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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.

Optimising antibiotic administration is critical when dealing with pathogens with reduced susceptibility. Vancomycin activity is dependent on the area under the concentration-time curve over 24 h at steady-state divided by the minimum inhibitory concentration (AUC/MIC), making continuous infusion (CI) or conventional twice daily administration pharmacodynamically equipotent. Because CI facilitates drug administration and serum level monitoring, we have implemented a protocol for CI of vancomycin by: (i) examining whether maintaining stable serum concentrations (set at 25-30 mg/L based on local susceptibility data of Gram-positive target organisms) can be achieved in patients suffering from difficult-to-treat infections; (ii) assessing toxicity (n = 94) and overall efficacy (n = 59); and (iii) examining the correlation between AUC/MIC and the clinical outcome in patients for whom vancomycin was the only active agent against a single causative pathogen (n = 20). Stable serum levels at the expected target were obtained at the population level (loading dose 20mg/kg; infusion of 2.57 g/24 h adjusted for creatinine clearance) for up to 44 days, but large intrapatient variations required frequent dose re-adjustments (increase in 57% and decrease in 16% of the total population). Recursive partitioning analysis of AUC/MIC ratios versus success or failure suggested threshold values of 667 (total serum level) and 451 (free serum level), corresponding to organisms with a MIC>1 mg/L. Nephrotoxicity potentially related to vancomycin was observed in 10% of patients, but treatment had to be discontinued in only two of them.

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.

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.

Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae are a major public health concern. We previously demonstrated the presence of ESBL-producing Enterobacteriaceae in sachet-packaged water bags sold in Kinshasa, the Democratic Republic of the Congo. In complement to the previous study, we aimed to assess the presence of ESBL-producing Enterobacteriaceae in waste waters in Kinshasa.Enterobacteriaceae isolates recovered from environmental water samples were screened and phenotypically confirmed as ESBL-producers by disk diffusion according to Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI M100-S21). Final identification to the species level and further antimicrobial susceptibility testing were carried out with MicroScan® NBC42 panels and the identification of bla (ESBL) coding genes was performed by a commercial multiplex ligation polymerase chain reaction (PCR) microarray (Check-Points CT 101, Wageningen, the Netherlands). Overall, 194 non-duplicate Enterobacteriaceae were recovered from several sewer and river sites in nine out of 24 municipalities of Kinshasa. Fourteen isolates (7.4 %) were confirmed as ESBL-producers, the main species being Enterobacter cloacae (46.6 %) and Klebsiella pneumoniae (40.0 %). Associated resistance to both aminoglycoside and fluoroquinolone antibiotics was observed in ten isolates; the remaining isolates showed co-resistance to either fluoroquinolone (n = 3) or to aminoglycoside (n = 1) alone. All but one isolate carried bla (CTX-M) genes belonging to the CTX-M-1 group. ESBL-producing Enterobacteriaceae are increasingly being reported from various sources in the community. The present results suggest that ESBL-producing Enterobacteriaceae are widespread in the environment in the community of Kinshasa. Cities in Central Africa should be added to the map of potentially ESBL-contaminated environments and highlight the need to reinforce safe water supply and public sanitation.

