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

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In many countries the high rate of clarithromycin resistance no longer allows its empirical use in standard anti-H pylori regimens. The knowledge of outpatient antibiotic consumption may provide a simple tool to predict the susceptibility of H pylori to quinolones and to macrolides and to adapt the treatment strategies.

OBJECTIVES: To determine the point prevalence of carbapenem-non-susceptible Enterobacteriaceae (CNSE) and carbapenemase-producing Enterobacteriaceae (CPE) isolates among hospitalized patients in Belgium. METHODS: Twenty-four hospital-based laboratories prospectively collected 200 non-duplicated Enterobacteriaceae isolates from clinical specimens of hospitalized patients over a 2 month period. All isolates were screened locally for decreased susceptibility to carbapenem drugs using a disc diffusion method according to CLSI interpretative criteria. CNSE strains were referred centrally for confirmation of carbapenemase by phenotypic and molecular testing. RESULTS: From February to April 2012, 158 of the 4564 screened Enterobacteriaceae isolates were categorized as non-susceptible to carbapenems, resulting in a point prevalence of CNSE of 3.5% (95% CI: 2.9%-4.2%; range per centre: 0.5%-8.5%). Of the 125 referred CNSE isolates, 11 Klebsiella pneumoniae isolates [OXA-48 (n = 7), KPC type (n = 3) and NDM type (n = 1)], 1 OXA-48-positive Escherichia coli isolate and 1 KPC-positive Klebsiella oxytoca isolate were detected in eight hospitals. None of the 72 carbapenem-non-susceptible Enterobacter spp. isolates were confirmed as CPE. The minimal estimated point prevalence of CPE isolates was 0.28% (13/4564; 95% CI: 0.13%-0.44%) overall (range per centre: 0%-1.5%). CONCLUSIONS: Despite the overall low prevalence of CNSE found in this study, the detection of CPE isolates in one-third of the participating centres raises concerns and highly suggests the spread and establishment of CPE in Belgian hospitals.

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.

The objective of this study was to examine whether asymptomatic colonization with MDRB would affect outcomes in older patients one year after hospitalization in a geriatric ward. Patient samples were analyzed to identify specific MDRBs, including methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase-producing Enterobaceriaceae (ESBLE), and vancomycin-resistant enterococci (VRE). Among 337 patients screened at hospital admission, 62 (18%) carried one or more MDRB isolates (MRSA: n=23; ESBLE: n=39; VRE: n=2). At 12 months after admission, 320 patients were interviewed by telephone (17 patients lost to follow up) to assess all-cause mortality, nursing home admissions, functional decline, and hospital readmissions. All-cause mortality rates were similar in MDRB carriers (34%; n=61) and non-carriers (30%; n=259) (P=0.512). Nursing home admission, functional decline, and hospital readmission did not differ between the two groups. In this population, predictors of mortality were: male gender (P=0.002), cognitive disorders at admission (P=0.028), low pre-albumin level at admission (P=0.048), a high level of co-morbidities (P=0.002), and a history of an acute condition in the three months prior to initial hospital admission (P=0.024). In conclusion, in this cohort of older patients, asymptomatic MDRB colonization was not significantly associated with adverse health outcomes at a one-year follow-up after hospitalization. The potential limitations of the study were the small sample size, relatively high mortality rate, and lack of MDRB reassessment during the follow-up.

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BACKGROUND: Recommendations were applied before and during the Belgian pandemic (2009) H1N1 influenza wave at a university hospital (420 beds), for optimizing isolation processes and therapeutic management of possible and confirmed infected cases. METHODS: All patients presenting to the Emergency Department (ED) between August 1st and December 31st 2009 were screened for ILI symptoms, and were isolated for clinical assessment in case of positive screening. Patients categorized as possible influenza cases and who required hospitalization were isolated in dedicated wards. Specific diagnostic algorithms were implemented. Medical charts were retrospectively reviewed and matched with results of the microbiology laboratory. Patient's characteristics were analyzed, the contribution of laboratory diagnosis on therapy and lengh of stay (LOS) in isolation was also assessed. RESULTS: 310 patients out of 6068 had a positive screening for ILI, of these, 265 were retained as possible influenza cases and 139 required hospitalization. Twenty-eight children (8 requiring hospitalization) and 20 hospitalized adult patients had confirmed influenza infection. Five adult patients were admitted to the intensive care unit (ICU), 3 requiring extracorporeal membrane oxygenation (ECMO). There was no death related to the new influenza strain. The majority of confirmed patients were diagnosed during the Belgian epidemic wave, with a sensitivity of antigen detection of 50% in children and 35% in adults comparatively to real-time PCR (RT-PCR). CONCLUSIONS: The impact of (2009) H1N1 pandemic influenza remained limited, except for ICU patients requiring ECMO. Implementation of screening, isolation, and virological diagnosis processes led to significant improvement of patient management.

