We screened 349 isolates of P. aeruginosa from cystic fibrosis (CF+) and non-cystic fibrosis (CF-) patients for the auxotrophy. Fourteen (4.0%) were auxotrophic and among them only one was recovered from CF-patient showing that this characteristic is strongly associated with cystic fibrosis. In total, a requirement for 5 different compounds (or combination) was verified and, of these, methionine was the most common single amino acid required. Only one auxotrophic isolate was no able to produce biofilm in vitro.

Biofilm production is an important mechanism that allows microbes to escape host defences and antimicrobial therapy. Vancomycin has been used largely for the treatment of methicillin-resistant staphylococcal infections. Here, we determined the minimal inhibitory concentration (MIC) and minimal biofilm eradication concentration (MBEC) for 82 Staphylococcus species isolated from central venous catheters (CVC). Our results showed that the 41 strong and moderate-biofilm-producing isolates presented a higher MBEC/MIC ratio for vancomycin than the 24 weak-biofilm-producing isolates, illustrating the importance of biofilm production ability and the difficulty in treating biofilm-related infections. The MBEC was significantly higher in moderate-biofilm-producing isolates than in weak-biofilm-producing isolates (p < 0.001) and in strong-biofilm-producing isolates than in weak-biofilm-producing isolates (p = 0.001). The correlation between the MIC and the MBEC was poor. Based on our results, we recommend that bacterial biofilms be suspected in all cases of CVC infection.

INTRODUÇÃO: Colonização pulmonar bacteriana é a principal causa de morbidade em fibrose cística (FC). Patógenos como Staphylococcus aureus são muito bem adaptados ao ambiente pulmonar e podem persistir por anos no mesmo paciente. Determinantes genéticos desta bactéria, como presença de SCCmec emergiram recentemente como um problema nesta população de pacientes. MÉTODOS: Foram comparados isolados de Staphylococcus aureus obtidos de diferentes materiais clínicos, de pacientes com e sem FC atendidos em um hospital de referência em tratamento de fibrocísticos de acordo com o tipo de SCCmec e o perfil de susceptibilidade aos antimicrobianos. RESULTADOS: Foram coletados 364 Staphylococcus aureus, um isolado por paciente, sendo 164 (45%) de pacientes com FC. Entre estes pacientes, 57/164 (44,5%) eram MRSA, e entre pacientes não fibrocísticos, MRSA compreendiam 89/200 (35%). Foram encontrados outros patógenos, associados ao Staphylococcus aureus, em 38 pacientes com FC. Todos 57 MRSA de pacientes com FC possuíam o cassete de multiresistência tipo III. Por outro lado, 31/89 MRSA de pacientes não fibrocísticos possuíam SCCmec tipo I (35%) e 44/89 possuíam tipo III (49%). O perfil de susceptibilidade aos antimicrobianos foi similar entre pacientes com e sem FC. CONCLUSÕES: A alta prevalência do SCCmec de multiresistência tipo III entre pacientes com FC comparado com pacientes sem FC em nossa instituição pode dificultar o controle da progressão da doença, feito através da antibioticoterapia, e que promove a sobrevivência deste tipo de paciente.

INTRODUCTION: Bacterial colonization of the lungs is the main cause of morbidity in cystic fibrosis (CF). Pathogens such as Staphylococcus aureus are very well adapted to the pulmonary environment and may persist for years in the same patient. Genetic determinants of these bacteria, such as the presence of SCCmec have recently emerged as a problem in this population of patients. METHODS: Staphylococcus aureus isolates obtained from different clinical materials coming from CF and non-CF patients attended at a cystic fibrosis reference hospital were compared according to SCCmec type and antibiotic susceptibility profile. RESULTS: Three hundred and sixty-four single-patient Staphylococcus aureus isolates were collected, of which 164 (45%) were from CF patients. Among the latter, 57/164 (44.5%) were MRSA, and among the non-CF patients, 89/200 (35%) were MRSA. Associated pathogens were found in 38 CF patients. All 57 MRSA from CF patients harbored the multiresistant cassette type III. In contrast, 31/89 MRSA from non-CF patients harbored SCCmec type I (35%) and 44/89 harbored type III (49%). The antibiotic susceptibility pattern was similar between CF and non-CF patients. CONCLUSIONS: The high prevalence of multiresistant SCCmec type III among CF patients compared with non-CF patients in our institution may make it difficult to control disease progression through antibiotic therapy for promoting the survival of this kind of patient.

