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OBJETIVO: Avaliar a suscetibilidade, in vitro, de Staphylococcus coagulase negativa (SCoN), isolados da conjuntiva e córnea, à meticilina, às fluoroquinolonas e aos aminoglicosídeos. MÉTODOS: Foram analisadas retrospectivamente 707 amostras oculares de SCoN quanto à suscetibilidade aos antimicrobianos pelo teste de disco difusão, durante o período de janeiro de 2000 a dezembro de 2003. RESULTADOS: Houve um aumento do número de SCoN em isolados da conjuntiva (n=57, ano de 2000 e n=153, ano de 2003) e da córnea (n=28, ano de 2000 e n=78, ano de 2003). A freqüência de SCoN resistentes à meticilina isolados da conjuntiva e da córnea, aumentou (1,8 a 19,6 por cento e 14,3 a 29,3 por cento respectivamente) durante o período avaliado. Não houve diferença estatisticamente significante nos anos estudados, nos percentuais de SCoN resistentes às fluoroquinolonas, nas conjuntivas (ofloxacina: 1,8 a 7,8 por cento e ciprofloxacina: 3,5 a 9,2 por cento) e nas córneas (ofloxacina: 14,3 a 9,0 por cento e ciprofloxacina:14,3 a 10,3 por cento). Avaliando-se os resultados das amostras isoladas da conjuntiva, observou-se um aumento na resistência à tobramicina: 15,8 a 34,6 por cento; e à gentamicina: 10,5 a 25,5 por cento; mas não houve mudança no perfil de resistência das amostras da córnea à tobramicina: 28,6 a 26,9 por cento e à gentamicina: 21,4 a 23,1 por cento). CONCLUSÃO: Houve diminuição na suscetibilidade in vitro dos SCoN para meticilina, tobramicina e gentamicina. As fluoroquinolonas, representadas pela ofloxacina e ciprofloxacina, demonstraram ter um padrão estável de suscetibilidade in vitro.

PURPOSE: To assess the in vitro susceptibility of conjunctival and corneal coagulase negative Staphylococcus (CoNS) to methicillin, fluoroquinolones and aminoglycosides. METHODS: A total of 707 conjunctival and corneal CoNS disk diffusion test results were retrospectively analyzed, from January 2000 through December 2003. RESULTS: From 2000 to 2003, there was an increase in number of CoNS isolated from conjunctiva (n=57 to n=153) and cornea (n=28 to n=78). The frequency of conjunctival and corneal methicillin-resistant CoNS also increased (1.8 to 19.6 percent and 14.3 to 29.3 percent, respectively). There was no statistically significant difference between fluoroquinolones-resistant CoNS percentages in conjunctiva (ofloxacin: 1.8 to 7.8 percent and ciprofloxacin: 3.5 to 9.2 percent) and cornea (ofloxacin: 14.3 to 9.0 percent and ciprofloxacin: 14.3 to 10.3 percent). Evaluating the results of the conjunctival samples, there was increased resistance to tobramycin (15.8 to 34.0 percent) and to gentamycin (10.5 to 25.5 percent). There was no change in resistance of corneal isolates to tobramycin (28.6 to 26.9 percent) and to gentamycin (21.4 to 23.1 percent). CONCLUSIONS: there was a decrease in in vitro CoNS susceptibility to methicillin, tobramycin and gentamycin. Fuoroquinolones represented by ofloxacin and ciprofloxacin demonstrated stable in vitro susceptibility.

