A. C. Fluit

Epidemiology and Susceptibility of 3,051 Staphylococcus aureus Isolates from 25 University Hospitals Participating in the European SENTRY Study

ABSTRACT A total of 3,051 methicillin-susceptible Staphylococcus aureus (MSSA) isolates and methicillin-resistant S. aureus (MRSA) isolates in Europe were compared. MRSA isolates constituted 25% of all isolates and were more prevalent in southern Europe. MRSA isolates appeared to be more prevalent in intensive care units than in outpatient departments. Only a small minority of MSSA isolates were multidrug resistant, whereas the majority of MRSA isolates were multidrug resistant.

Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci

ABSTRACT To compare commonly used phenotypic methods with genotypic identification methods 47 clinical isolates of coagulase-negative staphylococci (CONS), 10 CONS ATCC strains, and a Staphylococcus aureus clinical isolate were identified using the API Staph ID test, BD Phoenix Automated Microbiology System, and 16S rRNA gene and tuf gene sequencing. When necessary part of the sodA gene was sequenced for definitive identification. The results show that tuf gene sequencing is the best method for identification of CONS, but the API Staph ID test is a reasonably reliable phenotypic alternative. The performance of the BD Phoenix Automated Microbiology System for identification of CONS is poor. The present study also showed that although genotypic methods are clearly superior to phenotypic identifications, a drawback of sequence-based genotypic methods may be a lack of quality of deposited sequences in data banks. In particular, 16S rRNA gene sequencing suffers from the lack of high quality among sequences deposited in GenBank. Furthermore, genotypic identification based on 16S rRNA sequences has limited discriminating power for closely related Staphylococcus species. We propose partial sequencing of the tuf gene as a reliable and reproducible method for identification of CONS species.

Activities of the Glycylcycline Tigecycline (GAR-936) against 1,924 Recent European Clinical Bacterial Isolates

ABSTRACT The in vitro activities of tigecycline against 1,924 clinical isolates were examined. The new glycylcycline exhibited excellent activity against all gram-positive cocci (MICs at which 90% of the isolates tested were inhibited [MIC90s], ≤1 μg/ml). In addition, it was also very potent against most members of the Enterobacteriaceae, with most MIC90s being ≤2 μg/ml. Among the nonfermenters, Acinetobacter spp. and Stenotrophomonas maltophilia are included in the in vitro spectrum of tigecycline activity.

Presence of Integron-Associated Resistance in the Community Is Widespread and Contributes to Multidrug Resistance in the Hospital

ABSTRACT Integrons are strongly associated with the multidrug resistance seen in gram-negative bacilli in the hospital environment. No data, however, are available on their prevalence in the community. This study is the first to show that integrons are widespread in Enterobacteriaceae in the community and that integron-associated resistance genes in the community constitute a substantial reservoir for multidrug resistance in the hospital.

Frequency of Isolation of Pathogens from Bloodstream, Nosocomial Pneumonia, Skin and Soft Tissue, and Urinary Tract Infections Occurring in European Patients

Abstract The frequency of isolation of pathogens that cause different types of infections is an important guide for empiric therapy. As part of the SENTRY Antimicrobial Surveillance Program, the frequency of isolation of different bacterial species from bloodstream, nosocomial pneumonia, skin and soft tissue, and urinary tract infections occurring in European patients was determined. A total of 15,704 isolates were collected in 1997 and 1998 from 24 university hospitals in 14 European countries: 9,194 from bloodstream, 2,052 from nosocomial pneumonia, 2,320 from skin and soft tissue, and 2,138 from urinary tract infections. More than 95% of all bacterial infections were caused by only 15 different genera. Staphylococcus spp. and Escherichia spp. accounted for more than 50% of the infectious isolates, with the exception of those obtained from cases of nosocomial pneumonia. In the latter type of infection, isolates belonging to these two genera were responsible for 30% of the infections. An analysis at the individual species level showed that Escherichia coli caused a large proportion of bloodstream and urinary tract infections (20.8% and 49.3% of isolates, respectively). Staphylococcus aureus was the main causative species for nosocomial pneumonia and skin and soft tissue infections (21.5% and 37.4% of isolates, respectively). In addition, Pseudomonas aeruginosa played an important role in all types of infection analyzed.

