Objectives: The aim of this study is to report the occurrence of the first outbreak of food poisoning caused by SalmonellaAlachua in Brazil, as well as the antimicrobial susceptibility and the genetic relatedness of SalmonellaAlachua strains isolated from clinical and food samples.Material and methods: To elucidate the outbreak, an epidemiological investigation was carried out, and two samples of common food were tested - mayonnaise salad and galinhada(a traditional Brazilian dish of chicken and rice) - according to the Compendium of methods for the microbiological examination of foods. Five stool samples were tested employing classic methods for the isolation and identification of enterobacteria. Strains of Salmonella were characterized for antibiotic susceptibility according to the Clinical and Laboratory Stan- dards Institute guidelines (2013), and submitted to pulsed-field gel electrophoresis analysis, performed according to the Centers for Disease Control and Prevention PulseNet protocol.Results: A total of 94 people were interviewed after ingesting the food, 66 of whom had become ill. A 60-year old female patient who was hospitalized in a serious condition, developed septic shock and died two days after consuming the food. The presence of SalmonellaAlachua was confirmed in all the analyzed stool samples, and in the two types of food. The five strains showed higher than minimum inhibitory concentration values of nalidixic acid (≥256 µg/mL) and reduced ciprofloxacin susceptibility (minimum inhibitory concentration = 0.5 µg/mL). The pulsed-field gel electrophoresis analysis revealed indistinguishable patterns in SalmonellaAlachua strains isolated from clinical and food samples.Conclusion: The data presented herein confirm the foodborne disease outbreak. They also allowed for the identification of the source of infection, and suggest that products from poultry are potential reservoirs for this serotype, reinforcing the importance of warning consumers about the danger of possible contamination.

A identificação da Escherichia coli requer conhecimento sobre os sorotipos e fatores de virulência prevalentes permitindo a classificação em patogênico/não patogênico. No entanto, algumas destas bactérias não expressam o antígeno flagelar in vitro. Neste caso, o PCR-restriction fragment length polymorphism (RFLP-PCR) e o sequenciamento do gene fliC podem ser adequados para a identificação desses antígenos, substituindo a sorologia tradicional. Nesta pesquisa foram estudadas 17 amostras de E. coli isoladas de animais e que apresentavam antígeno H não tipável (HNT). Os antígenos H foram caracterizados por PCR-RFLP e sequenciamento do gene fliC. Três novos genes da flagelina foram identificados, para os quais anti-soros específicos foram obtidos. A técnica PCR-RFLP mostrou-se mais rápida que a sorotipagem do antígeno H em E. coli, fornecendo informações sobre algumas características desses antígenos e indicou a presença de novos genes fliC.

Identification of Escherichia coli requires knowledge regarding the prevalent serotypes and virulence factors profiles allows the classification in pathogenic/non-pathogenic. However, some of these bacteria do not express flagellar antigen invitro. In this case the PCR-restriction fragment length polymorphism (RFLP-PCR) and sequencing of the fliC may be suitable for the identification of antigens by replacing the traditional serology. We studied 17 samples of E. coli isolated from animals and presenting antigen H nontypeable (HNT). The H antigens were characterized by PCR-RFLP and sequencing of fliC gene. Three new flagellin genes were identified, for which specific antisera were obtained. The PCR-RFLP was shown to be faster than the serotyping H antigen in E. coli, provided information on some characteristics of these antigens and indicated the presence of new genes fliC.

Escherichia coli has been isolated frequently, showing flagellar antigens that are not recognized by any of the 53 antisera, provided by the most important reference center of E. coli, The International Escherichia and Klebsiella Center (WHO) of the Statens Serum Institute, Copenhagen, Denmark. The objective of this study was to characterize flagellar antigens of E. coli that express non-typeable H antigens. The methods used were serology, PCR-RFLP and DNA sequencing. This characterization was performed by gene amplification of the fliC (flagellin protein) by polymerase chain reaction in all 53 standards E.coli strains for the H antigens and 20 E. coli strains for which the H antigen was untypeable. The amplicons were digested by restriction enzymes, and different restriction enzyme profiles were observed. Anti-sera were produced in rabbits, for the non-typeable strains, and agglutination tests were carried out. In conclusion,the results showed that although non-typeable and typable H antigens strains had similar flagellar antigens, the two types of strains were distinct in terms of nucleotide sequence, and did not phenotypically react with the standard antiserum, as expected. Thirteen strains had been characterized as likely putative new H antigen using PCR-RFLP techniques, DNA sequencing and/or serology.