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PURPOSE: To evaluate different methods of oxacillin susceptibility testing of ocular isolates, considering polymerase chain reaction (PCR) as the 'gold standard', and to compare the in vitro susceptibility to oxacillin with that of other antimicrobials used in ophthalmologic practice. METHODS: The Vitek gram-positive identification card was used to identify ocular coagulase negative Staphylococcus species. The presence of the mecA gene was determined by the polymerase chain reaction assay with a combination of two primer sets (mecA and 16S rRNA) in a single region. Results were analyzed and compared with other oxacillin susceptibility methods: PBP2a detection by rapid slide latex agglutination test (SLA); oxacillin E-test; the Vitek automated gram-positive susceptibility card (GPS-105); the oxacillin salt agar screening test (OSAS) at a concentration of 6.0, 1.0 and 0.75 microg oxacillin per ml and the cefoxitin disk diffusion test (CDD). Automated susceptibility was also determined to other antimicrobial agents (fluoroquinolones, penicillin G, amoxicillin-ampicillin, cefazolin, ampicillin-sulbactam, erythromycin, clindamycin, gentamicin, tetracycline, trimethoprim-sulfamethoxazole, vancomycin and rifampin. RESULTS: Of the 69 CoNS isolates tested, 71% were mecA-positive and 29% mecA-negative. All methods tested had a statistically significant agreement with polymerase chain reaction. There was a tendency of positive polymerase chain reaction predomination among the S. epidermidis isolates in comparison to non-epidermidis isolates, although this was not statistically significant (78.3% vs. 56.5%; chi2= 2.54; P= 0.11). The oxacillin salt agar screening test (0.75 microg oxacillin/ml) showed the best performance, with 100% sensitivity and negative predictive value; 95% specificity and 98% positive predictive value. Using the E-test, the mecA-positive isolates were statistically significantly more resistant to ciprofloxacin, ofloxacin, gatifloxacin and moxifloxacin (P= 0.002; P= 0.008; P= 0.002 and P= 0.003, respectively). There was a statistically significant higher proportion of resistance of the coagulase negative Staphylococcus mecA-positives for: penicillin G, amoxicillin-ampicillin, cefazolin, ampicillin-sulbactam, erythromycin, clindamycin, gentamicin and tetracycline (P< or =0.05). All coagulase negative Staphylococcus species were susceptible to vancomycin and there was no statistically significant correlation between the mecA-positive isolates and resistance to trimethoprim-sulfamethoxazole or to rifampin. CONCLUSIONS: In the present study, we found that the E-test and the oxacillin salt agar screening test S (0.75 microg oxacillin per ml), when compared with polymerase chain reaction, were the most accurate currently available methods to phenotypically detect oxacillin resistance of coagulase negative Staphylococcus species. This study demonstrated that a good option for screening of ocular isolates for oxacillin resistance in the microbiology laboratory is the cefoxitin disk diffusion test and the automated Vitek system. We believe it is important to have available methods that accurately detect methicillin resistance of the less commonly encountered species, chiefly because of their increasing importance as opportunistic pathogens.

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PURPOSE: To evaluate different methods of oxacillin susceptibility testing of ocular isolates, considering polymerase chain reaction (PCR) as the 'gold standard', and to compare the in vitro susceptibility to oxacillin with that of other antimicrobials used in ophthalmologic practice. METHODS: The Vitek gram-positive identification card was used to identify ocular coagulase negative Staphylococcus species. The presence of the mecA gene was determined by the polymerase chain reaction assay with a combination of two primer sets (mecA and 16S rRNA) in a single region. Results were analyzed and compared with other oxacillin susceptibility methods: PBP2a detection by rapid slide latex agglutination test (SLA); oxacillin E-test; the Vitek automated gram-positive susceptibility card (GPS-105); the oxacillin salt agar screening test (OSAS) at a concentration of 6.0, 1.0 and 0.75 æg oxacillin per ml and the cefoxitin disk diffusion test (CDD). Automated susceptibility was also determined to other antimicrobial agents (fluoroquinolones, penicillin G, amoxicillin-ampicillin, cefazolin, ampicillin-sulbactam, erythromycin, clindamycin, gentamicin, tetracycline, trimethoprim-sulfamethoxazole, vancomycin and rifampin. RESULTS: Of the 69 CoNS isolates tested, 71 percent were mecA-positive and 29 percent mecA-negative. All methods tested had a statistically significant agreement with polymerase chain reaction. There was a tendency of positive polymerase chain reaction predomination among the S. epidermidis isolates in comparison to non-epidermidis isolates, although this was not statistically significant (78.3 percent vs. 56.5 percent; chi2= 2.54; P= 0.11). The oxacillin salt agar screening test (0.75 æg oxacillin/ml) showed the best performance, with 100 percent sensitivity and negative predictive value; 95 percent specificity and 98 percent positive predictive value. Using the E-test, the mecA-positive isolates were statistically significantly more...

