Coagulase-negative staphylococci (CoNS) are now recognized as the aetiological agents of an important range of infections in humans. Most developed countries have reported an increase in CoNS infections in hospitalized patients that are resistant to meticillin and other antibiotics. Staphylococcal cassette chromosome mec (SCCmec) typing is essential for understanding the molecular epidemiology of meticillin-resistant Staphylococcus strains. SCCmec elements are currently classified into types I to VI based on the characteristics of the mec and ccr gene complexes and are further classified into subtypes according to their 'junkyard DNA' region. We evaluated the distribution of SCCmec types in CoNS from patients attending the Hospital de Clínicas de Porto Alegre over the period August 2004-December 2005. Among the 129 bloodstream isolates, 36 (27.9 %) harboured SCCmec type I, 4 (3.0 %) harboured SCCmec type II, 67 (52 %) harboured SCCmec type III, 1 (0.8 %) harboured SCCmec type IV and 4 (3.0 %) harboured SCCmec types I and III. Seventeen isolates were not typable. Identification of CoNS at the species level indicated that Staphylococcus epidermidis was the most common species, with 87 isolates, followed by Staphylococcus haemolyticus (15), Staphylococcus hominis (13), Staphylococcus capitis (12) and Staphylococcus sciuri (1). SCCmec type III was the most prevalent among isolates of S. epidermidis (52 %). Among these strains, 30 (23 %) harboured a modified SCCmec type III which contained an additional dcs region in comparison with regular type III. SCCmec type III was also highly prevalent (75 %) among S. capitis isolates. The predominant SCCmec type found among S. haemolyticus isolates was type I. However, all four isolates harbouring SCCmec type II belonged to S. haemolyticus. Our results indicate that SCCmec type III was the most prevalent among the CoNS. Isolates with SCCmec type III were more resistant to non-b-lactam antimicrobials than isolates harbouring SCCmec types I, II and IV, although the increase in resistance was statistically significant only for clindamycin (P50.021), rifampicin (P50.010) and levofloxacin (P50.005).

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The prevalence of the ermA, ermB and ermC genes were 29.6%, 17.1% and 0.66% respectively, and constitutive resistance was the most frequent as compared to the other two phenotypes.

Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.

Biofilm resistance mechanisms are multifactorial and vary from one organism to another. The purpose of this study was to investigate the efficacy of linezolid against indwelling device-related meticillin-resistant Staphylococcus epidermidis (MRSE) biofilm, and compare this with other antimicrobials. MICs, minimum biofilm inhibitory concentrations (MBICs) and minimum biofilm eradication concentrations (MBECs) were determined by the microtitre plate method. Fourteen and thirteen isolates from patients with indwelling device-related bacteraemia (IDB) and indwelling device colonization not associated with bacteraemia, respectively, were assessed. High MBIC was associated with a high intensity of biofilm formation (gentamicin r50.796; linezolid r50.477; rifampicin r50.634; tigecycline r50.410; and vancomycin r50.771), but this correlation was not observed with MBEC. Linezolid demonstrated better in vitro antimicrobial activity than other antimicrobials (MBIC -gentamicin P,0.001, rifampicin P50.019, vancomycin P50.008; MBEC -gentamicin P,0.001, rifampicin P50.002, vancomycin P,0.001). Biofilm growth inhibition was strongly associated with biofilm formation intensity; however, biofilm eradication was not cell number dependent. MRSE biofilm eradication would represent a huge advance for IDB, although high concentrations of gentamicin, linezolid, rifampicin, tigecycline and vancomycin were required for that. In general, linezolid reached better in vitro concentrations and was demonstrated to be highly active against MRSE biofilms by inhibiting their growth during biofilm formation.

Biofilm-forming staphylococci are known for being opportunistic and invasive pathogens that cause severe disease, mostly catheter-related infections. Early detection and pathogenic strains carrying highly transferable resistance cassettes epidemiology are essential for infection spread control. Hence, this study was designed to evaluate staphylococci biofilm formation and SCCmec typing. Biofilm production and SCCmec typing were evaluated using a semi-quantitative method based on microtiter plates and a multiplex PCR for types, I-V, respectively. Blood cultures and peripheral intravenous device (IVD) staphylococci were consecutively enrolled and allocated into two different groups (invasive and colonizing) based on clinical and microbiological criteria. Seventy-four invasive and 30 colonizing isolates from distinct patients were studied. Vancomycin was the most administrated antimicrobial agent among these patient's treatments. Biofilm formation was observed in 89% of invasive and 64% of colonizing isolates (p < 0.05). There was significant difference regarding SCCmec typing between colonizing and invasive isolates when harboring SCCmec types IV or V (p < 0.05), but no correlation between biofilm intensity and SCCmec types was verified. The SCCmec elements spread are still ongoing and for that reason, antimicrobial resistance evolution in invasive and colonizing biofilm-forming staphylococci is highly relevant.

In the past two decades the members of the genus Enterococcus have emerged as important nosocomial pathogens worldwide. In the present study, we evaluated the antimicrobial resistance and genotypic characteristics of 203 Enterococcus spp. recovered from different clinical sources from two hospitals in Porto Alegre, Rio Grande do Sul, Brazil. The species were identified by conventional biochemical tests and by an automated system. The genetic diversity of E. faecalis presenting high-level aminoglycoside resistance (HLAR) was assessed by pulsed-field gel electrophoresis of chromosomal DNA after SmaI digestion. The E. faecalis was the most frequent specie (93.6%), followed by E. faecium (4.4%). The antimicrobial resistance profile was: 2.5% to ampicillin, 0.5% to vancomycin, 0.5% teicoplanin, 33% to chloramphenicol, 2% to nitrofurantoin, 66.1% to erythromycin, 66.5% to tetracycline, 24.6% to rifampicin, 30% to ciprofloxacin and 87.2% to quinupristin-dalfopristin. A total of 10.3% of the isolates proved to be HLAR to both gentamicin and streptomycin (HLR-ST/GE), with 23.6% resistant only to gentamicin (HLR-GE) and 37.4% only to streptomycin (HLR-ST). One predominant clonal group was found among E. faecalis HLR-GE/ST. The prevalence of resistance among beta-lactam antibiotics and glycopeptides was very low. However, in this study there was an increased number of HLR Enterococcus which may be spreading intra and inter-hospital.