Impact of mutations in hVISA isolates on decreased susceptibility to vancomycin, through population analyses profile - area under curve (PAP-AUC).

We analyzed sequences of graSR, vraSR, walKR and rpoB genes in hVISA from Brazil. Five isolates showed mutations in at least one gene. rpoB H481N and graS T224I were the most frequent mutations, followed by graR D148Q and walK A468T. Our study reinforces the heterogeneity of genetic patterns among hVISA.

Molecular detection of virulence factors among food and clinical Enterococcus faecalis strains in South Brazil.

The present report aimed to perform a molecular epidemiological survey by investigating the presence of virulence factors in E. faecalis isolated from different human clinical (n = 57) and food samples (n = 55) in Porto Alegre, Brazil, collected from 2006 to 2009. In addition, the ability to form biofilm in vitro on polystyrene and the ß-haemolytic and gelatinase activities were determined. Clinical strains presented a higher prevalence of aggregation substance (agg), enterococcal surface protein (esp) and cytolysin (cylA) genes when compared with food isolates. The esp gene was found only in clinical strains. On the other hand, the gelatinase (gelE) and adherence factor (ace) genes had similar prevalence among the strains, showing the widespread occurrence of these virulence factors among food and clinical E. faecalis strains in South Brazil. More than three virulence factor genes were detected in 77.2% and 18.2% of clinical and food strains, respectively. Gelatinase and ß-haemolysin activities were not associated with the presence of gelE and cylA genes. The ability to produce biofilm was detected in 100% of clinical and 94.6% of food isolates, and clinical strains were more able to form biofilm than the food isolates (Student's t-test, p < 0.01). Results from the statistical analysis showed significant associations between strong biofilm formation and ace (p = 0.015) and gelE (p = 0.007) genes in clinical strains. In conclusion, our data indicate that E. faecalis strains isolated from clinical and food samples possess distinctive patterns of virulence factors, with a larger number of genes that encode virulence factors detected in clinical strains.

Genomic analysis of pandemic and post-pandemic influenza A pH1N1 viruses isolated in Rio Grande do Sul, Brazil.

During the 2009 influenza A pH1N1 pandemics in Brazil, the state that was most affected was Rio Grande do Sul (RS), with over 3,000 confirmed cases, including 298 deaths. While no cases were confirmed in 2010, 103 infections with 14 deaths by pH1N1 were reported in 2011. Genomic analysis of the circulating viruses is fundamental for understanding viral evolution and supporting vaccine development against these pathogens. This study investigated whole genomes of six pH1N1 virus isolates from pandemic and post-pandemic periods in RS, Brazil. Phylogenetic analysis using the concatenated genome segments demonstrated that at least two lineages of the virus co-circulated in RS during the 2009 pandemic period. Moreover, our analysis showed that the post-pandemic pH1N1 virus from 2011 constitutes a distinct clade whose ancestor belongs to clade 7. All six isolates contained amino acid substitutions in their proteins when compared to the archetype strains California/04/2009 and California/07/2009. The 2011 isolates contained more amino acid substitutions, and most of their genes were under purifying selection. Based on the amino acid substitutions in HA epitopes from strains isolated in RS, Brazil, in silico analysis predicted a decrease in vaccine efficacy against post-pandemic strains (median 31.562 %) in relation to pandemic ones (median 39.735 %).

Molecular detection of virulence factors among food and clinical Enterococcus faecalis strains in South Brazil

The present report aimed to perform a molecular epidemiological survey by investigating the presence of virulence factors in E. faecalis isolated from different human clinical (n = 57) and food samples (n = 55) in Porto Alegre, Brazil, collected from 2006 to 2009. In addition, the ability to form biofilm in vitro on polystyrene and the β-haemolytic and gelatinase activities were determined. Clinical strains presented a higher prevalence of aggregation substance (agg), enterococcal surface protein (esp) and cytolysin (cylA) genes when compared with food isolates. The esp gene was found only in clinical strains. On the other hand, the gelatinase (gelE) and adherence factor (ace) genes had similar prevalence among the strains, showing the widespread occurrence of these virulence factors among food and clinical E. faecalis strains in South Brazil. More than three virulence factor genes were detected in 77.2% and 18.2% of clinical and food strains, respectively. Gelatinase and β-haemolysin activities were not associated with the presence of gelE and cylA genes. The ability to produce biofilm was detected in 100% of clinical and 94.6% of food isolates, and clinical strains were more able to form biofilm than the food isolates (Student's t-test, p < 0.01). Results from the statistical analysis showed significant associations between strong biofilm formation and ace (p = 0.015) and gelE (p = 0.007) genes in clinical strains. In conclusion, our data indicate that E. faecalis strains isolated from clinical and food samples possess distinctive patterns of virulence factors, with a larger number of genes that encode virulence factors detected in clinical strains.