OBJETIVOS: Avaliar os diferentes métodos de suscetibilidade à oxacillina, em isolados oculares, considerando a reação em cadeia da polimerase (PCR) como "padrão-ouro" e comparar a suscetibilidade in vitro para outros antimicrobianos de uso oftalmológico. MÉTODOS: O sistema automatizado Vitek foi utilizado para identificar as diferentes espécies de Staphylococcus coagulase negativo (SCoN). A presença do gene mecA foi determinado pela reação em cadeia da polimerase com a combinação de 2 "primer" sets (mecA e 16S rRNA) em uma única região. Estes resultados foram analisados e comparados com outros métodos de suscetibilidade à oxacilina: detecção da proteína PBP2a pelo teste de aglutinação em látex (SLA); E-test oxacilina; o sistema automatizado Vitek (GPS-105); o teste de triagem em ágar (OSAS) com oxacilina nas concentrações de 6,0, 1,0 e 0,75 æg oxacilina por ml e o teste de disco difusão com cefoxitina (CDD). A suscetibilidade automatizada foi obtida para os seguintes agentes antimicrobianos: fluorquinolonas, penicilina G, amoxicilina-ampicilina, cefazolina, ampicilina-sulbactam, eritromicina, clindamicina, gentamicina, tetraciclina, sulfametoxazol-trimetoprima, vancomicina e rifampicina. RESULTADOS: Dos 69 Staphylococcus coagulase negativo testados, 71 por cento foram mecA-positivos e 29 por cento, mecA-negativos. Todos os métodos testados apresentaram concordância estatisticamente significante com a reação em cadeia da polimerase. Houve tendência à predominância da positividade da reação em cadeia da polimerase entre os S. epidermidis comparado aos não-epidermidis, embora sem significância estatistica (78,3 por cento vs. 56,5 por cento; chi2= 2,54; p=0,11). O teste de triagem em ágar (0,75 æg oxacilina/ml) apresentou a melhor performance com resultados de: 100 por cento de sensibilidade e valor preditivo negativo, 95 por cento de especificidade e 98 por cento de valor preditivo positivo. Os isolados mecA-positivos foram estatisticamente...

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PURPOSE: To evaluate the fluoroquinolone susceptibilities of ocular isolate coagulase-negative staphylococci (CoNS), identified at the Microbiology Laboratory - UNIFESP. DESIGN: Experimental laboratory investigation. METHODS: The minimum inhibitory concentrations (MICs) of 21 strains of methicillin-resistant coagulase-negative staphylococci (MRCoNS) and 22 methicillin-sensitive coagulase-negative staphylococci (MSCoNS) to ciprofloxacin, ofloxacin, gatifloxacin and moxifloxacin were determined, using the E-test method standardized by the Clinical and Laboratory Standards Institute (CLSI/NCCLS). RESULTS: The MIC90s (microg/ml) for the second generation of tested fluoroquinolones were higher than the fourth generation, especially for the methicillin-resistant coagulase-negative staphylococci group. CONCLUSION: Our results indicate that methicillin-sensitive coagulase-negative staphylococci are more susceptible to quinolones than are methicillin-resistant coagulase-negative staphylococci and that fourth generation fluoroquinolones appear to be more potent, affecting even coagulase-negative staphylococcal strains resistant to second generation fluoroquinolones.

