Increasing incidence of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in Belgian hospitals.

Carbapenemase-producing Enterobacteriaceae are increasingly reported worldwide. The aim of the study was to determine the incidence and molecular epidemiology of carbapenemase-producing (CP) Escherichia coli and Klebsiella pneumoniae (CP-E/K) in Belgium. Eleven hospital-based laboratories collected carbapenem non-susceptible (CNS) isolates of E. coli and K. pneumoniae detected in clinical specimens from January 2013 to December 2014. All CNS strains were tested for carbapenemase production and typed by multilocus sequence typing (MLST) for a 6-month period as part of the European Survey on Carbapenemase-Producing Enterobacteriaceae in Europe (EuSCAPE) structured survey. In addition, an equal number of carbapenem-susceptible isolates collected were preserved as a control group for risk factor analysis. The overall incidence rate of CP-E/K isolates in hospitals increased from 0.124 in 2013 to 0.223 per 1000 admissions in 2014. From November 2013 to April 2014, 30 CP K. pneumoniae [OXA-48 (n = 16), KPC (n = 13), OXA-427 (n = 1)] and five CP E. coli [OXA-48 (n = 3), NDM (n = 1), OXA-427 (n = 1)] isolates were detected in ten hospitals. The 16 OXA-48-producing K. pneumoniae strains were distributed into eight sequence types (STs), while the 13 KPC-producing K. pneumoniae clustered into three STs dominated by ST512 (n = 7) and ST101 (n = 5). Compared to controls, we observed among CP-E/K carriers significantly higher proportion of males, respiratory origins, previous hospitalization, nosocomial setting, and a significantly lower proportion of bloodstream infections. Our study confirms the rapid spread of CP-E/K in Belgian hospitals and the urgent need for a well-structured and coordinated national surveillance plan in order to limit their dissemination.

Infection due to travel-related carbapenemase-producing Enterobacteriaceae, a largely underestimated phenomenon in Belgium.

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) are emerging worldwide, representing a major threat for public health. Early CPE detection is crucial in order to prevent infections and the development of reservoirs/outbreaks in hospitals. In 2008, most of the CPE strains reported in Belgium were imported from patients repatriated from abroad. Actually, this is no longer the case. OBJECTIVES AND METHODS: A surveillance was set up in Belgian hospitals (2012) in order to explore the epidemiology and determinants of CPE, including the link with international travel/hospitalization. The present article describes travel-related CPE reported in Belgium. Different other potential sources for importation of CPE are discussed. RESULTS: Only 12% of all CPE cases reported in Belgium (2012-2013) were travel related (with/without hospitalization). This is undoubtedly an underestimation (missing travel data: 36%), considering the increasing tourism, the immigration from endemic countries, the growing number of foreign patients using scheduled medical care in Belgium, and the medical repatriations from foreign hospitals. The free movement of persons and services (European Union) contributes to an increase in foreign healthcare workers (HCW) in Belgian hospitals. Residents from nursing homes located at the country borders can be another potential source of dissemination of CPE between countries. Moreover, the high population density in Belgium can increase the risk for CPE-dissemination. Urban areas in Belgium may cumulate these potential risk factors for import/dissemination of CPE. CONCLUSIONS: Ideally, travel history data should be obtained from hospital hygiene teams, not from the microbiological laboratory. Patients who received medical care abroad (whatever the country) should be screened for CPE at admission.

Evaluation of several phenotypic methods for the detection of carbapenemase-producing Pseudomonas aeruginosa.

