Characterization of CMY-2-type beta-lactamase-producing Escherichia coli isolated from chicken carcasses and human infection in a city of South Brazil.

BACKGROUND: Food-producing animals, mainly poultry, have been associated with the maintenance and dissemination of antibiotic-resistant bacteria, such as plasmid-mediated AmpC (pAmpC)-producing Enterobacteriaceae, to humans, thus impacting food safety. Many studies have shown that Escherichia coli strains isolated from poultry and humans infections share identical cephalosporin resistance, suggesting that transmission of resistance from poultry meat to humans may occur. The aim of this study was to characterize pAmpC-producing E. coli strains isolated from chicken carcasses and human infection in a restrict area and to determine their antimicrobial resistance profiles, and molecular type by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). RESULTS: A total of 14 pAmpC-producing E. coli strains were isolated, including eight strains from chicken carcasses and six strains from human infections (from urine, tissue and secretion). The blaCMY-2 gene was identified in all pAmpC-producing E. coli strains by polymerase chain reaction (PCR) and DNA sequencing. High percentages of strains resistant to tetracycline, nalidixic acid and sulfamethoxazole-trimethoprim (78-92%) were detected, all of which were considered multidrug-resistant. Among the non-beta-lactam resistance genes, the majority of the strains showed tetA, tetB, sulI and sulII. No strain was considered an extended-spectrum beta-lactamases (ESBL) producer, and the blaTEM-1 gene was found in 2 strains isolated from human infection. Six strains from chicken carcasses and four strains from humans infections were linked to an ISEcp1-like element. Through MLST, 11 sequence types were found. Three strains isolated from human infection and one strain isolated from chicken carcasses belonged to the same sequence type (ST354). However, considerable heterogeneity between the strains from chicken carcasses and humans was confirmed by PFGE analysis. CONCLUSION: This study showed the prevalence of E. coli strains producing blaCMY-2 linked to ISEcp1 that were present in both chickens and humans in a restricted area. Our results also suggest the presence of a highly diverse strains that harbor pAmpC, indicating no clonal dissemination. Therefore, continuous monitoring and comparative analyses of resistant bacteria from humans and food-producing animals are needed.

Genetic analysis of enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid reveals a new deletion within the EAF probe sequence among O119 typical EPEC strains.

BACKGROUND: Enteropathogenic Escherichia coli (EPEC) are classified into typical and atypical strains based on the presence of the E. coli adherence factor (EAF) plasmid. The EAF plasmid contains the bfp (bundle-forming pilus) operon and the perABC (plasmid encoded regulator) gene cluster. A 1-kb cryptic region of EAF plasmid has been widely used as a genetic probe for EPEC detection. However, some EPEC strains may harbor an EAF plasmid lacking the EAF probe sequence, which makes the differentiation between typical and atypical a complex task. In this study, we report the genetic analysis of the EAF plasmid-encoded genes in a collection of EPEC clinical isolates. METHODS: A total of 222 EPEC clinical isolates, which were previously classified as typical (n=70) or atypical (n=152) by EAF probe reactivity, were screened for the presence of different EAF sequences by PCR and DNA hybridization. RESULTS: All typical strains possessed intact bfpA and perA genes, and most of them were positive in the PCR for EAF probe sequence. However, a subset of 30 typical strains, 22 of which belonged to O119 serogroup, presented a 1652 pb deletion in the region between 1093-bp downstream perC and 616-bp of the EAF fragment. The bfpA, bfpG, and per genes were found in all typical strains. In addition, 32 (21%) atypical strains presented the perA gene, and 20 (13.2%) also presented the bfpA gene. Among the 32 strains, 16 belonged to the O119:H2, O119:HND, and ONT:HND serotypes. All 32 atypical strains contained perA mutation frameshifts and possessed an IS1294 element upstream of the per operon as detected by PCR followed by restriction fragment length polymorphism (RFLP) typing and multiplex PCR. Among the 20 bfpA probe-positive strains, eight O119 strains possessed deletion in the bfp operon at the 3'end of bfpA due to an IS66 element. CONCLUSION: Our data show that typical O119 strains may contain a deletion within the EAF probe sequence not previously reported. This new finding suggests that care should be taken when using the previously described EAF PCR assay in epidemiological studies for the detection of typical O119 strains. In addition, we were able to confirm that some atypical strains carry vestiges of the EAF plasmid.

Antibacterial activity of extracellular compounds produced by a Pseudomonas strain against methicillin-resistant Staphylococcus aureus (MRSA) strains.