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 bla GES-12 and a likely chromosomally located novel variant bla GES-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 bla GES-12. Cloning of the blaGES genes in Escherichia coli 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. Copyright © 2010, American Society for Microbiology. All Rights Reserved., SCOPUS: ar.j, info:eu-repo/semantics/published

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

The aim of this study was to evaluate the performance of Brilliance(TM) ESBL Agar (OX; Oxoid, Basingstoke, United Kingdom), a novel chromogenic agar for the selective isolation and the presumptive identification of extended-spectrum beta-lactamases (ESBL)-producing Enterobacteriaceae. A panel of 200 clinical Gram-negative Enterobacteriaceae and non-fermenting isolates with defined resistance mechanisms was inoculated onto OX and onto ChromID(TM) ESBL Agar (BM; bioMérieux, Marcy l'Etoile, France) chromogenic medium in a first part of the study to evaluate the growth selectivity and the chromogenic features of these two media. Of the 156 Enterobacteriaceae challenge isolates, 8 fully susceptible isolates were inhibited, all 98 ESBL producers were detected and 50 isolates harboring other resistance mechanisms were recovered on both chromogenic agars. In a second phase, 528 clinical samples (including 344 fecal specimens) were plated onto OX, BM and MacConkey agar with a ceftazidime disk (MCC) for the screening of ESBL-producing Enterobacteriaceae. Growth on at least one medium was observed in 144 (27%) of the clinical samples screened. A total of 182 isolates including 109 (60%) Enterobacteriaceae were recovered and 70 of these (from 59 specimens) were confirmed as ESBL-producing isolates. The sensitivities were 74.6%, 94.9% and 94.9% for MCC, BM and OX respectively. The specificities by specimens reached 94.9%, 95.5% and 95.7% for MCC, BM and OX respectively when only colored colonies were considered on the two selective chromogenic media. The high negative predictive value (99.3%) found for OX suggests that this medium may constitute an excellent screening tool for rapid exclusion of patients not carrying ESBL producers.

During a PCR-based surveillance study of beta-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 PFGE analysis. Three different alleles (the plasmid-located blaGES-11 and blaGES-12 and a likely chromosomal-located novel variant blaGES-14) were detected as part of a class 1 integron also including 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 E. coli 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.

The objective of this prospective study was to assess the prevalence of Exophiala dermatitidis in respiratory secretions of patients with cystic fibrosis (CF) and to identify risk factors for its presence. The results of all cultures performed over a 2-year period in non lung-transplant patients in our CF clinic were included in the study. Samples consisted of sputum (whenever possible) or deep pharyngeal aspirate after a session of physiotherapy. Specimens were inoculated onto Sabouraud gentamicin-chloramphenicol agar (SGCA) medium (Becton-Dickinson) and incubated at 35°C for 2 days and then at ambient temperature (15-25°C) for 3 weeks. The whole study group included 154 patients (mean age ± SD: 18.5 y ± 11.69). E. dermatitidis was isolated from 58 specimens (2.8%) of nine patients (5.8%) out of total of 2065 cultures prepared during the study period. All E. dermatitidis culture-positive patients were pancreatic insufficient and ≥12 y of age. Almost all (8/9) were homozygous for the F508 del mutation. Aspergillus fumigatus colonization and genotype seemed to be predisposing factors. No other significant characteristic was identified in this group, either in terms of predominant bacterial pathogen or treatment. A distinct comparative study performed over 3 months in our laboratory revealed that the use of SGCA yielded identical isolation rates of E. dermatitidis as erythritol-chloramphenicol agar (ECA).