Frequent contact with human waste and liquid manure from intensive livestock breeding, and the increased loads of antibiotic-resistant bacteria that result, are believed to be responsible for the high carriage rates of ESBL-producing E. coli found in birds of prey (raptors) in Central Europe. To test this hypothesis against the influence of avian migration, we initiated a comparative analysis of faecal samples from wild birds found in Saxony-Anhalt in Germany and the Gobi-Desert in Mongolia, regions of dissimilar human and livestock population characteristics and agricultural practices. We sampled a total of 281 wild birds, mostly raptors with primarily north-to-south migration routes. We determined antimicrobial resistance, focusing on ESBL production, and unravelled the phylogenetic and clonal relatedness of identified ESBL-producing E. coli isolates using multi-locus sequence typing (MLST) and macrorestriction analyses. Surprisingly, the overall carriage rates (approximately 5%) and the proportion of ESBL-producers among E. coli (Germany: 13.8%, Mongolia: 10.8%) were similar in both regions. Whereas blaCTX-M-1 predominated among German isolates (100%), blaCTX-M-9 was the most prevalent in Mongolian isolates (75%). We identified sequence types (STs) that are well known in human and veterinary clinical ESBL-producing E. coli (ST12, ST117, ST167, ST648) and observed clonal relatedness between a Mongolian avian ESBL-E. coli (ST167) and a clinical isolate of the same ST that originated in a hospitalised patient in Europe. Our data suggest the influence of avian migratory species in the transmission of ESBL-producing E. coli and challenge the prevailing assumption that reducing human influence alone invariably leads to lower rates of antimicrobial resistance. © 2012 Guenther et al.

In this retrospective study, the clinical profiles of patients carrying healthcare-associated ESBL-producing Enterobacteriacae is characterized by a high prevalence rate of several major co-morbidities and potential known risk factors. Both, the length of hospital stay and overall hospital mortality rates were particularly high. A prospective case-control matched study should be designed and performed in order to control for possible inclusion bias.

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.

The susceptibilities of 824 Bacteroides fragilis group isolates against nine antibiotics were evaluated in a Europe-wide study involving 13 countries. Species determination, by different methods, was carried out on all but one isolate. Resistance rates were evaluated according to species and geographical areas via CLSI and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints. The present data were compared with those obtained 10 and 20 years ago at a European level. High-level resistance (MIC ≥ 64 mg/L) to ampicillin was observed in 44.5% of the strains, which is a significant increase relative to 20 years ago (16%). Piperacillin/tazobactam was more active than amoxicillin/clavulanic acid (3.1% and 10.4% resistance, respectively), again with a resistance increase relative to earlier studies. Dramatic increases in resistance were observed for cefoxitin, clindamycin and moxifloxacin, with rates of 17.2%, 32.4% and 13.6%, respectively. The lowest resistances were found for imipenem, metronidazole and tigecycline (1.2%, <1% and 1.7%). Nonsusceptible strains to imipenem and metronidazole were more resistant to other anti-anaerobic drugs. Differences were detected between geographical areas, with higher resistance rates for moxifloxacin in Scandinavian countries (21.4%) than in Mediterranean countries (5.4%), whereas, for clindamycin, the resistance rates were higher in Mediterranean (41.8%) and lower in Scandinavian countries (22.5%). Piperacillin/tazobactam resistance was also higher in Scandinavian countries.

Age-related changes of gene expression contribute to the physiological alteration observed with human ageing. Herein the abundance of a selection of 148 transcripts involved in immunosenescence and stress response was compared in total RNA of PBMC of healthy young to middle-age probands (35.0 +/- 6.5 year old) and healthy old probands (82.5 +/- 6.8 year old). This study provides a list of 16 differentially abundant transcripts species in the healthy old probands. Thus these changes of abundance can be considered as easily accessible biomarkers of ageing. Some of these differential abundances like CD28, CD69, LCK (decreased abundance in old subjects), CD86, cathepsin D, H and S (increased abundance in old subjects) might explain biochemical and cytochemical changes observed at the protein level in the immune system and thus might correspond to regulatory processes affecting the ageing process. Indeed these changes reflect the low-grade proinflammatory status observed in old persons and suggest a hypo-responsiveness of T-cells together with an increase in antigen presentation potential. In addition, among the differentially abundant transcripts were transcripts involved in the oxidative stress response HMOX1 and HSPA6 mRNAs were found as more abundant in PBMC from elderly subjects.

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.

