imipenem, while GES-12 showed the highest specific activity against ceftazidime. This report highlights the emergence of various blaGES-like genes, especially those conferring carbapenem resistance in A. baumannii and its importation in Western Europe from Middle Eastern countries.

Temocillin and piperacillin/tazobactam resistance by disc diffusion as antimicrobial surrogate markers for the detection of carbapenemase-producing Enterobacteriaceae in geographical areas with a high prevalence of OXA-48 producers

OBJECTIVES To assess the performance of the agar disc diffusion method for the detection of carbapenemase-producing Enterobacteriaceae (CPE) referred to the national reference laboratories (NRLs) in Belgium and France. METHODS All Enterobacteriaceae isolates referred to the NRLs for the confirmation of CPE in 2012 were included. The inhibition zone diameters of meropenem, piperacillin/tazobactam and temocillin using CLSI disc diffusion methodology were recorded. Phenotypic and molecular detection of carbapenemases was performed on all isolates. RESULTS A total of 1354 Enterobacteriaceae isolates, including 435 (32.1%) confirmed CPE isolates [OXA-48 (n = 323), KPC (n = 60), VIM (n = 32) and NDM (n = 20)] and 919 carbapenemase-negative isolates, were tested. Using recommended interpretative criteria, non-susceptibility to meropenem had poor sensitivity (52.0% by CLSI susceptibility breakpoint and 80.0% by EUCAST screening breakpoints), while non-susceptibility to piperacillin/tazobactam (according to CLSI breakpoint) or to temocillin (according to Fuchs, Barry, Thornsberry et al. Eur J Clin Microbiol 1985; 4: 30-3) was highly sensitive (99.8% and 98.2%, respectively) but poorly specific (29.4% and 42.9%, respectively) for the detection of CPE. Temocillin diameters <12 mm alone had high specificity (90.0%) and the combination of temocillin diameters ≥12 mm with piperacillin/tazobactam diameters ≥16 mm observed in 40% of all referred isolates displayed excellent negative predictive value (99.2%). CONCLUSIONS In geographical areas with a high prevalence of OXA-48 producers, recommended meropenem susceptibility or screening breakpoints failed to detect CPE in a large proportion of isolates. The combination of modified zone diameter cut-offs for piperacillin/tazobactam (≥16 mm) and temocillin (≥12 mm) can be used to rule out the presence of carbapenemase and avoid unnecessary additional testing for confirmation of CPE.

Evaluation of two new commercial immunochromatographic assays for the rapid detection of OXA-48 and KPC carbapenemases from cultured bacteria.

BACKGROUND Rapid detection and confirmation of carbapenemases remains very challenging for diagnostic laboratories. OBJECTIVES The objective of this study was to assess the performance of two new immunochromatographic (IC) commercial assays for the rapid detection of OXA-48-producing and KPC-producing Enterobacteriaceae in pure bacterial isolates. METHODS A panel of 92 bacterial isolates predominantly including carbapenem-non-susceptible Enterobacteriaceae with previously defined carbapenem resistance mechanisms was tested. Then, 342 consecutive carbapenem-non-susceptible Enterobacteriaceae isolates referred to the reference laboratory were investigated prospectively in parallel with other phenotypic tests and with multiplex PCR and sequencing as the gold standard. RESULTS In the collection panel, each of the two IC assays correctly detected all 30 OXA-48-like-producing isolates and 25 KPC-producing isolates, whatever the species, their association with other β-lactamases and the level of resistance to carbapenems. All other carbapenemase producers and all non-carbapenemase-producing isolates yielded negative results with both tests. In the prospective evaluation, all OXA-48-like-producing Enterobacteriaceae isolates (n = 130) and KPC-producing Enterobacteriaceae isolates (n = 33) were correctly detected by the individual IC assays, while 179 non-OXA-48-like-producing and non-KPC-producing strains (137 non-carbapenemase producers and 42 isolates belonging to other carbapenemase family types) yielded negative results. Thus, each assay yielded 100% sensitivity and 100% specificity for the detection of OXA-48-like or KPC enzymes, respectively, at 15 min. CONCLUSIONS The two IC assays allow rapid and reliable direct confirmation of OXA-48 and KPC carbapenemases from culture colonies and appear to be very useful additions to the existing tests, obviating the need for more costly characterization by molecular amplification methods.