Objectives: Non-tuberculous mycobacteria (NTM) were pathogens of growing importance in non-paediatric cystic fibrosis (CF) patients. In our centre, the prevalence of NTM in these patients observed (0.5%) during the last decade was markedly inferior to those reported in the literature ranging from 6.5% to 24%. The aim of this study was to screen for NTM in adult patients in our centre with 3 different decontamination methods for mycobacterial cultures in order to determine whether the choice of the decontamination technique of samples may have an impact on their recovery in our centre. Methods: Between January and June 2009, consecutive sputum samples from adult patients with clinical suspicion of NTM infection (respiratory function degradation without other microbiologic explanation) were included in this study. 3 different decontamination protocols were used: N-acetyl-l-cysteine (NALC)-NaOH (DCT1), NALC-NaOH-oxalic acid 5% (DCT2) and DTT-Chlorhexidine 1% (DCT3). Decontaminated specimens were than cultured onto both solid Loewenstein-Jensen slants (LJ; BioRad) and liquid MGIT (BD) culture. A sample was considered NTM negative if no NTM isolate was recovered after a standard 42-day incubation protocol. Results: 36 sputum samples were collected from 26 adult patients during the study period. The median age of patients was 26 years. 88% of the patients received antimicrobial active against mycobacteria for more than 24 months before sputum collection (61% of the patients had fluoroquinolones and 88% had macrolides). No NTM was detected in the 36 specimens with none of the three decontamination techniques. Conclusions: Long-term use of macrolides and fluoroquinolones may be an explanation for the absence of recovery of NTM in these CF patients. Further studies in other CF centres prescribing similar antimicrobial treatment regimens are warranted to support this observation.

Objectives : Early detection of Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) infection in cystic fibrosis (CF) patients remains a challenge for the microbiology lab. The aim of this study was to compare commercial selective media to standard culture media for the recovery and the presumptive identification of PA and of SA directly from sputum collected in these patients. Methods: 67 sputa from 67 ambulatory CF patients were cultured onto 6 selective media including MacConkey agar (MC; home-made), Pseudomonas Isolation Agar (PABD; Becton-Dickinson fig 1), ChromID PA agar (PABM; bioMérieux – fig 2), a colistin aztreonam blood agar plate (CAP, Oxoid), CHROMagar SA medium (SABD; BD – fig 3) and ChromID SA agar (SABM; bioMérieux – fig 4). After up to 7 days of incubation at 35°C, colonies suggestive of PA or SA according to the plates reading guidelines provided by the manufacturers were selected for phenotypic identification. Isolates showing atypical testing results were further submitted for molecular identification by 16S rRNA sequencing. Conclusions : The use of specific selective media could increase the recovery of PA and SA isolates in CF respiratory samples and could lead to an earlier management and antimicrobial therapy for these colonized or infected patients. Nevertheless, the relatively low specificity of these media doesn’t exempt the microbiologist from usual confirmative test.

Campylobacter fetus is an opportunist Gram-negative bacillus. The most frequent clinical manifestation is bacteriemia but it can also be responsable for soft tissue infections, endovascular infections, meningitis, peritonitis and thrombophlebitis. Campylobacter fetus cellulitis has been described, but rarely identified in subcutaneous puncture samples. We report a case of an immunocompromised patient with Campylobacter fetus bacteriemia associated with a soft tissue infection whose subcutaneous puncture also revealed the bacteria.

Backgrounds and aims: Extended-spectrum beta-lactamases (ESBL) producing gram-negative bacteria are isolated with increasing frequency in pediatric populations. We report here for the first time in Belgium the emergence of Enterobacteriaceae carrying GES-type ESBL enzymes in a pediatric ward. Methods: All patients admitted to this 14-bed ward mainly occupied by children hospitalized for liver transplantation were screened for multi-resistant gram negative bacteria in stools additionally to clinical samples. Identification and susceptibility testing of isolates were performed by BD Phoenix® automate and ESBL production was confirmed by double discs synergy test using Rosco® tablets. All phenotypic ESBL-producing strains were characterized by PCR targeting various ESBL genes. Results: From January 2007 to January 2009, of the 2424 samples (562 stools) collected in 520 children, 50 ESBL-producing isolates (prevalence of 9.5%) were recovered including 10 GES-producing strains from 7 patients (6 fecal carriers). The GES ESBLs were found in 6 different Enterobacteriaceae species (two patients were colonized by more than one species) ; all isolates displayed identical resistance patterns. All patients but one were admitted for liver transplantation and originated from different countries of Eastern Europe. Patients and their families also stayed in the same hospital linked residency. Conclusions: The diversity of the species, the multiple origins of the patients and the local clustering together highly suggest both cross-transmission and horizontal gene transfer of these GES plasmid-borne ESBL enzymes. Although no infection was diagnosed, GES ESBL-producing isolates may lead to outbreaks and represent a threat for fragile immunosuppressed patients such as liver transplanted children.

