Evaluation of tests to predict metallo-ß-lactamase in cystic fibrosis (CF) and non-(CF) Pseudomonas.

Double disks synergy test (DDST) and combined disks test (CD) were evaluated to predict the presence of metallo-ß-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.

Evaluation of tests to predict metallo-β-lactamase in cystic fibrosis (CF) and non-(CF) Pseudomonas

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.

Evaluation of the Gram stain of sputa from cystic fibrosis patients to predict the presence of Staphylococcus aureus.

We evaluated the presence of Gram-positive cocci in cluster, seen in Gram-stained smears of cystic fibrosis (CF) sputa versus the microbiological culture for the prediction of the presence of S. aureus. Gram stain provided low accuracy (69.2%; odds ratio, 4.8; 95% confidence interval, 61.3-76.1) for predicting S. aureus in CF sputum.

The presence of quorum-sensing genes in Pseudomonas isolates infecting cystic fibrosis and non-cystic fibrosis patients.

Ninety-one Pseudomonas aeruginosa isolates recovered from cystic fibrosis and non-cystic fibrosis patients were evaluated regarding the ability to form biofilm and acyl-homoserine lactones production and for the presence of five quorum-sensing (QS) regulatory genes (lasI, lasR, rhlI, rhlR, and vfr). Most isolates (90.1 %) presented all five QS genes. Five isolates shown to be lasI/lasR-deficient were not able to produce biofilm in vitro. Moreover, one isolate harboring all five QS genes was also not able to form a biofilm. The function of rhlR gene may be compensated by the las QS system. However, in our study, all isolates which were deficient for the rhlR gene were also deficient for the lasI/lasR system. This may point to some hierarchy in QS regulation which may pose a potential for controlling biofilm infections due to P. aeruginosa.

Detection of methicillin-resistant Staphylococcus aureus in clinical specimens from cystic fibrosis patients by use of chromogenic selective agar.

We evaluated the use of a chromogenic selective medium (MRSA ID) as a useful tool for the detection of methicillin-resistant Staphylococcus aureus (MRSA) in cystic fibrosis (CF) patient samples. Fifty-four MRSA isolates were detected by MRSA ID, while only 24/54 (44%) (odds ratio [OR], 2.79; 95% confidence interval [CI], 1.63 to 4.76) were detected by conventional methods. A chromogenic selective medium for MRSA detection may improve its surveillance in CF patients.

Cystic and non-cystic fibrosis Pseudomonas aeruginosa isolates are not differentiated by the quorum-sensing signaling and biofilm production.

The capability for biofilm and quorum-sensing (QS) signaling production among Pseudomonas aeruginosa isolates were evaluated. A total of 231 isolates were recovered from sputa of cystic fibrosis (CF, n = 104) and non-CF (non-CF, n = 127) patients. One hundred ninety-seven (85.3%; 95% CI 80.1-89.3%) were biofilm producers and 157 (68%; 95% CI 61.7-73.6%) were weak QS-producing. No difference was observed between CF and non-CF isolates regarding the ability to produce biofilm and QS-signaling. Interestingly, the degree of QS production appears to be related to the degree of biofilm production. Thus, blocking QS pathways may be crucial in the prevention and treatment of biofilm-related infections.

Nutritional requirement among Pseudomonas aeruginosa isolates recovered from respiratory clinical specimens at a tertiary hospital from South of Brazil

We screened 349 isolates of P. aeruginosa from cystic fibrosis (CF+) and non-cystic fibrosis (CF-) patients for the auxotrophy. Fourteen (4.0 percent) 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 using distinct media and antimicrobial susceptibility profile of Pseudomonas aeruginosa.

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 using distinct media and antimicrobial susceptibility profile of Pseudomonas aeruginosa

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.

High vancomycin resistance among biofilms produced by Staphylococcus species isolated from central venous catheters

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.

Nutritional requirement among Pseudomonas aeruginosa isolates recovered from respiratory clinical specimens at a tertiary hospital from South of Brazil.

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.

Identification and detection of methicillin resistance in Non-Epidermidis coagulase-negative staphylococci

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 percent of the isolates. All methods gave 100 percent sensitivity, based on presence of the mecA gene. The specificity of the cefoxitin disk was 100 percent, while the oxacillin disk gave a specificity of 91 percent and agar dilution oxacillin gave a specificity of 88 percent. 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.

Evaluation of the accuracy of various phenotypic tests to detect oxacillin resistance in coagulase-negative staphylococci

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 percent) harbored the mecA gene. Using 30µg-cefoxitin and 1µg-oxacillin disks we obtained 100 and 98.3 percent accuracy, respectively. In addition, when cefoxitin was used as marker in an agar dilution method, the higher accuracy (99.4 percent) 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.

Clonal types and antimicrobial resistance profiles of methicillin-resistant Staphylococcus aureus isolates from hospitals in south Brazil / Tipos de clones e perfis de resistência antimicrobiana de Staphylococcus aureus resistentes à meticilina isolados de hospitais no sul do Brasil

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.

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.

Evaluation of urinalysis parameters to predict urinary-tract infection

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 percent. A combination between intense bacteriuria (microscopic analysis) and >20 leukocytes per µL of urine (flow cytometry) gave a higher accuracy (97.3 percent). 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.

Evaluation of oxacillin and cefoxitin disks for detection of resistance in coagulase negative staphylococci

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 percent 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 mug) and cefoxitin (30 mug) 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 percent) and oxacillin MIC (97.8 percent), 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.

Use of the D test method to detect inducible clindamycin resistance in coagulase negative staphylococci (CoNS)

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.

Bacteremia por Rhodococcus equi em paciente com síndrome da imunodeficiência adquirida: relato de caso / Bacteremia due to Rhodococcus equi in a patient with acquired immunodeficiency syndrome: case report

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.

Prevalência de Cryptococcus neoformans nos pombos urbanos da cidade de Porto Alegre, Rio Grande do Sul / Prevalence of Cryptococcus neoformans in urban pigeons of Porto Alegre (RS), Brazil

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 30,5ºC. Após cinco dias, colônias mucóides foram subcultivadas para identificação através de provas morfofisiológicas. O fungo C. neoformans foi isolado em 88 (100 por cento) das amostras avaliadas, confirmando sua ocorrência nos pombos que habitam as praças da cidade.