The purpose of this investigation was to compare several phenotypic methods, including combined disk tests (CDT) containing metallo-ß-lactamase (MBL) inhibitors or cloxacillin, and the Carba NP test for the detection of carbapenemase-producing Pseudomonas aeruginosa (CPPA). A new CDT using imipenem (10 µg) ± cloxacillin 4,000 µg and the Carba NP test were evaluated to detect CPPA. In addition, four commercially available combined disks containing a carbapenem and ethylene-diamine-tetra-acetic acid (EDTA) or dipicolinic acid (DPA) as the inhibitor were tested in order to detect MBL-positive P. aeruginosa. All these phenotypic methods were evaluated on 188 imipenem non-susceptible P. aeruginosa (CPPA, n = 75) isolates divided into 26 well-characterized collection strains and 162 non-duplicate clinical isolates referred to the national reference laboratory in 2013. For the total of 188 isolates tested, CDT containing EDTA or DPA displayed high sensitivities (99%) and specificities (95%) for detecting MBL-producing isolates. CDT with cloxacillin showed a sensitivity and specificity of 97%/96% compared to 88%/99% for the Carba NP test in order to detect CPPA. For the 162 clinical isolates, CDT containing EDTA or DPA displayed a high negative predictive value (NPV) (99%) for detecting MBL-producing isolates. CDT with cloxacillin showed an NPV of 98%, compared to 95% for the Carba NP test in order to detect CPPA. In our setting, CDT associating imipenem ± EDTA or ± DPA performed best for the detection of MBL-producing P. aeruginosa. Imipenem/imipenem-cloxacillin test yielded good NPV to exclude the presence of MBL in imipenem non-susceptible isolates.

Prevalence and distribution of beta-lactamase coding genes in third-generation cephalosporin-resistant Enterobacteriaceae from bloodstream infections in Cambodia.

Resistance to third-generation cephalosporins in Gram-negative bacteria is emerging in Asia. We report the prevalence and distribution of extended-spectrum beta-lactamase (ESBL), AmpC beta-lactamase and carbapenemase-coding genes in cefotaxime-resistant Enterobacteriaceae isolates from bloodstream infections (BSI) in Cambodia. All Enterobacteriaceae isolated from BSI in adult patients at Sihanouk Hospital Centre of HOPE, Phnom Penh, Cambodia (2007-2010) were assessed. Antimicrobial susceptibility testing was carried out by disc diffusion and MicroScan according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Screening for ESBL, plasmidic AmpC and carbapenemase-coding genes was performed by multiplex polymerase chain reaction (PCR) sequencing assays. Identification of the ST131 clone was performed in all CTX-M-positive Escherichia coli, using PCR targeting the papB gene. Out of 183 Enterobacteriaceae, 91 (49.7 %) isolates (84 BSI episodes) were cefotaxime-resistant: E. coli (n = 68), Klebsiella pneumoniae (n = 17) and Enterobacter spp. (n = 6). Most episodes were community-acquired (66/84; 78.3 %). ESBLs were present in 89/91 (97.8 %) cefotaxime-resistant isolates: 86 (96.6 %) were CTX-M, mainly CTX-M-15 (n = 41) and CTX-M-14 (n = 21). CTX-M of group 1 were frequently associated with TEM and/or OXA-1/30 coding genes and with phenotypic combined resistance to ciprofloxacin, sulphamethoxazole-trimethoprim and gentamicin (39/50, 78.0 %). Plasmidic AmpC (CMY-2 and DHA-1 types) were found alone (n = 2) or in combination with ESBL (n = 4). Eighteen E. coli isolates were identified as B2-ST131-O25B: 11 (61.1 %) carried CTX-M-14. No carbapenemase-coding genes were detected. ESBL among Enterobacteriaceae from BSI in Cambodia is common, mainly associated with CTX-M-15 and CTX-M-14. These findings warrant urgent action for the containment of antibiotic resistance in Cambodia.

Trends in production of extended-spectrum beta-lactamases among Enterobacteriaceae of clinical interest: results of a nationwide survey in Belgian hospitals.

