In this study, we aimed to evaluate the performance of different phenotypic tests to detect carbapenem-resistant Enterobacteriaceae. Three different phenotypic methods were evaluated: (1) combined-disk test of meropenem plus phenylboronic acid or EDTA reading after 24 h and 48 h; (2) selective/chromogenic read after 24 h and after 48 h; and (3) overnight selective enrichment broth containing 10 µg ertapenem disk followed by culture on MacConkey agar. A positive result in at least one of the methods was submitted to PCR for blaNDM-1, blaOXA-48, blaKPC, blaSPM-1, blaIMP, and blaGES detection. Carbapenem-resistant Enterobacteriaceae was detected in 31 (30.4%) of 102 rectal swabs evaluated. All isolates showed to be KPC-2-producing organisms. Results showed excellent agreement among the evaluated tests (positive and negative) (kappa = 0.88). It is important to state that combined-disk test with phenylboronic acid is not suitable for bacterial identification/isolation. Conversely, selective/chromogenic agar after 48h of incubation showed to be a useful tool, with the advantage of presumptive bacterial identification.

Double disks synergy test (DDST) and combined disks test (CD) were evaluated to predict the presence of metallo-β-lactamase in 70 Pseudomonas aeruginosa isolates recovered from cystic fibrosis and non-cystic fibrosis patients. DDST CAZ-EDTA 1 cm and CD IMP-EDTA tests showed the best accuracy (94.3%). Furthermore, for other combinations, accuracy unsatisfactory was obtained.

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 for bacterial survival and its occurrence together with antimicrobial resistance represents a challenge for clinical management. Here, we evaluated the ability for biofilm production among P. aeruginosa isolates from patients with or without cystic fibrosis (CF) using two distinct media, besides determining the antimicrobial susceptibility profile of these isolates for eight antimicrobial agents. The ability for biofilm production when TSB medium was used was higher than when used CF sputum media (p = 0.0198). However, P. aeruginosa isolates from CF have demonstrated similar performance for biofilm production, independently of the medium used. Besides, among the biofilm-producing isolates, those recovered from CF were more resistant to the carbapenems (meropenem and imipenem) agents than those isolates from non-CF isolates.

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.

The NCCLS (2004) presented a new methodology to detect, by disk-diffusion agar, oxacillin-resistance using a cefoxitin disk. We identified coagulase-negative staphylococci (SCoN) to the species level and compared the use of cefoxitin disks (30 µg) with oxacillin disks (1 µg), agar dilution (minimum inhibitory concentration of oxacillin) and mecA gene detection in isolates of coagulase-negative bacteria other than Staphylococcus epidermidis (SCoNne). A total of 238 SCoNne was evaluated; oxacillin-resistance (the mecA gene) was detected in 71% of the isolates. All methods gave 100% sensitivity, based on presence of the mecA gene. The specificity of the cefoxitin disk was 100%, while the oxacillin disk gave a specificity of 91% and agar dilution oxacillin gave a specificity of 88%. We conclude that the cefoxitin disk is an efficient test, and it is an easy method for use in clinical laboratories to detect oxacillin-resistance in staphylococci.

No presente estudo foram avaliados o perfil de macrorrestrição do DNA e tipos de SCCmec para nove MRSA multirresistentes selecionados. Além disso, susceptibilidade a 12 agentes antimicrobianos pelo teste de disco-difusão foi avaliada para 68 isolados de MRSA. Os isolados foram obtidos de hemoculturas de pacientes hospitalizados de três hospitais de Porto Alegre, Brasil. PFGE e PCR para detecção do gene mecA e para os tipos genéticos SCCmec I, II, III e IV foram realizados em nove isolados selecionados que apresentaram susceptibilidade somente a vancomicina, teicoplanina e linezolida. Dois perfis clonais, com cinco subtipos, foram demonstrados entre os isolados analisados. Oito isolados apresentaram SCCmec tipo III e um isolado não foi caracterizado quanto ao tipo de SCCmec. O conhecimento do tipo de SCCmec bem como dos perfis clonais e de susceptibilidade aos antimicrobianos entre isolados de S. aureus é essencial, principalmente, para a prevenção e controle da disseminação da resistência antimicrobiana.

In the present study were evaluated the DNA macrorestriction profile and SCCmec types for nine multi-resistant MRSA selected. Also antimicrobial susceptibility testing by disk diffusion method was evaluated for 68 MRSA isolates against 12 antimicrobial agents. The isolates were recovered from blood culture collected from hospitalized patients in three hospitals of Porto Alegre, Brazil. PFGE and PCR for mecA and SCCmec I, II, III, IV types genes were done on selected nine isolates with susceptibility only to vancomycin, teicoplanin and linezolid. Two clone profiles, with five subtypes, were demonstrated among multi-resistant MRSA analyzed. Eight isolates showed harbor SCCmec type III and one isolate was not typeable. The knowledge of SCCmec type, clone and antimicrobial profiles among S. aureus is essential mainly to prevention and control of dissemination of the antimicrobial resistance.