Nas últimas duas décadas os membros do gênero Enterococcus emergiram como importantes patógenos nosocomiais ao redor do mundo. No presente estudo, nós avaliamos a resistência antimicrobiana e as características genotípicas de 203 Enterococcus spp. obtidos de diferentes fontes clínicas em dois hospitais de Porto Alegre, Rio Grande do Sul, Brasil. As espécies foram identificadas por testes bioquímicos convencionais e por um sistema automatizado. A diversidade genética de E. faecalis demonstrando resistência à altos níveis de aminoglicosídeos (HLAR) foi avaliada através da análise do DNA cromossômico após digestão com a enzima SmaI, seguido por eletroforese em campo pulsado. O E. faecalis foi a espécie mais freqüente (93,6%), seguido por E. faecium (4,4%). O perfil de resistência antimicrobiana foi: 2,5% para ampicilina, 0,5% para vancomicina, 0,5% para teicoplanina, 33% para cloranfenicol, 2% para nitrofurantoína 66,1% para eritromicina, 66,5% para tetraciclina, 24,6% para rifampicina, 30% para ciprofloxacino e 87,2% para quinupristina-dalfopristina. Um total de 10,3% dos isolados apresentaram HLAR para ambos gentamicina e estreptomicina (HLR-ST/GE), sendo 23,6% resistentes somente a gentamicina (HLR-GE) e 37,4% somente a estreptomicina (HLR-ST). Um grupo clonal predominante foi encontrado em E. faecalis HLR-GE/ST. A prevalência de resistência a antibióticos ²-lactâmicos, e em particular aos glicopeptídeos, foi muito baixa. Entretanto, neste estudo, houve um número crescente de Enterococcus HLAR que podem estar se disseminando intra e interhospitais.

In the past two decades the members of the genus Enterococcus have emerged as important nosocomial pathogens worldwide. In the present study, we evaluated the antimicrobial resistance and genotypic characteristics of 203 Enterococcus spp. recovered from different clinical sources from two hospitals in Porto Alegre, Rio Grande do Sul, Brazil. The species were identified by conventional biochemical tests and by an automated system. The genetic diversity of E. faecalis presenting high-level aminoglycoside resistance (HLAR) was assessed by pulsed-field gel electrophoresis of chromosomal DNA after SmaI digestion. The E. faecalis was the most frequent specie (93.6%), followed by E. faecium (4.4%). The antimicrobial resistance profile was: 2.5% to ampicillin, 0.5% to vancomycin, 0.5% teicoplanin, 33% to chloramphenicol, 2% to nitrofurantoin, 66.1% to erythromycin, 66.5% to tetracycline, 24.6% to rifampicin, 30% to ciprofloxacin and 87.2% to quinupristin-dalfopristin. A total of 10.3% of the isolates proved to be HLAR to both gentamicin and streptomycin (HLRST/GE), with 23.6% resistant only to gentamicin (HLR-GE) and 37.4% only to streptomycin (HLRST). One predominant clonal group was found among E. faecalis HLR-GE/ST. The prevalence of resistance among beta-lactam antibiotics and glycopeptides was very low. However, in this study there was an increased number of HLR Enterococcus which may be spreading intra and inter-hospital.

