Insights about the epidemiology of Salmonella Typhimurium isolates from different sources in Brazil using comparative genomics.

BACKGROUND: Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) is an important zoonotic agent worldwide. The aim of this work was to compare genetically 117 S. Typhimurium isolated from different sources over 30 years in Brazil using different genomics strategies. RESULTS: The majority of the 117 S. Typhimurium strains studied were grouped into a single cluster (≅ 90%) by the core genome multilocus sequence typing and (≅ 77%) by single copy marker genes. The phylogenetic analysis based on single nucleotide polymorphism (SNP) grouped most strains from humans into a single cluster (≅ 93%), while the strains isolated from food and swine were alocated into three clusters. The different orthologous protein clusters found for some S. Typhimurium isolated from humans and food are involved in metabolic and regulatory processes. For 26 isolates from swine the sequence types (ST) 19 and ST1921 were the most prevalent ones, and the ST14, ST64, ST516 and ST639 were also detected. Previous results typed the 91 S. Typhimurium isolates from humans and foods as ST19, ST313, ST1921, ST3343 and ST1649. The main prophages detected were: Gifsy-2 in 79 (67.5%) and Gifsy-1 in 63 (54%) strains. All of the S. Typhimurium isolates contained the acrA, acrB, macA, macB, mdtK, emrA, emrB, emrR and tolC efflux pump genes. CONCLUSIONS: The phylogenetic trees grouped the majority of the S. Typhimurium isolates from humans into a single cluster suggesting that there is one prevalent subtype in Brazil. Regarding strains isolated from food and swine, the SNPs' results suggested the circulation of more than one subtype over 30 years in this country. The orthologous protein clusters analysis revealed unique genes in the strains studied mainly related to bacterial metabolism. S. Typhimurium strains from swine showed greater diversity of STs and prophages in comparison to strains isolated from humans and foods. The pathogenic potential of S. Typhimurium strains was corroborated by the presence of exclusive prophages of this serovar involved in its virulence. The high number of resistance genes related to efflux pumps is worrying and may lead to therapeutic failures when clinical treatment is needed.

Genotyping of Campylobacter coli strains isolated in Brazil suggests possible contamination amongst environmental, human, animal and food sources.

Campylobacter coli and Campylobacter jejuni are two of the most common causative agents of food-borne gastroenteritis in numerous countries worldwide. In Brazil, campylobacteriosis is under diagnosed and under-reported, and few studies have molecularly characterized Campylobacter spp. in this country. The current study genotyped 63 C. coli strains isolated from humans (n512), animals (n521), food (n510) and the environment (n520) between 1995 and 2011 in Brazil. The strains were genotyped using pulsed-field gel electrophoresis (PFGE), sequencing the short variable region (SVR) of the flaA gene ( flaA-SVR) and high-resolution melting analysis (HRMA) of the clustered regularly interspaced short palindromic repeat (CRISPR) locus to better understand C. coli genotypic diversity and compare the suitability of these three methods for genotyping this species. Additionally, the discrimination index (DI) of each of these methods was assessed. Some C. coli strains isolated from clinical and non-clinical origins presented ≥80 % genotypic similarity by PFGE and flaA-SVR sequencing. HRMA of the CRISPR locus revealed only four different melting profiles. In total, 22 different flaA-SVR alleles were detected. Of these, seven alleles, comprising gt1647­gt1653, were classified as novel. The most frequent genotypes were gt30 and gt1647. This distribution reveals the diversity of selected Brazilian isolates in comparison with the alleles described in the PubMLST database. The DIs for PFGE, flaA­SVR sequencing and CRISPR-HRMA were 0.986, 0.916 and 0.550, respectively. PFGE and flaA-SVR sequencing were suitable for subtyping C. coli strains, in contrast to CRISPR-HRMA. The high genomic similarity amongst some C. coli strains confirms the hypothesis that environmental and food sources potentially lead to human and animal contamination in Brazil.

Diagnóstico laboratorial da coqueluche: freqüência do isolamento de Bordetella pertussis de amostras clínicas, por meio da técnica de cultura realizada nos laboratórios regionais do Instituto Adolfo Lutz, São Paulo, Brasil / Pertussis diagnosis: frequency of Bordetella pertussis detection from clinical samples by the culture method performed at regional laboratories of the Instituto Adolfo Lutz, São Paulo, Brazil

