Evaluation of rep-PCR/DiversiLab versus PFGE and spa typing in genotyping methicillin-resistant Staphylococcus aureus (MRSA).

Pulsed-field gel electrophoresis (PFGE) is the 'gold standard' for genotyping of methicillin-resistant Staphylococcus aureus (MRSA); however, the DiversiLab (DL) system, based on rep-PCR, is faster, simpler and could be better adapted to daily routine hospital work. We genotyped 100 MRSA isolates using PFGE, DL, and spa typing, and evaluated the discriminatory power of each technique and the correlation between them by Simpson's index(SI) and adjusted Rand coefficient (ARI), respectively. The isolates were from clinical samples from eight hospitals in Extremadura (Spain) during 2010. DL separated the 100 MRSA into 18 patterns, with 69% of the isolates grouped into four predominant patterns. spa typing reported 17 spa types, classifying 69% of MRSA into two major types (t067 and t002). PFGE revealed the existence of 27 patterns, gathering 54% of MRSA into three pulse types (E8a, E7a and E7b). SI values were 0.819, 0.726, 0.887 and 0.460 for DL, spa typing, PFGE and CC-BURP, respectively. ARI values of DL over PFGE, spa typing and CC-BURP were 0.151, 0.321 and 0.071, respectively. DL has less discriminatory power than PFGE but more than spa typing. The concordance of DL with PFGE is low, primarily because DL does not discriminate between the three predominant MRSA pulse types in our environment.

An analysis of the association between genotype and antimicrobial resistance in methicillin-resistant Staphylococcus aureus clinical isolates.

Genotyping methods are useful resources for the surveillance, detection, prevention and control of multidrug-resistant nosocomial agents, such as methicillin-resistant Staphylococcus aureus (MRSA). An understanding of the association between genotype and antibiotic susceptibility in MRSA clones may be useful in the surveillance of MRSA and to avoid inappropriate treatment future resistance. We genotyped MRSA clinical isolates from the Extremadura region of Spain using pulsed field electrophoresis (PFGE) and analyzed the spectrum of antibiotic susceptibility for each isolate to determine whether resistance is associated with specific genotypes. PFGE revealed six major genotypes: E8a (25%), E7b (17%), E7a (12%), E8B (8%), E10 (6%), and E20 (4%). Isolates with the genotypes E8a and E10 exhibit higher resistance ratios for levofloxacin than isolates with the other major pulsotypes. Similar results were obtained for isolates with the E20 pulsotype with respect to mupirocin. Although we identified no vancomycin-, tigecycline-, linezolid- or daptomycin-resistant strains, we observed significant differences in the mean MIC values obtained for some of these drugs among the major genotypes. Specifically, isolates with the E7b, E8b, and E20 genotypes have signif-icantly higher MICs of tigecycline, vancomycin and linezolid, respectively, than the most sensitive pulsotypes. Isolates with the E8b profile also exhibit a significantly higher rate of re-duced vancomycin susceptibility (RVS) (i.e., MIC between 1 and 2 mg/L) than clones with the E10 and E8a profiles. In conclusion, we report associations between genotype and antibiotic sensitivity that should be considered in programs for monitor-ing and controlling MRSA in health care settings.

[State of infection caused by methicillin-resistant Staphylococcus aureus (MRSA) in Extremadura: susceptibility, clonality and role of community-associated MRSA]. / Situación actual de la infección por Staphylococcus aureus resistente a meticilina en Extremadura: sensibilidad, clonalidad, y protagonismo de la adquisición extrahospitalaria.

The correct surveillance and control of infection caused by methicillin-resistant Staphylococcus aureus (MRSA) needs of update knowledge of its specific properties in each place. Our study aims to describe the current characteristics of infection due to MRSA in Extremadura. During 2010, 309 MRSA were collected from clinical samples in our region. A susceptibility test that included 17 antibiotics tested by AST -588 card Vitek 2 ® and E -test method was performed on all isolates. A sample of 100 strains, selected by stratified random sampling, were genotyped by pulsed field electrophoresis (PFGE). The prevalence of MRSA in Extremadura was 20.2%. Don Benito-Villanueva area showed the most prevalence and a higher incidence. Merida reported the most favourable situation, with a relatively low ratios of prevalence and incidence. The community acquired reached 44 % in the region, showing predominantly in less populated areas (Navalmoral and Coria). The most common multiresistant pattern was tobramycin-levofloxacin-erythromycin (44%), followed tobramycin-erythromycin-clindamycin (20%). No linezolid, daptomycin and tigecycline resistant strains were observed, but 42 % of the MRSA strains showed decreased susceptibility vancomycin (DSV). PFGE analysis reported 27 genotypes, with 3 major genotypes: E8a (25%), E7b (17%) and E7a (12%). The post-hoc statistical analysis did not reveal significant differences in the distribution of genotypes between different areas. However it revealed some trends that should be considered.