BACKGROUND: The emergence of multidrug-resistant bacteria is a world health problem. Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA) strains, is one of the most important human pathogens associated with hospital and community-acquired infections. The aim of this work was to evaluate the antibacterial activity of a Pseudomonas aeruginosa-derived compound against MRSA strains. METHODS: Thirty clinical MRSA strains were isolated, and three standard MRSA strains were evaluated. The extracellular compounds were purified by vacuum liquid chromatography. Evaluation of antibacterial activity was performed by agar diffusion technique, determination of the minimal inhibitory concentration, curve of growth and viability and scanning electron microscopy. Interaction of an extracellular compound with silver nanoparticle was studied to evaluate antibacterial effect. RESULTS: The F3 (ethyl acetate) and F3d (dichloromethane- ethyl acetate) fractions demonstrated antibacterial activity against the MRSA strains. Phenazine-1-carboxamide was identified and purified from the F3d fraction and demonstrated slight antibacterial activity against MRSA, and synergic effect when combined with silver nanoparticles produced by Fusarium oxysporum. Organohalogen compound was purified from this fraction showing high antibacterial effect. Using scanning electron microscopy, we show that the F3d fraction caused morphological changes to the cell wall of the MRSA strains. CONCLUSIONS: These results suggest that P. aeruginosa-produced compounds such as phenazines have inhibitory effects against MRSA and may be a good alternative treatment to control infections caused by MRSA.

Impact of electroacupuncture on quality of life for patients with Relapsing-Remitting Multiple Sclerosis under treatment with immunomodulators: a randomized study.

BACKGROUND: Multiple sclerosis (MS) is a complex autoimmune disease mediated by an immune response to central nervous system antigens. Modern immunomodulatory therapies, however, do not ameliorate many of the symptoms, such as pain and depression. Patients thus seek alternative treatments, such as acupuncture, although the benefits of such treatments have not been objectively evaluated. The present study was thus designed to evaluate the effect of the use of acupuncture in the alleviation of the symptoms of patients with MS. METHODS: Thirty-one patients with Relapsing-Remitting Multiple Sclerosis undergoing treatment with immunomodulators were randomly distributed into sex-stratified experimental and placebo groups in a patient- and evaluator-blind design; they received either true or sham electroacupuncture during regular visits to the doctor in the university hospital outpatient clinic. Standardized questionnaires were used to evaluate the effect of electroacupuncture on the quality of life of these patients. Initial and follow-up assessment included the evaluation of clinical status (Expanded Disability Status Scale), pain (Visual Analogue Scale) and quality of life (Functional Assessment of multiple Sclerosis) to ascertain the impact of electroacupuncture on the quality of life of these patients. RESULTS: Electroacupuncture improved various aspects of quality of life, including a reduction in pain and depression. The self-report scales were more sensitive to improvement than was the more objective clinical measure. CONCLUSION: This paper provides evidence that electroacupuncture can significantly improve the quality of life of such patients. The results suggest that the routine use of a self-report scale evaluating quality of life should be included in regular clinical evaluations in order to detect changes more rapidly. TRIAL REGISTRATION: RBR-58yq52.

Shigella sonnei genome sequencing and phylogenetic analysis indicate recent global dissemination from Europe.

Shigella are human-adapted Escherichia coli that have gained the ability to invade the human gut mucosa and cause dysentery(1,2), spreading efficiently via low-dose fecal-oral transmission(3,4). Historically, S. sonnei has been predominantly responsible for dysentery in developed countries but is now emerging as a problem in the developing world, seeming to replace the more diverse Shigella flexneri in areas undergoing economic development and improvements in water quality(4-6). Classical approaches have shown that S. sonnei is genetically conserved and clonal(7). We report here whole-genome sequencing of 132 globally distributed isolates. Our phylogenetic analysis shows that the current S. sonnei population descends from a common ancestor that existed less than 500 years ago and that diversified into several distinct lineages with unique characteristics. Our analysis suggests that the majority of this diversification occurred in Europe and was followed by more recent establishment of local pathogen populations on other continents, predominantly due to the pandemic spread of a single, rapidly evolving, multidrug-resistant lineage.

Molecular typing and biological characteristics of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Brazil.

The present study had as objective to evaluate the genotypic diversity and biological characteristics, such as hemolysin, protease, elastase of 56 clinical strains of Pseudomonas aeruginosa isolated from 13 cystic fibrosis (CF) patients attending at the School Hospital of Campinas State University (UNICAMP), Brazil. Genotypic diversity has been determined by Ribotyping (RT) and the pattern of the enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) of each strain. The production of elastase was significantly different only among mucoid and nonmucoid isolates. Joint results obtained by (RT) and ERIC-PCR methods were able to discriminate all strains isolated from both the same and different patients. Additionally, we observed four strain clusters with low diversity. The most infective strains were located in just two clusters. These results suggest that either there is a strong selection towards a specific genotype or that specific isolates could be responsible for the initial and subsequent colonization processes. More studies are necessary to know if these conclusions can be generalized for the general CF population.

