Comparison of the Salmonella/Microsome Microsuspension Assay with the new Microplate Fluctuation Protocol for Testing the Mutagenicity of Environmental Samples

The objective of this study was to compare the responses of the Salmonella/microsome microsuspension assay with the new microplate fluctuation protocol (MPF) for the evaluation of the mutagenic activity of environmental samples. Organic extracts of total particulate atmospheric air samples, surface waters, and effluents were tested in dose-response experiments. The assays were performed with strain TA98 in the absence and presence of S9 mix. Both protocols produced similar results, despite the fact that the maximum score of the MPF is limited to 48 wells, whereas in the regular plate assay it is possible to count up to 1,500 colonies using an automatic counter. Similar sensitivities based on the lowest dose that resulted in a positive response were obtained for both assays. The MPF procedure is less laborious (e.g., all-liquid format, use of multi-channel pipettors) and allows for automation of the pipetting and dispensing steps, thus, reducing time of the analysis which is particularly important in environmental quality monitoring programs or in effect-directed analysis. The results show that the MPF procedure is a promising tool to test environmental samples for mutagenic activity. Environ. Mol. Mutagen. 51:31-38, 2010. (C) 2009 Wiley-Liss, Inc.

OCCURRENCE AND ANTIBIOTIC SENSIBILITY PROFILE OF Salmonella spp. ISOLATED FROM PORK MEAT CUTS COMMERCIALIZED IN THE OPEN MARKETS IN PELOTAS, RS (BRAZIL)

The objective of the present study was to evaluate the occurrence of Salmonella spp. in 15 samples of pork meat cuts (T-bone, shank, sausage and ribs) commercialized in open markets of Pelotas (RS, Brazil) and verify the prevalent serovars, and test the isolates profile of sensitivity to several antibiotics of importance in medicine (nalidixic acid, ampicillin, aztreonam, kanamycin, carbenicillin, cephalothin, cefoxitin, ceftriaxone, ciprofloxacin, chloramphenicol, gentamicin, sulfonamide, tetracycline and trimetoprina). Twelve samples (80%) were contaminated by Salmonella enterica, serovars Infantis, Derby, Panama and Typhimurium. All isolates were susceptible to trimetoprin, aztreonam, ciprofloxacin, ceftriaxone and cefoxitin. For the other antibiotics, the pattern of sensitivity varied as serovar. In addition, 39.1% of isolates showed up to be multiresistant.

Cloning, expression and purification of a glycosylated form of the DNA-binding protein Dps from Salmonella enterica Typhimurium

Dps, found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress and/or nutrient-limited environment. Dps has been shown to accumulate during the stationary phase, to bind to DNA non-specifically, and to form a crystalline structure that compacts and protects the chromosome. Our previous results have indicated that Dps is glycosylated at least for a certain period of the bacterial cell physiology and this glycosylation is thought to be orchestrated by some factors not yet understood, explaining our difficulties in standardizing the Dps purification process. In the present work, the open reading frame of the dps gene, together with all the upstream regulatory elements, were cloned into a PCR cloning vector. As a result, the expression of dps was also controlled by the plasmid system introduced in the bacterial cell. The gene was then over-expressed regardless of the growth phase of the culture and a glycosylated fraction was purified to homogeneity by lectin-immobilized chromatography assay. Unlike the high level expression of Dps in Salmonella cells, less than 1% of the recombinant protein was purified by affinity chromatography using jacalin column. Sequencing and mass spectrometry data confirmed the identity of the dps gene and the protein, respectively. In spite of the low level of purification of the jacalin-binding Dps, this work shall aid further investigations into the mechanism of Dps glycosylation. (C) 2008 Elsevier Inc. All rights reserved.

