Ariane Deplano

High Interlaboratory Reproducibility of DNA Sequence-Based Typing of Bacteria in a Multicenter Study

ABSTRACT Current DNA amplification-based typing methods for bacterial pathogens often lack interlaboratory reproducibility. In this international study, DNA sequence-based typing of the Staphylococcus aureus protein A gene (spa, 110 to 422 bp) showed 100% intra- and interlaboratory reproducibility without extensive harmonization of protocols for 30 blind-coded S. aureus DNA samples sent to 10 laboratories. Specialized software for automated sequence analysis ensured a common typing nomenclature.

Antimicrobial Resistance of Old and Recent Staphylococcus aureus Isolates from Poultry: First Detection of Livestock-Associated Methicillin-Resistant Strain ST398

ABSTRACT The susceptibilities of 12 antimicrobial agents for two collections of Staphylococcus aureus, isolated in the 1970s and in 2006 from poultry, were determined. For eight antibiotics, the percentage of resistance was significantly higher in the recent isolates. Ten recent isolates were methicillin resistant and had spa types t011 and t567, belonging to multilocus sequence type 398. This is the first report of “livestock-associated” methicillin resistant S. aureus from healthy poultry.

Evaluation of Molecular Typing Methods in Characterizing a European Collection of Epidemic Methicillin-Resistant Staphylococcus aureus Strains: the HARMONY Collection

ABSTRACT We analyzed a representative sample of methicillin-resistant Staphylococcus aureus (MRSA) from 11 European countries (referred to as the HARMONY collection) using three molecular typing methods used within the HARMONY group to examine their usefulness for large, multicenter MRSA surveillance networks that use these different laboratory methodologies. MRSA isolates were collected based on their prevalence in each center and their genetic diversity, assessed by pulsed-field gel electrophoresis (PFGE). PFGE groupings (≤3 bands difference between patterns) were compared to those made by sequencing of the variable repeats in the protein A gene spa and clonal designations based on multilocus sequence typing (MLST), combined with PCR analysis of the staphylococcal chromosome cassette containing the mec genes involved in methicillin resistance (SCCmec). A high level of discrimination was achieved using each of the three methodologies, with discriminatory indices between 89.5% and 91.9% with overlapping 95% confidence intervals. There was also a high level of concordance of groupings made using each method. MLST/SCCmec typing distinguished 10 groups containing at least two isolates, and these correspond to the majority of nosocomial MRSA clones described in the literature. PFGE and spa typing resolved 34 and 31 subtypes, respectively, within these 10 MRSA clones, with each subtype differing only slightly from the most common pattern using each method. The HARMONY group has found that the methods used in this study differ in their availability and affordability to European centers involved in MRSA surveillance. Here, we demonstrate that the integration of such technologies is achievable, although common protocols (such as we have developed for PFGE) may also be important, as is the use of centralized Internet sites to facilitate data analysis. PFGE and spa-typing data from analysis of MRSA isolates from the many centers that have access to the relevant equipment can be compared to reference patterns/sequences, and clonal designations can be made. In the majority of cases, these will correspond to those made by the (more expensive) method of choice—MLST/SCCmec typing—and these alternative methods can therefore be used as frontline typing systems for multicenter surveillance of MRSA.

Pseudomonas aeruginosa and Enterobacteriaceae bacteremia after biliary endoscopy: An outbreak investigation using DNA macrorestriction analysis

PURPOSE An outbreak of gram-negative bacteremia in patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) was investigated to determine the sources of infection and to control transmission. PATIENTS, METHODS, AND RESULTS The incidence of post-ERCP bacteremia increased from 1.6% (60 of 3,696) procedures to 3.6% (53 of 1,454) procedures (relative risk 2.3, p < 0.0001) after endoscopes were processed in a new automated disinfector. Bacteremia involved nine species of Pseudomonas and Enterobacteriaceae, which were also isolated from processed endoscopes. Seven epidemic strains with highly related genomic macrorestriction profiles each infected 2 or more patients, accounting for 29 (55%) episodes of post-ERCP bacteremia. Strains recovered from endoscopes and from the disinfector were associated with 22 (42%) and 5 (9%) bacteremic episodes respectively. Effective endoscope disinfection was achieved by cleansing and disinfection of a blind channel not processed in the disinfector, additional isopropanol-air flush of all channels, and auto-disinfection of the disinfector. In the following period, the incidence of post-ERCP bacteremia returned to the pre-epidemic rate (1.7%, p = 0.0001). CONCLUSION Bacterial genome fingerprinting by macrorestriction analysis enabled delineation of a multi-strain outbreak of post-ERCP bacteremia. Cross-contamination, and to a lesser extent, common-source contamination, appeared related to inadequate disinfection of endoscopes processed in an automated disinfector.