Rapid and reliable detection of methicillin-resistant Staphylococcus aureus (MRSA) carriers is crucial for control of MRSA nosocomial transmission. We aimed to evaluate the performance of the GeneXpert real-time PCR system using the Xpert MRSA assay on a collection of 40 representative Belgian MRSA strains and for MRSA screening of geriatric inpatients. Double nasal swabs were used: the first swab for the Xpert MRSA assay and the second for culture onto chromogenic selective medium and enrichment broth. All but 1 of the 40 collection strains were recognized as MRSA by the Xpert MRSA assay. Nares swabs were prospectively collected from 246 inpatients including 25 nasal MRSA carriers. Compared with enriched cultures, the sensitivity, the specificity, and the positive and negative predictive values of the Xpert MRSA assay were 69.2%, 97.7%, 78.3%, and 96.3% respectively. The 7 evaluable false-negative results according to the assay were due to its possible lack of sensitivity (n = 3) and to the occurrence of a Belgian MRSA clone carrying a particular staphylococcal chromosomal cassette mec (SCCmec) type IV variant (n = 4) not targeted by the current Xpert MRSA assay. Because of the evolution of SCCmec in MRSA, new primers should be designed and further studies are warranted to ensure continuous monitoring of this assay.

Lyme neuroborreliosis (LNB) represents the second most frequent manifestation of Lyme disease (LD) in Europe after cutaneous involvement. In the USA, LNB represents the third most frequent manifestation of LD after cutaneous involvement and arthritis. The scope of this article is, in the light of recent publications, to review the specific manifestations of LNB in children including predictive models, and to discuss diagnosis criteria, new diagnostic tools and new therapeutic options. Differences in disease patterns between the USA and Europe are also highlighted.

We have investigated the antimicrobial resistance of verocytotoxin-producing Escherichia coli (VTEC) strains isolated from humans, animals, food and environment in Belgium. Resistance was more frequent in human non-O157 as compared to O157 strains from humans or other sources and within non-O157 VTEC, intimin-positive strains were more resistant than intimin-negative strains. We also report the first VTEC producing an IncI1 plasmid-borne blaTEM-52 extended-spectrum beta-lactamase previously associated with Salmonella enterica and E. coli isolates from different origins.

Objectives Treatment of chronic or recurrent Staphylococcus aureus infections may require using antibiotics with activity against intracellular multiresistant organisms. Quinupristin/dalfopristin (3:7) has been examined in this context. Methods Quinupristin and dalfopristin were used separately or mixed. Strains used were: (i) methicillin-susceptible and -resistant S. aureus (MSSA and MRSA); (ii) one vat(B) MSSA and msr(A/B) MRSA; (iii) erm(A)(+) [MSSA, MRSA, vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA)]; and (iv) one erm(A/B)(+) cfr(+) MRSA resistant to quinupristin, dalfopristin and their combination. Assessment of activity was determined by: (i) MICs (CLSI method); and (ii) concentration-response curves in broth and after phagocytosis by THP-1 macrophages, with descriptors of the model (E(min)) and the pharmacodynamic response [maximal relative efficacy (E(max)), relative potency (EC(50)) and apparent static concentration (C(static))]. Results erm(A)-positive strains were all susceptible to quinupristin/dalfopristin (except strain CM05), with MICs not adversely influenced by acid pH or by the MRSA, VISA or VRSA character of the strain. In concentration-response experiments, quinupristin/dalfopristin showed similar patterns for all strains (except strain CM05), with a >3 log(10) cfu decrease in broth and a 1.3 [erm(A) strain] to 2.6 [fully susceptible, vat(B) and msr(A/B) strains] log(10) cfu decrease for intracellular bacteria at the maximal extracellular concentration tested (25 mg/L). Maximal extracellular and intracellular activity was obtained for a quinupristin/dalfopristin ratio of 3:7. For strain CM05, quinupristin/dalfopristin was static in all conditions. Conclusions Based on historical comparisons with rifampicin, fluoroquinolones, lipoglycopeptides and other antistaphylococcal drugs with a large accumulation in eukaryotic cells, quinupristin/dalfopristin appears to be one of the most active antibiotics against intr

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.