Y155H amino acid substitution in influenza A(H1N1)pdm09 viruses does not confer a phenotype of reduced susceptibility to neuraminidase inhibitors.

The Y155H amino acid substitution in the neuraminidase gene (NA) has previously been associated with highly reduced inhibition by neuraminidase inhibitors in the seasonal H1N1 influenza A virus which circulated in humans before the 2009 pandemic. During the 2012/13 epidemic season in Spain, two A(H1N1) pdm09 viruses bearing the specific Y155H substitution in the NA were detected and isolated from two patients diagnosed with severe respiratory syndrome and pneumonia requiring admission to the intensive care unit. Contrary to what was observed in the seasonal A(H1N1) viruses, neither of the Y155H A(H1N1) pdm09 viruses described here showed a phenotype of reduced inhibition by NAIs as determined by the neuraminidase enzyme inhibition assay (MUNANA). High-throughput sequencing of the NA of both Y155H viruses showed that they were composed to >99% of H155 variants. We believe that this report can contribute to a better understanding of the biological significance of amino acid substitutions in the neuraminidase protein with regard to susceptibility of influenza viruses to neuraminidase inhibitors. This is of critical importance for optimal management of influenza disease patients.

Outbreak of multiresistant Acinetobacter baumannii in a polyvalent intensive care unit: clinical, epidemiological analysis and PFGE-printing evolution.

An outbreak of multidrug-resistant Acinetobacter baumannii (MRAB) occurred over the course of a 27-week period in our adult polyvalent intensive care unit (ICU). Twenty-one patients were affected, and 72 strains were identified from different clinical samples. The strains were resistant to all antibiotics except for colistin and ampicillin/sulbactam. Forty-nine MRAB strains collected from 18 patients were analysed by pulsed-field gel electrophoresis (PFGE). This analysis revealed four highly-related PFGE types (genetic similarity index >90%) termed 1, 2, 3 and 4, that were isolated in 13, seven, one, and three patients, respectively. A single PFGE type was identified from five of ten patients with successive isolation of MRAB; in the other five patients, two or three PFGE types were detected. This suggested phased evolution of PFGE types 2, 3 and 4 from PFGE type 1. Global mortality was high (13 patients; 62%). Non-survivors had higher APACHE II scores than survivors on the date that MRAB was isolated (OR = 1.57; 95% CI [1.02, 2.44]). The outbreak was controlled after implementation of an extensive infection control program.

Moxifloxacin and biofilm production by coagulase-negative staphylococci.

The in vitro activity of moxifloxacin against 41 strains of coagulase-negative staphylococci was determined. A relationship between the activity of moxifloxacin and biofilm formation was detected. Biofilm-producing strains were more resistant to moxifloxacin than biofilm-negative strains. Our global results obtained with six strains of Staphylococcus epidermidis showed that subinhibitory concentrations of moxifloxacin did not significantly modify biofilm formation. On the other hand, moxifloxacin concentrations of 2, 10, 50 and 100 x MIC produced a log decrease in viable count (included in a biofilm) of 0.20, 0.37, 1.10 and 1.69, respectively.

Effect of ritonavir and saquinavir on Candida albicans growth rate and in vitro activity of aspartyl proteinases.

An in vitro study to evaluate the antifungal effect and activity of aspartyl proteinases of the HIV-proteinase inhibitors ritonavir and saquinavir was conducted. Ritonavir diminished the growth rate of Candida albicans as well as the activity of its secreted aspartyl proteinases (Saps) in a nitrogen-limited medium, yeast carbon base and bovine serum albumin (YCB-BSA). This inhibition occurred in a dose-dependent fashion; with 8 mg l(-1) of ritonavir a partial growth inhibition (44%) was produced. The growth rate of C. albicans in medium with saquinavir was similar to that seen in the control, and Sap activity was inhibited only at high concentrations. In conventional medium (RPMI-1640), which does not induce the production of yeast proteases, no inhibitory effect was detected with either HIV-protease inhibitor.