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PURPOSE: To evaluate the fluoroquinolone susceptibilities of ocular isolate coagulase-negative staphylococci (CoNS), identified at the Microbiology Laboratory - UNIFESP. DESIGN: Experimental laboratory investigation. METHODS: The minimum inhibitory concentrations (MICs) of 21 strains of methicillin-resistant coagulase-negative staphylococci (MRCoNS) and 22 methicillin-sensitive coagulase-negative staphylococci (MSCoNS) to ciprofloxacin, ofloxacin, gatifloxacin and moxifloxacin were determined, using the E-test method standardized by the Clinical and Laboratory Standards Institute (CLSI/NCCLS). RESULTS: The MIC90s (µg/ml) for the second generation of tested fluoroquinolones were higher than the fourth generation, especially for the methicillin-resistant coagulase-negative staphylococci group. CONCLUSION: Our results indicate that methicillin-sensitive coagulase-negative staphylococci are more susceptible to quinolones than are methicillin-resistant coagulase-negative staphylococci and that fourth generation fluoroquinolones appear to be more potent, affecting even coagulase-negative staphylococcal strains resistant to second generation fluoroquinolones.

OBJETIVOS: Avaliar a suscetibilidade a fluorquinolonas dos Staphylococcus coagulase-negativo (SCoN) identificados no Laboratório de Microbiologia Ocular da Unifesp. MÉTODOS: Foi determinada a concentração inibitória mínima de 21 cepas de SCoN meticilina-resistentes e 22 meticilina-sensíveis para ciprofloxacina, ofloxacina, gatifloxacina e moxifloxacina, utilizando o E-test estandartizado pelo CLSI/NCCLS. RESULTADOS: Os MIC90 (µg/ml) de 43 SCoN isolados para fluorquinolonas de segunda geração foram maiores do que os de quarta geração, principalmente para o grupo dos meticilina-resistentes. CONCLUSÃO: Nossos resultados indicam que Staphylococcus coagulase-negativo meticilina-sensíveis são mais suscetíveis às quinolonas do que os Staphylococcus coagulase-negativo meticilina-resistentes, fluorquinolonas de quarta geração parecem ser mais potentes, cobrindo inclusive cepas de Staphylococcus coagulase-negativo resistentes à segunda geração de fluorquinolonas.

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PURPOSE: To compare the in vitro susceptibility profiles of bacterial ocular isolates and to determine minimum inhibitory concentrations (MICs) of gatifloxacin and moxifloxacin (fourth-generation fluoroquinolones) versus ciprofloxacin and ofloxacin (second-generation fluoroquinolones). METHODS: Gram-positive and gram-negative isolates were recovered from cases of keratitis, conjunctivitis, and endophthalmitis between 2002 and 2004 and were identified and extracted from the Microbiology Data Bank of the Federal University of São Paulo, São Paulo, Brazil. The comparison of in vitro MIC and susceptibility profiles for ofloxacin, ciprofloxacin, gatifloxacin, and moxifloxacin in gram-positive and gram-negative (n = 219) isolates was performed using the E test method. RESULTS: The fourth-generation fluoroquinolones were statistically more potent than the second generations for gram-positive bacteria. The MIC90 level was lower for moxifloxacin than that for gatifloxacin against Staphylococcus aureus, methicillin-susceptible coagulase-negative Staphylococcus (CoNS), and S. pneumoniae, whereas the levels were equal against S. viridans and the gatifloxacin MIC90 was lower in methicillin-resistant CoNS. There was no statistically significant difference between moxifloxacin and gatifloxacin when the permutation method from the MULTTEST procedure (SAS proc multtest) was used to obtain the adjusted P value. MIC90 for ciprofloxacin was lower in gram-negative bacteria. MIC90 for ofloxacin was higher against Haemophilus spp. and Moraxella spp. Ciprofloxacin was the most statistically potent fluoroquinolone for Pseudomonas spp. Ciprofloxacin was statistically just as potent as gatifloxacin for the other gram-negative isolates. CONCLUSION: From susceptibility profiles achieved with in vitro testing, the fourth-generation fluoroquinolones may offer some advantages over the currently available fluoroquinolones; however, a combination of the pharmacodynamics and pharmacokinetics of the drug, infection site, and the MIC is needed to predict the in vivo efficacy and best clinical applicability.