Pseudomonas aeruginosa causes severe nosocomial pneumonia in Intensive Care Unit (ICU) patients, with an increased prevalence of multiresistant strains. We examined the impact of the use of antipseudomonal antibiotic(s) on the susceptibility of P. aeruginosa isolated from ICU patients with clinically suspected hospital-acquired pneumonia collected in five teaching hospitals (110 non-duplicate initial isolates; 62 clonal pairs of initial and last isolates during treatment). Minimum inhibitory concentrations (MICs) were determined for amikacin, ciprofloxacin, meropenem, piperacillin/tazobactam (TZP), cefepime and ceftazidime (used in therapy) as well as five reporter antibiotics (aztreonam, colistin, gentamicin, piperacillin and ticarcillin) using Clinical and Laboratory Standards Institute (CLSI) methodology. Susceptibility was assessed according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) and CLSI breakpoints. Resistance rates prior to treatment exceeded 25% for cefepime, ceftazidime, piperacillin, ticarcillin and aztreonam (EUCAST and CLSI) and for gentamicin, TZP and colistin (EUCAST only). The highest rates of cross-resistance were noted for ceftazidime and cefepime and the lowest rate for amikacin. Mean MIC values were systematically higher in isolates from patients previously exposed (1 month) to the corresponding antibiotic. For clonal pairs, a systematic increase in MIC between initial and last isolates (significant for amikacin, cefepime, meropenem and TZP) was noted. There was a significant correlation between the use of antibiotics (adjusted for respective proportional use of each drug) and loss of susceptibility at the population level when using EUCAST breakpoints. The high level of resistance of P. aeruginosa in ICU patients with nosocomial pneumonia as well as its further increase during treatment severely narrows the already limited therapeutic options. Further observational studies and the development of early diagnosis for

Objectives. - The aim of the study was to evaluate the antibiotic resistance in noninvasive clinical isolates of Streptococcus pneumoniae collected in Belgium during winter 2008-2007. Method. - Four hundred and forty eight unduplicated isolates collected by 15 laboratories were tested by microdilution following CLSI. Results. - Insusceptibility rates (I + R) were as follows: penicillin G (PEN) 11.6% (4.0% R), ampicillin 11.4% (4.0% R), amoxicillin + /-clavulanic acid 0, cefaclor 10.3% (9.6% R), cefuroxime 9.2% (8.7% R), cefuroxime-axetil 8.7% (7.8% R), cefotaxime, ceftazidime and cefepime 2.0% (0% R), imipenem 2.5% (0% R), ciprofloxacin and ofloxacin 5.1% (0.4% R), levofloxacin 0.7% (0.4% R), moxifloxacin 0.4% (0.2% R), erythromycin (ERY) 29.7% (29.2% R), azithromycin 29.7% (28.8% R), telithromycin 0%, clindamycin 26.3% (25.4% R) and tetracycline (TET) 21.9% (16.5% R). From 2001 to 2008, a significant decrease in penicillinin-susceptibility (21.0% to 11.6%). penicillin-resistance (9.7% to 4.0%) and ciprofloxacin-insusceptibility (11.2% to 5.1%) was found. Cross-resistance between penicillin and other betalactams in penicillin-insusceptible isolates was incomplete: all these isolates remained fully susceptible to amoxicillin. Erythromycin-insusceptibility was significantly higher in children than in adults (43.9%/27.4%), while penicillin-insusceptibility significantly higher in Brussels than in the Flanders (22.9%/8.1%). The commonest resistance phenotype was ERY-TET (12.7%) followed by ERY (7.4%) and PEN-ERY-TET (5.8%). Capsular types 19(25%), 14(19.3%), 23 (15.4%) and 15 (13.5%) were the most important in penicillin-insusceptible. Conclusion. - We noted a decrease in resistance to the majority of the compounds. Insusceptibility rates were higher in children than in adults and the difference between the north and the south of Belgium became less marked. (C) 2009 Elsevier Masson SAS. All rights reserved.

