Marta Inês Cazentini Medeiros

Etiology of acute diarrhea among children in Ribeirão Preto-SP, Brazil

To study the main enteropathogens causing diarrhea in the region of Ribeirão Preto regarding serogroups and serotypes, the feces of 1836 children under 10 years old, from both sexes, attack of acute gastroenteritis, were analysed during a period of 4 years in Adolfo Lutz Institute - Ribeirão Preto, SP. The pathogens identified by standard methods were the following: Escherichia coli, Salmonella spp., Shigella spp., Campylobacter spp., Yersinia spp., and Cryptosporidium spp. Positive samples were 22.8% (419) with 1.7% association of pathogens. Larger isolates were mainly from children 0 to 11 months old. Enteropathogenic E. coli (EPEC) was most frequent (8.7%) with predominance of serogroup O119 (40.2%), followed by Shigella (6.2%), 63.6% of which S. sonnei.

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

In the study of conjunctivitis outbreaks occurring from September 1994 to September 1996 in the region of Ribeirão Preto, conjunctival exudates of 92 patients were cultivated in Instituto Adolfo Lutz Laboratory I, Ribeirão Preto. Most cases occurred in the age range 2-7 years. The etiological agents which were most frequently isolated from the analyzed cases were: Streptococcus pneumoniae and Haemophilus influenzae, in 40.22% and 21.74%, respectively. 51.35% of the S. pneumoniae isolated strains were not typable. The oxacillin-resistant S. pneumoniae strains were submitted to the minimum inhibitory concentration test (MIC) and three of them presented intermediate resistance, whereas only one was highly resistant to penicillin.

MLVA typing reveals higher genetic homogeneity among S. Enteritidis strains isolated from food, humans and chickens in Brazil in comparison to the North American Strains

Salmonella Enteritidis (S. Enteritidis) is a major causative agent of food-borne gastroenteritis associated with the consumption of contaminated poultry products. In this study we used multilocus variable number of tandem repeats (VNTRs) analysis (MLVA) to discriminate a total of 188 S. Enteritidis strains recovered from human (n=67), food (n=61) and chickens (n=60) during a 24 year period (1986 through 2010) in Brazil. MLVA profiles of the 188 strains from Brazil were compared to the MLVA profiles of 100 human clinical (n=52) and poultry-associated (n=48) strains isolated in North America between 1986 and 2008. MLVA typing led to classification of the 288 strains from Brazil and North America into two major clusters named A and B with 35% of similarity. Cluster A consisted of a vast majority of strains isolated from North America (n=71) and only three strains isolated from Brazil which included two pre-pandemic strains (SE5 and SE4). In contrast, cluster B consisted of all of the post-pandemic strains isolated from Brazil (n=185) and fewer strains isolated from North America (n=29). In general, MLVA typing showed that the North American strains were more genetically diverse whereas Brazilian strains were more genetically clonal. The clustering of pre-pandemic strains from Brazil with the North American strains suggests the possibility that the pre-pandemic strains were more likely genetically diverse; however after 1993 a new and prevalent subtype of S. Enteritidis was introduced in this country. This is the first study describing MLVA genotyping of the S. Enteritidis strains isolated from Brazil.

Molecular characterization of Salmonella Typhimurium isolated in Brazil by CRISPR-MVLST

CRISPR-multi-locus virulence sequence typing (CRISPR-MVLST) was performed to type 92 S. Typhimurium strains isolated from humans and food sources between 1983 and 2013 in Brazil and assess the suitability of this methodology comparing it with PFGE already used for subtyping the same strains. Among the 92 S. Typhimurium strains studied, we identified 25 CRISPR1 alleles, 27 CRISPR2 alleles, 2 fimH alleles and 3 sseL alleles showing that the genetic variability is much higher in the CRISPRs loci than in the virulence genes. The CRISPR-MVLST analysis provided similar results to the PFGE previously published used to type the same set of strains, demonstrating that CRISPR-MVLST is a very efficient approach for subtyping S. Typhimurium serovar and can be used to complement and validate results obtained by the PFGE methodology.

Genotypic diversity, pathogenic potential and the resistance profile of Salmonella Typhimurium strains isolated from humans and food from 1983 to 2013 in Brazil

Salmonella enterica subsp. enterica serovar Typhimurium is one of the leading serovars that causes salmonellosis worldwide. However, few studies have molecularly characterized S. Typhimurium strains in Brazil. In this study, we genotyped 92 S. Typhimurium strains isolated from humans (43) and food (49) between 1983 and 2013 in Brazil using PFGE, multiple-locus variable number of tandem repeats analysis (MLVA) and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Moreover, we assessed the frequency of 12 virulence markers by PCR and the resistance profile against 12 antimicrobials. More than 85.8% of the strains studied carried 11 of the virulence markers or more. Thirty-three strains (25%) were multidrug resistant (MDR). The 92 S. Typhimurium studied were grouped by PFGE as PFGE-A, PFGE-B1 and PFGE-B2; by MLVA as MLVA-A, MLVA-B1 and MLVA-B2; and, finally, by ERIC-PCR as ERIC-A and ERIC-B. The strains isolated from humans before the mid-1990s were allocated to all clusters. The strains isolated from humans after the mid-1990s were distributed in the PFGE-B1, MLVA-B1, MLVA-B2 and ERIC-A clusters. The strains isolated from food were distributed in all clusters, except in PFGE-B2. All typing results suggested that the S. Typhimurium strains of human clinical origin isolated before the mid-1990s were genetically more diverse, which might indicate the selection of a more adapted S. Typhimurium subtype after Salmonella Enteritidis became the most prevalent serovar in Brazil. Regarding strains isolated from food, the results suggest the current circulation of more than one subtype. Furthermore, the high frequency of virulence genes and the presence of MDR strains reinforces their potential hazard for humans and the risk of their presence in foods in Brazil.