In this study, we determined the accuracy of phenotypic tests (cefoxitin agar dilution, 30µg-cefoxitin and 1µg-oxacillin disks) to detect the oxacillin resistance/mecA gene among coagulase-negative staphylococci (CoNS) isolates. The presence of the mecA gene was detected by PCR technique (gold standard). A total of 176 CoNS isolates from blood of hospitalized patients were evaluated. Of these, 138 (78.4%) harbored the mecA gene. Using 30µg-cefoxitin and 1µg-oxacillin disks we obtained 100 and 98.3% accuracy, respectively. In addition, when cefoxitin was used as marker in an agar dilution method, the higher accuracy (99.4%) was established with 8mg cefoxitin per mL breakpoint. Thus, despite of the agar dilution method using cefoxitin as a marker not being standard for this detection, our results suggested that it is an excellent alternative to detect the oxacillin resistance/mecA gene among CoNS isolates.

We evaluated the performance of automated-flow cytometry, urinalysis dipsticks and microscopic urine sediment analysis as predictors of urinary tract infection. Urine cultures were used as a reference method for comparison. Six-hundred-seventy-five urine samples from hospitalized and not hospitalized patients attended at Hospital Mãe de Deus, Porto Alegre, in south Brazil, were included in the study. Among the individual measures analyzed, intense bacteriuria in the microscopic analysis of urinary sediment gave an accuracy of 92.9%. A combination between intense bacteriuria (microscopic analysis) and >20 leukocytes per µL of urine (flow cytometry) gave a higher accuracy (97.3%). We conclude that though it is laborious, microscopic urinalysis is a good analytical tool. Taken together with flow cytometry and dipsticks, we obtained a clinically-acceptable prediction of urinary-tract infection.

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.

According to the National Committee for Clinical Laboratory Standards (NCCLS, 2004), a method to evaluate the inducible clindamycin resistance in accordance with an approach of the disks of erythromycin and clindamycin - the D test - has been reported. We analyzed the performance of this method in 200 coagulase negative staphylococci (CoNS) strains obtained from blood cultures of hospitalized patients at a general hospital in Southern Brazil. Twenty-seven clinical isolates with suitable profile (erythromycin-resistant and clindamycin-susceptible) were evaluated for the D test realization. Thus, only 5 CoNS were D test positive. The D test method showed to be simple and an important technique in the detection of inducible clindamycin resistance.

Rhodococcus equi é um importante agente de infecções zoonóticas, podendo causar sérias infecções em humanos, principalmente em pacientes imunocomprometidos. Neste estudo, nós relatamos o caso de uma bacteremia fatal devido a Rhodococcus equi em paciente com síndrome da imunodeficiência adquirida (HIV positivo).

Rhodococcus equi is an important agent for zoonotic infections, and may cause serious infections in humans, especially immunocompromised patients. In this study, a case of fatal bacteremia due to Rhodococcus equi in a patient with acquired immunodeficiency syndrome (HIV positive) is reported.

Cryptococcus neoformans, levedura encapsulada, é o agente etiológico da criptococose em humanos e animais. A variedade neoformans, encontrada em fontes ambientais, incluindo fezes de pombos, é importante causa de mortalidade em indivíduos imunodeprimidos em todo o mundo. Contudo, ainda não há estudos sobre sua ecologia na cidade de Porto Alegre, onde se tem registro da ocorrência de casos humanos dessa micose. Para pesquisar fontes saprofíticas de C. neoformans na cidade de Porto Alegre, foram coletadas 88 amostras de excretas de pombos em distintas praças da cidade. Suspensões das amostras em salina estéril foram semeadas em placas com meio ágar Sabouraud e incubadas em estufa a temperaturas entre 29 e 30,5°C. Após cinco dias, colônias mucóides foram subcultivadas para identificação através de provas morfofisiológicas. O fungo C. neoformans foi isolado em 88 (100%) das amostras avaliadas, confirmando sua ocorrência nos pombos que habitam as praças da cidade.

Cryptococcus neoformans is an encapsulated yeast, causing cryptococcosis in human beings and animals. The variety neoformans, found in environmental sources, including pigeon excrements, is an important cause of mortality in immunocompromised individuals in the whole world. However, there are still no studies on its ecology in the city of Porto Alegre, where register of the occurrence of human cases of this mycosis is found. To study the saprophitic sources of C. neoformans in the city of Porto Alegre, 88 samples of avian excreta have been collected in distinct squares. Suspension of the samples in sterile saline solution had been planted in plates with Sabouraud agar and incubated in stove at 29-30.5°C. After five days mucoid colonies were subcultived for identification through morphologic and physiologic tests. C. neoformans was isolated in 88 (100%) of the samples, proving its occurrence in the pigeons that inhabit the squares of that city.