Identification of Lactobacillus sp. strains by phenotypic methods may lead to doubtful results possibly interfering in the reliability of the epidemiological and probiotics studies. Therefore this study aimed to determine the best methodology for the identification of the large diversity of lactobacilli species found in the vagina by comparing two techniques, one based on their biochemical profile and other employing molecular biology. A carbohydrate fermentation test (API 50 CH) was compared with multiplex polymerase chain reaction (PCR) for the identification of species of vaginal lactobacilli from 135 healthy women. The kappa index was used to evaluate agreement between the methods. Using the molecular technique, L. crispatus (32.6%), L. jensenii (25%) and L. gasseri (20.6%) were the most frequent species. However, using the biochemical technique, the most frequent species were: L. acidophilus (34.8%), L. crispatus (27.2%) and L. fermentum (13%). Although L. acidophilus was the most frequent specie found by biochemical tests, no strain of this microorganism was detected by PCR. Agreement between the methods was low for identification of all the most common species. Although rates of L. crispatus detected were similar using both methods (32.6% and 27.2%), agreement between them was relatively low (kappa = 0.52). Conclusions: Our results confirmed the limitation of the biochemical method and the applicability of a previously published molecular method (Multiplex PCR) for the identification of lactobacilli in the vaginal tract, focusing on further necessity of its improvement for also targeting L. vaginalis and L. iners.

Amostras de Escherichia coli, isoladas de pacientes do sexo feminino com quadro clínico de cistite, foram caracterizadas quanto à presença de fatores de virulência associados à formação de biofilme e ao agrupamento filogenético. Os resultados da reação em cadeia da polimerase demonstraram que todas as amostras foram positivas para o gene fimH (fímbria do tipo1), 91 amostras foram positivas para o gene fliC (flagelina) 50 amostras positivas para o gene papC (fímbria P), 44 amostras positivas para o gene kpsMTII (cápsula) e 36 amostras positivas para o gene flu (antígeno 43). Os resultados dos ensaios de quantificação da formação de biofilme demonstraram que 44 amostras formaram biofilme em microplacas de poliestireno e 56 amostras apresentaram resultado ausente/fraco. Também confirmamos a incidência das amostras de Escherichia coli no grupo filogenético B2 e D.

Escherichia coli samples isolated from female patients with cystitis were characterized with regard to the presence of virulence factors associated with biofilm formation and phylogenetic groupings. Polymerase chain reaction results demonstrated that all the samples were positive for the gene fimH (type 1 fimbriae), 91 for fliC (flagellins), 50 for papC (P fimbriae), 44 for kpsMTII (capsules) and 36 for flu (antigen 43). The results from assays to quantify the biofilm formation demonstrated that 44 samples produced biofilm on polystyrene microplates and 56 samples produced weak or no biofilm. We also confirmed that Escherichia coli samples were present in phylogenetic groups B2 and D.

Adesinas (Fímbria P, fímbria S, fímbria do tipo 1 e a adesina afimbrial), toxinas (α-hemolisina e o fator necrosante citotóxico do tipo 1), sistemas de captação de ferro (aerobactina), e mecanismos de defesa do hospedeiro (cápsula ou lipopolissacarídeo) são prevalentes em amostras de Escherichia coli associadas a infecções do trato urinário. O objetivo deste trabalho foi caracterizar genotipicamente 162 amostras de Escherichia coli uropatogênica (UPEC) de pacientes com cistite através do ensaio da reação em cadeia da polimerase. Foram realizados três ensaios de PCR multiplex para os seguintes fatores de virulência: papC, papE/F, alelos de papG, fimH, sfa/foc, afaE, hly, cnf-1, usp, cdtB, iucD, e kpsMTII. Os resultados da PCR identificaram, 158 amostras fimH (97,5%), 86 amostras kpsMTII (53,1%), 53 amostras papC/papEF/papG (32,7%), 45 amostras sfa (27,8%), 42 amostras iucD (25,9%), 41 amostras hly (25,3%), 36 amostras usp (22,2%), 30 amostras cnf-1 (18,5%) e 10 amostras afa (6,2%). Nenhuma amostra foi positiva para o gene cdtB. Neste trabalho, demonstramos que podemos encontrar múltiplas adesinas em uma única amostra e que diferentes genes de fatores de virulência podem ser encontrados em associação.

Adhesins (P-fimbriae, S-fimbriae, type 1 fimbriae and afimbrial adhesin), toxins (α-hemolysin and cytotoxic necrotizing factor type 1), iron acquisition systems (aerobactin) and host defense avoidance mechanisms (capsule or lipopolysaccharide) have been shown to be prevalent in Escherichia coli strains associated with urinary tract infections. In this work, 162 Uropathogenic Escherichia coli (UPEC) strains from patients with cystitis were genotypically characterized by polymerase chain reaction (PCR) assay. We developed three multiplex PCR assays for virulence-related genes papC, papE/F, papG alleles, fimH, sfa/foc, afaE, hly, cnf-1, usp, cdtB, iucD, and kpsMTII, all of them previously identified in UPEC strains. The PCR assay results identified 158 fimH (97.5%), 86 kpsMTII (53.1%), 53 papC/papEF/papG (32.7%), 45 sfa (27.8%), 42 iucD (25.9%), 41 hly (25.3%), 36 usp (22.2%), 30 cnf-1(18.5%) and 10 afa (6.2%) strains. No strain was positive for cdtB. In this work, we also demonstrated that adhesins may be multiple within a single strain and that several virulence genes can occur combined in association.