OBJECTIVES: to assess the frequency and diversity of extended-spectrum ß-lactamases (ESBLs) in Enterobacteriaceae isolates in Belgium. METHODS: during 2006 and 2008, non-duplicate clinical isolates of Enterobacteriaceae resistant to ceftazidime and/or cefotaxime were collected in 100 Belgian hospitals. ESBL production was confirmed by phenotypic and genotypic tests. MICs of 13 antimicrobial agents were determined by Etest. ESBL-encoding genes were identified by PCR sequencing and the bla(CTX-M) environment was characterized by PCR mapping. Selected isolates were genotyped by PFGE, multilocus sequence typing analysis and phylogenetic grouping by PCR. RESULTS: overall, 733 isolates were confirmed as ESBL producers. Carbapenems and temocillin were active against ≥ 95% of all tested isolates. Co-resistance to co-trimoxazole and to ciprofloxacin was found in almost 70% and 80% of the strains, respectively. Overall, Escherichia coli (49%), Enterobacter aerogenes (32%) and Klebsiella pneumoniae (9%) represented the most prevalent species. Isolates harboured predominantly TEM-24 (30.7%), CTX-M-15 (24.2%) and TEM-52 (12.1%). Compared with 2006, the proportion of CTX-M-type enzymes increased significantly in 2008 (54% versus 23%; P < 10(-6)), mostly linked to a rising proportion of CTX-M-15-producing E. coli. TEM-24 decreased (19% in 2008 versus 43% in 2006; P < 10(-6)) during the same period, while the prevalence of TEM-52 remained unchanged (10% in 2008 versus 14% in 2006; not significant). Over 80% of the CTX-M-15-producing E. coli isolates clustered into a single PFGE type and phylogroup B2, corresponding to the sequence type (ST) 131 clone. Intra- and inter-species gene dissemination (CTX-M-15, CTX-M-2 and CTX-M-9) and wide epidemic spread of the CTX-M-15-producing E. coli ST131 clone in several Belgian hospitals were observed. CONCLUSIONS: the rapid emergence of multiresistant CTX-M-15-producing E. coli isolates is of major concern and highlights the need for further surveillance in Belgium.

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

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 manufacturer's 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 manufacturer's 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.

Detection and characterization of class A extended-spectrum-beta-lactamase-producing Pseudomonas aeruginosa isolates in Belgian hospitals.

OBJECTIVES: To investigate the presence of extended-spectrum beta-lactamases (ESBLs) among Pseudomonas aeruginosa clinical isolates referred to two Belgian reference laboratories. METHODS: Antibiograms were analysed for P. aeruginosa isolates referred between 2004 and 2008. Isolates resistant to ceftazidime (MIC > 8 mg/L) and with a positive double-disc synergy test between ceftazidime and clavulanate were serotyped and screened for the presence of ESBL-encoding genes. Genes encoding metallo-beta-lactamases (bla(MBL)) were sought by PCR in ESBL-producing isolates with positive imipenem/EDTA synergy tests. PFGE of SpeI-digested genomic DNA was used to compare isolates and selected strains were characterized by multilocus sequence typing. RESULTS: Forty-eight (2.2%) of 2150 P. aeruginosa isolates were confirmed as class A ESBL-producing isolates by molecular testing. bla(BEL) and bla(PER) alleles were detected, respectively, in 39 and 10 P. aeruginosa isolates originating from 16 hospitals (two isolates were simultaneously positive for BEL and PER). Fifteen of the isolates were found to co-produce ESBLs and VIM carbapenemases. These strains were pan-resistant and remained susceptible only to colistin (MICs

Molecular characterization of AmpC-producing Escherichia coli clinical isolates recovered at two Belgian hospitals.