Epidemiology of multidrug-resistant microorganisms among nursing home residents in Belgium.

Objectives A national survey was conducted to determine the prevalence and risk factors of methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamases-producing Enterobacteriaceae (ESBLE) and vancomycin-resistant enterococci (VRE) among nursing home residents in Belgium. Methods A random stratified, national prevalence survey was conducted in nursing home residents who were screened for carriage of ESBLE, MRSA and VRE by multisite enriched culture. Characteristics of nursing homes and residents were collected by a questionnaire survey and were analysed by multilevel logistic regression analysis. Results Of 2791 screened residents in 60 participating nursing home, the weighted prevalence of ESBLE and MRSA carriage were 6.2% (range: 0 to 20%) and 12.2% (range: 0 to 36%), respectively. No cases of VRE were found. No relationship was found between ESBLE and MRSA prevalence rates within nursing homes and the rate of co-colonization was very low (0.8%). Geographical variations in prevalence of MRSA and ESBLE and in distribution of ESBL types in nursing home residents paralleled that of acute hospitals. Risk factors of ESBLE carriage included previously known ESBLE carriage, male gender, a low level of mobility and previous antibiotic exposure. Risk factors for MRSA colonization were: previously known MRSA carriage, skin lesions, a low functional status and antacid use. Conclusions A low prevalence of ESBLE carriage was found in nursing home residents in Belgium. The prevalence of MRSA carriage decreased substantially in comparison to a similar survey conducted in 2005. A low functional status appeared as a common factor for ESBLE and MRSA carriage. Previous exposure to antibiotics was a strong predictor of ESBLE colonization while increased clustering of MRSA carriage suggested the importance of cross-transmission within nursing homes for this organism. These results emphasize the need for global coordination of the surveillance of MDRO within and between nursing homes and hospitals.

Comparative Evaluation of Two Chromogenic Tests for Rapid Detection of Carbapenemase in Enterobacteriaceae and in Pseudomonas aeruginosa Isolates

ABSTRACT We compared the performance of the Carba NP test and the Rosco Rapid CARB screen kit for detecting carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa. Both tests are rapid and highly sensitive; however, the Carba NP test showed superior specificity, and several uninterpretable results were observed with the Rapid CARB screen.

Nosocomial infections caused by multidrug-resistant Pseudomonas putida isolates producing VIM-2 and VIM-4 metallo-β-lactamases

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Evaluation of the βLacta Test, a Rapid Test Detecting Resistance to Third-Generation Cephalosporins in Clinical Strains of Enterobacteriaceae

ABSTRACT For decades, third-generation cephalosporins (3GC) have been major drugs used to treat infections due to Enterobacteriaceae; growing resistance to these antibiotics makes the rapid detection of such resistance important. The βLacta test is a chromogenic test developed for detecting 3GC-resistant isolates from cultures on solid media within 15 min. A multicenter prospective study conducted in 5 French and Belgian hospitals evaluated the performance of this test on clinical isolates. Based on antibiotic susceptibility testing, strains resistant or intermediate to cefotaxime or ceftazidime were classified as 3GC resistant, and molecular characterization of this resistance was performed. The rates of 3GC resistance were 13.9% (332/2,387) globally, 9.4% in Escherichia coli (132/1,403), 25.6% in Klebsiella pneumoniae (84/328), 30.3% in species naturally producing inducible AmpC beta-lactamases (109/360), and 5.6% in Klebsiella oxytoca and Citrobacter koseri (7/124). The sensitivities and specificities of the βLacta test were, respectively, 87.7% and 99.6% overall, 96% and 100% for E. coli and K. pneumoniae, and 67.4% and 99.6% for species naturally producing inducible AmpC beta-lactamase. False-negative results were mainly related to 3GC-resistant strains producing AmpC beta-lactamase. Interestingly, the test was positive for all 3GC-resistant extended-spectrum beta-lactamase-producing isolates (n = 241). The positive predictive value was 97% and remained at ≥96% for prevalences of 3GC resistance ranging between 10 and 30%. The negative predictive values were 99% for E. coli and K. pneumoniae and 89% for the species producing inducible AmpC beta-lactamase. In conclusion, the βLacta test was found to be easy to use and efficient for the prediction of resistance to third-generation cephalosporins, particularly in extended-spectrum beta-lactamase-producing strains.