Salmonella antibiotic-mutant strains reduce fecal shedding and organ invasion in broiler chicks

We investigated the exposure to antibiotics in the production of antibiotic-mutant strains of Salmonella. Ten isolates of poultry origin were assayed for antibiotic susceptibilities. One strain of Salmonella Enteritidis, one of Salmonella Heidelberg, and one of Salmonella Typhimurium were selected to induce antimicrobial resistance. Each strain was exposed to high concentrations of streptomycin, rifampicin, and nalidixic acid, respectively. Parent and antibiotic-mutant strains were assayed for antibiotic susceptibilities using a commercial microdilution test and the disk susceptibility test. The strains were assessed for virulence genes and evaluated for fecal shedding, cecal colonization, organ invasion, and mean Salmonella counts after inoculation in 1-day-old chicks. The study revealed that exposure to high concentrations of streptomycin produced the antibiotic-mutant strain SE/LABOR/USP/08 and the exposure to rifampicin produced the antibiotic-mutant SH/LABOR/USP/08. These strains showed significantly reduced fecal shedding (P = 0.05) and organ invasion, persisting less than the parental strains and showing no clinical signs in inoculated chicks. High concentrations of nalidixic acid produced the antibiotic-mutant strain ST/LABOR/USP/08, which did not show any differences compared with the parent strain. Likewise, SE/LABOR/USP/08 did not show the expression of plasmid-encoded fimbriae (pefA) and plasmid virulence protein (spvC), suggesting that after exposure to streptomycin, the parent isolate lost the original gene expression, reducing fecal shedding and organ invasion in inoculated chicks.

Prevention of Salmonella Typhimurium colonization and organ invasion by combination treatment in broiler chicks

The effects in broiler chicks of treatment with a competitive exclusion (CE) product, an experimental dietary probiotic, and the abiotic beta-glucan on cecal colonization, organ invasion, and serum and intestinal IgG and IgA levels to Salmonella challenge was evaluated. Four groups of 1-d-old chicks were treated by oral gavage on d 1 with an appropriate dose of a commercial CE product. Three groups received daily doses of probiotic, beta-glucan, or both, for 6 d. Three other groups were fed daily from d 1 onwards with probiotic, beta-glucan, or both. Subgroups of 30 chicks from each group were challenged on d 1, 9, 16, or 23 with 10(7) cfu/mL of Salmonella Typhimurium (1769NR) and killed 7 d later. Control groups were maintained untreated and remained unchallenged (negative control), or were challenged with Salmonella Typhimurium (1769NR; positive control), as described above. Cecum, liver, and spleen samples were examined for the presence of Salmonella, whereas serum and intestinal fluid samples were assayed for total antibody (IgG and IgA) concentrations. Data were analyzed by 1-way ANOVA, and means were compared using Duncan`s multiple range test. In comparison with other treatments, those involving CE product and beta-glucan, with or without probiotic during the first week, resulted in a superior inhibition of cecal colonization and organ invasion by Salmonella and also offered a higher level of protection (P < 0.05). During the second week, treatments containing experimental dietary probiotic and beta-glucan, with or without CE product, resulted in an inhibition of liver invasion (P < 0.05). The IgA levels were significantly higher (P < 0.05) in intestinal fluid compared with serum, whereas IgG had low levels. The results in the first and third week indicate that combination treatments involving CE product, probiotic, and beta-glucan are a more effective control of Salmonella colonization than the corresponding individual preparations.

Metabolic activation of polycyclic aromatic hydrocarbons and other procarcinogens by cytochromes P450 1A1 and P4501B1 allelic variants and other human cytochromes P450 in Salmonella typhimurium NM2009

