Pseudomonas aeruginosa: study of Antibiotic Resistance and Molecular Typing in Hospital Infection Cases in a Neonatal Intensive Care Unit from Rio de Janeiro City, Brazil

This study had the objective of to analyze the demographic and bacteriologic data of 32 hospitalized newborns in an neonatal intensive care unit of a public maternity hospital in Rio de Janeiro city, Brazil, seized by Pseudomonas aeruginosa sepsis during a period ranged from July 1997 to July 1999, and to determine the antimicrobial resistance percentage, serotypes and pulsed field gel electrophoresis (PFGE) patterns of 32 strains isolated during this period. The study group presented mean age of 12.5 days, with higher prevalence of hospital infection in males (59.4%) and vaginal delivery (81.2%), than females (40.6%) and cesarean delivery (18.8%), respectively. In this group, 20 (62.5%) patients received antimicrobials before positive blood cultures presentation. A total of 87.5% of the patients were premature, 62.5% presented very low birth weight and 40.6% had asphyxia. We detected high antimicrobial resistance percentage to b-lactams, chloramphenicol, trimethoprim/sulfamethoxazole and tetracycline among the isolated strains. All isolated strains were classified as multi-drug resistant. Most strains presented serotype O11 while PFGE analysis revealed seven distinct clones with isolation predominance of a single clone (75%) isolated from July 1997 to June 1998.

Evaluation of adherence, hemagglutination, and presence of genes codifying for virulence factors of Acinetobacter baumannii causing urinary tract infection

Acinetobacter baumannii is a strictly aerobic bacterium which causes severe infections, however its pathogenic characteristics are not well defined. Thirteen A. baumannii strains isolated from urine of hospitalized and nonhospitalized patients with different ages were investigated for the presence of virulence factors. The isolates belonged to biotypes 2, 6, and 9 and were sensitive to imipenem. The majority of them showed resistance to amikacin, ceftazidime, ceftriaxone, ciprofloxacin, gentamicin, norfloxacin, and trimethoprim-sulfamethoxazole. None of A. baumannii strains presented genes codifying for 17 different virulence factors previously described in uropathogenic Escherichia coli, when tested by polymerase chain reaction (PCR). Nine isolates agglutinated human group AB erythrocytes, in presence of mannose, but none of them agglutinated group O erythrocytes. Adherence to polystyrene was observed in 7 isolates, and this result did not correlate with that obtained in hemagglutination assay. All the isolates were able to grow in iron-limiting conditions, showing that A. baumannii produces some type of siderophore. However, the genes iutA and fyuA, from iron uptake system of E. coli and Yersinia sp., respectively, were not present in the isolates, suggesting the presence of a different type of siderophore. The fimbriae of A. baumannii strains that mediates the adherence are possibly mannose-resistant, eventhough the mechanism of adherence to human epithelial cells still remains to be elucidated.

Antimicrobial susceptibility patterns of unusual nonfermentative gram-negative bacilli isolated from Latin America: report from the SENTRY Antimicrobial Surveillance Program (1997-2002)

The antimicrobial susceptibility of 176 unusual non-fermentative gram-negative bacilli (NF-GNB) collected from Latin America region through the SENTRY Program between 1997 and 2002 was evaluated by broth microdilution according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations. Nearly 74% of the NF-BGN belonged to the following genera/species: Burkholderia spp. (83), Achromobacter spp. (25), Ralstonia pickettii (16), Alcaligenes spp. (12), and Cryseobacterium spp. (12). Generally, trimethoprim/sulfamethoxazole (MIC50, < 0.5 µg/ml) was the most potent drug followed by levofloxacin (MIC50, 0.5 µg/ml), and gatifloxacin (MIC50, 1 µg/ml). The highest susceptibility rates were observed for levofloxacin (78.3%), gatifloxacin (75.6%), and meropenem (72.6%). Ceftazidime (MIC50, 4 µg/ml; 83.1% susceptible) was the most active beta-lactam against B. cepacia. Against Achromobacter spp. isolates, meropenem (MIC50, 0.25 µg/ml; 88% susceptible) was more active than imipenem (MIC50, 2 µg/ml). Cefepime (MIC50, 2 µg/ml; 81.3% susceptible), and imipenem (MIC50, 2 µg/ml; 81.3% susceptible) were more active than ceftazidime (MIC50, >16 µg/ml; 18.8% susceptible) and meropenem (MIC50, 8 µg/ml; 50% susceptible) against Ralstonia pickettii. Since selection of the most appropriate antimicrobial agents for testing and reporting has not been established by the NCCLS for many of NF-GNB species, results from large multicenter studies may help to guide the best empiric therapy.

