Characterization of MLS(b) resistance among Staphylococcus aureus and Staphylococcus epidermidis isolates carrying different SCCmec types.

This work characterizes MLS(b) resistance in 39 methicillin-resistant Staphylococcus aureus (MRSA) and 32 Staphylococcus epidermidis (MRSE) isolates. Of 21 erm(A) gene encoding MRSA isolates, 71.4% carried SCCmecIII, whereas of 12 isolates carrying the erm(C) gene, 83.3% carried SCCmecIV. Among the 25 MRSE isolates positive for the erm(C) gene, 80% had SCCmecIV or nontypeable cassettes. Isolates carrying these genes had MIC(90) ≥ 256 µg/mL to erythromycin and clindamycin. The msr(A) gene was associated with a low MIC(90) to these drugs. The erm(A) gene was associated with SCCmecIII in MRSA isolates, whereas the erm(C) gene was associated with SCCmecIV in both MRSA and MRSE isolates.

A Brazilian lineage of Staphylococcus lugdunensis presenting rough colony morphology may adhere to and invade lung epithelial cells.

Staphylococcus lugdunensis is an unusually virulent coagulase-negative species, which causes serious infection similar to S. aureus. We evaluated the expression of virulence factors such as S. lugdunensis synergistic haemolysin (SLUSH), fibrinogen-binding protein (Fbl), biofilm production and biofilm-production-related genes in 23 S. lugdunensis clinical isolates and one type strain that had been previously characterized for their genotypes. In addition, the biofilm composition and the ability of isolates to adhere to and invade human epithelial lung cells were also investigated. The PCR method used detected the presence of slush and intercellular adhesin (ica) virulence genes in all isolates. All isolates produced the Fbl protein and, with the exception of the type strain, all isolates produced the SLUSH haemolysin. Fourteen (60.9 %) isolates produced biofilms. The detachment assay, using sodium metaperiodate or proteolytic enzymes to analyse the biofilm composition, showed protein-mediated biofilms in two representative isolates, one for each colony type (rough and smooth). All strongly biofilm-producing isolates, including three with rough colony morphology, had the same prevalent PFGE pattern. However, among the representative strains tested, only the S. lugdunensis isolate that formed rough colonies was able to adhere to and invade A549 cell monolayers in the same quantities as those observed with S. aureus isolates (P = 1.000). No significant adhesion or invasion was observed for the other isolates in comparison with the S. aureus isolate, independent of biofilm production or clonality. Our results could explain the incredible ability of this pathogen to cause infections that are as aggressive as S. aureus. In addition, the ability of S. lugdunensis to adhere to and invade eukaryotic cells was also noticed for isolates with rough colony morphology, reinforcing the increased virulence in this species.

Pentaclethra macroloba tannins fractions active against methicillin-resistant staphylococcal and Gram-negative strains showing selective toxicity

The ethanol extract of the vegetal species Pentaclethra macroloba (Willd.) Kuntze, Fabaceae, was fractioned and the antibacterial activity was determined. The active ethyl acetate (ea) fraction showed activity against Gram-positive (Staphylococcus spp. and Enterococcus spp.) and Gram-negative (Pseudomonas aeruginosa, Acinetobacter spp. and Klebsiella pneumoniae) multiresistant bacteria. Gallic acid derivatives were identified as the main compounds in inactive subfractions from the ea fraction, while the active one afforded ellagic acid as the major constituent when submitted to acid hydrolysis reaction, which suggests the presence of hydrolysable tannins. The minimum bactericidal concentration analysis showed a bactericide mechanism of action for the tannin subfraction found. The antibacterial mechanism of action of the active tannin subfraction against S. aureus reference strains (ATCC 29213 e 33591) was proposed adopting an in vitro assay of protein synthesis inhibition. For this, bacterial cells were labeled with [35S] methionine in the presence of the subfraction. The protein synthesis inhibition was observed at 256 µg/mL of this subfraction. At this concentration it did not present cytotoxicity in eukaryotic cells by the neutral red technique, suggesting selective toxicity. The present study is the first in vitro investigation of the antibacterial properties of tannin fractions obtained from a polar extract of P. macroloba.