Virulence-associated genes, antimicrobial resistance and molecular typing of Salmonella Typhimurium strains isolated from swine from 2000 to 2012 in Brazil

The aims of this study were to assess the pathogenic potential, antimicrobial resistance and genotypic diversity of Salmonella Typhimurium strains isolated in Brazil from swine (22) and the surrounding swine environment (5) from 2000 to 2012 and compare them to the profiles of 43 human strains isolated from 1983 to 2010, which had been previously studied.

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 1940s. However, since the early 1980s 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.

Phenotypic and genotypic characterization of Rhodococcus equi isolated from sputum

INTRODUCTION Rhodococcus equi is an opportunistic pathogen, causing rhodococcosis, a condition that can be confused with tuberculosis. Often, without identifying M. tuberculosis, physicians initiate empiric treatment for tuberculosis. R. equi and M. tuberculosis have different susceptibility to drugs. Identification of R. equi is based on a variety of phenotypic, chromatographic, and genotypic characteristics. OBJECTIVE This study aimed to characterize bacterial isolates from sputum samples suggestive of R. equi. METHODS The phenotypic identification included biochemical assays; thin-layer chromatography (TLC) and polymerase chain reaction (PCR) were used for genotypic identification. RESULTS Among 78 Gram-positive and partially acid-fast bacilli isolated from the sputum of tuberculosis-suspected patients, 51 were phenotypically and genotypically characterized as R. equi based on literature data. Mycolic acid analysis showed that all suspected R. equi had compounds with a retention factor (R(f)) between 0.4-0.5. Genotypic characterization indicated the presence of the choE gene 959bp fragments in 51 isolates CAMP test positive. Twenty-two CAMP test negative isolates were negative for the choE gene. Five isolates presumptively identified as R. equi, CAMP test positive, were choE gene negative, and probably belonged to other bacterial species. CONCLUSIONS The phenotypic and molecular techniques used constitute a good methodological tool to identify R. equi.

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

Nasopharyngeal carriage of Haemophilus influenzae (Hi) was studied in 114 healthy children < 3 years old, attending day-care centers (DCCs) in Ribeirao Preto, State of Sao Paulo, Brazil. Biotype, serotype (by specific antisera and PCR) and antibiotic susceptibility to 14 antibiotics of each isolate were determined. Carriage rates of Hi were 72.0%. Isolates belonged to biotype II (36.5%), I (21.5%), V (18.2%) and III (16.1%). The prevalence of encapsulated Hi carriers was 3.2% for type f, 1.0% for type b, 1.0% for type d and 1.0% for type e. Resistances to trimethoprim-sulphamethoxazole and ampicillin were 46.2% and 10.7% respectively. Multidrug resistance was found in 14 (15.0%) of the isolates tested. Among the isolates, 13.9% were b-lactamase producers; there were no b-lactamase negative ampicillin resistant isolates. DCCs are niches with a high potential for the spread of microorganisms and should be continuously monitored to detect elimination or replacement of H. influenzae type b colonization.

Molecular and phenotypic characterization of strains of Shigella sonnei isolated over 31 years suggests the circulation of two prevalent subtypes in São Paulo State, Brazil.

Shigella sonnei is an important causative agent of bacillary dysentery worldwide that has recently emerged in developing countries. However, there are few studies that have characterized strains ofS. sonnei isolated in Brazil. The aims of this study were to assess the presence of 12 virulence genes, the antimicrobial resistance profile against 16 drugs and the genotypic diversity of strains of S. sonnei isolated in this country. Seventy-two strains of S. sonnei isolated from human diarrhoeic faeces in São Paulo State, Brazil from 1983-2014 were studied. All of the strains contained the ipaH, iuc and sigA genes. The ipaBCD gene was detected in 19 % of the strains, the ial and virF genes in 18 % and the sen gene in 10 % of the strains. The set1A, set1B, pic,sepA and sat genes were not detected. A total of 42 (58.3 %) strains were resistant to trimethoprim-sulfamethoxazole. Thirty (41.6 %) strains were resistant to tetracycline. The S. sonnei strains were grouped in two clusters called A and B by PFGE and ERIC-PCR, and the majority of the strains comprised in each cluster presented ≥80 % similarity. In conclusion, the pathogenic potential of the strains studied was highlighted by the presence of important virulence genes. The high rates of resistance to trimethoprim-sulfamethoxazole and tetracycline are alarming once those drugs can be used in the treatment of shigellosis. The PFGE and ERIC-PCR results suggest that there are two prevalent subtypes in the studied strains of S. sonnei that differed little over 31 years and have been contaminating humans and causing diseases in São Paulo State, Brazil.