Shigella in Brazilian children with acute diarrhoea: prevalence, antimicrobial resistance and virulence genes

Diarrhoeal disease is still considered a major cause of morbidity and mortality among children. Among diarrhoeagenic agents, Shigella should be highlighted due to its prevalence and the severity of the associated disease. Here, we assessed Shigella prevalence, drug susceptibility and virulence factors. Faeces from 157 children with diarrhoea who sought treatment at the Children's Hospital João Paulo II, a reference children´s hospital in Belo Horizonte, state of Minas Gerais, Brazil, were cultured and drug susceptibility of the Shigella isolates was determined by the disk diffusion technique. Shigella virulence markers were identified by polymerase chain reaction. The bacterium was recovered from 10.8% of the children (88.2% Shigella sonnei). The ipaH, iuc, sen and ial genes were detected in strains isolated from all shigellosis patients; set1A was only detected in Shigella flexneri. Additionally, patients were infected by Shigella strains of different ial, sat, sen and set1A genotypes. Compared to previous studies, we observed a marked shift in the distribution of species from S. flexneri to S. sonnei and high rates of trimethoprim/sulfamethoxazole resistance.

Determinação simultânea de resíduos de sulfametoxazol e trimetoprima em superfícies de equipamentos de produção

A cleaning validation method was developed and validated, based on swabbing sampling and simultaneous chromatographic determination of sulfamethoxazole (SMX) and trimethoprim (TMP) residues. The method presented limits of detection of 0.06 mg mL-1 for SMX and 0.09 mg mL-1 for TMP. It was considered selective, precise, accurate and robust according to the guidelines from ANVISA, the Brazilian regulatory agency, and International Conference on Harmonization. Mean swab recovery factors of 98.5% for SMX and 97.7% for TMP were obtained for spiked stainless steel plates. The method was successfully applied to the assay of actual swab samples collected from eleven points on an equipment surface.

Desenvolvimento de métodos de análise por CLAE-UV para os antimicrobianos tetraciclina, sulfametoxazol e trimetoprima utilizando materiais à base de sílica como sistemas de pré-concentração

This paper evaluates the adsorption capacity of zirconocene-based silica materials in the pre-concentration of antimicrobians (tetracycline, sulfamethoxazole and trimethoprim) in aqueous medium. These materials were prepared by grafting the zirconocene onto silicas pre-treated at different temperatures. The retention capacity of these materials was evaluated by off line SPE and HPLC-UV and the proposed methodology was validated in ultrapure, tap and river water. The recovery for tetracycline was 72% (in the solid phase A) and, for sulfamethoxazole and trimethoprim was 68 and 95% in the commercial C18, respectively. The target antimicrobians were not detected in the Arroio Dilúvio (Porto Alegre - RS).

High prevalence of methicillin resistance and PVL genes amongStaphylococcus aureus isolates from the nares and skin lesions of pediatric patients with atopic dermatitis

Staphylococcus aureus is highly prevalent among patients with atopic dermatitis (AD), and this pathogen may trigger and aggravate AD lesions. The aim of this study was to determine the prevalence of S. aureus in the nares of pediatric subjects and verify the phenotypic and molecular characteristics of the isolates in pediatric patients with AD. Isolates were tested for antimicrobial susceptibility, SCCmectyping, and Panton-Valentine Leukocidin (PVL) genes. Lineages were determined by pulsed-field gel electrophoresis and multilocus sequence typing (MLST). AD severity was assessed with the Scoring Atopic Dermatitis (SCORAD) index. Among 106 patients, 90 (85%) presented S. aureus isolates in their nares, and 8 also presented the pathogen in their skin infections. Two patients had two positive lesions, making a total of 10 S. aureusisolates from skin infections. Methicillin-resistant S. aureus(MRSA) was detected in 24 (26.6%) patients, and PVL genes were identified in 21 (23.3%), including 6 (75%) of the 8 patients with skin lesions but mainly in patients with severe and moderate SCORAD values (P=0.0095). All 24 MRSA isolates were susceptible to trimethoprim/sulfamethoxazole, while 8 isolates had a minimum inhibitory concentration (MIC) to mupirocin >1024 μg/mL. High lineage diversity was found among the isolates including USA1100/ST30, USA400/ST1, USA800/ST5, ST83, ST188, ST718, ST1635, and ST2791. There was a high prevalence of MRSA and PVL genes among the isolates recovered in this study. PVL genes were found mostly among patients with severe and moderate SCORAD values. These findings can help clinicians improve the therapies and strategies for the management of pediatric patients with AD.