Research of Salmonella spp. and evaluation of pathogenicity, cytotoxicity of Escherichia coli isolates proceeding from sparrows (Passer domesticus) / Pesquisa de Salmonella spp. e avaliação da patogenicidade, citotoxidade de isolados de Escherichia coli procedentes de pardais (Passer domesticus)

The aim of this study was to research the occurrence of Salmonella spp. and Escherichia coli in feces samples of sparrows, as well as to identify the pathogenicity, cytotoxicity and sensitivity profile of the isolates to antimicrobial use. Two hundred and twenty eight sparrows were captured in eight farms. The in vitro pathogenicity test was performed by the isolates culture on congo red-magnesium oxalate Agar, whilst the in vivo pathogenicity test was performed in one day-old chicks. In order to study the cytotoxic effects of indicators, samples were inoculated into Vero cells. The results obtained for Escherichia coli isolation confirmed the presence of this microorganism in 30 (13.2%) of the evaluated samples. Out of those isolates, 10 (33.3%) presented the capacity of absorbing ongo red. As for in vivo pathogenicity a 68.0% of mortality rate of the evaluated samples was observed. Out of 20 isolates tested for cytotoxin production, none of them presented cytotoxic effect in the Vero cells. The Salmonella spp was isolated only in one sample (0.04%), and it was identified as Salmonella enterica subspecies houtenae. Results obtained through this research indicate the need for new studies to identify other virulence factors of E. coli samples and to delineate the phylogenetic profile of the isolates in order to establish a relation with colibacillosis outbreaks in chickens and broilers in the studied region, as well as to analyze the critical points in the aviculture productive chain to identify the source of Salmonella enterica subspecies houtenae.

Objetivou-se com este estudo pesquisar a ocorrência de Salmonella spp. e Escherichia coli em amostras de fezes de pardais, além de avaliar a patogenicidade, citotoxicidade e perfil de sensibilidade dos isolados frente a antimicrobianos. Foram capturados 228 pardais em oito granjas. O teste de patogenicidade in vitro foi realizado por meio do cultivo dos isolados em ágar oxalato de magnésio acrescido de vermelho de congo, enquanto o teste de patogenicidade in vivo foi realizado em pintos de um dia. Para o estudo dos indicadores dos efeitos citotóxicos, as amostras foram inoculadas em células Vero. Os resultados obtidos quanto ao isolamento de Escherichia coli confirmaram a presença deste microorganismo em 30 (13,2%) amostras analisadas. Destes isolados, dez (33,3%) apresentaram capacidade de absorção do vermelho congo. Quanto à patogenicidade in vivo observou-se uma taxa de mortalidade de 68,0% das amostras analisadas. Dos 20 isolados testados quanto à produção de citotoxina, nenhum apresentou efeito citotóxico nas células Vero. Obteve-se o isolamento de Salmonella spp. em apenas uma amostra (0,04%), sendo tipificada em Salmonella enterica subespécie houtenae. Os resultados obtidos nesta pesquisa indicam a necessidade da realização de novos estudos para identificar outros fatores de virulência das amostras de E. coli e traçar o perfil filogenético dos isolados para estabelecer uma relação com surtos de colibacilose em galinhas e frango de corte na região estudada, além de analisar os pontos críticos na cadeia produtiva da avicultura para identificar a origem da Salmonella enterica subespécie houtenae.

Resistência antimicrobiana e perfil plasmidial de Escherichia coli isolada de frangos de corte e poedeiras comerciais no Estado de Pernambuco / Antimicrobial resistance and plasmidial profile of Escherichia coli strain isolated from broilers and commercial layers in the state of Pernambuco, Brazil