A variety of polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, arylamines, heterocyclic amines, and nitroarenes, were incubated with cDNA-based recombinant (Escherichia coli or Trichoplusia ni) systems expressing different forms of human cytochrome P450 (P450 or CYP) and NADPH-P450 reductase using Salmonella typhimurium, tester strain NM2009, and the resultant DNA damage caused by the reactive metabolites was detected by measuring expression of umu gene in the cells. Recombinant (bacterial) CYP1A1 was slightly more active than any of four CYP1B1 allelic variants, CYP1B1*1, CYP1B1*2, CYP1B1*3, and CYP1B1*6, in catalyzing activation of chrysene-1,2-diol, benz[a]anthracene-trans-1,2-, 3,4-, 5,6-, and 8,9-diol, fluoranthene-2,3-diol, dibenzo[a]pyrene, benzo[c]phenanthrene, and dibenz[a,h]anthracene and several arylamines and heterocyclic amines, whereas CYP1A1 and CYP1B1 enzymes had essentially similar catalytic specificities toward other procarcinogens, such as (+)-, (-)-, and (+/-)-benzo[a]pyrene-7,8-diol, 5-methylchrysene-1,2-diol, 7,12-dimethylbenz[a]anthracene-3,4-diol, dibenzo[a,l]pyrene-11,12-diol, benzo[b]fluoranthene-9,10-diol, benzo[c]chrysene, 5,6-dimethylchrysene-1,2-diol, benzo[c]phenanthrene-3,4-diol, 7,12-dimethylbenz[a]anthracene, benzo[a]pyrene, 5-methylchrysene, and benz[a]anthracene. We also determined activation of these procarcinogens by recombinant (T. ni) human P450 enzymes in S. typhimurium NM2009. There were good correlations between activities of procarcinogen activation by CYP1A1 preparations expressed in E. coli and T. ni cells, although basal activities with three lots of CYP1B1 in T. ni cells were very high without substrates and NADPH in our assay system. Using 14 forms of human P450S (but not CYP1B1) (in T. ni cells), we found that CY1P1A2, 2C9, 3A4, and 2C19 catalyzed activation of several of polycyclic aromatic hydrocarbons at much slower rates than those catalyzed by CYP1A1 and that other enzymes, including CYP2A6, 2B6, 2C8, 2C18, 2D6, 2E1, 3A5, 3A7, and 4A11, were almost inactive in the activation of polycyclic aromatic hydrocarbons examined here.

Metabolic activation of heterocyclic amines and other procarcinogens in Salmonella typhimurium umu tester strains expressing human cytochrome P4501A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4 and human NADPH-P450 reductase and bacterial O-acetyltransferase

We investigated roles of different forms of cytochrome P450 (P450 or CYP) in the metabolic activation of heterocyclic amines (HCAs) and other procarcinogens to genotoxic metabolite(s) in the newly developed umu tester strains Salmonella typhimurium (S. typhimurium) OY1002/1A1, OY1002/1A2, OY1002/1B1, OY1002/2C9, OY1002/2D6, OY1002/2E1 and OY 1002/3A4. which express respective human P450 enzymes and NADPH-cytochrome P350 reductase (reductase) and bacterial O-acetyltransferase (O-AT). These strains were established by introducing two plasmids into S. typhimurium TA 1535, one carrying both P450 and the reductase cDNA in a bicistronic construct under control of an IPTG-inducible double me promoter and the other, pOA 102, carrying O-AT and umuClacZ fusion genes. Expression levels of CYP were found to range between 35 to 550 nmol/l cell culture in the strains tested. O-AT activities in different strains ranged from 52 to 135 nmol isoniazid acetylated/min/mg protein. All HCAs tested, and 2-aminoanthracene and 2-aminofluorene exhibited high genotoxicity in the OY1002/1A2 strain, and genotoxicity of 2-amino-3-methylimidazo [4,5-f]quinoline was detected in both the OY1002/1A1 and OY1002/1A2 strains. 1-Amino-1,4-dimethyl-5H-pyrido[4.3-b]-indole and 3-amino-1-methyl-5H-pyrido[4,3-b]-indole were activated in the OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4 strains. Aflatoxin B-1 exhibited genotoxicity in the OY1002/1A2, OY1002/1A1, and OY1002/3A4 strains. beta -Naphthylamine and benzo[a]pyrene did not exhibit genotoxicity in any of the strains. These results suggest that CYP1A2 is the major cytochrom P450 enzyme involved in bioactivation of HCAs. (C) 2001 Elsevier Science B.V. All rights reserved.