Biofilm formation on polystyrene under different temperatures by antibiotic resistant Enterococcus faecalis and Enterococcus faecium isolated from food.

The ability of antibiotic resistant E. faecalis and E. faecium isolated from food to form biofilm at different temperatures in the absence or presence of 0.75% glucose was evaluated. A synergistic effect on biofilm at 10 °C, 28 °C, 37 °C and 45 °C and glucose was observed for E. faecalis and E. faecium.

Biofilm formation on polystyrene under different temperatures by antibiotic resistant Enterococcus faecalis and Enterococcus faecium isolated from food

The ability of antibiotic resistant E. faecalis and E. faecium isolated from food to form biofilm at different temperatures in the absence or presence of 0.75% glucose was evaluated. A synergistic effect on biofilm at 10 °C, 28 °C, 37 °C and 45 °C and glucose was observed for E. faecalis and E. faecium.

Species distribution and antimicrobial susceptibility of enterococci isolated from broilers infected experimentally with Eimeria spp and fed with diets containing different supplements

Resistant bacteria in animal can be spread to environment and to humans. Poultry feed and infections caused by Eimeria spp. are important factors in determining the intestinal microbial communities. The aim of this study was to verify the prevalence of species and antimicrobial susceptibility of Enterococcus isolated from broilers fed with different supplements and infected experimentally with Eimeria spp. Broilers were divided in eight groups, fed with diets supplemented with a combination of antimicrobial, ionophore-coccidiostatics, probiotic, essential oil. At 14 days old all birds, except the control, received a solution containing oocysts of Eimeria spp. Samples of cloacal swabs from broilers were collected. A total of 240 Enterococcus sp. strains were isolated, confirmed genus by PCR, classified as species, tested for antimicrobial susceptibility and screened by PCR for the presence of tet(L), tet(M) and erm(B) genes. The overall distribution of species isolated from fecal samples was E. faecalis (40 percent), followed by E. casseliflavus/E. gallinarum (10.8 percent), E. mundtii (10.8 percent), E. faecium (10.8 percent), E. columbae (5.8 percent) and E. gallinarum (4.2 percent). Changes in the composition or frequency of Enterococcus species were observed in all dietary supplementation. Antimicrobial susceptibility tests showed resistance phenotypes a range of antibiotics, especially used in humans such as, streptomycin, penicillin, rifampicin and vancomycin. There was no correlation between different supplementation for broilers and antimicrobial resistance and the presence of tet(M), tet(L) and erm(B) genes. Dietary supplementation had effect on the Enterococcus sp. colonization, but did not have significant effect on the phenotype and genotype of antimicrobial resistance in enterococci.

Species distribution and antimicrobial susceptibility of enterococci isolated from broilers infected experimentally with Eimeria spp and fed with diets containing different supplements.

Resistant bacteria in animal can be spread to environment and to humans. Poultry feed and infections caused by Eimeria spp. are important factors in determining the intestinal microbial communities. The aim of this study was to verify the prevalence of species and antimicrobial susceptibility of Enterococcus isolated from broilers fed with different supplements and infected experimentally with Eimeria spp. Broilers were divided in eight groups, fed with diets supplemented with a combination of antimicrobial, ionophore-coccidiostatics, probiotic, essential oil. At 14 days old all birds, except the control, received a solution containing oocysts of Eimeria spp. Samples of cloacal swabs from broilers were collected. A total of 240 Enterococcus sp. strains were isolated, confirmed genus by PCR, classified as species, tested for antimicrobial susceptibility and screened by PCR for the presence of tet(L), tet(M) and erm(B) genes. The overall distribution of species isolated from fecal samples was E. faecalis (40%), followed by E. casseliflavus/E. gallinarum (10.8%), E. mundtii (10.8%), E. faecium (10.8%), E. columbae (5.8%) and E. gallinarum (4.2%). Changes in the composition or frequency of Enterococcus species were observed in all dietary supplementation. Antimicrobial susceptibility tests showed resistance phenotypes a range of antibiotics, especially used in humans such as, streptomycin, penicillin, rifampicin and vancomycin. There was no correlation between different supplementation for broilers and antimicrobial resistance and the presence of tet(M), tet(L) and erm(B) genes. Dietary supplementation had effect on the Enterococcus sp. colonization, but did not have significant effect on the phenotype and genotype of antimicrobial resistance in enterococci.