A coqueluche é uma doença respiratória infecciosa aguda, causada por Bordetella pertussis, cocobacilos Gram-negativos fastidiosos com requisitos nutricionais complexos. Recentemente foi descrito em vários países o aumento do número de casos da doença - com o acometimento de populações vacinadas - e da sua incidência em adolescentes e adultos. No Brasil, a coqueluche só se tornou doença de notificação compulsória a partir de 2001. O presente trabalho avaliou o índice de positividade no diagnóstico da coqueluche por meio da técnica de cultura de secreção de nasofaringe, realizada segundo Regan e Lowe, 1997, e também a sua freqüência em diferentes faixas etárias, nas regiões de abrangência dos Laboratórios Regionais do Instituto Adolfo Lutz, São Paulo, SP, no período de 2001 a 2005. Os resultados mostraram aumento no número de amostras positivas a partir de 2004, o que pode ter ocorrido em função da maior conscientização dos profissionais envolvidos na investigação e notificação da doença.O maior número de amostras positivas deu-se na faixa etária de 0 a 6 meses (65,0%), com predomínio da faixa de 1 a 3 meses. A taxa de positividade em adolescentes e adultos foi de 19,4%. Os dados obtidos são relevantes para a melhoria do diagnóstico da coqueluche na região estudada e indicam a necessidade de informações adicionais para dar suporte às estratégias de prevenção e prover um melhor entendimento da doença no país.

Pertussis or whooping cough is an acute respiratory infectious disease caused by Bordetella pertussis, a small Gram-negative coccobacillus which has complex nutritional requirements. Several countries have recently reported an increase in pertussis in immunized populations, accompanied by a significant increase among adolescents and adults. In Brazil, pertussis had its epidemiological surveillance established only in 2001. This study evaluated the indices of positivity on culture method performed according to Regan & Lowe, 1997, for pertussis diagnosis in the regions enclosed by the regional laboratories of the Instituto Adolfo Lutz - São Paulo state, Brazil, from 2001 to 2005. An increase in the number of positive samples was observed since 2004, which could be due to a better identification of the disease by health workers involved on its investigation, diagnosis, and notification. According to these results, the young infants presented the highest positivity rate (age 0-6 months, 65.0%). The older age groups (adolescents and adults) showed 19.4% of positivity. These data are relevant for monitoring pertussis diagnosis and for conducting epidemiological studies in the studied regions, and also to provide support to prevention programs.

Phenotypical and genotypical characterization of Bordetella pertussis strains isolated in São Paulo, Brazil, 1988-2002.

Whooping cough or pertussis was a major cause of childhood morbidity and mortality in the world until the introduction of a whole-cell vaccine in the 1940's. However, since the early 1980's whooping cough cases have increased in many countries, becoming an important problem of public health. This increase may be due to accuracy of laboratory diagnosis and reporting of the disease, a decline in immunity over time, demographic changes, and adaptation of the bacterial population to vaccine-induced immunity. The purpose of this study was to analyze phenotypically and genotypically a collection of 67 Bordetella pertussis isolates recovered during the period 1988-2002 in São Paulo State, Brazil to determine their characteristics and relatedness. All isolates were submitted to susceptibility testing to erythromycin, serotyping, and 56 isolates were analyzed by Pulsed Field Gel Electrophoresis (PFGE). All isolates were susceptible to erythromycin and the majority of them belonged to serotype 1,3. The 56 isolates were classified into 11 PFGE profiles according to the differences in banding patterns. Although more than 60% of the isolates were recovered from patients aged less than three months, almost 15% of them were isolated from adolescents/adults evidencing the increase in the incidence of pertussis among this age group.

Phenotypical and genotypical characterization of Bordetella pertussis strains isolated in São Paulo, Brazil, 1988-2002

Whooping cough or pertussis was a major cause of childhood morbidity and mortality in the world until the introduction of a whole-cell vaccine in the 1940's. However, since the early 1980's whooping cough cases have increased in many countries, becoming an important problem of public health. This increase may be due to accuracy of laboratory diagnosis and reporting of the disease, a decline in immunity over time, demographic changes, and adaptation of the bacterial population to vaccine-induced immunity. The purpose of this study was to analyze phenotypically and genotypically a collection of 67 Bordetella pertussis isolates recovered during the period 1988-2002 in São Paulo State, Brazil to determine their characteristics and relatedness. All isolates were submitted to susceptibility testing to erythromycin, serotyping, and 56 isolates were analyzed by Pulsed Field Gel Electrophoresis (PFGE). All isolates were susceptible to erythromycin and the majority of them belonged to serotype 1,3. The 56 isolates were classified into 11 PFGE profiles according to the differences in banding patterns. Although more than 60 percent of the isolates were recovered from patients aged less than three months, almost 15 percent of them were isolated from adolescents/adults evidencing the increase in the incidence of pertussis among this group of age.