Eficácia e segurança de Sultamicilina (Ampicilina/Sulbactam) e Amoxacilina/Clavulanato no tratamento das infecções de via aéreas superiores em adultos: um estudo multicêntrico, aberto e randomizado

As IVAS em crianças e adultos são os motivos mais freqüentes de consulta médica e os que mais demandam o uso de antibióticos. A crescente resistência bacteriana causada pela produção das beta-lactamases constitui um dos mais sérios problemas atuais. A Sultamicilina é uma pró-droga dupla da ampicilina e do sulbactam, um potente inibidor de beta-lactamases que pode fazer frente a estas dificuldades. OBJETIVO: avaliar a eficácia, segurança e tolerabilidade da Ampicilina/Sulbactan comparada à Amoxacilina/Ácido Clavulânico no tratamento de IVAS, em adultos. METODOLOGIA: 102 pacientes com diagnóstico de IVAS foram randomizados em dois grupos recebendo Ampicilina/Sulbactan ou Amoxacilina/Clavulanato por 10 dias. Foram avaliados 10 e 30 dias após para análise da resposta terapêutica. RESULTADOS: Não houve diferença entre os grupos com relação à proporção de pacientes curados ao final do tratamento (visita 2) ou do estudo (visita 3). No grupo que recebeu Amoxacilina/Clavulanato, as proporções de cura foram de 61.7% e 93.2% nas visitas 2 e 3, comparadas a 64.4% e 97.4%, respectivamente, no grupo que recebeu Ampicilina/Sulbactan. A proporção de pacientes que experimentou pelo menos um evento adverso foi semelhante nos dois grupos (p = 0.940). A diarréia foi significativamente mais freqüente no grupo Amoxacilina-Clavulanato (70.6%) do que no grupo Ampicilina/Sulbactan (29.4%), (p=0.0164). CONCLUSÕES: A Ampicilina/Sulbactan é tão segura e eficaz quanto a Amoxacilina/Clavulanato no tratamento empírico de IVAS em adultos. A ocorrência significativamente menor de quadros de diarréia no grupo recebendo Ampicilina/Sulbactan necessita confirmação em estudos posteriores.

Infecções cervicais profundas: análise de 80 casos

As infecções cervicais profundas são afecções graves que acometem os diversos espaços do pescoço. A mais temível complicação é a mediastinite necrosante descendente, que necessita de diagnóstico precoce e tratamento, por vezes, agressivo. OBJETIVOS: Analisar os 80 casos tratados de infecção cervical profunda e propor uma diretriz de conduta. MATERIAL E MÉTODO: Os autores realizaram um estudo retrospectivo de 80 casos de infecções cervicais profundas tratados no período de junho de 1997 a junho de 2003. RESULTADOS: As causas odontogênicas e amigdalianas foram as mais comumente encontradas. Os espaços mais acometidos foram o submandibular e parafaríngeo. Os principais microorganismos envolvidos foram o Staphylococcus aureus e o Streptococcus sp. CONCLUSÃO: O manejo da via aérea difícil nas infecções cervicais profundas deve ter cuidado especial, quando da indicação cirúrgica, de preferência com intubação sob visão endoscópica e sem uso de miorrelaxantes. Drenagem cirúrgica ampla permanece o tratamento padrão das infecções cervicais profundas. A tomografia computadorizada é o exame de escolha para o diagnóstico das infecções cervicais profundas. Essas infecções apresentam alta morbimortalidade, quando associada ao choque séptico e à mediastinite. Nossa mortalidade foi de 11,2% e dos cinco doentes com mediastinite apenas um sobreviveu.

Otimizando o diagnóstico para o tratamento adequado das principais infecções agudas em otorrinopediatria: tonsilite, sinusite e otite média

