Antonia Maria de Oliveira Machado

Phenotypic and molecular characterization of Salmonella Enteritidis strains isolated in São Paulo, Brazil

In São Paulo State, Brazil, the epidemic increase in isolation of Salmonella Enteritidis has been observed since 1994. A total of 105 S. Enteritidis strains (72 from human and 33 from non-human sources) isolated during the period 1975-1995, previously characterized by phage typing, was analyzed by antimicrobial susceptibility, plasmid profile, and ribotyping. Over 70% of the strains were susceptible to all antimicrobial agents tested, however, multiple resistance to antimicrobials was observed among the studied strains, mainly those from hospitalized patients. Phage type 8 (PT-8) was predominant among the strains isolated during the period of 1975-1992, but in the following years, PT-4 was the most frequent phage type identified. Seven different plasmid profiles were detected and 96% of the isolates harbored a plasmid of approximately 36 MDa. Ribotyping discriminated fourteen ribotypes (R1 to R14) among the strains examined. By analysis of dendrogram the strains were included in three groups with similarity level of 60%. The obtained results indicate that, a single ribotype (R11), determined for PT-4 strains isolated from 1993, characterizes the epidemic clone of S. Enteritidis in our region.

Detection of carbapenemase activity directly from blood culture vials using MALDI-TOF MS: a quick answer for the right decision

OBJECTIVES Recently, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was successfully applied for the detection of carbapenemase activity directly from Gram-negative colonies. Based on this principle, we evaluated the performance of MALDI-TOF MS for rapid detection of carbapenemase activity directly from positive blood culture vials. METHODS A total of 100 blood culture vials were randomly selected. MALDI-TOF MS carbapenemase assay results were confirmed by the detection of carbapenemase-encoding genes. RESULTS A total of 110 bacterial isolates were recovered. The MALDI-TOF MS carbapenemase assay identified 21 of 29 (72.4%) of the carbapenemase-producing isolates directly from the blood culture vials, especially those encoding KPC-2 (100%) and SPM-1 (100%), after a 4 h incubation period. Although the majority of OXA-23-producing Acinetobacter baumannii isolates were not identified on day 1, all isolates were identified as carbapenemase producers directly from the colony on the next day. CONCLUSIONS The MALDI-TOF MS carbapenemase assay is a feasible and rapid test to identify carbapenemase activity directly from blood culture vials. It may contribute to faster readjustment of empirical antimicrobial therapy and implementation of infection control measures.

Clinical culture surveillance of carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter species in a teaching hospital in Sao Paulo, Brazil: a 7-year study.

Carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter species are worrisome nosocomial pathogens. After introduction of a preventive program involving clinical surveillance culture to reduce the spread of those pathogens, we observed an 80% decrease in the percentage of cultures that yielded carbapenem-resistant Acinetobacter isolates. The percentage of cultures that yielded carbapenem-resistant P. aeruginosa remained relatively stable during the intervention.

Enterococcus faecalis resistant to vancomycin and teicoplanin (VanA phenotype) isolated from a bone marrow transplanted patient in Brazil

We report for the first time in Brazil, a patient from whom an Enterococcus faecalis VanA phenotype was isolated. Glycopeptide resistance is not commonly observed in Enterococcus faecalis, so this finding is of great concern since this species is responsible for 90% of enterococcal infections in Brazil. The isolate was recovered from a surveillance rectal swab culture from a patient with acute lymphocytic leukemia (ALL). Identification to the species level was performed by conventional biochemical tests and Vitek GPI cards. Antimicrobial susceptibility testing was evaluated by use of broth microdilution and Etest (AB BIODISK, Solna, Sweden) methods. The isolate was identified as E. faecalis and was considered resistant to both vancomycin (MIC, > 256 microg/mL) and teicoplanin (MIC, 256 microg/mL). The isolate also showed high level resistance to gentamicin and streptomycin (MICs, > 1024 microg/mL), but was considered susceptible to ampicillin (MIC, 4 microg/mL). Although the frequency of enterococcal infections is very low in most Latin America countries, the finding of glycopeptide (VanA) resistance in E. faecalis increases concern about apreading antimicrobial resistance in this region.