Embora existam linhagens de Escherichia coli não patogênicas para aves, muitas outras possuem a capacidade de causar sérios danos à saúde das mesmas, sendo capazes de ocasionar diferentes tipos de processos infecciosos. As linhagens patogênicas são denominadas Avian Pathogenic Escherichia coli (APEC), possuindo genes relacionados ao processo de patogênese em epissomos (plasmídios) ou no cromossomo. A presença de plasmídios, contendo genes de resistência a antibióticos em linhagens aviárias, patogênicas ou não, indicam a possibilidade de transferência gênica lateral entre diferentes tipos de linhagens facilitando também a transferência de genes de patogenicidade ou virulência. Objetivou-se com este estudo avaliar o perfil de sensibilidade a antibióticos (13) de diferentes amostras (35) de E. coli isoladas de aves comerciais do Estado de Pernambuco apresentando, ou não, sinais clínicos de processos infecciosos e correlacionar esta resistência com a presença de plasmídios. Os testes utilizados demonstraram que 94,28% dos isolados foram resistentes a três ou mais antibióticos, com a lincomicina apresentando o maior percentual de resistência (100%). Na Concentração Inibitória Mínima (CIM) observou-se multirresistência a vários antimicrobianos. A presença de plasmídios foi detecada em 80,0% (28/35) dos isolados, com 16 isolados apresentando plasmídios com peso molecular aproximado de 88 MDa. Também foi verificada a presença de linhagens apresentando plasmídios de vários tamanhos. Concluiu-se que isolados de E. coli resistentes a antimicrobianos utilizados na avicultura estão presentes no Estado de Pernambuco, tanto em frangos de corte quanto em poedeiras comerciais. A presença de plasmídios detectados na maioria dos isolados pode estar associada à resistência aos antimicrobianos e sugere a presença de possíveis genes relacionados à patogenicidade. Monitorar a resistência a antibióticos em bactérias isoladas de animais torna-se um fator determinante para eleição e êxito do tratamento, bem como a possibilidade de eliminação daquelas que possuem plasmídios para se evitar a transferência de genes relacionados à patogenicidade.

Although exist poultry non-pathogenic Escherichia coli strains, many others have capacity to impose serious damages to this birds, being able to cause different infectious diseases. Pathogenic strains are termed Avian pathogenic Escherichia coli (APEC) strains. APEC strains harbor chromossomal and plasmid pathogenicity-related genes. The presence of resistance plasmids in avian E. coli strains could facilitate horizontal tranfer of virulence gene between pathogenic and non pathogenic strains. The aim of this paper was to determine the resistance level to 13 different antibacterial drugs of avian E. coli strains (35) isolated from commercial poultry of Pernambuco State, Brazil, and to correlate the detected resistance level to the presence of plasmids. The results show that 94.28% of strains were resistant to at least three different antibacterial drugs with the highest percentage to lincomycin. The Minimal Inibitory Concentration (MIC) showed that multi- resistance to various antibacterial drugs was present in these strains. Plasmids of several sizes, including plasmids of approximately 88Mda were detected in most of the studied strains. The results herein obtained suggest that the high resistance level observed could be due to the presence of plasmids, what could facilitate the transfer of pathogenicity related genes among pathogenic and non pathogenic strains; it is necessary to take a constant survey on the resistance level to antimicrobial drugs of avian E. coli strains to reach a better control of APEC strains and avoid transfer of pathogenicity related genes between strains.

Molecular typing and biological characteristics of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Brazil

The present study had as objective to evaluate the genotypic diversity and biological characteristics, such as hemolysin, protease, elastase of 56 clinical strains of Pseudomonas aeruginosa isolated from 13 cystic fibrosis (CF) patients attending at the School Hospital of Campinas State University (UNICAMP), Brazil. Genotypic diversity has been determined by Ribotyping (RT) and the pattern of the enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) of each strain. The production of elastase was significantly different only among mucoid and nonmucoid isolates. Joint results obtained by (RT) and ERIC-PCR methods were able to discriminate all strains isolated from both the same and different patients. Additionally, we observed four strain clusters with low diversity. The most infective strains were located in just two clusters. These results suggest that either there is a strong selection towards a specific genotype or that specific isolates could be responsible for the initial and subsequent colonization processes. More studies are necessary to know if these conclusions can be generalized for the general CF population.

Typing of avian pathogenic Escherichia coli strains by REP-PCR

A técnica de REP (Repetitive extragenic palindrome)-PCR foi utilizada para avaliar a variabilidade genética de 49 amostras de Escherichia coli patogênicas para aves (APEC), isoladas de aves de corte (frangos) em diferentes surtos de septicemia (n=24), síndrome da cabeça inchada (n=14) e onfalite (n=11). Trinta amostras comensais, isoladas de frangos sem sinais de doença, foram utilizadas como controle. A análise do perfil eletroforético obtido por reação de REP-PCR utilizando DNA purificado das amostras evidenciou a amplificação de 0 a 15 bandas de DNA com pesos moleculares variando entre 100 pb e 6.1 Kb. A análise deste padrão permitiu a construção de um dendrograma demonstrando o agrupamento das 79 amostras em 49 perfis distintos. Embora a técnica de REP-PCR tenha apresentado grande poder discriminatório, as amostras patogênicas e não patogênicas não foram discriminadas entre si assim como não foi observado o agrupamento de amostras causadoras do mesmo tipo de doença. Por outro lado, demonstramos recentemente que outras técnicas tais como ERIC-PCR e a análise de isoenzimas foram eficientes quando utilizadas para esta mesma finalidade. Concluindo, REP-PCR parece não ser uma técnica eficiente e universal para discriminar entre amostras APEC. Porém, a estrutura clonal populacional obtida com o uso de REP-PCR não deve ser desprezada, particularmente se considerarmos que os mecanismos de patogenicidade de APEC ainda não são completamente conhecidos.

