Polyphasic characterisation of Burkholderia cepaciacomplex species isolated from children with cystic fibrosis

Cystic fibrosis (CF) patients with Burkholderia cepacia complex (Bcc) pulmonary infections have high morbidity and mortality. The aim of this study was to compare different methods for identification of Bcc species isolated from paediatric CF patients. Oropharyngeal swabs from children with CF were used to obtain isolates of Bcc samples to evaluate six different tests for strain identification. Conventional (CPT) and automatised (APT) phenotypic tests, polymerase chain reaction (PCR)-recA, restriction fragment length polymorphism-recA, recAsequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) were applied. Bacterial isolates were also tested for antimicrobial susceptibility. PCR-recA analysis showed that 36 out of the 54 isolates were Bcc. Kappa index data indicated almost perfect agreement between CPT and APT, CPT and PCR-recA, and APT and PCR-recA to identify Bcc, and MALDI-TOF and recAsequencing to identify Bcc species. The recAsequencing data and the MALDI-TOF data agreed in 97.2% of the isolates. Based on recA sequencing, the most common species identified were Burkholderia cenocepacia IIIA (33.4%),Burkholderia vietnamiensis (30.6%), B. cenocepaciaIIIB (27.8%), Burkholderia multivorans (5.5%), and B. cepacia (2.7%). MALDI-TOF proved to be a useful tool for identification of Bcc species obtained from CF patients, although it was not able to identify B. cenocepacia subtypes.

Quando e como valorizar culturas de urina polimicrobianas no laboratório de microbiologia clínica / When and how to enhance polymicrobial urine cultures at the laboratory of clinical microbiology

INTRODUÇÃO: Na maioria dos laboratórios de Microbiologia considera-se contaminação uma cultura de urina com mais de um morfotipo colonial, ignorando-se o desenvolvimento ou solicitando-se novo material. Raramente o isolado é considerado significativo. OBJETIVOS: Com a finalidade de estudar as infecções urinárias polimicrobianas, no período de agosto de 2003 a janeiro de 2004, na cidade de Tubarão, Santa Catarina, foram selecionadas 117 amostras de urina de pacientes internados no Hospital Nossa Senhora da Conceição, de ambos os sexos, com idades que variavam de 14 a 98 anos. MÉTODOS: Realizou-se uma análise minuciosa dos prontuários dos pacientes. Procedeu-se o Gram da gota de urina não centrifugada e a cultura com alça calibrada de 1 ou 10 μl em ágar CLED (cistina-lactose deficiente em eletrólitos). Todas as culturas foram repetidas com nova coleta de urina com supervisão direta. Os clínicos aguardaram a segunda coleta (confirmatória) para iniciar a antibioticoterapia. Descartaram-se os pacientes que iniciaram a antibioticoterapia imediatamente após a primeira coleta ou que estavam utilizando antimicrobianos. RESULTADOS: Obteve-se o total de seis (12,8 por cento) culturas polimicrobianas confirmadas, em um universo de 47 amostras com crescimento bacteriano estudadas. Os resultados foram compatíveis com as indicações clínicas. CONCLUSÃO: É importante ressaltar a correlação entre os achados laboratoriais e as indicações clínicas dos pacientes. Recomenda-se avaliar criteriosamente isolados polimicrobianos em amostras de urina, sejam ambulatoriais ou hospitalares.

INTRODUCTION: In most microbiology laboratories, a urine culture is regarded as contaminated when there is more than one colonial morphotype, thus either it is ignored or a new sample is requested. The isolate is rarely considered significant. OBJECTIVES: In order to study the polymicrobial urinary tract infections, 117 urine samples were selected from inpatients of both genders aged from 14 to 98 years old at Nossa Senhora da Conceição Hospital, Tubarão city, Santa Catarina, Brazil, from August 2003 to January 2004. METHODS: A detailed analysis of patients' records was carried out. Gram test of non-centrifuged urine drop and culture with calibrated loop of 1 or 10 microliters in CLED agar were performed. All cultures were repeated with new urine collection under direct supervision. Physicians waited for the second collection (confirmatory test) to initiate antibiotic therapy. Patients that had started antibiotic therapy immediately after the first collection or those that were using antibiotics were discarded. RESULTS: Six polymicrobial cultures were confirmed from a total of 47 samples with bacterial growth. The results were consistent with clinical indications. CONCLUSION: It is important to emphasize the correlation between laboratory findings and patients' clinical indications. It is recommended that polymicrobial isolates in urine samples from both out-patients and inpatients should be thoroughly investigated.