Molecular Characterization of an Epidemic Clone of Panantibiotic-Resistant Pseudomonas aeruginosa

ABSTRACT We describe the molecular characterization of a multiresistant Pseudomonas aeruginosa clone causing an outbreak in the intensive care unit (ICU) of a tertiary-care university hospital. Analysis included antimicrobial susceptibility profile, O-serotyping, pulsed-field gel electrophoresis, and amplified fragment length polymorphism. Resistance mechanisms were characterized, including production of naturally occurring and acquired β-lactamases, porin expression, and efflux pump systems. Eighteen patients were colonized or infected with multiresistant P. aeruginosa. Multiresistant P. aeruginosa was panresistant to penicillins, cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones and remained susceptible only to colistin. Sixteen isolates (89%) belonged to serotype O:11, pulsed-field gel electrophoresis type A1, and amplified fragment length polymorphism type A. Resistance characterization of this epidemic clone showed an overexpression of the chromosomal cephalosporinase AmpC combined with decreased expression of porin OprD and the absence of metallo-β-lactamase or extended-spectrum beta-lactamase. An upregulation of the MexXY efflux system due to an agrZ mutation in the mexZ repressor was detected. This epidemic clone was restricted to the ICU and was not found elsewhere in hospital. Contamination of the ICU environment and the hands of an ICU nurse with this clone suggests possible hand-borne transmission. Implementation of contact precautions effectively controlled transmission of the epidemic clone. This study illustrates the ability of multiresistant P. aeruginosa to cause an outbreak with significant morbidity and mortality and underscores the need to identify clonal outbreaks, which require targeted infection control measures.

High occurrence of methicillin-resistant Staphylococcus aureus ST398 in equine nasal samples

Methicillin-resistant Staphylococcus aureus (MRSA) infections do occur in equine patients. Little is known, however, about their origin and the general equine MRSA colonization status. In West European horses in particular, neither the colonization rate nor the present strains or their antimicrobial susceptibility patterns are known. In the present study, a sample of 110 (Belgian, French, Dutch and Luxemburg) horses presented at a Belgian equine clinic was screened for nasal MRSA carriage. An indirect culturing protocol using a 0.001% colistin and nalidixic acid containing broth was compared to a direct agar method. Phenotypic identification following growth on a chromogenic MRSA screening agar (ChromID MRSA) was combined with genotypic analysis (PCR, PFGE, SCCmec, spa, and MLST typing). Antimicrobial susceptibility was tested through disk diffusion. Twelve (10.9%) horses carried MRSA, with the enrichment protocol resulting in a significantly higher isolation rate. None of the isolated strains were typeable through SmaI PFGE. They all harboured SCCmec type IVa or V and belonged to spa type t011 or t1451 of the ST398 lineage. All isolates were tetracycline resistant and sulfonamide and enrofloxacin susceptible. Macrolide, lincosamide, trimethoprim and aminoglycoside susceptibility varied and in total five different antimicrobial resistance patterns were distinguished. These results show that ST398 is certainly present in West European horses. Due to its known interspecies transmission and the structure of the equine industry, the presence of this clone in horses poses a substantial health hazard for both animals and humans.