OBJECTIVE: To characterize the genetic determinants associated with an AmpC phenotype in clinical Escherichia coli isolates. METHODS: E. coli strains recovered at two Belgian hospitals between 2004 and 2006 were selected on the basis of an AmpC-producing phenotype. Plasmid-mediated cephalosporinases coding genes and the sequence of chromosomal ampC genes were identified by PCR/sequencing. The isolates were submitted to phylotyping and genotyping analysis using rep-PCR (Diversilab) and PFGE. A novel chromosomal ampC gene was cloned. RESULTS: Eighty-three out of 6850 E. coli isolates were selected. Seventy-two isolates were found to overexpress their chromosomal cephalosporinases while 11 contained plasmid-mediated cephalosporinases. Among chromosomal AmpC overproducers, 12 were extended-spectrum AmpC (ESAC) expressing isolates which all displayed reduced susceptibility to cefepime. Cloning of a new ESAC allele suggested that L293P mutation was responsible of the extension of the hydrolysis spectrum to cefepime. AmpC overproducers, including ESAC producers, predominantly belonged to phylogenetic group A and B1, while plasmid-mediated AmpC-producing isolates preferentially belong to phylogroup B2 and D. According to rep-PCR, the majority of the E. coli isolates belonging to phylogroup A were clonally related which was further confirmed by PFGE for the 11 ESAC expressing isolates. CONCLUSIONS: Chromosomal AmpC overproduction was the most common resistance mechanism, and the occurrence of ESAC was found to be as frequent as plasmid-mediated cephalosporinases. The detection of a new ESAC allele, of an ESAC producing strain belonging to phylogroup D and the existence of a clonal relationship between ESAC producing strains underline the need for study of the clinical relevance of this mechanism of resistance.

Antimicrobial susceptibility of anaerobic bacteria in Belgium as determined by E-test methodology.

The objective was to collect recent data on the antibiotic susceptibility of clinically significant anaerobes in Belgium. A total of 333 anaerobic clinical isolates from various body sites were prospectively collected between 2005 and 2007 at two tertiary care hospitals in Belgium. The minimal inhibitory concentrations (MICs) were determined using the E-test method for nine anti-anaerobic antibiotics. Sixty-one percent of the isolates were beta-lactamase producers, which explains the poor activity of penicillin. Amoxicillin/clavulanic acid, piperacillin/tazobactam, metronidazole and meropenem were very active against most anaerobes, but around 10% of the Bacteroides fragilis group strains were non-susceptible to the two beta-lactam/beta-lactamase inhibitors. No resistance was observed to metronidazole, while 3% of the Bacteroides spp. had decreased susceptibility to meropenem (MIC > or = 4 mg/L). Cefoxitin, clindamycin and moxifloxacin were less active, with 33%, 52% and 57% of the B. fragilis group being non-susceptible respectively. Tigecycline showed consistently good activity against most anaerobes with MIC(50) and MIC(90) of 0.25 and 2 mg/L. Metronidazole, amoxicillin/clavulanate, piperacillin/tazobactam and meropenem remain good empirical choices when anaerobes are expected in our setting. Because of the occurrence of resistance to most classes of current anti-anaerobic antibiotics, it is recommended that the antimicrobial resistance patterns be monitored regularly in order to guide empirical therapy.

Evaluation of a new meropenem-EDTA double-ended Etest strip for the detection of the cfiA metallo-beta-lactamase gene in clinical isolates of Bacteroides fragilis.

Thirty-five Bacteroides fragilis clinical isolates with varying susceptibility to meropenem were analysed with a prototype of a double-ended Etest strip containing meropenem +/- EDTA, designed for the detection of the CfiA metallo-beta-lactamase. Phenotypic results obtained with this new Etest strip were related to the genotype and compared to the results of the Etest containing imipenem +/- EDTA. Whereas the Etest with imipenem +/- EDTA only allowed detection of isolates with high-level resistance (both MICs of imipenem and meropenem >32 mg/L), reflecting the possible underestimation of CfiA prevalence in B. fragilis, the Etest with meropenem +/- EDTA proved to be more accurate, particularly for isolates with low-level carbapenem resistance, suggesting its potential for broader detection of CfiA production.

Design and validation of a low density array (Nosochip) for the detection and identification of the main pathogenic bacteria and fungi responsible for nosocomial pneumonia.