Biomarkers indicating biological age are of significant interest for prevention, diagnosis and monitoring (and the treatment) of age-related diseases. We previously reported an alteration of serum N-glycan profile in old humans using "DNA Sequencer Adapted-Fluorophore Assisted Carbohydrate Electrophoresis" (DSA-FACE). To validate the shift in serum N-glycan profile during ageing, we studied serum N-glycan profiles in different age groups of healthy volunteers, patients with dementia, and patients with Cockayne syndrome, a genetic DNA repair disorder involving neurodegeneration and premature ageing. We found that the log of the ratio of two glycans (NGA2F and NA2F), named GlycoAgeTest, remained steady up to the age of 40years and thereafter gradually increased to reach its highest level in nonagenarians. Patients with dementia or Cockayne syndrome had a higher GlycoAgeTest level than age-matched healthy individuals. We thus demonstrate that the value of GlycoAgeTest is better than chronological age for estimating the physiological age of a human individual, and that it could be used as an ageing biomarker for healthy humans. Our data indicate that the GlycoAgeTest could be used as a non-invasive surrogate marker for general health, for forecasting disease progression during ageing, and for monitoring the efficacy of anti-ageing food compounds.

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.

BACKGROUND:: A prediction model based on clinical and cerebrospinal fluid (CSF) analysis has been proposed for the differentiation of Lyme meningitis (LM) from non-Lyme aseptic meningitis (NLAM) in the United States. No similar model has ever been proposed for European patients. The objective of our study was to develop a prediction model to differentiate LM from NLAM based on clinical and CSF biologic data. METHODS:: The medical charts of all children older than 2 years of age admitted to our hospital from 1996 through 2006 with a definite diagnosis of LM were analyzed and compared retrospectively with those having a diagnosis of NLAM. Chart review included the duration of symptoms, the presence of cranial neuropathy, and CSF analysis. RESULTS:: A total of 93 patients were included (LM: 26 patients; NLAM: 67 patients) in the study. Patients with LM had statistically more frequent cranial neuropathy (73% vs. 4%), displayed a longer duration of symptoms before admission (8.8 vs. 1.8 days), had a higher CSF protein (71 vs. 38 mg/d), and had a lower percentage of neutrophil cells in the CSF (3.4% vs. 51%) than patients with NLAM. A predicted probability was derived from these 4 variables. At a cutoff point of >0.432, the model had a negative predictive value of 100% and a positive predictive value of 92.3%, with a sensitivity of 100% and a specificity of 97%. CONCLUSIONS:: We report the first European prediction model for LM. Owing to its high negative predictive value, this model may assist physicians in managing aseptic meningitis (AM) while awaiting serologic tests, especially in Lyme endemic regions.

S. aureus is an opportunistic intracellular organism. Although poorly accumulating in eukaryotic cells, beta-lactams show activity against intracellular MSSA if exposure times and drug concentrations are sufficient. Intraphagocytic MRSA are susceptible to penicillins and carbapenems because acidic pH favors the acylation of PBP 2a by these beta-lactams through pH-induced conformational change. Ceftobiprole, showing almost similar in vitro activities against MRSA and MSSA in broth, was examined for intracellular activity (THP-1 macrophages, keratinocytes) against a panel of hospital-acquired and community-acquired MRSA (MICs: 0.5-2.0 mg/L at pH 7.4 and 0.25-1.0 mg/L at pH 5.5) vs. MSSA isolates, with measurement of the key pharmacological descriptors of relative efficacy (Emax), relative potency (E50), and static concentration (Cs). All strains showed sigmoidal dose-responses with Emax at about 1 log10 CFU decrease from post-phagocytosis inoculum, and E50 and Cs at 0.2 to 0.3 x and 0.6 to 0.9 x the MIC, respectively. Ceftobiprole effectively competed with Bocillin FL (a fluorescent derivative of penicillin V) for binding to PBP 2a at both pH 5.5 and 7.4. In contrast, cephalexin, cefuroxime, cefoxitin, or ceftriaxone (i) were less potent in PBP 2a competitive binding assays; (ii) showed only partial restoration of activity towards MRSA in broth at acidic pH; (iii) were collectively less effective against MRSA in THP-1 macrophages and ineffective in keratinocytes. The improved activity of ceftobiprole towards intracellular MRSA vs. conventional cephalosporins, can be explained, at least in part, by its greater ability to bind to PBP 2a not only at neutral but also at acidic pH.

