Vanessa Vankerckhoven

Development of a Multiplex PCR for the Detection of asa1, gelE, cylA, esp, and hyl Genes in Enterococci and Survey for Virulence Determinants among European Hospital Isolates of Enterococcus faecium

ABSTRACT A multiplex PCR for the simultaneous detection of five virulence genes (asa1, gelE, cylA, esp, and hyl) in enterococci was developed. The presence of these genes was investigated in 153 clinical and 118 fecal Enterococcus faecium isolates from inpatients at an increased risk of developing infections (such as patients in intensive care units and hematology wards) from 13 hospitals in eight European countries. Of the 271 E. faecium isolates, 135 were vancomycin resistant E. faecium (VREF) isolates and 136 were vancomycin susceptible E. faecium (VSEF) isolates. Susceptibilities to ampicillin, gentamicin, streptomycin, vancomycin, teicoplanin, ramoplanin, quinupristin-dalfopristin, and linezolid were tested by the microdilution method. Overall, the prevalence of esp was significantly higher (P = 0.03) in clinical VREF isolates (92%) than in fecal VREF isolates (73%). In Italy, the prevalence of esp was significantly higher (P = 0.02) in VREF isolates (91%) than in VSEF isolates (68%), whereas in the United Kingdom, hyl was significantly more prevalent (P = 0.01) in VREF isolates (71%) than in VSEF isolates (29%). No significant differences were found for the other countries. Pulsed-field gel electrophoresis was used to check the clonality among the strains tested and showed the spread of two center-specific (esp-positive) VREF clones in Italy and one center-specific (hyl-positive) clone in the United Kingdom. These clones were resistant to ampicillin, gentamicin, and streptomycin. The multiplex PCR reported in this study is a convenient and rapid method for the simultaneous detection of the virulence genes asa1, gelE, cylA, esp, and hyl in enterococci. Molecular analysis showed the intrahospital spread of esp-positive VREF clones (in Italy) and hyl-positive VREF clones (in the United Kingdom); the role of hyl remains to be elucidated.

Differences in gut microbiota composition between obese and lean children: a cross-sectional study.

BackgroundAn altered gut microbiota composition has recently been linked to obesity. The principal aim of this study is to investigate and compare the gut microbiota composition in obese and lean children. Secondly, associations between analysed gut bacterial species, dietary compounds, energy intake and biochemical blood parameters are evaluated.MethodsIn this prospective cross-sectional study, 26 overweight/obese (mean BMI: 28.7 ± 6.5) and 27 lean (mean BMI: 16.5 ± 2.1) children aged 6 to 16 were included. Faecal samples were collected and subjected to selective plating and quantitative real-time PCR (qPCR) in order to determine the concentrations of bacterial species belonging to the genera: Bacteroides, Bifidobacterium, Clostridium, Staphylococcus and Lactobacillus. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was applied for an in-depth identification of species of Bacteroides fragilis group. Differences in the concentrations of gut bacterial species between obese and lean children were statistically analysed using Mann Whitney U test. Subsequently, random forest analysis and multiple linear regression analysis were performed in order to test associations between gut bacterial species, dietary compounds and blood parameters.ResultsObese children showed an elevated Firmicutes-to-Bacteroidetes ratio compared with lean children. Furthermore, low relative proportions of B. vulgatus and high concentrations of Lactobacillus spp. were observed in the obese microbiota. In all children, Staphylococcus spp. were positively associated with energy intake. Additionally, in obese children, Lactobacillus spp. were positively associated with plasma hs-CRP.ConclusionsOur findings corroborate a significant difference in the gut microbiota composition of important bacterial species between obese and lean children. In future, non-invasive manipulation of gut microbiota composition in early infancy could offer a new approach to manage childhood obesity and associated disorders.