Methicillin-resistant Staphylococcus epidermidis carrying biofilm formation genes: detection of clinical isolates by multiplex PCR.

Staphylococcus epidermidis is the most prevalent coagulase-negative Staphylococcus (CNS) and is a major cause of hospital bacteremia. Based on 18 reference strains and 149 Staphylococcus clinical strains, used in a novel multiplex PCR method, the aim of this study was to identify S. epidermidis with respect to the sequence of three genes: recN, which encodes a recombination/repair protein, mecA (methicillin resistance), and icaAB, which is involved in biofilm formation. Amplicons of 219 bp (S. epidermidis-recN gene), 154 bp (mecA gene), and 546 bp (icaAB genes) were obtained. Reliable results were achieved for 100% of the evaluated strains, suggesting that this new multiplex-PCR approach could be useful for the accurate identification of methicillin-resistant S. epidermidis with the potential to produce biofilm.

Methicillin-resistant Staphylococcus epidermidis carrying biofilm formation genes: detection of clinical isolates by multiplex PCR

Staphylococcus epidermidis is the most prevalent coagulase-negative Staphylococcus (CNS) and is a major cause of hospital bacteremia. Based on 18 reference strains and 149 Staphylococcus clinical strains, used in a novel multiplex PCR method, the aim of this study was to identify S. epidermidis with respect to the sequence of three genes: recN, which encodes a recombination/repair protein, mecA (methicillin resistance), and icaAB, which is involved in biofilm formation. Amplicons of 219 bp (S. epidermidis-recN gene), 154 bp (mecA gene), and 546 bp (icaAB genes) were obtained. Reliable results were achieved for 100% of the evaluated strains, suggesting that this new multiplex-PCR approach could be useful for the accurate identification of methicillin-resistant S. epidermidis with the potential to produce biofilm (AU)

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Characterization of virulence factors and clonal diversity of Enterococcus faecalis isolates from treated dental root canals.

The high prevalence of Enterococcus faecalis in root canal treated teeth with post-treatment disease, as evidenced by both molecular and traditional culturing methods, suggests that this species may be a key player in endodontic treatment failure. This study aimed to detect virulence factors by phenotypic and western blotting tests, and virulence genes by PCR from 20 clinical strains of E. faecalis isolated from treated root canals of teeth with (10) or without (10) apical periodontitis. Moreover, genomic diversity of these strains was assessed by pulsed-field gel electrophoresis (PFGE) and rep-PCR. All 20 strains presented the gelE gene (gelatinase), but 10 of them did not hydrolyze gelatin. Seven of the 10 gelatinase-producing isolates were recovered from root canals with lesions, which suggests a role for this virulence factor in the pathogenesis of post-treatment disease. The esp gene was expressed only in cases where gelatinase production was negative. The other virulence genes were found in 90% (efaA and ace genes), 45% (agg gene) and 95% (cpd gene) of the E. faecalis isolates. As for PFGE and rep-PCR, no specific clonal type of E. faecalis was found in association with teeth with or without disease, revealing the interindividual clonal diversity of endodontic infections.

Comparison of endodontic bacterial community structures in root-canal-treated teeth with or without apical periodontitis.

Bacterial occurrence in treated root canals, even in patients without post-treatment apical periodontitis, raises the possibility that factors other than mere bacterial presence can be determinants for a favourable outcome of endodontic treatment. Because these factors may be related to the bacterial communities colonizing the root canal, including virulence, density and interactions, the objective of this study was to compare the community structures found in root-canal-treated teeth with (12 samples) and without (11 samples) apical periodontitis lesions by means of a PCR-denaturing gradient gel electrophoresis fingerprinting approach. Results confirmed a polymicrobial composition even in treated patients without post-treatment disease. A large microbial community diversity was observed for treated teeth both with or without disease, but no specific pattern was detected for diseased teeth. Nevertheless, the number of bands from samples with apical periodontitis lesions was statistically significantly higher (P=0.04) than that from samples collected from root-canal-treated teeth without post-treatment apical periodontitis. Furthermore, predominant bands in samples from patients with apical disease were also observed.