A Gardnerella vaginalis e as infecções do trato urinário / The Gardnerella vaginalis and the urinary tract infections

As infecções do trato urinário (ITUs) estão entre as mais frequentes nos seres humanos. São causadas por grande variedade de uropatógenos habituais, porém podem ser provocadas por alguns micro-organismos fastidiosos, como a Gardnerella vaginalis, que é uma bactéria anaeróbia facultativa, observada sob a forma de cocobacilos Gram-variáveis. Ela habita a mucosa vaginal e eventualmente pode ocasionar ITUs. O isolamento pode ser realizado em amostras de urina utilizando o ágar CNA (colistina e ácido nalidíxico), com incubação de 48 a 72 horas em atmosfera rica em CO2. O exame de Gram de urina não centrifugada pode auxiliar o microbiologista na identificação das amostras nas quais uma bactéria fastidiosa seja o agente causal, já que a visualização de inúmeras células epiteliais, a ausência de leucócitos e a presença de mais de um tipo morfológico sugerem contaminação da amostra. Como estudos comprovam a incidência de G. vaginalis entre os agentes causadores de ITUs, o isolamento em uroculturas não deve ser desprezado. A interpretação clínica do crescimento da G. vaginalis é de difícil avaliação, sendo imprescindível a troca de informações entre o laboratório de microbiologia clínica e a equipe médica, investigando a presença de sinais e sintomas que possam estar associados à ITU.

Urinary tract infections are among the most recurrent in human beings. They are caused by a wide variety of usual uropathogens, although they may be caused by some fastidious micro-organisms, such as Gardnerella vaginalis. It is a facultative anaerobe, found in the form of Gram-variable coccobacilli. It inhabits the vaginal mucosa and occasionally may cause urinary tract infections. The isolation may be performed on urine samples using CNA agar, incubated for 48-72 hours in atmosphere rich in CO2. The Gram examination of non-centrifuged urine may aid the microbiologist in identifying samples in which a fastidious bacterium is the causative agent, since the visualization of numerous epithelial cells, the absence of leukocytes and the presence of more than one morphological type suggest sample contamination. As studies show the Gardnerella vaginalis incidence among the causative agents of urinary tract infections, the isolation in urine cultures can not be overlooked. The clinical interpretation of the Gardnerella vaginalis growth is difficult to assess, thus being essential the information exchange between the clinical microbiology laboratory and medical staff in order to investigate the presence of signs and symptoms that may be associated with urinary tract infections.

Occurrence of extended-spectrum beta-lactamases in Enterobacteriaceae isolated from hospitalized patients in Curitiba, southern Brazil

Production of extended-spectrum beta-lactamases (ESBL) by enterobacteria is an important resistance mechanism against antimicrobial beta-lactamics. We tested 498 bacterial strains isolated from two tertiary-care teaching hospitals for ESBL production, using screening breakpoints for aztreonam and third generation cephalosporins, according to CLSI recommendations. Among these isolates, 155 were positive for the ESBL screening test, and 121 (78 percent) were confirmed by the clavulanic acid combination disk method. We found a high frequency of ESBL (24 percent) among Enterobacteriaceae, with a frequency of 57.4 percent for Klebsiella pneumoniae, 21.4 percent for Klebsiella oxytoca, and 7.2 percent for E. coli. In other members of Enterobacteriaceae, non-Klebsiella and non-E. coli, the prevalence was 21.6 percent. Ceftriaxone and cefotaxime showed a higher sensitivity in the screening test (99.2 percent) when compared to ceftazidime, aztreonam and cefpodoxime. However, cefotaxime/cefotaxime plus clavulanic acid showed a higher sensitivity in the confirmatory test (96.7 percent).