Oxacilin-resistant Coagulase-negative staphylococci (CoNS) bacteremia in a general hospital at São Paulo city, Brasil / Bacteremias por Staphylococcus coagulase negativos oxacilina resistentes em um hospital na cidade de São Paulo, Brasil

In the last decades, coagulase-negative staphylococci (CoNS), especially Staphylococcus epidermidis have become an important cause of bloodstream infections. In addition, rates of methicillin-resistance among CoNS have increased substantially, leading to the use of glicopeptides for therapy. The objective of this study was to evaluate eleven consecutives clinically relevant cases of oxacillin-resistant CoNS bacteremia in a general hospital localized in São Paulo city, Brazil. Five different species were identified by different phenotypic methods, including S. epidermidis (5), S. haemolyticus (3), S. hominis (1), S. warneri (1) and S. cohnii subsp urealyticus (1). A variety of Pulsed Field Gel Electrophoresis profiles was observed by macrorestriction DNA analysis in S. epidermidis isolates, but two of three S. haemolyticus isolates presented the same profile. These data indicated the heterogeneity of the CoNS isolates, suggesting that horizontal dissemination of these microorganisms in the investigated hospital was not frequent. One S. epidermidis and one S. haemolyticus isolates were resistant to teicoplanin and susceptible to vancomycin. The selective pressure due to the use of teicoplanin in this hospital is relevant.

Staphylococcus coagulase negativos (SCoN), especialmente Staphylococcus epidermidis tem se tornado causa importante de infecções da corrente circulatória nas últimas décadas. Além disso, percentuais de resistência a meticilina entre os SCoN têm aumentado significativamente, levando ao uso de glicopeptídeos nestes pacientes. O objetivo deste estudo foi avaliar onze casos consecutivos de bacteremia clinicamente relevantes por SCoN oxacilina resistentes em um hospital localizado na cidade de São Paulo, Brasil. Cinco diferentes espécies foram identificadas por diferentes métodos fenotípicos, incluindo S. epidermidis (5), S. haemolyticus (3), S. hominis (1), S. warneri (1) e S. cohnii subsp urealyticus (1). Diferentes perfis eletroforéticos obtidos pela técnica de "Pulsed Field Gel Electrophoresis" foram observados na análise da macrorestrição do DNA nos isolados de S. epidermidis, mas dois dos três isolados de S. haemolyticus apresentaram o mesmo perfil. Esses dados indicam uma heterogeneidade nos isolados SCoN, sugerindo que a disseminação horizontal no hospital investigado não é freqüente. Um isolado de S. epidermidis e um de S. haemolyticus foram resistentes à teicoplanina e sensíveis à vancomicina. Observa-se a relevância da pressão seletiva pelo uso de teicoplanina nos pacientes deste hospital.

Oxacilin-resistant Coagulase-negative staphylococci (CoNS) bacteremia in a general hospital at São Paulo city, Brasil.

In the last decades, coagulase-negative staphylococci (CoNS), especially Staphylococcus epidermidis have become an important cause of bloodstream infections. In addition, rates of methicillin-resistance among CoNS have increased substantially, leading to the use of glicopeptides for therapy. The objective of this study was to evaluate eleven consecutives clinically relevant cases of oxacillin-resistant CoNS bacteremia in a general hospital localized in São Paulo city, Brazil. Five different species were identified by different phenotypic methods, including S. epidermidis (5), S. haemolyticus (3), S. hominis (1), S. warneri (1) and S. cohnii subsp urealyticus (1). A variety of Pulsed Field Gel Electrophoresis profiles was observed by macrorestriction DNA analysis in S. epidermidis isolates, but two of three S. haemolyticus isolates presented the same profile. These data indicated the heterogeneity of the CoNS isolates, suggesting that horizontal dissemination of these microorganisms in the investigated hospital was not frequent. One S. epidermidis and one S. haemolyticus isolates were resistant to teicoplanin and susceptible to vancomycin. The selective pressure due to the use of teicoplanin in this hospital is relevant.