In vitro antimicrobial activity of linezolid tested against vancomycin-resistant enterococci isolated in Brazilian hospitals

The emergence of vancomycin-resistant enterococci (VRE) has been described recently in Brazil. This is in contrast to the USA and Europe, where the VRE appeared in the late 1980s. The progressive increase in VRE isolation poses important problems in the antimicrobial therapy of nosocomial infections. Treatment options and effective antimicrobial agents for VRE are often limited and the possibility of transfer of vancomycin genes to other Gram-positive microorganisms continues. In the search for antimicrobial agents for multiresistant Gram-positive cocci, compounds such as linezolid and quinupristin/dalfopristin have been evaluated. The present study was conducted to evaluate the in vitro activity of the oxazolidinone linezolid and 10 other antimicrobial agents, including quinupristin-dalfopristin, against multiresistant enterococci isolated in Brazilian hospitals. Thirty-three vancomycin resistant isolates (17 Enterococcus faecium and 16 E. faecalis), were analyzed. Strains were isolated from patients at São Paulo Hospital, Oswaldo Cruz Hospital, Hospital do Servidor Público Estadual, Santa Marcelina Hospital, Santa Casa de Misericórdia de São Paulo, and Hospital de Clínicas do Paraná. The samples were tested by a broth microdilution method following the National Committee for Clinical Laboratory Standards (NCCLS) recommendations. All isolates were molecular typed using pulsed-field gel electrophoresis (PFGE). Linezolid was the most active compound against these multiresistant enterococci, showing 100% inhibition at the susceptible breakpoints. Quinupristin/dalfopristin and teicoplanin showed poor activity against both species. The molecular typing results suggest that there has been interhospital spread of vancomycin resistant E. faecium and E. faecalis among Brazilian hospitals. The results of this study indicate that linezolid is an appropriate therapeutic option for the treatment of vancomycin-resistant enterococci infections in Brazil.

New Approach for Diagnosis of Candidemia Based on Detection of a 65-Kilodalton Antigen

ABSTRACT Nosocomial candidiasis is a major concern in tertiary care hospitals worldwide. This infection generally occurs in patients with degenerative and neoplastic diseases and is considered the fourth most frequent cause of bloodstream infections. Diagnosis of candidemia or hematogenous candidiasis has been problematic because clinical signs and symptoms are nonspecific, leading to delays in diagnosis and, consequently, delays in appropriate antifungal therapy. We developed an inhibition enzyme-linked immunosorbent assay (ELISA) for detection of a 65-kDa antigen in an experimental model of candidemia and for diagnosis of patients in intensive care units (ICUs) with suspected candidemia. An anti-65-kDa monoclonal antibody was tested for detection of the 65-kDa antigen produced by Candida albicans, Candida tropicalis, and Candida parapsilosis in murine candidemia models. The 65-kDa antigen was detected in sera at concentrations ranging from 0.012 to 3.25 μg/ml. A total of 20 human patients with candidemia were then evaluated with the inhibition ELISA using sequential sera. Sixteen (80%) patients had the 65-kDa antigen in concentrations ranging from 0.07 to 5.0 μg/ml. Sequential sera from patients with candidemia presented three different patterns of antigenemia of the 65-kDa molecule: (i) total clearance of antigenemia, (ii) initial clearance and relapse of antigenemia, and (iii) partial clearance of antigenemia. Our results indicate detection of the 65-kDa protein may be a valuable tool for the diagnosis of candidemia by C. albicans, C. tropicalis, and C. parapsilosis.

Influenza virus and proteolytic bacteria co-infection in respiratory tract from individuals presenting respiratory manifestations

A role for proteolytic bacteria in the exacerbation of influenza virus has been shown in natural hosts such as pigs and humans. Four hundred seven samples were collected from the respiratory tract of individuals presenting clinical manifestations, during influenza season (2003-2005) in São Paulo City. The aim of this study was to evaluate the incidence of determined bacteria co-infecting virus in human respiratory tract. Tests, such as bacteriological, immunofluorescence (IF), RT/PCR and hemagglutination (HA) were used for bacterial and viral investigation. Thirty seven (9.09%) positive for influenza virus were screened by IF. The RT/PCR confirmed the presence of influenza virus in these samples. Bacterial and agar casein tests demonstrated that 18 (48.64%) individuals were infected with proteolytic bacteria such as Staphylococcus spp., Streptococcus spp. and Pseudomonas spp. Among these samples, 13 (35.13%) were co-infected with influenza A virus. Influenza type B, co-infecting bacteria were found in five (13.51%) samples. In vitro the S. aureus protease increased the influenza HA titer after contact for 30 min at 25 masculineC. Results revealed the occurrence of co-infection with proteolytic bacteria and influenza in the evaluated individuals. This finding corroborates that virus versus bacteria synergism could be able to potentiate respiratory infection, increasing damage to hosts.

Influence of Culture Media on Detection of Carbapenem Hydrolysis by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT In this study, we evaluated the influence of distinct bacterial growth media on detection of carbapenemase hydrolysis by matrix-assisted laser desorption ionization–time of flight mass spectrometry. False-negative results were observed for OXA-25-, OXA-26-, and OXA-72-producing Acinetobacter baumannii isolates grown on MacConkey agar medium. The other culture media showed 100% sensitivity and 100% specificity for detecting carbapenemase.