A coqueluche ou pertussis foi a maior causa de morbidade e mortalidade infantil em todo o mundo até a introdução de uma vacina na década de 1940. Entretanto, desde a década de 1980, a coqueluche tornou-se, em muitos países , um importante problema de saúde pública. Este acontecimento pode ser atribuído à melhoria do diagnóstico laboratorial e da notificação da doença, declínio da imunidade no decorrer do tempo, mudanças demográficas ou adaptação da população bacteriana à imunidade induzida pela vacina. O objetivo deste estudo foi analisar as características fenotípicas e genotípicas de uma coleção de 67 cepas de Bordetella pertussis isoladas no período 1988-2002 em São Paulo, Brasil. Todas as cepas foram submetidas à determinação do perfil de resistência à eritromicina, à sorotipagem e 56 cepas à eletroforese em campo pulsado (PFGE). Todas as cepas foram sensíveis à eritromicina e a maioria delas pertencia ao sorotipo 1,3. As 56 cepas foram classificadas em 11 perfis de PFGE com base nas diferenças no padrão de bandas. Embora mais de 60 por cento das cepas tenham sido isoladas de crianças com menos de três meses de idade, cerca de 15 por cento delas era de adolescentes/adultos evidenciando um aumento da incidência da coqueluche nesse grupo etário.

Nasopharyngeal colonization by Haemophilus influenzae in children attending day-care centers, in Ribeirão Preto, State of São Paulo, Brazil

A colonização da nasofaringe por Haemophilus influenzae (Hi) foi estudada em 114 crianças saudáveis com menos de 3 anos de idade e que freqüentam creches (day-care centers DCC) em Ribeirão Preto, estado de São Paulo, Brasil. Para cada uma das cepas isoladas foram determinados o biótipo, o sorotipo (por antisoro especifico e PCR) e a sensibilidade a 14 antibióticos. A freqüência de colonização por Hi foi de 72,0 por cento. As cepas isoladas foram identificadas como pertencentes aos biótipos II (36,5 po cento), I (21,5 por cento), V (18,2 por cento) e III (16,1 por cento). A freqüência encontrada de cepas encapsuladas foi de 3,2 por cento para o tipo f, 1,0 por cento para o tipo b, 1,0 por cento para o tipo d e 1,0 por cento para o tipo e. A resistência para trimetoprim-sulfametoxazole e ampicilina foi de 46,2 por cento e 10,7 por cento respectivamente. Resistência múltipla foi encontrada em 14 (15,0 por cento) das cepas analisadas. 13,9 por cento das cepas analisadas eram produtoras de b-lactamase, e não foi recuperada nenhuma cepa b-lactamase negativa e ampicilina resistente. DCCs são considerados locais de risco, com um alto potencial de disseminação de microrganismos e por isto devem ser continuadamente monitorados com a finalidade de detectar a eliminação da colonização da nasofaringe por cepas H. influenzae tipo b das crianças que freqüentam DCC, ou detectar a sua substituição por outro tipo de cepa.

Comparison of two slide agglutination serotyping methods and PCR-based capsule typing for the characterization of Haemophilus influenzae serotypes

Noventa e três isolados nasofaringeanos de H. influenzae foram sorotipados através de 2 métodos de aglutinação em lamina (SAST 1 e SAST 2) e os resultados foram comparados com a sorotipagem por PCR. SAST 1 apresentou uma baixa correlação com os resultados obtidos por PCR (75,2 por cento) enquanto que SAST 2 mostrou uma melhor concordância com os resultados da técnica molecular (93,5 por cento). Estes resultados indicam que SAST 2 pode ser um método alternativo para a correta detecção de H. influenzae tipo b.

Streptococcus pneumoniae and Haemophilus influenzae as etiological agents of conjunctivitis outbreaks in the region of Riveiräo Preto, SP, Brazil

No estudo de surtos de conjuntivite ocorridos no periodo de setembro de 1994 a setembro de 1996, na regiäo de Ribeirao Preto, foram semeadas no Instituto Adolfo Lutz Laboratorio I, Ribeiräo Preto, exsudatos conjuntivais de 92 pacientes, sendo que a maioria dos casos estava na faixa etaria de 2-7 anos. Os agentes etiologicos mais frequentemente isolados foram dos casos analisados foram: Streptococcus pneumoniae e Haemophilus influenzae em 40,22 por cento e 21,74 por cento respectivamente. 51,35 por cento das cepas de S. pneumoniae isoladas foram näo tipaveis. As cepas de S. pneumoniae oxacilina resistente foram submetidas ao teste de concentracao inibitoria minima (CIM), sendo que tres apresentaram resistencia intermediaria e apenas uma foi altamente resistente a penicilina