The aim of this study was to be able to amplify and to detect on one array 27 different etiologic agents found in nosocomial pneumonia, some being phylogenetically closely related and others very distant. The assay is based on the use of consensus primers combined with the identification of the resulting amplicons by hybridization on specific capture probes present on an array. Three genes were necessary in order to cover the different pathogens. We took a redundancy of at least two positive spots to confirm the identity of each species. Each probe was present in triplicate on the array. The detection limit was between 10 and 1,000 DNA copies in the assay depending on the bacteria and the probe. The assay was also specific when tested both on reference collection strains corresponding to the 27 species of interest and on 57 other bacterial species of the normal human flora. Accuracy of the assay was assessed on 200 clinical isolates and some polymorphisms were indeed observed for 5 species. Effectiveness of the assay was preliminarily validated on 25 endotracheal aspirates and sputum samples, and the results were in accordance either with the cell culture or with the sequencing. Polybacterial infections were well detected in three samples. The results show that a combination of appropriate polymerase chain reaction (PCR) and redundancy of signals on the array allows specific screening of bacteria belonging to different species and genus and even fungi. The results open the way for a possible molecular detection of bacteria in the clinical diagnostic setting.

In vitro activity of temocillin against prevalent extended-spectrum beta-lactamases producing Enterobacteriaceae from Belgian intensive care units.

Temocillin is a narrow spectrum penicillin with high stability to most beta-lactamases including AmpC types and extended-spectrum types (ESBLs). We have analysed its in vitro activity against 652 clinical isolates of Enterobacteriaceae prospectively collected from patients hospitalised in intensive care units at seven different university hospitals in Belgium in 2005. Strains were screened for ESBL production using cefotaxime and ceftazidime screen agar plates and by double ESBL E-tests. The MIC of temocillin and of five comparators was determined using the E-test method. ESBLs were characterized at one central laboratory by isoelectric focusing, PCR for bla genes of the SHV, TEM, and CTX-M families, and by DNA sequencing. The prevalence of ESBL-producing Enterobacteriaceae averaged 11.8% and ranged between 3.0 and 29% in the different hospitals. Meropenem exhibited the highest in vitro activity overall (mode MIC 0.064 microg; MIC(90); 0.19 microg/ml), whereas ceftazidime (MIC(90) > 256 microg/ml) and ciprofloxacin (MIC(90) > 32 microg/ml) scored the worst. Temocillin was active against more than 90% of the isolates including most AmpC- and ESBL-producing isolates. These data indicate the well preserved activity of temocillin over the years against Enterobacteriaceae and show the wide variation in prevalence of ESBL-producing Enterobacteriaceae isolates in Belgian intensive care units. Prospective clinical studies are, however, needed to validate the usefulness of temocillin in the treatment of microbiologically documented infections caused by ESBL- and/or AmpC- overproducing nosocomial Enterobacteriaceae pathogens.

Determination of antimicrobial susceptibility patterns of Nocardia spp. from clinical specimens by Etest.

Susceptibilities to 11 antimicrobial agents were determined by Etest for 93 Nocardia isolates from clinical specimens and 15 type strains belonging to different Nocardia spp. All isolates were susceptible to trimethoprim-sulphamethoxazole, amikacin and linezolid, but susceptibilities of the various Nocardia spp. to beta-lactams, aminoglycosides, ciprofloxacin and clarithromycin varied markedly. Overall, there was a good correlation between the drug resistance patterns and the species identification established by conventional phenotypic tests and 16S rDNA sequencing. Among the different species encountered, Nocardia farcinica and Nocardia brasiliensis displayed the most multiresistant profiles, with resistance to imipenem occurring mainly among isolates of N. brasiliensis and Nocardia abscessus. The species variability in susceptibility profiles and the numerous recent taxonomic changes means that in-vitro susceptibility tests may be a complementary tool for the identification of Nocardia isolates from human clinical specimens. Further studies on a larger number of species from more diverse geographical sources, including species that are found less commonly among clinical isolates, are required to validate and extend the results.