Acute endophthalmitis following intravitreal bevacizumab (Avastin) injection.

PURPOSE: To report two cases of acute endophthalmitis following intravitreal bevacizumab injection. METHODS: Two patients with exudative age-related macular degeneration were treated sequentially with an intravitreal injection of bevacizumab and developed signs of severe but painless infectious endophthalmitis 2 days later. Vitreous samples were obtained, followed by the injection of vancomycin 1 mg/0.1 ml and ceftazidime 2.25 mg/0.1 ml. Pulsed-field gel electrophoresis (PFGE) was used to determine whether the isolated microorganisms were the same. RESULTS: Coagulase-negative staphylococci were identified and isolated from the vitreous specimen of both patients. PFGE revealed different patterns of banding, excluding that interpatient contamination occured. CONCLUSIONS: Infectious endophthalmitis is a potential complication of intravitreal bevacizumab injection.

Variations of agar screen tests for detection of methicillin resistance in staphylococci: focus on cefoxitin.

Members of the genus Staphylococcus are among the most important human pathogens, and strains demonstrating resistance to methicillin are an increasing problem worldwide, both within and outside of hospital environments. The objective of this study was to evaluate the use of variations of agar screening tests with cefoxitin and oxacillin to detect methicillin resistance in staphylococcal isolates. The agar screening test with cefoxitin (4 microg/ml) showed 99.4% accuracy for detecting both S. aureus and coagulase-negative staphylococci. The performance of the agar screening test with cefoxitin (4 microg/ml) either equaled or was superior to the other agar screening test variations evaluated and can be used to characterize the presence of the mecA gene among staphylococcal species.

An outbreak of catalase-negative methicillin-resistant Staphylococcus aureus.

The wide dissemination of a major epidemic methicillin-resistant Staphylococcus aureus (MRSA) clone in Brazilian hospitals (Brazilian clone) limits the value of molecular typing techniques such as pulsed-field gel electrophoresis (PFGE) for outbreak investigation. We report the first outbreak of a catalase-negative strain of MRSA, which was initially detected by the unusual result of this phenotypical test. The outbreak occurred in the Hospital Sanatorinhos de Carapicuíba, a 237-bed secondary hospital located in São Paulo, Brazil. From May to August 2002, a total of 11 MRSA isolates were recovered from four patients in the intensive care unit. All the isolates were catalase negative and susceptible only to vancomycin and linezolid. Three of the four patients eventually died. Molecular typing demonstrated an indistinguishable PFGE pattern among the 11 isolates, with similarities to the Brazilian clone and the hospital's usual MRSA strain. This report emphasizes the importance of an uncommon phenotypical result as a marker for initiating an outbreak investigation and should encourage clinical laboratories to recognize and report such isolates.

Avidity of IgG for rubella: an evaluation of the need for implementation at the Materno-Infantil Presidente Vargas Hospital in Porto Alegre, Rio Grande do Sul, Brazil.

Rubella serum assays performed in the laboratory of the Materno-Infantil Presidente Vargas Hospital (HMIPV) from 1998 to 2002 were reviewed to determine if IgG avidity assays should be implemented. IgG was determined using the Enzyme Linked Fluorescent Assay, ELFA, VIDAS system, bioMerieux or the Microparticle Enzyme Immunoassay, MEIA, Axsym system, Abbott, and IgM was determined using the ELFA, VIDAS system, bioMerieux, a capture format assay. Specific IgG was assayed in 2,863 samples, with positive results for 84% of the patients, for the most part with high levels of antibodies. IgM was assayed in 2,851 samples, being positive in 14 (0.49%) and inconclusive in 25 (0.88%). Serology for toxoplasmosis was also positive or inconclusive in 5 patients. After a cost-effectiveness analysis, it was decided not to implement avidity assays, considering that the HMIPV is a public institution, with limited funding. Difficulties concerning the integration of the Clinical Pathology Service with the Clinical Staff of the institution were also considered.