Coagulase-negative Staphylococcus spp. was considered nonpathogenic until the emergence of multiresistance and the demonstration of their participation as infectious agents. In Brazil, oxacillin resistance may be present in over 80% of isolates, and the Clinical and Laboratory Standards Institute standardized a disk-diffusion method to predict this resistance in Staphylococcus. The aim of this study was to evaluate the variability among commercial disks of oxacillin (1 µg) and cefoxitin (30 µg) widely used in clinical laboratories of microbiology, compared with mecA gene and minimum inhibitory concentration (MIC) of oxacillin. The use of oxacillin and cefoxitin disks simultaneously allowed the detection of important differences, particularly, in less frequent species such as S. cohnii, S. haemolyticus, S. saprophyticus, and S. sciuri. Disks of cefoxitin of the brand 2 displayed good correlation with the mecA gene (98.7%) and oxacillin MIC (97.8%), while major discrepancies were observed using disks of brand 1. One of the critical points in the diffusion disk test is the quality of the disks: the use of better quality disks associated with molecular methods lead to better results to define the best antibiotic therapy.

Este estudo foi desenvolvido para comparar métodos de detecção e para estimar a prevalência de Klebsiella spp e E.coli produtoras de beta-lactamases de espetro ampliado (ESBL) em um Hospital Universitário no sul do Brasil. A correlação genética, determinada através de método molecular de tipagem, entre as amostras de K. pneumoniae também foi determinada. A produção de ESBL foi investigada em 95 amostras de Klebsiella spp e E.coli obtidas de pacientes no Hospital de Clínicas de Porto Alegre usando-se: medida do diâmetro a zona de inibição (KB), dupla-difusão de disco (DD), valores de concentração inibitória mínima da ceftazidima (MIC CAZ), aumento do diâmetro da zona de inibição com adição de clavulanato (CAZ/CAC) e a relação entre o MIC da ceftazidima/MIC ceftazidima com clavulanato (MIC CAZ/CAC). A tipagem molecular foi realizada utilizando-se o método de macrorestrição de DNA e eletroforese em campo pulsado (PFGE). O método KB apresentou as maiores taxas de produção de ESBL (> 70% para Klebsiella e 59% para E.coli) contrastando com os outros métodos (p< 0,05). Os métodos confirmatórios (DD, MIC CAZ e MIC CAZ/CAC) indicaram a produção de ESBL em 8 a 13% de E.coli e em 33 a 40% para as espécies de Klebsiella. Portanto, o método KB é útil apenas como método de triagem devido aos diversos resultados considerados falso-positivos. A tipagem molecular realizada em 17 amostras de K.pneumoniae ESBL indicou não existência de relação clonal. Este estudo encontrou uma boa correlação entre os métodos confirmatórios de detecção de ESBL embora os métodos que avaliam a inibição da enzima pelo clavulanato pareçam ser mais específicos. A alta prevalência de Klebsiella ESBL em nosso hospital provavelmente se deve a seleção individual de cepas resistentes do que a transmissão de uma cepa comum.

His study was performed to compare the methods of detection and to estimate the prevalence of extended-spectrum beta-lactamases (ESBL) among Klebsiella spp and E.coli in a university hospital in southern Brazil. We also used a molecular typing method to evaluate the genetic correlation between isolates of ESBL K.pneumoniae. Production of ESBL was investigated in 95 clinical isolates of Klebsiella spp and Escherichia coli from Hospital de Clínicas de Porto Alegre, using Kirby-Bauer zone diameter (KB), double-disk diffusion (DD), breakpoint for ceftazidime (MIC CAZ), increased zone diameter with clavulanate (CAZ/CAC) and ratio of ceftazidime MIC/ceftazidime-clavulanate MIC (MIC CAZ/CAC). Molecular typing was performed by DNA macrorestriction analysis followed by pulsed-field gel electrophoresis. The KB method displayed the highest rates of ESBL (up to 70% of Klebsiella and 59% of E.coli), contrasting with all the other methods (p < 0.05). The confirmatory methods (DD, MIC CAZ, CAZ/CAC and MIC CAZ/CAC) showed a range of ESBL production from 8 to 13% for E.coli and from 33 to 40% for Klebsiella species. Therefore, the KB method was useful only as a screening method as it provided several false positive results. Molecular typing of 17 ESBL K.pneumoniae indicated that the isolates had no clonal relation. We found a good correlation among the confirmatory methods for ESBL detection although the methods which evaluate inhibition of the beta-lactamase by clavulanate appeared to be more specific. The high prevalence of ESBL Klebsiella in our hospital is probably due to individual selection of resistant strains rather than the transmission of a common strain.