Evaluation of the Magnetic Immuno PCR assay for rapid detection ofSalmonella

A new technique, the Magnetic Immuno PCR Assay (MIPA), has been developed for the detection ofSalmonella. The assay utilizes magnetic particles coated with monoclonal antibodies againstSalmonella to extract these bacteria from the sample. Trapped bacteria are lysed, and the supernatant, which contains bacterial DNA, is then subjected to the polymerase chain reaction (PCR) using primers from theSalmonella typhimurium origin of DNA replication to amplify a 163 bp region. The specificity of the primer set was tested in the PCR; amplification occurred with all 25Salmonella strains tested but not with 19 other species ofEnterobacteriaceae tested. A sensitivity of 100 cfuSalmonella typhimurium was achieved for the MIPA by visualization of the amplified products by ethidiumbromide stained agarose gel electrophoresis. A tenfold higher sensitivity was obtained by Southern blotting of the amplified products. The presence of 107 cfuEscherichia coli did not interfere with these detection levels. The MIPA thus specifically detected 100 cfu ofSalmonella within 5 h and may be potentially useful for rapid detection ofSalmonella in clinical specimens and food.

Frequency of Isolation and Antimicrobial Resistance of Gram-Negative and Gram-Positive Bacteria from Patients in Intensive Care Units of 25 European University Hospitals Participating in the European Arm of the SENTRY Antimicrobial Surveillance Program 1997-1998

Abstract.A total of 3,981 isolates from patients treated at intensive care units were collected in 25 European university hospitals during 1997 and 1998 as part of the SENTRY Antimicrobial Surveillance Program. Overall, the most important species isolated were Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, coagulase-negative staphylococci (CNS), Enterobacter spp., Haemophilus influenzae, Streptococcus pneumoniae, and Enterococcus faecalis. Thirty-nine percent of all Staphylococcus aureus isolates were resistant to oxacillin. All Staphylococcus aureus isolates were fully susceptible to linezolid and vancomycin. Moreover, all CNS isolates were susceptible to vancomycin and minocycline. All Enterococcus faecalis isolates were susceptible to vancomycin, and 99% of these isolates were also susceptible to ampicillin. The antimicrobial agents most effective against Pseudomonas aeruginosa isolates were amikacin, piperacillin/tazobactam, meropenem, and cefepime, with 87, 85, 84, and 83% of isolates being susceptible, respectively. Escherichia coli isolates were fully susceptible to carbapenems, and at least 99% of these isolates were susceptible to ceftriaxone, cefepime, and amikacin. The Enterobacter spp. were also highly susceptible to carbapenems, amikacin, and cefepime, with 99, 97, and 96% of isolates being susceptible, respectively. Haemophilus influenzae was susceptible to most of the antibiotics tested. Only 68% of the pneumococcal isolates were fully susceptible to penicillin, yet 100% were susceptible to a number of fluoroquinolones and vancomycin. There are still sufficient treatment options for patients infected with the most important bacterial species involved in infections in intensive care units. However, the situation for patients with Pseudomonas aeruginosa infections is critical.

Rapid detection of TEM, SHV and CTX-M extended-spectrum β-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis

OBJECTIVES Fast and adequate detection of extended-spectrum beta-lactamases (ESBLs) is crucial for infection control measures and the choice of antimicrobial therapy. The aim of this study was to develop and evaluate a novel ESBL assay using ligation-mediated amplification combined with microarray analysis to detect the most prevalent ESBLs in Enterobacteriaceae: TEM, SHV and CTX-M. METHODS Analysis of the Lahey database revealed that the vast majority of TEM and SHV ESBLs differ from non-ESBL variants in three amino acid positions. TEM ESBLs have at least one of the following amino acid substitutions: R164S/H/C, G238D/N/S and E104K. In SHV ESBLs, one or more of the following substitutions is observed: D179A/N/G, G238S/A and E240K. Oligonucleotide probes were designed to detect these substitutions, covering 95% of ESBL TEM variants and 77% of ESBL SHV variants. In addition, probes were designed to distinguish between CTX-M groups 1, 2, 9 and 8/25. For evaluation of the assay, 212 Enterobacteriaceae isolates with various beta-lactamases were included (n = 106 ESBL positive). RESULTS The sensitivity of the microarray was 101/106 (95%; 95% CI 89%-98%), and the specificity 100% (95% CI 97%-100%) using molecular characterization of ESBLs by PCR and sequencing as reference. Assay performance time was 8 h for 36 isolates. CONCLUSIONS This novel commercially available DNA microarray system may offer an attractive option for rapid and accurate detection of CTX-M, TEM and SHV ESBL genes in Enterobacteriaceae in the clinical laboratory.

Evaluation of the DiversiLab System for Detection of Hospital Outbreaks of Infections by Different Bacterial Species

ABSTRACT Many bacterial typing methods are specific for one species only, time-consuming, or poorly reproducible. DiversiLab (DL; bioMérieux) potentially overcomes these limitations. In this study, we evaluated the DL system for the identification of hospital outbreaks of a number bacterial species. Appropriately typed clinical isolates were tested with DL. DL typing agreed with pulsed-field gel electrophoresis (PFGE) for Acinetobacter (n = 26) and Stenotrophomonas maltophilia (n = 13) isolates. With two exceptions, DL typing of Klebsiella isolates (n = 23) also correlated with PFGE, and in addition, PFGE-nontypeable (PFGE-NT) isolates could be typed. Enterobacter (n = 28) results also correlated with PFGE results; also, PFGE-NT isolates could be clustered. In a larger study (n = 270), a cluster of 30 isolates was observed that could be subdivided by PFGE. The results for Escherichia coli (n = 38) correlated less well with an experimental multilocus variable number of tandem repeats analysis (MLVA) scheme. Pseudomonas aeruginosa (n = 52) showed only a limited number of amplification products for most isolates. When multiple Pseudomonas isolates were assigned to a single type in DL, all except one showed multiple multilocus sequence types. Methicillin-resistant Staphylococcus aureus generally also showed a limited number of amplification products. Isolates that belonged to different outbreaks by other typing methods, including PFGE, spa typing, and MLVA, were grouped together in a number of cases. For Enterococcus faecium, the limited variability of the amplification products obtained made interpretation difficult and correlation with MLVA and esp gene typing was poor. All of the results are reflected in Simpsons index of diversity and adjusted Rands and Wallaces coefficients. DL is a useful tool to help identify hospital outbreaks of Acinetobacter spp., S. maltophilia, the Enterobacter cloacae complex, Klebsiella spp., and, to a somewhat lesser extent, E. coli. In our study, DL was inadequate for P. aeruginosa, E. faecium, and MRSA. However, it should be noted that for the identification of outbreaks, epidemiological data should be combined with typing results.

mecA Gene Is Widely Disseminated in Staphylococcus aureus Population

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important causes of hospital infections worldwide. High-level resistance to methicillin is caused by the mecA gene, which encodes an alternative penicillin-binding protein, PBP 2a. To determine the clonal relationships between methicillin-susceptible S. aureus (MSSA) and MRSA, we typed 1,069 S. aureus isolates (493 MSSA isolates and 576 MRSA isolates), collected mainly in North American and European hospitals between the 1960s and the year 2000, using pulsed-field gel electrophoresis and ribotyping. Of 10 widespread S. aureus lineages recognized, 8 had corresponding mecA-positive strains. Multiresistant MRSA strains are found in hospitals worldwide, while unrelated and more susceptible strains represent less than 1% of the MRSA population. This supports the hypothesis that horizontal transfer plays an important role in the dissemination of the mecA gene in the S. aureus population.