In the present study the repetitive extragenic palindromic (REP) polymerase chain reaction (PCR) technique was used to establish the clonal variability of 49 avian Escherichia coli (APEC) strains isolated from different outbreak cases of septicemia (n=24), swollen head syndrome (n=14) and omphalitis (n=11). Thirty commensal strains isolated from poultry with no signs of these illnesses were used as control strains. The purified DNA of these strains produced electrophoretic profiles ranging from 0 to 15 bands with molecular sizes varying from 100 bp to 6.1 kb, allowing the grouping of the 79 strains into a dendrogram containing 49 REP-types. Although REP-PCR showed good discriminating power it was not able to group the strains either into specific pathogenic classes or to differentiate between pathogenic and non-pathogenic strains. On the contrary, we recently demonstrated that other techniques such as ERIC-PCR and isoenzyme profiles are appropriate to discriminate between commensal and APEC strains and also to group these strains into specific pathogenic classes. In conclusion, REP-PCR seems to be a technique neither efficient nor universal for APEC strains discrimination. However, the population clonal structure obtained with the use of REP-PCR must not be ignored particularly if one takes into account that the APEC pathogenic mechanisms are not completely understood yet.

Sorotipagem de amostras de Streptococcus suis isoladas de suínos em granjas dos Estados de Säo Paulo, Minas Gerais e Paraná / Serotyping of Streptococcus suis strains isolated from pigs in the States of Säo Paulo, Minas Gerais e Paraná, Brazil

Infecçöes causadas por Streptococcus suis säo muito comuns em países onde a indústria de carne suína é desenvolvida. Estas infecçöes estäo relacionadas a casos clínicos de broncopneumonia, meningite, artrite, pericardite, miocardite, endocardite, poliserosite fibrinosa, septicemia, rinite e aborto. Esta bactéria também foi descrita como patógeno de ruminantes e humanos. No Brasil há evidências clínicas da existência de processos infecciosos causados por S. suis afetando mais de 50 por cento das granjas em Estados como Säo Paulo, Minas Gerais e Paraná. No presente estudo foram isoladas 51 amostras de S. suis de granjas do Estados acima referidos, coletadas de diferentes casos clínicos como septicemia, meningite, artrite e pneumonia, tendo sido obtidas ou em cultura pura ou como patógeno de maior predominância nos tecidos de suínos. Este material foi semeado em Columbia ágar sangue adicionado de 5 por cento de sangue bovino e incubado a 37°C por 24 horas. Para a identificaçäo bioquímica as colônias que apresentavam a-hemólise, bem como as amostras padräo, foram submetidas a testes convencionais para a confirmaçäo da espécie S. suis, tais como: hidrólise de arginina, teste de Voges-Proskauer, e produçäo de ácido a partir de vários carboidratos (inulina, salicina, trealose, lactose, sacarose, sorbitol, manitol e glicerol). As amostras também foram testadas para habilidade de crescimento em meio de TSA com 6,5 por cento de NaCl e para a produçäo de amilase. Todas as amostras que fizeram parte desta pesquisa foram testadas pelo sistema Api 20 Strep para confirmaçäo dos resultados obtidos nos testes convencionais. Para a sorotipagem foram produzidos antissoros de 1 a 8. Outras amostras näo pertencentes a estes sorotipos também foram sorotipadas. O antissoro produzido em coelhos foi titulado pelo teste de aglutinaçäo em tubo com 2-mercaptoetanol e pelo teste de reaçäo capsular e, quando adequados, foram usados no teste de co-aglutinaçäo, para a sorotipagem das amostras de S. suis. A sorotipagem das 51 amostras isoladas mostraram os seguintes resultados: 30 (58,8 por cento) foram classificadas como sorotipo 2, 11 (21,6 por cento) das amostras como sorotipo 3, sete (13,72 por cento) como sorotipo 7, duas (3,92 por cento) como sorotipo 1 e uma amostra como pertencente ao sorotipo14 (1,96 por cento). Este é o primeiro relato do isolamento de um grande número de amostras de S. suis no Brasil, de casos típicos de processos infecciosos causados por esta bactéria