Avidity of IgG for rubella: an evaluation of the need for implementation at the Materno-Infantil Presidente Vargas Hospital in Porto Alegre, Rio Grande do Sul, Brazil

Rubella serum assays performed in the laboratory of the Materno-Infantil Presidente Vargas Hospital (HMIPV) from 1998 to 2002 were reviewed to determine if IgG avidity assays should be implemented. IgG was determined using the Enzyme Linked Fluorescent Assay, ELFA, VIDAS® system, bioMérieux or the Microparticle Enzyme Immunoassay, MEIA, Axsym® system, Abbott, and IgM was determined using the ELFA, VIDAS® system, bioMérieux, a capture format assay. Specific IgG was assayed in 2,863 samples, with positive results for 84 percent of the patients, for the most part with high levels of antibodies. IgM was assayed in 2,851 samples, being positive in 14 (0.49 percent) and inconclusive in 25 (0.88 percent). Serology for toxoplasmosis was also positive or inconclusive in 5 patients. After a cost-effectiveness analysis, it was decided not to implement avidity assays, considering that the HMIPV is a public institution, with limited funding. Difficulties concerning the integration of the Clinical Pathology Service with the Clinical Staff of the institution were also considered.

Evaluation of an automated system for the identification and antimicrobial susceptibility testing of enterococci.

The performance of a new version of an automated system panel, the Positive Combo Panel Type 11 of MicroScan WalkAway 96 (WA96; Dade Behring) was evaluated and compared to that of reference methods for the identification and for antimicrobial susceptibility testing of the different enterococcal species. A total of 376 enterococcal isolates were tested. The MicroScan WA96 correctly identified 99.6% (266/267), 78.3% (18/23) and 68.6% (59/86) of Enterococcus faecalis, Enterococcus faecium and species other than E. faecalis and E. faecium, respectively. Although low probability of accurate identification was obtained for 37 (9.8%) strains, the system indicated that supplementary tests were necessary for precise identification of 8 (9.3%) among the 86 strains included in the non-faecalis/non-faecium group and of 3 (13.0%) among the E. faecium isolates. In comparison to the agar screening method, the percentage of agreement for detection of resistance markers by the automated system was 90.2% (37/41) for ampicillin, 90.6% (48/53) for high-level resistance to streptomycin (HLRS), 96.4% (80/83) for high-level resistance to gentamicin (HLRG), and 100% (14/14) for vancomycin. The results indicate that the MicroScan WA96 performed well for the identification of E. faecalis and typical E. faecium isolates, and for the detection of resistance to vancomycin and HLRG. However, the system still needs further improvement in order to provide reliable results for the characterization of the other enterococcal species, including atypical variants of E. faecium.

Hematological alterations in rats from xylene and benzene.

Benzene and xylene are used by the chemical industry as raw materials for the manufacturing of paints, insecticides, gums, resins and other compounds. Through its myelotoxic actions, benzene produces hematologic changes ranging from pancytopenia to total bone marrow aplasia. The present investigation studied the possible effects of xylene on rat peripheral blood and compared these effects to those produced by benzene. Thirty-six male Wistar rats were injected s.c. with 2 ml benzene, xylene or saline/kg body weight 3 times a week at 2-3 d intervals for 5 w. The animals were lightly anesthetized and blood was collected by puncture of the retro-orbital plexus before the first administration of the solvents on d 7, 14, 21 and 35 of dosing, and 14 d after dosing was discontinued. Xylene induced leukocytosis as an increase in absolute neutrophil numbers, whereas benzene caused leukocytopenia due to decreases in absolute lymphocyte number. The 2 solvents reduced erythrocyte counts, hematocrit and hemoglobin. Platelet counts were high throughout the dosing with benzene and xylene. Fourteen d after discontinuation of xylene dosing, the rats recovered their initial hematologic values, whereas the animals dosed with benzene did not fully recover leukocytes and erythrocytes. The intoxications with benzene and xylene were not solely hematologic since there was also growth retardation, as shown by reduced weight gain, which did not recover after dosing ceased.