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.

Accuracy of phenotypic methicillin susceptibility methods in the detection of Staphylococcus aureus isolates carrying different SCCmec types.

A total of 138 isolates, 118 methicillin-resistant Staphylococcus aureus (MRSA) isolates (staphylococcal cassette chromosome type II, 20 isolates, type III, 39 isolates and type IV, 59 isolates) and 20 methicillin-sensitive S. aureus isolates were evaluated by phenotypic methods: cefoxitin and oxacillin disk diffusion (DD), agar dilution (AD), latex agglutination (LA), oxacillin agar screening (OAS) and chromogenic agar detection. All methods showed 100% specificity, but only the DD tests presented 100% sensitivity. The sensitivity of the other tests ranged from 82.2% (OAS)-98.3% (AD). The LA test showed the second lowest sensitivity (86.4%). The DD test showed high accuracy in the detection of MRSA isolates, but there was low precision in the detection of type IV isolates by the other tests, indicating that the genotypic characteristics of the isolates should be considered.

Accuracy of phenotypic methicillin susceptibility methods in the detection of Staphylococcus aureus isolates carrying different SCCmec types

A total of 138 isolates, 118 methicillin-resistant Staphylococcus aureus (MRSA) isolates (staphylococcal cassette chromosome type II, 20 isolates, type III, 39 isolates and type IV, 59 isolates) and 20 methicillin-sensitive S. aureus isolates were evaluated by phenotypic methods: cefoxitin and oxacillin disk diffusion (DD), agar dilution (AD), latex agglutination (LA), oxacillin agar screening (OAS) and chromogenic agar detection. All methods showed 100 percent specificity, but only the DD tests presented 100 percent sensitivity. The sensitivity of the other tests ranged from 82.2 percent (OAS)-98.3 percent (AD). The LA test showed the second lowest sensitivity (86.4 percent). The DD test showed high accuracy in the detection of MRSA isolates, but there was low precision in the detection of type IV isolates by the other tests, indicating that the genotypic characteristics of the isolates should be considered.

Detecção de metalo-beta-lactamases em amostras hospitalares de Pseudomonas aeruginosa e Acinetobacter baumannii / Detection of metallo-beta-lactamases in hospital strains of Pseudomonas aeruginosa and Acinetobacter baumannii

INTRODUÇÃO E OBJETIVO: Entre os bacilos Gram-negativos não fermentadores da glicose, Pseudomonas aeruginosa e Acinetobacter baumannii são os agentes etiológicos de infecções hospitalares mais frequentes. A resistência aos carbapenemas entre esses patógenos está se tornando um problema terapêutico mundial e a produção de metalo-beta-lactamases (MBL) tem emergido como um dos mecanismos responsáveis por esta resistência. O presente estudo objetivou verificar a produção de MBL em amostras de P. aeruginosa e A. baumannii isoladas de pacientes internados no Hospital Estadual Azevedo Lima, localizado em Niterói-RJ. MATERIAL E MÉTODOS: Um total de 400 amostras (286 P. aeruginosa e 114 A. baumannii) foi identificado por meio de sistemas comerciais e sua susceptibilidade aos antimicrobianos foi testada por disco-difusão. As amostras resistentes a ceftazidima foram submetidas à avaliação da produção de MBL pelo método de disco-aproximação. RESULTADOS: A produção de MBL foi verificada em 49 amostras de P. aeruginosa (17,1 por cento) e em 20 amostras de A. baumannii (17,5 por cento). Grande parte das amostras de P. aeruginosa e de A. baumannii foi resistente a todos os antimicrobianos testados. CONCLUSÃO: A produção de MBL é um dos mecanismos de resistência aos carbapenemas e outros beta-lactâmicos entre amostras de P. aeruginosa e A. baumannii isoladas no hospital-alvo desta investigação. A rápida detecção deste fenótipo de resistência é essencial para implementar medidas rígidas de controle de infecção e permitir o início de terapia empírica adequada.

INTRODUCTION AND OBJECTIVE: P. aeruginosa and A. baumannii are the most prevalent etiological agents of hospital infections among non-fermentative gram-negative bacilli. Carbapenem resistance among these pathogens has become a therapeutic problem worldwide. Metallo-beta-lactamases (MBL) production has emerged as one of the mechanisms responsible for this resistance. The aim of the present study was to assess MBL production in clinical isolates of P. aeruginosa and A. baumannii from inpatients at Hospital Estadual Azevedo Lima, Niterói, RJ. MATERIAL AND METHODS: A total of 400 strains of P. aeruginosa (286) and A. baumannii (114) were identified through commercial systems. Antimicrobial susceptibility was tested by disk diffusion. Isolates resistant to ceftazidime were screened for MBL production by disk approximation test. RESULTS: MBL production was detected in 49 (17.1 percent) P. aeruginosa isolates and in 20 (17.5 percent) A. baumannii isolates. Most P. aeruginosa and A. baumannii samples were resistant to all antimicrobial agents. CONCLUSION: Production of MBL is one of the mechanisms of carbapenem resistance and other beta-lactams among P. aeruginosa and A. baumannii isolates in the investigated hospital. The rapid detection of this resistance phenotype is essential to implement strict infection control procedures and initiate appropriate empirical therapy.

