Keli Cristine Reiter

Evaluation of oxacillin and cefoxitin disks for detection of resistance in coagulase negative staphylococci

Coagulase-negative Staphylococcus spp. was considered nonpathogenic until the emergence of multiresistance and the demonstration of their participation as infectious agents. In Brazil, oxacillin resistance may be present in over 80% of isolates, and the Clinical and Laboratory Standards Institute standardized a disk-diffusion method to predict this resistance in Staphylococcus. The aim of this study was to evaluate the variability among commercial disks of oxacillin (1 microg) and cefoxitin (30 microg) widely used in clinical laboratories of microbiology, compared with mecA gene and minimum inhibitory concentration (MIC) of oxacillin. The use of oxacillin and cefoxitin disks simultaneously allowed the detection of important differences, particularly, in less frequent species such as S. cohnii, S. haemolyticus, S. saprophyticus, and S. sciuri. Disks of cefoxitin of the brand 2 displayed good correlation with the mecA gene (98.7%) and oxacillin MIC (97.8%), while major discrepancies were observed using disks of brand 1. One of the critical points in the diffusion disk test is the quality of the disks: the use of better quality disks associated with molecular methods lead to better results to define the best antibiotic therapy.

Correlation between biofilm formation and gelE, esp, and agg genes in Enterococcus spp. clinical isolates

Enterococci are a leading cause of nosocomial infections. Enterococcus faecalis is the species most frequently isolated, and it is commonly recovered from surgical wounds, intra-abdominal infections, the bloodstream, and especially urinary tract infections (UTIs).1,2

High biofilm production by invasive multiresistant staphylococci.

Reiter KC, Paim TGdS, de Oliveira CF, d’Azevedo PA. High biofilm production by invasive multiresistant staphylococci. APMIS 2011; 119: 776–81.

EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES

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 its storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.

Upregulation of icaA, atlE and aap genes by linezolid but not vancomycin in Staphylococcus epidermidis RP62A biofilms

Biofilms are complex bacterial structures protected by a self-produced polymer matrix that enables survival in hostile environments. Biofilms are more resistant to antibiotics than their planktonic counterparts and are therefore more difficult to eradicate. The aim of this study was to investigate the influence of vancomycin and linezolid on the maintenance of staphylococcal biofilms and their effect on the expression of biofilm-associated genes in Staphylococcus epidermidis. Pre-formed biofilms of S. epidermidis RP62A were challenged with linezolid and vancomycin at different concentrations as well as at their clinically relevant target concentration (15 mg/L) over time. Expression of icaA, atlE, aap, rnaIII, luxS, sarA, rsbU and icaR genes following 2h of exposure to these antibiotics was determined by quantitative PCR. Vancomycin did not significantly affect the biofilm under the tested conditions. However, linezolid affected the biofilm structure at concentrations of ≥ 2 mg/L (P<0.05); moreover, the exposure time to this antibiotic was a determinant for biofilm eradication. The level of transcription of icaA, aap and atlE increased by 5.18-, 2.58- and 3.06-fold, respectively, in biofilms exposed to linezolid, but no changes were observed for vancomycin. The other genes were not affected by these antibiotics. This study demonstrated that linezolid was effective in eradicating biofilms formed by S. epidermidis RP62A. Under the conditions tested, linezolid upregulated biofilm-associated genes probably due to the stress caused by low-dose antibiotic stimulation. In this study, linezolid showed better performance than vancomycin against staphylococcal biofilms.

Rifampicin fails to eradicate mature biofilm formed by methicillin-resistant Staphylococcus aureus

INTRODUCTIONnAntimicrobial activity on biofilms depends on their molecular size, positive charges, permeability coefficient, and bactericidal activity. Vancomycin is the primary choice for methicillin-resistant Staphylococcus aureus (MRSA) infection treatment; rifampicin has interesting antibiofilm properties, but its effectivity remains poorly defined.nnnMETHODSnRifampicin activity alone and in combination with vancomycin against biofilm-forming MRSA was investigated, using a twofold serial broth microtiter method, biofilm challenge, and bacterial count recovery.nnnRESULTSnMinimal inhibitory concentration (MIC) and minimal bactericidal concentration for vancomycin and rifampicin ranged from 0.5 to 1mg/l and 0.008 to 4mg/l, and from 1 to 4mg/l and 0.06 to 32mg/l, respectively. Mature biofilms were submitted to rifampicin and vancomycin exposure, and minimum biofilm eradication concentration ranged from 64 to 32,000 folds and from 32 to 512 folds higher than those for planktonic cells, respectively. Vancomycin (15mg/l) in combination with rifampicin at 6 dilutions higher each isolate MIC did not reach in vitro biofilm eradication but showed biofilm inhibitory capacity (1.43 and 0.56log10 CFU/ml reduction for weak and strong biofilm producers, respectively; p<0.05).nnnCONCLUSIONSnIn our setting, rifampicin alone failed to effectively kill biofilm-forming MRSA, demonstrating stronger inability to eradicate mature biofilm compared with vancomycin.