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PURPOSE: To assess the in vitro susceptibility of conjunctival and corneal coagulase negative Staphylococcus (CoNS) to methicillin, fluoroquinolones and aminoglycosides. METHODS: A total of 707 conjunctival and corneal CoNS disk diffusion test results were retrospectively analyzed, from January 2000 through December 2003. RESULTS: From 2000 to 2003, there was an increase in number of CoNS isolated from conjunctiva (n=57 to n=153) and cornea (n=28 to n=78). The frequency of conjunctival and corneal methicillin-resistant CoNS also increased (1.8 to 19.6% and 14.3 to 29.3%, respectively). There was no statistically significant difference between fluoroquinolones-resistant CoNS percentages in conjunctiva (ofloxacin: 1.8 to 7.8% and ciprofloxacin: 3.5 to 9.2%) and cornea (ofloxacin: 14.3 to 9.0% and ciprofloxacin: 14.3 to 10.3%). Evaluating the results of the conjunctival samples, there was increased resistance to tobramycin (15.8 to 34.0%) and to gentamycin (10.5 to 25.5%). There was no change in resistance of corneal isolates to tobramycin (28.6 to 26.9%) and to gentamycin (21.4 to 23.1%). CONCLUSIONS: there was a decrease in in vitro CoNS susceptibility to methicillin, tobramycin and gentamycin. Fuoroquinolones represented by ofloxacin and ciprofloxacin demonstrated stable in vitro susceptibility.

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O objetivo do presente trabalho foi caracterizar geneticamente estirpes de Campylobacter jejuni subsp. jejuni isoladas de humanos e de diferentes origens animais (bovinas, suínas, cães, primatas, javalis, suínos e aves de corte). Um total de 828 amostras (fezes, carcaças, fetos abortados e útero histerectomizado) foram analisadas por métodos de rotina bacteriológica e 36 estirpes de C. jejuni foram isoladas. Trinta estirpes de origem fecal humana foram obtidas de laboratórios de análises clínicas da cidade de São Paulo. As 66 estirpes de C. jejuni isoladas foram submetidas à caracterização genética. Oligonucleotídeos baseados no gene fla A foram usados na reação de polimerase em cadeia (PCR) e amplificou um fragmento de 702 pb. Os produtos obtidos pela PCR foram avaliados pelas técnicas de seqüenciamento e análise genealógica. Análise da variabilidade genética das 66 estirpes revelou 44 diferentes subtipos de C. jejuni. Um subtipo de origem humana apresentou seqüência idêntica à de C. jejuni depositada no GenBank (GENBANK acesso número AF050186). A subtipagem das estirpes de C. jejuni baseadas no seqüenciamento da região variável do gene fla A e na análise do alinhamento das seqüências pelo método da Máxima Parcimônia, mostraram-se altamente discriminatórios fornecendo melhores condições para a correta diferenciação entre estirpes originárias de surto e as isoladas esporadicamente. Este foi o primeiro estudo de subtipagem molecular de estirpes de C. jejuni de origem humana e animal utilizando a técnica do seqüenciamento com análise genealógica realizado no Estado de São Paulo, Brasil.

