Kátia Regina Netto dos Santos

Association of Enterococcus faecalis With Different Forms of Periradicular Diseases

Data from culture studies have revealed that Enterococcus faecalis is occasionally isolated from primary endodontic infections but frequently recovered from treatment failures. This molecular study was undertaken to investigate the prevalence of E. faecalis in endodontic infections and to determine whether this species is associated with particular forms of periradicular diseases. Samples were taken from cases of untreated teeth with asymptomatic chronic periradicular lesions, acute apical periodontitis, or acute periradicular abscesses, and from root-filled teeth associated with asymptomatic chronic periradicular lesions. DNA was extracted from the samples, and a 16S rDNA-based nested polymerase chain reaction assay was used to identify E. faecalis. This species occurred in seven of 21 root canals associated with asymptomatic chronic periradicular lesions, in one of 10 root canals associated with acute apical periodontitis, and in one of 19 pus samples aspirated from acute periradicular abscesses. Statistical analysis showed that E. faecalis was significantly more associated with asymptomatic cases than with symptomatic ones. E. faecalis was detected in 20 of 30 cases of persistent endodontic infections associated with root-filled teeth. When comparing the frequencies of this species in 30 cases of persistent infections with 50 cases of primary infections, statistical analysis demonstrated that E. faecalis was strongly associated with persistent infections. The average odds of detecting E. faecalis in cases of persistent infections associated with treatment failure were 9.1. The results of this study indicated that E. faecalis is significantly more associated with asymptomatic cases of primary endodontic infections than with symptomatic ones. Furthermore, E. faecalis was much more likely to be found in cases of failed endodontic therapy than in primary infections.

Antimicrobial Ellagitannin of Punica granatum Fruits

The ethyl acetate extract of Punica granatum fruits was fractionated by chromatographic techniques to afford the ellagitannin punicalagin. The substance was found to be active against methicillin-resistant Staphylococcus aureus strains and was identified by HPLC/UV and 1HNMR. The antibacterial assays which guided the isolation of the tannin were conducted using the disc diffusion method. Minimum inhibitory concentration (MIC) was determined by the dilution method according to NCCLS (National Committee for Clinical Laboratory Standards) procedure.

Detection of ileS-2 gene encoding mupirocin resistance in methicillin-resistant Staphylococcus aureus by multiplex PCR

The presence of the ileS-2 gene, responsible for mupirocin resistance, in clinical isolates of methicillin-resistant Staphylococcus aureus was determined by multiplex polymerase chain reaction. Three pairs of primers were used, which yielded specific fragments of femA (encoding a unique feature of S. aureus), mecA (encoding resistance to methicillin) and ileS-2 genes. The multiplex polymerase chain reaction system is an easy and time-saving technique that, together with a rapid method for DNA extraction by boiling, may be incorporated as a routine analysis in clinical diagnostic laboratories.

Clonal Composition of Staphylococcus aureus Isolates at a Brazilian University Hospital: Identification of International Circulating Lineages

ABSTRACT 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.

Emergence of High-Level Mupirocin Resistance in Methicillin-Resistant Staphylococcus aureus Isolated From Brazilian University Hospitals

Surveillance for methicillin-resistant Staphylococcus aureus (MRSA) was implemented in Rio de Janeiro and Uberlândia University Hospitals, which had different policies on use of mupirocin. One hundred fourteen multiresistant MRSA strains were isolated from 62 patients. Mupirocin resistance was observed in 63% of strains in Rio de Janeiro, where there was extensive use of topical mupirocin, and 6.1% in Uberlândia, where its use was rare.

Coagulase-Negative Staphylococci: Comparison of Phenotypic and Genotypic Oxacillin Susceptibility Tests and Evaluation of the Agar Screening Test by Using Different Concentrations of Oxacillin

ABSTRACT This study evaluated the oxacillin susceptibilities of 152 coagulase-negative staphylococcal (CoNS) strains of 12 species by disk diffusion; agar dilution; E-test; the slide latex agglutination test (Slidex MRSA Detection test; bioMérieux S/A, Paris, France); the agar screening test with 1, 2, 4, or 6 μg of oxacillin per ml and incubation for 24 or 48 h; and detection of the mecA gene by PCR. The results revealed that the agar screening test with 4 μg of oxacillin per ml and incubation for 48 h was superior to any single phenotype-based susceptibility assay, presenting a sensitivity and a specificity of 100% each. For the different methods evaluated, the sensitivities and specificities were as follows: for disk diffusion, 94.2 and 91.8%, respectively; for the agar dilution test 100 and 73.5%, respectively; for E-test, 100 and 71.4%, respectively; and for the slide latex agglutination test, 97.1 and 98%, respectively. A good correlation was observed between oxacillin susceptibility testing results and PCR results for Staphylococcus epidermidis, S. haemolyticus, S. hominis subsp. hominis, and all mecA-positive strains. However, at least 60% of the mecA-negative isolates of the species S. saprophyticus, S. cohnii subsp. urealyticum, S. lugdunensis, and S. sciuri were erroneously classified as oxacillin resistant by the agar dilution test. Conversely, the slide latex agglutination test presented a high sensitivity (97.1%) and a high specificity (98%) for all CoNS species. Our results demonstrated the accuracy of the agar screening test with 4 μg of oxacillin per ml and incubation for 48 h and the slide latex agglutination test for the appropriate detection of the oxacillin susceptibilities of CoNS isolates. Both assays are technically simple and can be easier to perform in routine laboratories than PCR.