Enhancement of antistaphylococcal activities of six antimicrobials against sasG-negative methicillin-susceptible Staphylococcus aureus: an in vitro biofilm model

This study was designed to evaluate antimicrobial activities against methicillin-susceptible Staphylococcus aureus in both sessile and planktonic forms and to detect genes associated with this biofilm phenotype. Minimal biofilm inhibition and eradication concentrations (MBIC and MBEC, respectively) were determined by an in vitro biofilm model, and icaA, atlA, and sasG genes were detected by polymerase chain reaction. Vancomycin and tigecycline presented better biofilm inhibitory activity (MBIC range: 4-8 μg/mL) (P ≤ 0.05) and lower MBEC/MIC ratios (P ≤ 0.001) than other antimicrobials. All isolates harbored icaA and atlA, whereas sasG was present only in strong biofilm formers (P ≤ 0.05). Interestingly, antimicrobial activities against sasG- weak biofilm formers were significantly higher than those against sasG+ strong biofilm formers (P ≤ 0.05), demonstrating that number of cells in a biofilm matrix affected the antimicrobial activity, which was also variable, and might be associated with specific genetic determinants. To our knowledge, this was the first study reporting the presence of sasG in clinical isolates of S. aureus in South America.

Imbalance in DNA repair machinery is associated with BRAFV600E mutation and tumor aggressiveness in papillary thyroid carcinoma

The involvement of alterations in MLH1, an essential mismatch repair component, in BRAFV600E mutated papillary thyroid carcinoma (PTC) has been suggested to be associated with features of tumor aggressiveness. Thirty-two PTC and surrounding normal thyroid tissues were evaluated for 11 representative DNA repair genes expression. BRAFV600E mutational status assessment and clinicopathological correlations were evaluated for their gene and protein expression. BRAFV600E PTC is associated with lower levels of XPD and MLH1 gene expression. Decrease in MLH1 and XPD mRNA levels in BRAFV600E PTC (but not their protein products) are associated with predictors of poor patient outcomes. Considering the complete subset of patients, MGMT and XRCC2 genes were shown down and upregulated, respectively, in PTC tissues. Low expression of MGMT gene and weak XRCC2 protein expression were correlated with characteristics of tumor aggressiveness. These results suggest that an imbalance in DNA repair gene expression in PTC is associated with aggressive clinicopathological features and BRAFV600E mutation.

Coagulase-negative staphylococci in southern brazil: Looking toward its high diversity

INTRODUCTIONnCoagulase-negative staphylococci (CoNS) are the most prevalent pathogens in nosocomial infections and may serve as a reservoir of mobile genetic elements such as the staphylococcal cassette chromosome mec (SCCmec) encoding methicillin resistance. Molecular characterization of SCCmec types combined with advanced molecular typing techniques may provide essential information for understanding the evolution and epidemiology of CoNS infections. We therefore aimed to investigate the SCCmec distribution, multidrug-resistance (MDR), and biofilm formation in CoNS blood culture isolates from a hospital in Southern Brazil.nnnMETHODSnWe analyzed 136 CoNS blood culture isolates obtained during 2002-2004 from patients admitted to a tertiary care hospital in Brazil. SCCmec types I to V were determined using multiplex PCR. The clonal relationship of Staphylococcus epidermidis was determined using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Molecular epidemiological data were interpreted along with data on biofilm formation, presence of the icaD gene, and MDR.nnnRESULTSnThe most prevalent species were S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis harboring mainly SCCmec types II, III, and V. Overall, the presence of multiple SCCmec was associated with non-MDR, except for S. epidermidis. S. epidermidis isolates showed a high prevalence of icaD, but had low phenotypic biofilm formation. PFGE and MLST revealed high genetic diversity in the S. epidermidis population.nnnCONCLUSIONSnOur results suggest a major shift in SCCmec types within a short period and reveal a different behavior of S. epidermidis with regard to the association between the presence of multiple SCCmec types and MDR profile.

Coexistence of virulence genes in methicillin-resistant Staphylococcus aureus clinical isolates

INTRODUCTIONnThe pathogenic versatility of Staphylococcus aureus is attributed to various virulence genes, including enterotoxins and hemolysins.nnnMETHODSnHere, the virulence genes in 177 nosocomial MRSA strains in Porto Alegre, Brazil were detected by PCR.nnnRESULTSnThe overall prevalence rates were as follows: sea, 4.5%; pvl, 18.6%; tst, 27.7%; hla, 87.6%; and hld, 90.4%. No strain contained all tested genes. However, there was frequent coexistence of tst with pvl and hla with hld (40.7% and 26.6%, respectively).nnnCONCLUSIONSnHorizontal transfer of virulence genes is very common in S. aureus, as suggested by the frequent coexistence of several virulence genes.