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The objective of the present trial was to characterize genetically strains of Campylobacter jejuni subsp. jejuni isolated from humans and several animal sources (bovines, swine, dogs, primates, wild boars and poultry). A total of 828 different animal samples (feces, carcass, aborted fetus and hysterectomized uterus) were analysed by means of routine bacteriological methods, and 36 C. jejuni strains were isolated. Thirty strains of human fecal origin were obtained in clinical analysis laboratories in the city of São Paulo. The 66 C. jejuni strains isolated were submitted to genetic characterization. Primers based on fla A gene were used in a polymerase chain reaction (PCR) and amplified a fragment of the 702 bp. PCR products were evaluated by means of sequencing and genealogic analysis. Genetic variability analysis of 66 strains showed 44 different subtypes of C. jejuni. One subtype was identical to a C. jejuni strain of human origin with the sequence in the GenBank (GENBANK accession number AF050186). Subtyping analysis of C. jejuni strains based on sequencing of the fla A gene variable region and analysis of sequence alignment by the Maximum Parsimony method showed to be highly discriminatory, providing the best conditions to differentiate strains involved in outbreaks from those sporadically isolated. This is the first study of molecular subtyping analysis of human and animal C. jejuni strains using sequencing technique and genealogic analysis in the state of São Paulo, Brazil.

O objetivo do presente trabalho foi caracterizar geneticamente estirpes de Campylobacter jejuni subsp. jejuni isoladas de humanos e de diferentes origens animais (bovinas, suínas, cães, primatas, javalis, suínos e aves de corte). Um total de 828 amostras (fezes, carcaças, fetos abortados e útero histerectomizado) foram analisadas por métodos de rotina bacteriológica e 36 estirpes de C. jejuni foram isoladas. Trinta estirpes de origem fecal humana foram obtidas de laboratórios de análises clínicas da cidade de São Paulo. As 66 estirpes de C. jejuni isoladas foram submetidas à caracterização genética. Oligonucleotídeos baseados no gene fla A foram usados na reação de polimerase em cadeia (PCR) e amplificou um fragmento de 702 pb. Os produtos obtidos pela PCR foram avaliados pelas técnicas de seqüenciamento e análise genealógica. Análise da variabilidade genética das 66 estirpes revelou 44 diferentes subtipos de C. jejuni. Um subtipo de origem humana apresentou seqüência idêntica à de C. jejuni depositada no GenBank (GENBANK acesso número AF050186). A subtipagem das estirpes de C. jejuni baseadas no seqüenciamento da região variável do gene fla A e na análise do alinhamento das seqüências pelo método da Máxima Parcimônia, mostraram-se altamente discriminatórios fornecendo melhores condições para a correta diferenciação entre estirpes originárias de surto e as isoladas esporadicamente. Este foi o primeiro estudo de subtipagem molecular de estirpes de C. jejuni de origem humana e animal utilizando a técnica do seqüenciamento com análise genealógica realizado no Estado de São Paulo, Brasil.

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Forty-six S. maltophilia isolates obtained from hospital clinical specimens were studied for protease (caseinase and elastase) production, hemolytic activity, adhesion to HEp-2 cells, plastic and glass. Susceptibility to antimicrobial agents was also evaluated. The majority of isolates were obtained from respiratory tract secretions of patients using medical devices. All the isolates grown overnight were able to hydrolyze casein at 30ºC and 37ºC. After 72h, all the isolates hydrolyzed elastase at 30ºC and 40 isolates (87 per cent) at 37ºC. Most of the isolates presented hemolytic activity after 96h of incubation at both temperatures. Rabbit blood showed the hightest hemolytic activity, after 96h 61(per cent) and 98(per cent) of tested isolates presented b-hemolysis at 30ºC and 37ºC, respectively. All isolates were susceptible to trimethoprim-sulfametoxazole and were resistant to most b-lactams tested. By the dilution method, S. maltophilia showed a high susceptibility to ticarcillin-clavulanate and a lower susceptibility to ciprofloxacin than the agar diffusion. The isolates showed adhesion to HEp-2 cells, plastic and glass. The proteolytic activities and adhesion to inanimate surfaces detected in S. maltophilia can be related to the pathogenesis of this bacterium and/or medical device colonization which favors the development of nosocomial infections.

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