Emergence of SCCmec type IV as the most common type of methicillin-resistant Staphylococcus aureus in a university hospital.

BACKGROUND: The epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically changed over the last decade by the emergence of community-associated MRSA (CA-MRSA). Recent studies indicate that these strains have already spread to hospitals. To evaluate if SCCmec type IV and Panton-Valentine leukocidin (PVL) are unambiguous markers of CA-MRSA, we analyzed 77 sporadic MRSA strains isolated, in our low MRSA incidence university hospital, from inpatients between 2000 and 2004. METHODS: MRSA strains were analyzed by staphylococcal cassette chromosome mmecec (SCCmec) typing, PCR for PVL genes and pulsed-field gel electrophoresis (PFGE). MRSA was classified in HA-MRSA or CA-MRSA according to Centers for Disease Control and Prevention (CDC) criteria. Antimicrobial susceptibility testing was performed using microbroth dilution method following CLSI recommendations. RESULTS: Among 77 sporadic single-patient strains, SCCmec types I-IV and four subtypes were identified. Type IV/IVA was most common (42.9%).The distribution of SCCmec types changed over the years. Type IV/IVA strains increased from 33.3% in 2000 to 57.9% in 2004. Type IV strains were resistant to ciprofloxacin in 81.8%, and in 9.1% to tobramycin while type IVA strains were 100% resistant to both antimicrobials. In contrast, non-type IV/IVA strains were resistant to ciprofloxacin in 86.4%, and in 75.0% to tobramycin. Only one strain was PVL positive and harbored SCCmec type III variant. By PFGE analysis, the 33 SCCmec type IV/IVA strains comprised 12 distinct genotypes. 36.4% of 11 CA-MRSA and 43.9% of 66 HA-MRSA harbored SCCmec type IV/IVA. CONCLUSION: Type IV/IVA has become the most common SCCmec type in inpatients of our university hospital. The SCCmec type IV/IVA is present in both CA-MRSA and HA-MRSA limiting its use as a marker for CA-MRSA.

Epidemiology of Clostridium difficile-associated disease at University Hospital Basel including molecular characterisation of the isolates 2006-2007.

A prospective study was conducted during a one-year period between 2006 and 2007 to describe the epidemiology of Clostridium difficile-associated disease (CDAD) at University Hospital Basel, Switzerland (UHBS) and to determine phenotypic and genotypic features of C. difficile strains isolated at the Microbiology Laboratory UHBS including strains from regional non-university hospitals. We prospectively identified 78 CDAD cases at UHBS with an incidence of 2.65/1,000 hospitalised patients or 2.3/10,000 patient-days. Sixteen patients (20.5%) were infected with clindamycin-resistant strains of PCR-ribotype 027 during an outbreak at the geriatric hospital. Among 124 single-patient isolates, 28 (22.6%) were resistant to moxifloxacin and 34 (27.4%) were resistant to clindamycin, but all remained susceptible to metronidazole and vancomycin. Of 102 toxigenic isolates, 19 (18.7%) had an 18-bp deletion in the tcdC gene, eight (7.8%) a 39-bp deletion, and one (1.0%) a 54-bp deletion. Genes for binary toxin were present in 27 (21.8%). PCR-ribotype 027 was associated with older age (median age 83.5 vs. 65.5 years, p < 0.0001) and longer duration of hospitalisation before onset of disease (median 15.5 vs. 9 days, p = 0.014) with a trend towards higher crude mortality, more severe disease, and previous use of macrolides compared to ribotype non-027. Overall, severe disease correlated with use of a nasogastric tube and surprisingly shorter duration of hospitalisation before onset of disease. Today, laboratory-based and epidemiological surveillance systems are required to monitor CDAD cases and emergence of new epidemic strains.

Simplifying the WHO 'how to hand rub' technique: three steps are as effective as six-results from an experimental randomized crossover trial.

OBJECTIVES: The World Health Organization (WHO) issued guidelines on hand hygiene recommending a six-step 'how to hand rub' technique for applying alcohol-based hand rub. However, adherence to all six steps is poor. We assessed a simplified three-step technique and compared it to the conventional WHO six-step technique in terms of bacterial count reduction on healthcare workers' hands. METHODS: Thirty-two participants were randomly assigned to clean their hands following the six-step 'how to hand rub' technique (WHO reference group) or a simplified three-step technique (intervention group). Assignments were reversed after 1 day. The degree of bacterial killing was assessed following the European norm for testing hand hygiene products. Hands were contaminated with Escherichia coli, and the mean logarithmic reduction in bacterial counts was compared between both techniques. RESULTS: Bacterial density before hand hygiene performance did not differ between the WHO reference group (median 6.37 log10 CFU, interquartile range (IQR) 6.19-6.54) and the intervention group (median 6.34 log10 CFU, IQR 6.17-6.60, p 0.513). After hand hygiene, the logarithmic reduction factor was higher in the intervention group (median 4.45, IQR 4.04-5.15) compared to the WHO reference group (median 3.91, IQR 3.69-4.62, p 0.021). CONCLUSIONS: The WHO six-step 'how to hand rub' technique can be simplified to a 3-step procedure based on the reduction of bacterial counts on healthcare workers' hands achieved under experimental conditions. The proposed technique is easier to perform and could improve adherence to the execution of hand hygiene action.