As sinusites, otites médias agudas e tonsilites são muito freqüentes em crianças. A maioria dessas infecções é causada por vírus, mas em geral, elas são tratadas com antibióticos. O uso inapropriado de antibióticos favorece a seleção, crescimento e disseminação de bactérias resistentes que colonizam as vias respiratórias, atingindo toda a comunidade. A emergência de bactérias resistentes dificulta os tratamentos das infecções respiratórias, sendo essencial desenvolver estratégias efetivas para restringir o uso de antibióticos sem prejudicar as crianças que realmente precisam desses medicamentos. OBJETIVO: Analisar os resultados de estudos randomizados e controlados sobre critérios clínicos e laboratoriais utilizados para diagnóstico e tratamento das tonsilites, sinusites e otites. MÉTODOS: Levantamento dos estudos randomizados e controlados sobre o tema, publicados no MEDLINE e SCIELO, de 2000 a 2006. CONCLUSÕES: Como a maioria dessas infecções evolui bem sem antibióticos, deve-se evitar o uso desses medicamentos a menos que a criança pertença aos grupos de alto risco para complicações ou apresente persistência ou piora dos sintomas com tratamento sintomático. É necessário que os médicos e leigos conheçam melhor a evolução natural das infecções respiratórias agudas e que seja garantido o acesso das crianças a serviços médicos de boa qualidade para orientação e reavaliação, quando necessária.

Infecções naturais de mamíferos silvestres em área endêmica de leishmaniose tegumentar do Estado de São Paulo, Brasil

Descreve-se a presença de foco de leishmaniose tegumentar no vale do rio Moji-Guaçú, em região comum aos municípios de Luiz Antonio, São Carlos, Rincão e Santa Rita do Passa Quatro, no Estado de São Paulo, Brasil. Os casos humanos apresentavam formas clínicas caracterizadas por lesões úlcero-vegetantes, de evolução lenta e pobres em parasitos. As investigações sobre infecção natural em animais silvestres levou ao isolamento de roedores, de três cepas em cultura, duas procedentes de Akodon arviculoides e uma de Oryzomys nigripes. As provas de inoculação em hamsters foram, até o momento, positivas para duas delas, mas com evolução lenta, com manifestações clínicas muito discretas e pobres em parasitos. Pelos dados disponíveis até o momento, parece tratar-se de cepas filiáveis à raça "lenta", à qual se atribui papel na etiologia da forma cutaneo-mucosa da leishmaniose.

Estudo sorológico de infecções ocasionadas por citomegalovírus

Estudou-se a distribuição etária e por sexo de anticorpos fixadores do complemento para citomegalovírus em 1294 soros de indivíduos do município de São Paulo. A uma porcentagem de positividade de 33%, nos primeiros quinze anos de vida, seguiu-se, após uma queda inicial no nível de anticorpos, um aumento progressivo até níveis de ordem de 70%. Não se observaram diferenças de positividade significativas em relação ao sexo. A comparação dos resultados obtidos com a reação de fixação do complemento e a reação de neutralização, numa amostra de 236 soros de indivíduos adultos, usando-se a cepa de citomegalovírus AD 169, evidenciou uma boa correlação.

Comportamento do Staphilococcus aureus causadores de infecções hospitalares, frente a atividade "in vitro" da fosfomicina

Estudou-se a atividade "in vitro" da fosfomicina em 337 amostras de Staphylococcus aureus coletados de infecções intra-hospitalares, obtendo-se 310 amostras sensíveis (91,9%). Comparando-se com outros antimicrobianos, concluiu-se que a atividade da fosfomicina foi superior a todos.

Meio modificado de cultura para caracterização de Salmonella lactose positiva

Foi desenvolvido um meio modificado de cultura para isolamento e caracterização de enterobactérias, visando especialmente salmonelas fermentadoras da lactose. No chamado "Meio modificado" as colônias das duas estirpes de Salmonella (lactose positivas e lactose negativas) apresentam a morfologia idêntica, o que não ocorre quando são empregados os meios rotineiros à base de lactose, para isolamento de enterobactérias. Esse meio é uma modificação do meio de Hektoen Enteric Agar, do qual retirou-se lactose e adicionou-se xilose e L-lisina. Foi verificado que há possibilidade de diferenciar-se os diversos grupos de enterobactérias, empregando um meio de cultura sem lactose e usando como sistema diferenciador xilose e L-lisina. O meio modificado foi também avaliado quantitativamente comparando o seu poder enriquecedor ou inibitório, ao dos meios de Hektoen Enteric Agar, Brilliant Green Agar e SS Agar para diferentes grupos de enterobactérias.