Detection of Staphylococcus lugdunensis by a new species-specific PCR based on the fbl gene.

Staphylococcus lugdunensis are unusually virulent coagulase-negative staphylococci associated with skin infections and endocarditis. We developed an accurate and simple PCR assay to identify S. lugdunensis isolates based on detection of the fbl gene, which encodes a fibrinogen-binding protein involved in pathogen adhesion. The PCR assay was established using 16 reference strains of different Staphylococcus species and further validated with a collection of 63 clinical staphylococcal isolates that were also phenotypically characterized. Reliable results for the detection of S. lugdunensis isolates were obtained for 100% of the strains evaluated, indicating that this PCR assay can be used in the routine of microbiology laboratories as one more tool for correct species differentiation.

Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus: methicillin-resistant isolates are detected directly in blood cultures by multiplex PCR.

In this study, we standardized and evaluated a multiplex-PCR methodology using specific primers to identify Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus and their methicillin-resistance directly from blood cultures. Staphylococci clinical isolates (149) and control strains (16) previously identified by conventional methods were used to establish the multiplex PCR protocol. Subsequently, this methodology was evaluated using a fast and cheap DNA extraction protocol from 25 staphylococci positive blood cultures. A wash step of the pellet with 0.1% bovine serum albumin (BSA) solution was performed to reduce PCR inhibitors. Amplicons of 154bp (mecA gene), 271bp (S. haemolyticus mvaA gene) and 108 and 124bp (S. aureus and S. epidermidis species-specific fragments, respectively) were observed. Reliable results were obtained for 100% of the evaluated strains, suggesting that this new multiplex-PCR combined with an appropriate DNA-extraction method could be useful in the laboratory for fast and accurate identification of three staphylococci species and simultaneously their methicillin resistance directly in blood cultures.

Molecular surveillance of methicillin-susceptible Staphylococcus aureus at a neonatal intensive care unit in Brazil.

BACKGROUND: We evaluated the relationship among hospital infection and colonization by methicillin-susceptible Staphylococcus aureus (MSSA), clonal spread, and associated risk factors in a neonatal intensive care unit (NICU) of the Uberlândia Federal University-affiliated hospital in Brazil. METHODS: Between February 2004 and June 2005, a longitudinal surveillance study was carried out in an NICU with neonates presenting infections, through both the NNIS system and S aureus punctual colonization prevalence inquests. RESULTS: The overall rate of infection incidence was 23/1000 patient-days. Of all the neonates assessed, 15 were infected and 15 colonized. Sepsis was the most frequent infection, whereas anterior nare was the most isolated site. Antibiotics use, central vascular catheter (CVC), and CVC use more than 7 days and its insertion by phlebotomy were the risk factors for colonization/infection. Molecular analysis showed polyclonal origin (12 genotypes), with predominance of a genotype ("B"), and clonal identity between colonization and infection samples. CONCLUSION: The analysis by means of classical epidemiology and molecular techniques pointed out that methicillin-susceptible Staphylococcus aureus infections were associated with previous colonization by the pathogen, with evidence of horizontal transmission within the unit.

Multiplex PCR assay to identify methicillin-resistant Staphylococcus haemolyticus.

Staphylococcus haemolyticus is the most frequently coagulase-negative Staphylococcus species associated with antimicrobial resistance isolated from nosocomial infections. We developed an accurate and simple multiplex PCR assay to identify methicillin-resistant S. haemolyticus (MRSH) isolates. We designed species-specific primers of the mvaA gene that encodes a 3-hydroxy-3-methylglutaryl coenzyme A involved in the mevalonate pathway of the microorganism. Simultaneously, mecA gene primers of methicillin resistance were also used. The PCR assay was established using 16 strains of different reference Staphylococcus species and validated with a collection of 147 clinical staphylococcal isolates that were also phenotypically characterized. Reliable results for the detection of MRSH isolates were obtained for 100% of the strains evaluated, showing that this PCR assay can be used for the routine microbiology laboratories. This is the first report using species-specific multiplex PCR to detect a single segment of S. haemolyticus associated with a segment of mecA gene.