Low sensitivity of fecal toxin A/B enzyme immunoassay for diagnosis of Clostridium difficile infection in immunocompromised patients.

The optimal approach in laboratory diagnosis of Clostridium difficile infection (CDI) is still not well defined. Toxigenic culture (TC) or alternatively fecal toxin assay by cell cytotoxicity neutralization assay are considered to be the reference standard, but these methods are time-consuming and labor intensive. In many medical centers, diagnosis of CDI is therefore still based on fecal toxin A/B enzyme immunoassay (EIA) directly from stool alone, balancing cost and speed against limited diagnostic sensitivity. The aim of the study was to assess in which patient population the additional workload of TC is justified. All consecutive stool specimens submitted for diagnosis of suspected CDI between 2004 and 2011 at a tertiary-care center were examined by toxin EIA and TC. Clinical data of patients with established diagnosis of CDI were collected in a standardized case-report form. From 12,481 stool specimens submitted to the microbiologic laboratory, 480 (3.8%) fulfilled CDI criteria; 274 (57.1%) were diagnosed by toxin EIA; and an additional 206 (42.9%) were diagnosed by TC when toxin EIA was negative. Independent predictors for negative toxin EIA but positive TC were high-dose corticosteroids (odds ratio (OR) 2.97, 95% confidence interval (CI) 1.50-5.90, p 0.002), leukocytopenia <1000/µL (OR 2.52, 95% CI 1.22-5.23, p 0.013) and nonsevere CDI (OR 2.21, 95% CI 1.39-3.50, p 0.001). There was no difference in outcomes such as in-hospital mortality and recurrence between both groups. In conclusion, negative toxin EIA does not rule out CDI in immunocompromised patients in the setting of relevant clinical symptoms. Methods with improved sensitivity such as TC or PCR should be used, particularly in this patient population.

Glutamine synthetase and heteroresistance in methicillin-resistant Staphylococcus aureus.

Inactivation of femC in methicillin-resistant Staphylococcus aureus (MRSA) results in lowered methicillin resistance and a reduction in the amidation of the iso-D-glutamate of the peptidoglycan stem peptide. The femC phenotype is due to insertional inactivation of the glutamine synthetase repressor gene glnR by Tn551, which has a polar effect on glutamine synthetase (glnA) transcription. The complete glutamine synthetase operon (glnRA) of S. aureus was cloned and sequenced, and its transcriptional start was determined. The deduced amino acid sequence of the staphylococcal glutamine synthetase showed 76% identity and 87% similarity to the Bacillus subtilis glutamine synthetase. The staphylococcal glnRA operon was shown to complement an Escherichia coli glutamine synthetase-negative mutant and to restore methicillin resistance in femC mutants. femC mutants revert to resistance in the presence of high concentrations of methicillin. These revertants, which still carried the femC lesion, were shown to retain the lowered amidation of the iso-D-glutamate peptidoglycan stem peptide. A new chromosomal locus hmrC was postulated to have mutated to allow expression of high methicillin resistance in these femC revertants. Although the highly resistant hmrC revertant resembled phenotypically the highly methicillin-resistant subclones occurring in heterogeneously resistant MRSA, we could show by transduction that the locus hmrC was distinct from chr*, a chromosomal site postulated to confer high methicillin resistance in heterogeneous MRSA. This suggests that S. aureus can adopt multiple ways to achieve high methicillin resistance.

Lif, the lysostaphin immunity factor, complements FemB in staphylococcal peptidoglycan interpeptide bridge formation.

The formation of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide chain needs FemA and FemB for the incorporation of glycines Gly2-Gly3, and Gly4-Gly5, respectively. The lysostaphin immunity factor Lif was able to complement FemB, as could be shown by serine incorporation and by an increase in lysostaphin resistance in the wild-type as well as in a femB mutant. However, Lif could not substitute for FemA in femA or in femAB-null mutants. Methicillin resistance, which is dependent on functional FemA and FemB, was not complemented by Lif, suggesting that serine-substituted side chains are a lesser substrate for penicillin-binding protein PBP2' in methicillin resistance.

Critical comparison of Giardia duodenalis from Australia and Switzerland using isoenzyme electrophoresis.

Isoenzyme electrophoresis using 13 enzyme systems was applied to 31 Australian and 7 Swiss isolates of Giardia of human, cat, cattle, dog, sheep and rat origin. The Portland (ATCC No. 30888) reference strain was also included. The 39 isolates were divided into 22 different zymodemes. These consisted of 19 zymodemes containing the P1 and Australian isolates and three zymodemes containing Swiss isolates only. Differences in enzyme profiles between zymodemes was measured by euclidean distance and it was found that Australian isolates of Giardia exhibited more variation than the Swiss isolates. Relationships between zymodemes determined by clustering analysis are discussed with particular reference to the zoonotic potential of Giardia.