European Surveillance of Antimicrobial Consumption (ESAC): quality appraisal of antibiotic use in Europe

OBJECTIVES To assess quality of outpatient antibiotic use in Europe in 2009 based on the 12 European Surveillance of Antimicrobial Consumption (ESAC) drug-specific quality indicators and to evaluate changes in quality between 2004 and 2009. METHODS Quality of outpatient antibiotic use in 2009 was compared between 32 countries by calculating the indicator values for 2009 for each of the 12 ESAC drug-specific quality indicators based on outpatient antibiotic use data expressed in defined daily doses per 1000 inhabitants per day (DID). For each of the indicators we grouped the 2009 indicator values into four quartiles. To evaluate changes in quality between 2004 and 2009, based on their respective indicator values, countries were also grouped according to the quartile distribution of the 2004 indicator values. Only countries able to deliver data for both years were included in this analysis. RESULTS In 2009 a difference in the quality of outpatient antibiotic use between Nordic and Southern European countries was observed. Quality of outpatient antibiotic use decreased between 2004 and 2009. In particular, there were increases in the quality indicators [J01F_DID], [J01M_DID], [J01CR_%] and [J01_B/N], i.e. the use of macrolides, lincosamides and streptogramins in DID, the use of quinolones in DID, the proportional use of combinations of penicillins, including β-lactamase inhibitors and the ratio of broad- to narrow-spectrum antibiotics. CONCLUSIONS Quality of outpatient antibiotic use in DID decreased between 2004 and 2009. A continuous effort to improve outpatient antibiotic consumption seems to be essential to reduce outpatient antibiotic use in general and the use of broad-spectrum antibiotics in particular.

European Surveillance of Antibiotic Consumption (ESAC) point prevalence survey 2008: paediatric antimicrobial prescribing in 32 hospitals of 21 European countries.

BACKGROUND Antimicrobials are the most common medicines prescribed to children, but very little is known about patterns of hospital paediatric antimicrobial prescribing. This study aimed at describing paediatric antimicrobial prescribing in European hospitals to identify targets for quality improvement. METHODS The European Surveillance of Antibiotic Consumption (ESAC) project (www.esac.ua.ac.be) collected data during 2 calendar weeks between May and June 2008 in 32 hospitals of 21 European countries with paediatric departments, using a standardized method. The ESAC point prevalence survey included all inpatient beds and identified all patients who were receiving systemic antimicrobials on the day of the survey or had received antimicrobial surgical prophylaxis on the previous day. RESULTS Of 1799 children, 583 (32%) received one or more antimicrobials (range 17%-100%). The indications were therapeutic in 71%, prophylactic in 26% and both indications in 3% of patients. The parenteral route was used in 82% of therapeutic indications and in 63% of prophylactic indications. Third-generation cephalosporins were the most prescribed antimicrobials for therapeutic indications (18%). A high proportion of treated children received antimicrobial combinations (37%). The most commonly treated diagnosis site was the respiratory tract for both therapeutic use (30%) and prophylaxis (25%). The duration of surgical prophylaxis was >1 day in 67%. CONCLUSIONS Targets identified for quality improvement of antimicrobial use in children included excessive use of antimicrobial combinations and a high proportion of parenteral antimicrobials, both of which require further investigation. Surgical prophylaxis for >1 day should also be curbed in order to achieve quality improvement.

European Surveillance of Antimicrobial Consumption (ESAC): outpatient parenteral antibiotic treatment in Europe