Background: Pseudomonas aeruginosa has been described as an uncommon cause of endocarditis in adults but to our knowledge no pediatric case has been reported in the literature for the past 20 years. Clinical case: We describe a 3-year-old boy diagnosed with acute lymphoblastic leukemia who first developed a fully susceptible Pseudomonas aeruginosa bacteremia while he was in neutropenia. An initial antibiotherapy was started using piperacillintazobactam and amikacin. Recurrent bacteremias occurred during the following 101 days despite central venous access device replacements and multiple courses of antibiotics, including meropenem, ceftazidime, ciprofloxacin, rifampicin and colimycin. Several cardiac ultrasound were performed but evidence of endocarditis was observed only after 60 days of bacteremia when both transoesophageal and transthoracic ultrasound showed the rupture of a tricuspid cord and the presence of a millimetric image on the atrial side of the tricuspid valve. Due to failure of medical treatment, cardiac surgery was performed. Peroperative findings confirmed a purulent granuloma and perforations on the tricuspid valve.The abcess was removed and the valve repaired. The valvular biopsy yielded Pseudomonas aeruginosa. Antibiotics were extended for 30 days after surgery with no further positive blood cultures at 5 months of follow-up. Conclusion: Pseudomonas aeruginosa is an exceptional cause of endocarditis in children. Prompt removal of central venous device should be performed in case of Pseudomonas aeruginosa bacteremia. Surgical intervention must be considered without delay if not responding to targeted antibiotics, particularly in leukemic patients with a greater risk of negative impact on their hematological outcome.

The genetic environment of the blaOXA-18 geneencoding a peculiar clavulanic acid-inhibited Ambler class D extended-spectrum beta-lactamase was determined from the prototype OXA-18-producing Pseudomonas aeruginosa MUS clinical isolate. An 8.2-kb genomic DNA fragment containing blaOXA-18 gene was cloned from P. aeruginosa MUS. While most oxacillinases are integron-located, blaOXA-18 lacked gene cassette specific features. It was bracketed by two duplicated sequences, containing ISCR19, a novel insertion sequence of the ISCR family of mobile elements, DeltaintI1, a truncated integrase gene and, a truncated Deltaaac6' Ib gene cassette. It is likely that ISCR19 was at the origin of the blaOXA-18 gene mobilization by a Rolling Circle (RC) transposition event followed by homologous recombination. Furthermore, analysis of the cloned genomic DNA fragment revealed the presence of the integron-containing blaOXA-20 gene. Concommitantly, three P. aeruginosa clinical isolates, displaying a synergy image by double-disk diffusion tests on cloxacillin-containing plates, were isolated from three patients hospitalized in different wards over a nine-month period at the Saint-Luc University hospital (Brussels, Belgium). These isolates were positive by PCR for blaOXA-18 andblaOXA-20 genes, genetically related to P. aeruginosa MUS by PFGE and carried the same blaOXA-18/blaOXA-20 associated genetic structures. This report characterized the genetic elements likely at the origin of blaOXA-18 gene mobilization in P. aeruginosa and suggests the spread of this type of OXA-ESBL in P. aeruginosa in the Saint-Luc University hospital of Brussels, Belgium.

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

Introduction: Through their oxidative metabolism, bacteria reduce the redox potential of their environment. It is thus possible to detect a bacterial growth by measuring the redox potential of the culture broth. Methods: We have determined the oxydoreduction curves (shape and kinetics) of different pathogens at serial initial concentrations. Material: We have used basic potentiometers associated to combined redox electrodes with an Ag/AgCl reference. Repeated measurements have been performed on a strirred Tryptic Soya broth maintained at 37°C. Results: We have shown that there is a linear correlation between the initial germs concentration and the time needed to observe a diminution of the potential (in mV). Additionally, the shape and kinetics of the curves are reproductible and specific to the microbial species. We have equally studied the effect of different concentrations of antibiotics on the variation of the redox potential. Conclusions: These preliminary studies show that there is a big “potential” for the use of electrochemistry for qualitative and quantitative diagnosis of a bacterial infection and its associated antibiotics resistances. If proven implementable, this technique would offer the consequent advantages of being low-cost and faster than actual diagnosis methods.

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.

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