Nocardia asteroides peritonitis during continuous ambulatory peritoneal dialysis

This paper reports a case of peritonitis by Nocardia asteroides during continuous ambulatory peritoneal dialysis in a man who had systemic lupus erythematous and chronic renal failure. Diagnosis was established by microscopic examination (Gram and Kinyoun) and culture of centrifuged dialysis fluid and the patient was treated with Trimethoprin-Sulfamethoxazole by intraperitoneal route.

Fatal disseminated paracoccidioidomycosis in a two-year-old child

A two year-old female child was admitted at the Pediatric Intensive Care Unit in a septic shock associated with a lymphoproliferative syndrome, with history of fever, adynamia and weight loss during the last two months. On admission, the main clinical and laboratory manifestations were: pallor, jaundice, disseminated enlarged lymph nodes, hepatosplenomegaly, crusted warts on face, anemia, eosinophilia, thrombocytopenia, increased direct and indirect bilirubin, alkaline phosphatase, and gammaglutamyl transpeptidase. A parenteral administration of fluids, dobutamine and mechanical ventilation was started, without improvement of the clinical conditions. A direct examination of exsudate collected from cervical lymph node revealed numerous oval-to-around cells with multiple budding, like a "pilot wheel" cell, suggesting Paracoccidioides brasiliensis. Even though treatment with intravenous sulfamethoxazole-trimethoprine was soon started, the child died 36 hours after hospital admission. Disseminated paracoccidioidomycosis was confirmed in the autopsy. This is the youngest case of paracoccidioidomycosis in children reported in the literature.

Mycophenolate mofetil may protect against Pneumocystis carinii pneumonia in renal transplanted patients

Pneumocystis carinii pneumonia (PCP) is usually prevented in transplanted patients by prophylactic trimethoprim-sulfamethoxazol (TMS). Mycophenolate mofetil (MMF) has been shown to have a strong protective effect against PCP in rats. This effect is also suggested in humans by the absence of PCP in patients receiving MMF. After January 1998 MMF has been used with no TMS prophylaxis in renal transplanted patients. In azathioprine (AZA) treated patients TMS prophylaxis was maintained. The incidence of PCP was analyzed in both groups. Data were collected in order to have a minimum 6-month follow-up. Two hundred and seventy-two patients were eligible for analysis. No PCP occurred either in patients under MMF without TMS prophylaxis nor in patients under AZA. MMF may have an effective protective role against PCP as no patient under MMF, despite not receiving TMS coverage, developed PCP. A larger, controlled, trial is warranted to consolidate this information.

Clinical guideline for diagnosis and management of melioidosis

Melioidosis is an emerging infection in Brazil and neighbouring South American countries. The wide range of clinical presentations include severe community-acquired pneumonia, septicaemia, central nervous system infection and less severe soft tissue infection. Diagnosis depends heavily on the clinical microbiology laboratory for culture. Burkholderia pseudomallei, the bacterial cause of melioidosis, is easily cultured from blood, sputum and other clinical samples. However, B. pseudomallei can be difficult to identify reliably, and can be confused with closely related bacteria, some of which may be dismissed as insignificant culture contaminants. Serological tests can help to support a diagnosis of melioidosis, but by themselves do not provide a definitive diagnosis. The use of a laboratory discovery pathway can help reduce the risk of missing atypical B. pseudomallei isolates. Recommended antibiotic treatment for severe infection is either intravenous Ceftazidime or Meropenem for several weeks, followed by up to 20 weeks oral treatment with a combination of trimethoprim-sulphamethoxazole and doxycycline. Consistent use of diagnostic microbiology to confirm the diagnosis, and rigorous treatment of severe infection with the correct antibiotics in two stages; acute and eradication, will contribute to a reduction in mortality from melioidosis.

Nocardiosis: an overview and additional report of 28 cases in cattle and dogs

Phenotypic characteristics, antimicrobial susceptibility profile, and clinical-epidemiological features of 28 Nocardia strains isolated from 19 cases of bovine mastitis, eight cutaneous-subcutaneous lesions and one case of pneumonia in dogs were evaluated. Microbiological, biochemical, cytological and scanning electron microscopy methods were used in diagnosis. Nocardia asteroides type IV, Nocardia otitidiscaviarum,Nocardia nova (type III) and Nocardia farcinica (type V) were isolated from bovine milk, bronchial lavage and/or cutaneous-subcutaneous abscesses in dogs. Nocardial bovine mastitis was diagnosed predominantly in clinical cases, in dairy herds with poor environmental hygienic conditions between milking and inappropriate intramammary therapy. Canine nocardiosis was observed commonly in animals co-infected with distemper virus. Sulphamethoxazole-trimethoprim (92.8%), amikacin (92.8%) and ceftiofur (92.8%) were the most effective drugs in 28 isolates. Multiple drug resistance to three or more and five or more antimicrobials was observed in ten (35.7%) and three (10.7%) strains, respectively, predominantly with use of cloxaxillin, cefoperazone and ampicillin. The species (type) classification, clinical-epidemiological characteristics, diagnosis, multiple-drug resistance and public health considerations in Nocardia strains isolated from cattle and dogs in Brazil are discussed, with special reference to report of bovine mastitis by N. otitidiscaviarum by first time in Brazil and the similarity between Nocardia species isolated from human and animal origin.