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.

Improved and rapid detection of methicillin-resistant Staphylococcus aureus nasal carriage using selective broth and multiplex PCR.

To improve efficiency in detecting nasal methicillin-resistant Staphylococcus aureus (MRSA), we evaluated a multiplex PCR using pre-enrichment of the specimen in selective broths, and compared it with detection performed by routine tests in hospital laboratories. Nasal swab specimens from 311 patients were inoculated onto mannitol-salt agar (MSA) at the hospital laboratories and in two Mueller-Hinton broths with 7% NaCl containing oxacillin at concentrations of 2 and 4 micro g/ml. Isolates on MSA were identified as MRSA by classical laboratory tests (coagulase and oxacillin disk diffusion tests). Oxacillin broth cultures were subcultured on blood agar and MRSA isolates were identified by coagulase and susceptibility tests, including agar dilution and the oxacillin-screening method (gold standard method). Simultaneously, multiplex-PCR was performed from the selective broths to detect S. aureus species-specific and mecA gene segments (OxMPCR method). Thirty-two S. aureus isolates were recovered: 29 (90.6%) were MRSA strains and 3 (9.4%) were oxacillin-susceptible isolates. Twenty-eight (96.5%) MRSA isolates were detected by OxMPCR, while 17 (58.6%) were identified by routine tests (P=0.002). This new method for detection of MRSA nasal carriers showed higher sensitivity and led to faster reporting--i.e., within 24 h--of results.

Clonal composition of Staphylococcus aureus isolates at a Brazilian university hospital: identification of international circulating lineages.

In only a few instances has the clonal composition of Staphylococcus aureus collections that include methicillin-susceptible S. aureus (MSSA) been extensively characterized. In order to investigate the clonal composition of MSSA and methicillin-resistant S. aureus (MRSA) and examine whether the infections diagnosed at our hospital were related to internationally distributed S. aureus lineages, we collected 89 clinical S. aureus isolates from patients at a public university hospital in Rio de Janeiro, Brazil, from September 1999 to June 2000. All S. aureus isolates were genotyped by pulsed-field gel electrophoresis and multilocus restriction fragment typing (MLRFT), and a subset (n = 17) was further characterized by multilocus sequence typing (MLST). The 34 MRSA isolates were additionally characterized by SCCmec typing. The MSSA population (n = 55) was grouped into 18 restriction fragment types (RFTs); of these, five RFTs accounted for 67% (37) of the MSSA isolates. MRSA isolates were clustered into only three RFTs (P = 0.02). The majority of MSSA RFTs were related to sequence type 30 (ST30) (12 isolates, 22%), ST1, ST188, and ST432 (6 isolates, 11% each). The predominant MRSA RFT comprised 31 (91%) of 34 isolates; four randomly selected isolates of this RFT were ST239, the previously described widely disseminated Brazilian clone. However, a fifth isolate belonging to this RFT was the ST644, a new single locus variant of ST239. By applying MLRFT and MLST, we found evidence for a clonal structure in MSSA isolates and detected the dissemination of MSSA clonal complexes 1, 5, 8, 30, and 45.

Genomic fingerprinting of bacteriocin-producer strains of Staphylococcus aureus.

Among 363 strains of Staphylococcus aureus, 21 were shown to produce bacteriocins (Bac), antimicrobial peptides with potential biotechnological applications. This collection includes strains which are either isolated from food, patients and healthy cattle, or are involved in subclinical bovine mastitis. From these 21 strains, 17 were shown to carry closely-related 8.0-kb Bac plasmids encoding bacteriocins either identical to or similar to aureocin A70, a bacteriocin able to inhibit strains of Listeria monocytogenes, a food-borne pathogen. Such findings prompted us to investigate the genetic relationships among these Bac+ strains. To obtain more discriminatory results, a combined analysis of AP-PCR, rep-PCR, and a modified PCR technique that we designated SD-PCR was employed. The 17 Bac+ strains harboring 8.0-kb Bac plasmids exhibited seven fingerprint patterns. One such genotype was composed of 8 out of the 11 strains associated with bovine mastitis, which suggests the prevalence of a clone of Bac+ strains involved in this animal infection carrying 8.0-kb Bac plasmids. Our data support the assumption that Bac+ strains of S. aureus carrying genetically related 8.0-kb Bac plasmids do not belong to a single clone. It seems, therefore, that 8.0-kb Bac plasmids have spread horizontally among different S. aureus strains. There also seems to be genetic diversity among the remaining Bac+ strains analyzed.