OBJECTIVES To assess the proportion of parenteral treatment of the total outpatient antibiotic use in Europe, and to identify the antibiotic groups and individual antibiotics most commonly administered in this way. METHODS Within the European Surveillance of Antimicrobial Consumption (ESAC; www.esac.ua.ac.be), using the anatomic therapeutic chemical (ATC) and defined daily dose (DDD) classification, data on outpatient use of antibacterials for systemic use (ATC J01), aggregated at the level of the active substance and expressed in DDD per 1000 inhabitants per day (DID; WHO version 2007), were extracted for 2006 by route of administration and by country. Parenteral use was expressed as a percentage of the total outpatient use in DID. RESULTS In 20 European countries, the total outpatient antibiotic use ranged from 27.91 DID in France to 9.58 DID in Russia. The proportion of outpatient parenteral antibiotic treatment ranged from 6.75% in Russia to 0.001% in Iceland. The three most commonly used antibiotic groups for parenteral treatment in Europe were the cephalosporins (J01D; 44.58%), the aminoglycosides (J01G; 25.27%) and the penicillins (J01C; 17.78%). Four antibiotics [gentamicin (J01GB03) 18.53%; ceftriaxone (J01DD04) 17.85%; cefazolin (J01DB04) 13.16%; and lincomycin (J01FF02) 5.47%] represented more than half of the use. CONCLUSIONS In all 20 European countries studied together, 2.04% of outpatient antibiotics were used for parenteral treatment. However, as for the total outpatient antibiotic use and the use of different antibiotic groups and antibiotics, there is a striking variation in the proportions of parenteral antibiotic use in Europe. More in-depth data on outpatient antibiotic use are needed to explain this variation.

Genotypic Diversity, Antimicrobial Resistance, and Virulence Factors of Human Isolates and Probiotic Cultures Constituting Two Intraspecific Groups of Enterococcus faecium Isolates

ABSTRACT The intraspecific relationships among a collection of Enterococcus faecium isolates comprising probiotic cultures and human clinical isolates were investigated through the combined use of two high-resolution DNA-fingerprinting techniques. In addition, the incidences of antimicrobial resistance and virulence traits were investigated. A total of 128 E. faecium isolates from human clinical or nonclinical sources or used as probiotic cultures were subjected to fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting and pulsed-field gel electrophoresis (PFGE) analysis of SmaI macrorestriction patterns. Susceptibilities to 16 antimicrobial agents were tested using broth microdilution, and the presence of the corresponding resistance genes was investigated using PCR. Multiplex PCR was used to detect the presence of the enterococcal virulence genes asa1, gelE, cylA, esp, and hyl. The results of the study showed that two intraspecific genomic groups (I and II) were obtained in FAFLP analysis. PFGE analysis demonstrated high variability within these two groups but also indicated that some probiotic cultures were indistinguishable and that a number of clinical isolates may be reisolations of commercial probiotic cultures. Compared to group II, which contained the majority of the probiotic isolates and fewer human clinical isolates, higher phenotypic and genotypic resistance frequencies were observed in group I. Two probiotic isolates were phenotypically resistant to erythromycin, one of which contained an erm(B) gene that was not transferable to enterococcal recipients. None of the probiotic E. faecium isolates demonstrated the presence of the tested virulence genes. The previously reported observation that E. faecium consists of two intraspecific genomic groups was further substantiated by FAFLP fingerprinting of 128 isolates. In combination with antimicrobial resistance and virulence testing, this grouping might represent an additional criterion in assessing the safety of new potential probiotic E. faecium isolates.

Indications for antimicrobial prescribing in European nursing homes : results from a point prevalence survey

In light of the emerging problem with multiresistant microorganisms in nursing homes (NHs), the European Surveillance of Antimicrobial Consumption NH subproject was set up to measure and describe antimicrobial use across Europe. The aim of this paper was to investigate the indications for antimicrobial use and hence identify targets for quality improvement.

Intraspecific Genotypic Characterization of Lactobacillus rhamnosus Strains Intended for Probiotic Use and Isolates of Human Origin

ABSTRACT A set of 118 strains of the species Lactobacillus rhamnosus was collected, including probiotic strains, research strains with potential probiotic properties, food starter cultures, and human isolates. The majority of the strains were collected from companies, hospitals, or culture collections or were obtained after contacting authors who reported clinical case studies in the literature. The present work aimed to reveal the genotypic relationships between strains of these diverse sources. All strains were initially investigated using fluorescent amplified fragment length polymorphism (FAFLP) with three different primer combinations. Numerical analysis of FAFLP data allowed (i) confirmation of the identification of all strains as members of L. rhamnosus and (ii) delineation of seven stable intraspecific FAFLP clusters. Most of these clusters contained both (potentially) probiotic strains and isolates of human origin. For each of the clusters, strains of different sources were selected for pulsed-field gel electrophoresis (PFGE) of macrorestriction fragments obtained with the enzymes NotI and AscI. Analysis of PFGE data indicated that (i) some (potentially) probiotic strains were indistinguishable from other probiotic strains, suggesting that several companies may use duplicate cultures of the same probiotic strain, and (ii) in a number of cases human isolates from sterile body sites were indistinguishable from a particular probiotic strain, suggesting that some of these isolates may be reisolations of commercial strains.