Comparison of M.I.C.E. and Etest with CLSI Agar Dilution for Antimicrobial Susceptibility Testing against Oxacillin-Resistant Staphylococcus spp

Objective The main objective of this study was to comparatively evaluate the performance of M.I.C.E. and Etest methodologies to that of agar dilution for determining the antimicrobial susceptibility profile of oxacillin-resistant Staphylococcus spp. Methods A total of 100 oxacillin-resistant Staphylococcus spp. isolates were collected from hospitalized patients at a teaching hospital. Antimicrobial susceptibility testing for vancomycin, teicoplanin and linezolid was performed using the reference CLSI agar dilution method (2009), Etest and M.I.C.E. methodologies. The MIC values were interpreted according to CLSI susceptibility breakpoints and compared by regression analysis. Results In general, the essential agreement (±1-log2) between M.I.C.E. and CLSI agar dilution was 93.0%, 84.0% and 77.0% for linezolid, teicoplanin and vancomycin, respectively. Essential agreement rates between M.I.C.E. and Etest were excellent (>90.0%) for all antibiotics tested. Both strips (M.I.C.E. and Etest) yielded two very major errors for linezolid. Unacceptable minor rates were observed for teicoplanin against CoNS and for vancomycin against S. aureus. Conclusions According to our results, linezolid and teicoplanin MICs against all staphylococci and S. aureus, respectively, were more accurately predicted by M.I.C.E. strips. However, the Etest showed better performance than M.I.C.E. for predicting vancomycin MICs against all staphylococci. Thus, microbiologists must be aware of the different performance of commercially available gradient strips against staphylococci.

Estudos dos fatores de risco pré-operatórios para bacteriobilia em doentes portadores de colecistite aguda calculosa

OBJECTIVE to determine an association between the preoperative clinical status and the result of bile and gallbladder wall cultures. MATERIAL AND METHODS 28 variables regarding history, physical examination and labatorial assessment in 38 patients with acute calculosis cholecystitis submitted to urgency surgery were prospectively studied during a 19-month period, between November 1995 and May 1997. Cultures for aerobic and anaerobic agents from both the gallbladder wall and the bile were performed, in three different culture media (BACTEC 9240, BHI and HEMOBAC). RESULTS bacteria were isolated in at least one culture medium, in 68.2% of the patients. At univariate analysis, five preoperative factors were identified as predictors of bactibilia: over 55 years of age, a greater than 0.4 degrees C difference in the axillary-rectal temperature, a greater than 12.000 cels/m3 blood leukocyte count, a greater than 75% neutrophil percentage and a greater than 4% rod neutrophil percentage. Owing to the small sample size, statistical significance of the series could not be noted by logistic regression, although a trend to preoperative determination could be observed in 98% of the subjects with positive culture, by means of the model based on age and percentage of rod neutrophil. By analyzing predictive factors jointly, it was noted that patients with more than one predictive factor have a significantly greater possibility to yielding positive culture when compared to those with up to one predictive factor for bactibilia. CONCLUSIONS We concluded that, in patients with acute calculosis cholecystitis, bactibilia may be predicted yet at the preoperative period, by using simple and easily obtained data.OBJECTIVE: to determine an association between the preoperative clinical status and the result of bile and gallbladder wall cultures. MATERIAL AND METHODS: 28 variables regarding history, physical examination and laboratorial assessment in 38 patients with acute calculosis cholecystitis submitted to urgency surgery were prospectively studied during a 19-month period, between November 1995 and May 1997. Cultures for aerobic and anaerobic agents from both the gallbladder wall and the bile were performed, in three different culture media (BACTEC 9240, BHI and HEMOBAC). RESULTS: bacteria were isolated in at least one culture medium, in 68.2% of the patients. At univariate analysis, five preoperative factors were identified as predictors of bactibilia: over 55 years of age, a greater than 0.4°C difference in the axillary-rectal temperature, a greater than 12.000 cels/m3 blood leukocyte count, a greater than 75% neutrophil percentage and a greater than 4% rod neutrophil percentage. Owing to the small sample size, statistical significance of the series could not be noted by logistic regression, although a trend to preoperative determination could be observed in 98% of the subjects with positive culture, by means of the model based on age and percentage of rod neutrophil. By analyzing predictive factors jointly, it was noted that patients with more than one predictive factor have a significantly greater possibility to yelding positive culture when compared to those with up to one predictive factor for bactibilia. CONCLUSIONS: We concluded that, in patients with acute calculosis cholecystitis, bactibilia may be predicted yet at the preoperative period, by using simple and easily obtained data.