Validation of Pulsed-Field Gel Electrophoresis and spa Typing for Long-Term, Nationwide Epidemiological Surveillance Studies of Staphylococcus aureus Infections

ABSTRACT Pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments has been used by the Belgian Reference Laboratory for Staphylococci for national hospital surveys of methicillin-resistant Staphylococcus aureus since 1992. The sequencing of the polymorphic X region of the protein A gene (spa typing) offers significant advantages over PFGE in terms of speed, ease of interpretation, and exportability. To validate its potential use for national surveillance, we evaluated the robustness of spa typing compared with that of PFGE based on a collection of 217 S. aureus strains representative of the Belgian S. aureus epidemiology during the last 13 years. spa typing and PFGE both showed high discriminatory power (discriminatory indexes of 0.98 and 0.96, respectively) and achieved high concordance (95.9%) in type classification. Both methods also showed good concordance with multilocus sequence typing (MLST) (95.5%). However, we observed occasional “violations” of MLST clonal complex assignment by spa typing. Our results suggest that both PFGE and spa typing are reliable methods for long-term, nationwide epidemiological surveillance studies. We suggest that spa typing, which is a single-locus-based method, should preferably be used in combination with additional markers, such as staphylococcal cassette chromosome mec typing or resistance or virulence gene detection.

Rapid emergence and spread of OXA-48-producing carbapenem-resistant Enterobacteriaceae isolates in Belgian hospitals

During a polymerase chain reaction (PCR)-based surveillance study of β-lactam resistance, 19 OXA-48-positive enterobacterial isolates were detected at nine Belgian hospitals from January 2010 to April 2011. Most cases were presumed to have been locally acquired and were detected in patients who had not travelled abroad. Clonally related outbreaks occurred in two different cities. The majority of isolates co-produced several β-lactamases as well as non-β-lactam resistance genes. This report highlights the rapid emergence and spread of OXA-48-producing Enterobacteriaceae in Belgium.

In Vitro Activities of Ceftobiprole, Tigecycline, Daptomycin, and 19 Other Antimicrobials against Methicillin-Resistant Staphylococcus aureus Strains from a National Survey of Belgian Hospitals

ABSTRACT The in vitro activities of 22 antimicrobial agents, including ceftobiprole, daptomycin, and tigecycline, against 511 methicillin-resistant Staphylococcus aureus (MRSA) isolates from 112 Belgian hospitals were studied by using the CLSI agar dilution method. Isolates were characterized by pulsed-field gel electrophoresis (PFGE) analysis and by PCR detection of determinants of resistance to aminoglycosides, macrolides-lincosamides-streptogramins, and tetracyclines. A representative set of isolates with different PFGE genotypes was further characterized by multilocus sequence typing, determination of staphylococcal cassette chromosome mec (SCCmec) type, and multiplex PCR for toxic shock syndrome type 1 (TSST-1) and Panton-Valentine leukocidin genes. MRSA isolates belonged to nine epidemic MRSA clones, of which sequence type 45 (ST45)-SCCmec IV and ST8-SCCmec IV were predominant, accounting for 49 and 20% of isolates, respectively. The distribution of antimicrobial resistance and TSST-1 genes was strongly linked to clonal types. Ceftobiprole, daptomycin, and tigecycline showed high activity against all isolates of these sporadic and epidemic MRSA clones, as indicated by MIC90s of 2 mg/liter, 0.5 mg/liter, and 0.25 mg/liter, respectively. The MIC distribution of daptomycin and tigecycline was not different in isolates with decreased susceptibility to glycopeptides or tetracyclines, respectively. Ceftobiprole MICs were not correlated with oxacillin and cefoxitin MICs. These data indicate excellent activity of the newly developed agents ceftobiprole, daptomycin, and tigecycline against MRSA isolates recently recovered from hospitalized patients in Belgium, supporting their therapeutic potential for nosocomial MRSA infections.

Inter-centre comparison of pulsed-field gel electrophoresis for the typing of methicillin-resistant Staphylococcus aureus

The results of pulsed-field gel electrophoresis (PFGE) of chromosomal DNA of the same 12 methicillin-resistant S. aureus (MRSA) strains of diverse geographical origin, performed in three different laboratories were compared; one laboratory used field-inversion gel electrophoresis (FIGE), one used contour clamped homogenous electrophoresis (CHEF) and one used both (all manufactured by BioRad Laboratories Inc., Hercules, CA, USA). No single method produced the maximum number of chromosomal fragments from all isolates. In only four instances were the same number of fragments identified by any two techniques. Although there were similar trends in strain identification the results showed many discrepancies even with a three-band difference rule to discriminate between strains. Plasmids in seven of the isolates produced a fragment, but this did not affect discrimination of the study isolates. There is a great need to standardise methodology and produce a standard set of strains to assist in this process.