European Surveillance of Antimicrobial Consumption (ESAC): outpatient cephalosporin use in Europe (1997–2009)

BACKGROUND Data on 13 years of outpatient cephalosporin use were collected from 33 European countries within the European Surveillance of Antimicrobial Consumption (ESAC) project, funded by the European Centre for Disease Prevention and Control (ECDC), and analysed in detail. METHODS For the period 1997-2009, data on outpatient use of systemic cephalosporins aggregated at the level of the active substance were collected using the Anatomical Therapeutic Chemical (ATC)/defined daily dose (DDD) method (WHO, version 2011) and expressed in DDD per 1000 inhabitants per day (DID). For detailed analysis of trends over time, seasonal variation and composition of outpatient cephalosporin use in 33 European countries, we distinguished between first-generation (J01DB), second-generation (J01DC), third-generation (J01DD) and fourth-generation (J01DE) cephalosporins. RESULTS Total outpatient cephalosporin use in 2009 varied from 8.7 DID in Greece to 0.03 DID in Denmark. In general, use was higher in Southern and Eastern European countries than in Northern European countries. Total outpatient cephalosporin use increased over time by 0.364 (SD 0.473) DID between 1997 and 2009. Cephalosporin use increased for half of the countries. Low-consuming Northern European countries and the UK further decreased their use. Second-generation cephalosporins increased by >20% in seven countries (mainly cefuroxime), coinciding with a decrease in first-generation cephalosporins. Substantial parenteral use of third-generation substances (mainly ceftriaxone) was observed in France, Italy and the Russian Federation. CONCLUSIONS Since 1997, the use of the older (narrow-spectrum) cephalosporins decreased in favour of the newer (i.e. broad-spectrum) cephalosporins in most countries. Extreme variations between European countries in cephalosporin use over time suggest that they are to a large extent inappropriately used.

European Surveillance of Antimicrobial Consumption (ESAC): outpatient use of tetracyclines, sulphonamides and trimethoprim, and other antibacterials in Europe (1997–2009)

BACKGROUND Data on more than a decade of outpatient use of tetracyclines, sulphonamides and trimethoprim, and other antibacterials in Europe were collected from 33 countries as part of the European Surveillance of Antimicrobial Consumption (ESAC) project, funded by the European Centre for Disease Prevention and Control (ECDC). METHODS For the period 1997-2009, data on outpatient use of systemic tetracyclines, sulphonamides and trimethoprim, and other antibacterials aggregated at the level of the active substance were collected and expressed in defined daily doses (DDD; WHO, version 2011) per 1000 inhabitants per day (DID). Using the Anatomical Therapeutic Chemical (ATC) classification, trends in the use of tetracyclines (J01A), sulphonamides and trimethoprim (J01E) and other antibacterials (J01X) over time, seasonal variation and composition of use were analysed. RESULTS In 2009, the variations in outpatient use of systemic tetracyclines, sulphonamides and trimethoprim, and other antibacterials between countries, and also in the composition of use over time, were huge. For tetracyclines a significant and for sulphonamides and trimethoprim a non-significant decrease in use was observed between 1997 and 2009 in Europe. The seasonal variation in their use significantly decreased over time. For the other antibacterials, no significant changes in the volume of use or its seasonal variation were seen. CONCLUSIONS As for all other major antibiotic subgroups, a striking variation in use and composition of use between countries in Europe was observed for outpatient use of tetracyclines, sulphonamides and trimethoprim, and other antibacterials. In combination with the decreasing use, especially of recommended substances, this represents an opportunity not only to reduce antibiotic use but also to improve its quality.