Selected Endodontic Pathogens in the Apical Third of Infected Root Canals: A Molecular Investigation

Bacteria located at the apical portion of the root canals are conceivably in a strategic position to induce damage to the periradicular tissues and resulting inflammatory diseases. This study sought to investigate the prevalence of 11 selected putative endodontic pathogens in the apical third of infected root canals associated with periradicular lesions. The apical root portion of 23 extracted teeth with carious pulpal exposures and attached periradicular lesions was sectioned, and the root canals were sampled for microbiological investigation. DNA was extracted from the samples and analyzed for the presence of 11 bacterial species using a nested polymerase chain reaction assay. The results showed that Pseuramibacter alactolyticus occurred in 10 cases (44%), Treponema denticola in 6 (26%), Fusobacterium nucleatum in 6 (26%), Porphyromonas endodontalis in 4 (17%), Filifactor alocis in 2 (9%), Dialister pneumosintes in 1 (4%), Porphyromonas gingivalis in 1 (4%), and Tannerella forsythensis in 1 (4%). No sample yielded Prevotella intermedia, Prevotella nigrescens, or Campylobacter rectus. Of the samples examined, 17 were positive for at least 1 of the target species. Occurrence of these bacterial species in the apical third of infected root canals suggests that they can be involved in causation of periradicular lesions.

DNA typing of methicillin-resistant Staphylococcus aureus : isolates and factors associated with nosocomial acquisition in two Brazilian university hospitals

Control and prevention of methicillin-resistant Staphylococcus aureus (MRSA) infections should include early identification of patients at higher risk of MRSA acquisition and analysis of isolates by discriminatory bacterial DNA typing methods. One hundred and three MRSA isolates cultured between Sept. 1994 and Sept. 1995 from 62 patients in two teaching hospitals (hospital 1, in Rio de Janeiro; hospital 2, in Minas Gerais) were tested for antimicrobial resistance and genomic DNA was analysed by pulsed-field gel electrophoresis (PFGE). Ten profiles were identified: A, B, C, I and J in hospital 1 and A, B, D, E, F, G and H in hospital 2. PFGE patterns A and B were isolated at both hospitals. The majority (80%) of isolates had similar PFGE patterns (type A). Subtype A1 was isolated at both hospitals, but was more frequent in hospital 2 (54%), while subtype A2 predominated in hospital 1 (63%). MRSA isolates were resistant to the majority of antimicrobial agents tested. However, susceptibility to vancomycin alone was found in 32% of the isolates at hospital 1, whereas 48% of isolates from hospital 2 were susceptible to both vancomycin and mupirocin, and 34% demonstrated susceptibility to vancomycin, mupirocin and chloramphenicol. Thirty-nine percent of all isolates were mupirocin-resistant, with 90% of these belonging to PFGE pattern A. Four main risk factors were associated with MRSA infection or colonisation which may be useful in the early identification of patients at risk: >7 days hospitalisation (95%), very dependent patients (84%), invasive procedures (79%) and recent antimicrobial therapy (79%). The data demonstrate that PFGE pattern A is disseminated in both hospitals. However, at both hospitals subtypes of pattern A and the other PFGE types were associated with different antibiotic resistance patterns.

Detection of Putative Oral Pathogens in Acute Periradicular Abscesses by 16S rDNA-Directed Polymerase Chain Reaction

A 16S rDNA-directed polymerase chain reaction method was used to assess the occurrence of four black-pigmented anaerobic rods, Treponema denticola, and Actinobacillus actinomycetemcomitans in acute periradicular abscesses. Pus was collected by aspiration from 10 cases diagnosed as acute abscesses of endodontic origin. DNA was extracted from the samples and analyzed using a polymerase chain reaction-based identification assay. The method allowed detecting black-pigmented anaerobes in 80% of the examined abscesses. Porphyromonas endodontalis was found in 70%, T. denticola in 50%, Porphyromonas gingivalis in 40%, and Prevotella intermedia in 10% of the cases. P. gingivalis was always found associated with P. endodontalis. Prevotella nigrescens and A. actinomycetemcomitans were not found in any pus sample. The high prevalence of P. endodontalis, T. denticola, and P. gingivalis suggests that they can play an important role in the etiology of acute periradicular abscesses.

Mupirocin for controlling methicillin-resistant Staphylococcus aureus: lessons from a decade of use at a university hospital.

BACKGROUND From 1990 to 1995 at Hospital Universitário Clementino Fraga Filho, patients colonized or infected with methicillin-resistant Staphylococcus aureus (MRSA) were treated with mupirocin to eliminate MRSA carriage. In 1995, 65% of MRSA patients at this hospital had mupirocin-resistant isolates. Starting in 1996, mupirocin use was restricted to patients colonized, but not infected, with MRSA. OBJECTIVES To describe the use of mupirocin for controlling MRSA over a decade and to analyze the molecular epidemiology of mupirocin-resistant MRSA infections at this hospital. SETTING A 490-bed, tertiary-care university hospital. METHODS The incidence densities of patients with MRSA and acquisition of mupirocin by the hospital were calculated for the period 1992-2001. S. aureus isolates from 1999-2000 were analyzed by pulsed-field gel electrophoresis. Mupirocin-resistant MRSA isolates from 1994-1995 and 1999-2000 were analyzed for ileS-2 gene background polymorphisms. RESULTS The incidence density of MRSA patients increased slightly over time, whereas the purchase of mupirocin decreased dramatically. Mupirocin-resistant MRSA infections decreased from 65% in 1994-1995 to 15% in 1999-2000. The MRSA Brazilian clone, detected in 1992, was still highly prevalent. The same ileS-2 encoding plasmid found in 1994-1995 persisted in three identical MRSA isolates from 1999-2000 belonging to the Brazilian clone. CONCLUSIONS After mupirocin use decreased, the ileS-2 encoding plasmid persisted in only a few Brazilian clone isolates. Our data on mupirocin-resistant MRSA incidence and mupirocin use strongly suggested that restricted use was related to decreased rates of mupirocin resistance at our hospital.