Susceptibility tests by disk diffusion and by E-test and molecular typing by macrorestriction analysis were performed to determine the relatedness of Pseudomonas aeruginosa isolates from three distinct hospitals. The resistance profile of 124 isolates to 8 antimicrobial agents was determined in three different hospitals, in Porto Alegre, Brazil. Frequencies of susceptibility ranged from 43.9% for carbenicillin to 87.7% for ceftazidime. Cross-resistance data of imipenem-resistant isolates indicated that most (70%) were also resistant to carbenicillin, although 30% remained susceptible to ceftazidime and cefepime. In general, susceptibility profiles were not able to determine relatedness among isolates of P. aeruginosa. On the other hand, molecular typing by macrorestriction analysis demonstrated high discriminatory power and identified 66 strains among 72 isolates of P. aeruginosa. Imipenem-susceptible isolates were all different. However, identical clones of imipenem-resistant isolates were found in two of the hospitals, despite variable response to other antibiotics. No clustering of infection among the different medical centers was observed. In conclusion, clones of P. aeruginosa did not spread among the different hospitals in our city even though related isolates of imipenem-resistant P. aeruginosa were found.

O sistema de lise-centrifugação (Isolator TM) tem sido considerado como método padrão para aumentar as taxas de diagnóstico de candidemia através de hemocultura. Neste estudo, o processo de concentração, segundo este sistema, foi comparado com métodos convencionais de cultivo em meio líquido e bifásico. Foram realizados testes "in vitro" utilizando Candida albicans em contagens de 100, 10, 1 e 0,5 células/ml de sangue. Culturas em ágar-chocolate foram realizadas a partir do sedimento obtido pela centrifugação do tubo de Isolator TM e do caldo das culturas convencionais após sua incubação por 24 horas a 35oC. Esfregaços corados pelo Gram, preparados a partir dos métodos convencionais, também foram observados em 24 horas. Foi possível detectar Candida albicans independentemente do número de células ou da metodologia utilizada. O tempo para diagnóstico também não foi diferente para os métodos comparados, já que blastoconídios e crescimento foram observados no mesmo prazo de tempo. Assim, sugerimos que o processo de concentração não é o maior fator responsável pelas taxas de recuperação de Candida albicans a partir do sangue obtidas pelo sistema IsolatorTM.

The lysis-centrifugation system (Isolator TM) is recognized as the standard method for recovery of Candida spp. from blood. In this study, the effect of the concentration process of this system was compared with conventional methods of blood culture using liquid and biphasic media. The tests were performed in vitro using Candida albicans in counts of 100, 10, 1, and 0.5 cells/ml of blood. Cultures onto chocolate agar were performed with the sediment obtained after centrifugation of IsolatorTM tube and with liquid and biphasic media after their incubation for 24 h at 35oC. Gram stain prepared from the conventional methods were also evaluated in the first 24 h. It was possible to detect Candida albicans in blood, regardless both the number of cells or methodology. As blastoconidia were observed in Gram stains at the same time that growth was noted, time for diagnosis was also not different for the compared methods. Therefore, we suggest that the process of concentration is not the single important factor responsible for the recovery rates of Candida albicans from blood by the IsolatorTM system.