Electrophoretic characterization of Giardia isolated from humans, cattle, sheep, and a dog in Switzerland.

Nine isolates of Giardia lamblia from humans, cattle, sheep, and 1 dog were compared by employing agarose gel isoenzyme electrophoresis and isoelectric focusing of total soluble cell protein on polyacrylamide gels. The banding patterns of the 14 enzymes examined showed remarkable similarities among the Swiss Giardia isolates. This was true also of the total soluble trophozoite proteins. The electrophoretic mobilities of most enzymes and other proteins obtained for the Swiss isolates were the same as those of 2 isolates from humans in other geographical areas, the WB and the Portland-1 strains. Only the human isolate CH-H2 could clearly be distinguished from all other isolates analyzed. The great biochemical similarities observed among the Swiss isolates contrast with the extensive heterogeneity previously demonstrated for G. lamblia by other investigators who used similar analytical techniques. These data are consistent with recent transmission studies of Giardia and suggest that in Switzerland domestic animals may serve as a reservoir of human Giardia infections and that cross-transmission between humans and animals is likely to occur.

Function of the mouse Mx1 protein is inhibited by overexpression of the PB2 protein of influenza virus.

The interferon-induced murine Mx1 protein possesses an intrinsic antiviral activity with selectivity for influenza viruses. Mx1 accumulates in the nucleus and inhibits the replication of influenza virus at the level of primary transcription. Simultaneous overexpression of the three influenza virus polymerase subunits via recombinant vaccinia virus vectors can titrate out the inhibitory action of Mx1 as determined by the amplification of a transfected recombinant viral reporter gene. A low degree of neutralization was also observed, when PB2 was overexpressed alone [Huang, T., Pavlovic, J., Staeheli, P., and Krystal, M. (1992) J. Virol. 66, 4154-4160]. We now employed a much simpler experimental setting which allowed us to directly measure the effect of PB2 on the antiviral activity of Mx1. We stably transfected a cell line derived from an A2G mouse (homozygous for a functional Mx1 gene) with expression vectors coding for cDNAs of the influenza virus polymerase subunits PB1 and PB2 or of the nucleoprotein (NP). Cells coexpressing Mx1 and PB1 or NP remained resistant to influenza virus infection whereas cells coexpressing Mx1 and PB2 became sensitive to influenza virus infection. The degree of neutralization of Mx1 activity by PB2 was dependent on the Mx1 concentration in the cell. Immunofluorescence analysis revealed that the nuclear localization of Mx1 and PB2 overlapped to a great extent. These findings support the view that Mx1 exerts its antiviral activity by interfering with the function of the influenza virus polymerase subunit PB2.

Molecular typing of methicillin-resistant Staphylococcus aureus: can PCR replace pulsed-field gel electrophoresis?

Pulsed-field gel electrophoresis (PFGE) is considered the "gold standard" for molecular typing of methicillin-resistant Staphylococcus aureus (MRSA). However, the method is time-consuming and expensive, and its discriminatory power may not be necessary in outbreak situations. We used a rapid multiplex PCR-based method with published primers and compared the results with those obtained by PFGE. A total of 75 clinical isolates were typed: 59 strains originated from our prospectively collected clinical strains and were epidemiologically unrelated; 16 strains came from an outbreak that was epidemiologically well defined in time and space. A primer mix of the spa gene, the coa gene, and the hypervariable region adjacent to mecA gene was used for multiplex PCR. Both PFGE and PCR clustered the 75 strains into 41 different genotypes. Concordance of the results was 100% for strains originating from the outbreak. Overall, both methods produced concordant results in 72% of cases. A total of 16% were clustered together by PFGE, but not by PCR and 12% were clustered together by PCR but not by PFGE, respectively. The turnaround time was only 8 h for PCR but 5 days for PFGE. This PCR-based method is excellent for rapid and inexpensive typing of MRSA in an outbreak setting, but the discriminatory power and reproducibility are still insufficient to replace PFGE in longitudinal studies in the endemic setting.

Cell wall monoglycine cross-bridges and methicillin hypersusceptibility in a femAB null mutant of methicillin-resistant Staphylococcus aureus.

The femAB operon is involved in the formation of the characteristic pentaglycine side chain of the staphylococcal peptidoglycan. Allele replacement of the femAB operon with the tetracycline resistance determinant tetK in a methicillin-resistant Staphylococcus aureus strain resulted in impaired growth, methicillin hypersusceptibility, and lysostaphin resistance. The usual pentaglycine cross-bridges were replaced by monoglycine bridges exclusively, and cross-linking of the peptidoglycan strands was drastically reduced. Complementation of the femAB null mutant by either femA or femAB resulted in the extension of the cross-bridges to a triglycine or a pentaglycine, respectively. This finding suggests that FemA is responsible for the formation of glycines 2 and 3, and FemB is responsible for formation of glycines 4 and 5, of the pentaglycine side chain of the peptidoglycan precursor. Moreover, it can be deduced that addition of the first glycine must occur by a femAB-independent mechanism.