Smqnr VARIANTS IN CLINICAL ISOLATES OF Stenotrophomonas maltophilia IN BRAZIL

SUMMARYStenotrophomonas maltophilia contains a novel chromosomally-encoded qnr gene named Smqnr that contributes to low intrinsic resistance to quinolone. We described Smqnr in 13 clinical isolates of S. maltophilia from two Brazilian hospitals, over a 2-year period. The strains were identified by API 20 NE (bioMérieux, France). Susceptibility by microdilution method to trimetroprim/sulfamethoxazole, ciprofloxacin, levofloxacin, minocycline, ceftazidime, chloramphenicol and ticarcillin/clavulanate was performed according to CLSI. PCR detection of Smqnr gene was carried out. The sequence of Smqnr was compared with those deposited in GenBank. Pulsed-field gel electrophoresis (PFGE) of all strains was performed. Thirteen Smqnr positives isolates were sequenced and three novel variants of Smqnr were identified. All 13 Smqnr isolates had distinguishable patterns by PFGE. This is the first report of Smqnr in S. maltophilia isolated in Brazil.

In vitro action of antiparasitic drugs, especially artesunate, against Toxoplasma gondii

INTRODUCTION: Toxoplasmosis is usually a benign infection, except in the event of ocular, central nervous system (CNS), or congenital disease and particularly when the patient is immunocompromised. Treatment consists of drugs that frequently cause adverse effects; thus, newer, more effective drugs are needed. In this study, the possible activity of artesunate, a drug successfully being used for the treatment of malaria, on Toxoplasma gondii growth in cell culture is evaluated and compared with the action of drugs that are already being used against this parasite. METHODS: LLC-MK2 cells were cultivated in RPMI medium, kept in disposable plastic bottles, and incubated at 36ºC with 5% CO2. Tachyzoites of the RH strain were used. The following drugs were tested: artesunate, cotrimoxazole, pentamidine, pyrimethamine, quinine, and trimethoprim. The effects of these drugs on tachyzoites and LLC-MK2 cells were analyzed using nonlinear regression analysis with Prism 3.0 software. RESULTS: Artesunate showed a mean tachyzoite inhibitory concentration (IC50) of 0.075µM and an LLC MK2 toxicity of 2.003µM. Pyrimethamine was effective at an IC50 of 0.482µM and a toxicity of 11.178µM. Trimethoprim alone was effective against the in vitro parasite. Cotrimoxazole also was effective against the parasite but at higher concentrations than those observed for artesunate and pyrimethamine. Pentamidine and quinine had no inhibitory effect over tachyzoites. CONCLUSIONS: Artesunate is proven in vitro to be a useful alternative for the treatment of toxoplasmosis, implying a subsequent in vivo effect and suggesting the mechanism of this drug against the parasite.

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase of isolates from the Amazon Region of Brazil

Since the late 1970s pyrimethamine-sulfadoxine (PS; FansidarTM Hoffman-LaRoche, Basel) has been used as first line therapy for uncomplicated malaria in the Amazon basin. Unfortunately, resistance has developed over the last ten years in many regions of the Amazon and PS is no longer recommended for use in Brazil. In vitro resistance to pyrimethamine and cycloguanil (the active metabolite of proguanil) is caused by specific point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR), and in vitro resistance to sulfadoxine has been associated with mutations in dihydropteroate synthase (DHPS). In association with a proguanil-sulfamethoxazole clinical trial in Brazil, we performed a nested mutation-specific polymerase chain reaction to measure the prevalence of DHFR mutations at codons 50, 51, 59, 108 and 164 and DHPS mutations at codons 436, 437, 540, 581 and 613 at three sites in the Brazilian Amazon. Samples from two isolated towns showed a high degree of homogeneity, with the DHFR Arg-50/Ile-51/Asn-108 and DHPS Gly-437/Glu-540/Gly-581 mutant genotype accounting for all infections in Peixoto de Azevedo (n = 15) and 60% of infections in Apiacás (n = 10), State of Mato Grosso. The remaining infections in Apiacás differed from this predominant genotype only by the addition of the Bolivia repeat at codon 30 and the Leu-164 mutation in DHFR. By contrast, 17 samples from Porto Velho, capital city of the State of Rondônia, with much in- and out-migration, showed a wide variety of DHFR and DHPS genotypes.