Mycobacterium interjectum is a rare causative agent of cervical lymphadenitis. We describe a 2-year-old girl with suspected tuberculous cervical lymphadenitis. Sequencing of the 16S rRNA gene allowed the correct identification of Mycobacterium interjectum. As yet, only nine case reports of infections due to M. interjectum in children have been reported in the literature, and in all of them a correct identification could only be obtained using gene sequencing.

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.

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.

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.

S. aureus invades eukaryotic cells. MRSA ATCC 33591 phagocytized by human THP-1 macrophages shows complete restoration of susceptibility to cloxacillin and meropenem, becoming indistinguishable from MSSA ATCC 25923 due to the acid pH prevailing in phagolysosomes (Lemaire et al., Antimicrob. Agents Chemother. 51:1627-32, 2007). We have examined whether this observation can be extended to (i) strains of current clinical and epidemiological interest (3 HA-MRSA; 2 CA-MRSA; 2 HA-MRSA with VISA phenotype; 1 HA-MRSA with VRSA phenotype; 1 animal [porcine] MRSA); (ii) activated THP-1 cells and non-professional phagocytes (keratinocytes, CALU-3 bronchial epithelial cells); (iii) other beta-lactams (imipenem, oxacillin, cefuroxime, cefepime). All strains showed (i) a marked reduction of MIC in broth at pH 5.5 vs. pH 7.4; (ii) sigmoidal dose-response curves to cloxacillin (0.01 to 100 MIC; 24 h incubation) after phagocytosis by THP-1 macrophages, indistinguishable from each other and from that of MSSA ATCC 25923 (relative potency [E50], 6.09 x MIC; CI: 4.50 to 8.25); relative efficacy [Emax] -0. 69 log cfu; CI: -0.79 to -0.58). Similar dose-response curves to cloxacillin were also observed for MSSA ATCC 25923 and MRSA ATCC 33591 after phagocytosis by activated THP-1 macrophages, keratinocytes, and Calu-3 cells. By contrast, there was a lesser restoration of susceptibility of MRSA ATCC 33591 to cefuroxime and cefepime after phagocytosis by THP-1 macrophages, even when data were normalized for differences in MICs. We conclude that the restoration of MRSA susceptibility to beta-lactams after phagocytosis is independent of the strain and the type of cells, but varies between beta-lactams.

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). ...

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.

Ecthyma gangrenosum is a cutaneous infection most commonly associated with Pseudomonas aeruginosa bacteraemia, but it may also be encountered after breakdown of mechanical defence barriers and local infection. The characteristic lesions of ecthyma gangrenosum are haemorrhagic blisters with surrounding erythema that rapidly evolve to necrotic ulcers. A high mortality rate is reported, especially when diagnosis is delayed and in the absence of appropriate therapy. Ecthyma gangrenosum usually occurs in critically ill and immunocompromised patients. Haematological malignancies and neutropaenia are the major risk factors, but other comorbidities (e.g. diabetes mellitus and malnutrition) have also been involved. Here, we report a case of ecthyma gangrenosum in a non-neutropaenic elderly patient with multiple comorbidities and review the literature on ecthyma gangrenosum.