Insights on the genomic diversity, virulence and resistance profile of a Campylobacter jejuni strain isolated from a hospitalized patient in Brazil.

Campylobacteriosis is currently recognized as one of the major causes of foodborne bacterial diseases worldwide. In Brazil, there is insufficient data to estimate the impact of Campylobacter in public health. The aim of this present study was to characterize a C. jejuni CJ-HBSJRP strain isolated from a hospitalized patient in Brazil by its ability to invade human Caco-2 epithelial cells, to survive in U937 human macrophages, and to assess its phenotypic antimicrobial resistance profile. In addition, prophages, virulence and antimicrobial resistance genes were search using whole-genome sequencing data. The genetic relatedness was evaluated by MLST and cgMLST analysis by comparison with 29 other C. jejuni genomes isolated from several countries. The CJ-HBSJRP strain showed an invasion percentage of 50% in Caco-2 polarized cells, 37.5% of survivability in U937 cells and was phenotypically resistant to ampicillin, ciprofloxacin and nalidixic acid. A total of 94 virulence genes related to adherence, biofilm, chemotaxis, immune modulation, invasion process, metabolism, motility and toxin were detected. The resistance genes blaOXA-605 (blaOXA-61), cmeB and mutations in the QRDR region of gyrA were also found and none prophages were detected. The MLST analysis showed 23 different STs among the strains studied. Regarding cgMLST analysis, the CJ-HBSJRP strain was genetically distinct and did not group closely to any other isolate. The results obtained reinforce the pathogenic potential of the CJHBSJRP strain and highlighted the need for more careful attention to Campylobacter spp. infections in Brazil since this pathogen has been the most commonly reported zoonosis in several countries worldwide.

Phylogenetic relationship and genomic characterization of Salmonella Typhimurium strains isolated from swine in Brazil.

Salmonella Typhimurium has been transmitted between humans and animals. Although, Brazil has been one of the largest pork meat exporters worldwide, there are few studies that characterized epidemiologically S. Typhimurium strains from swine. The aims of this work were to study the phylogenetic relationship of S. Typhimurium genomes isolated from swine in Brazil among themselves and with other genomes isolated from several sources and countries using wgMLST and cgMLST and to perform the search of Salmonella pathogenicity islands (SPIs). In addition, for S. Typhimurium strains from swine to compare the virulence and antimicrobial resistance genes by VFDB and ResFinder, genetic content by BLAST Atlas and orthologous proteins clusters by OrthoVenn. The constructed phylogenetic trees by wgMLST and cgMLST grouped the majority (92.3% and 80.7%, respectively) of the strains isolated from swine in Brazil into the same group. All the isolates contained important SPIs (SPI-1, SPI-2, SPI-3, SPI-5 and SPI-9). A total of 100 and 31 virulence and resistance genes were detected in the S. Typhimurium strains isolated from swine, respectively. The BLAST Atlas and orthologous proteins analysis found regions of phages and differences in metabolic, regulatory and cellular processes among S. Typhimurium LT2 and S. Typhimurium isolates from swine. In conclusion, molecular typing based in the wgMLST and cgMLST suggested that the S. Typhimurium isolates from swine studied were genetically related. The pathogenic potential of the strains studied was corroborated by the presence of important SPIs and virulence genes. The high number of antimicrobial resistance genes detected is worrying and reinforced their potential risk in swine in Brazil. The comparison by BLAST Atlas suggested differences in mobile genetic elements among S. Typhimurium LT2 and S. Typhimurium isolates from swine in Brazil. The orthologous proteins analysis revealed unique genes related to important cellular processes in the strains from swine.

Genetic diversity assessed using PFGE, MLP and MLST in Candida spp. candidemia isolates obtained from a Brazilian hospital.

Candida spp. are the main causative agents of invasive fungal infections in immunocompromised patients. Candidemia has attributable mortality rates of 15 to 35% and increases hospitalisation time and costs, thus making this disease a public health concern. This study aimed to use pulsed-field gel electrophoresis (PFGE), microsatellite length polymorphism (MLP) and multilocus sequence typing (MLST) to analyse the genetic relationships among 65 Candida spp. bloodstream isolates, including 35 Candida albicans, 15 Candida glabrata and 15 Candida tropicalis isolates, all of which were obtained from patients in a Brazilian hospital. Moreover, patient clinical data were assessed. All techniques resulted in high discriminatory indexes. C. albicans and C. tropicalis isolates showed high genetic variability, while C. glabrata isolates had relatively low genetic variability. Moreover, a cluster of C. glabrata isolates was identified in a hospital unit. New MLST sequence types, diploid sequence types and alleles are described. Relationships were not observed between the molecular typing results and clinical characteristics. The molecular typing of clinical strains increases our understanding of candidemia epidemiology and promotes the development of strategies that can reduce the incidence of this disease. Moreover, this study is the first to combine these techniques to genotype these three species in Brazil.

Antimicrobial Resistance Profiles and Phylogenetic Analysis of Campylobacter jejuni Strains Isolated in Brazil by Whole Genome Sequencing.

Aims: The objectives of this work were to use whole genome sequencing (WGS) to determine the antimicrobial resistance genotypes of 116 Campylobacter jejuni strains isolated in Brazil and to compare it with the results obtained by antimicrobial susceptibility testing (AST). In addition, WGS was used to uncover the phylogenetic relationship among those strains. Results: By AST, the C. jejuni strains resistant to ciprofloxacin, tetracycline, doxycycline, and erythromycin were 51 (44%), 41 (35.3%), 41 (35.3%), and 6 (5.2%), respectively. By WGS, the genes aph(3')III, aadE, blaOXA-449, blaOXA-184, blaOXA-61, and tet(O) were detected in 6 (5.2%), 3 (2.6%), 1 (0.9%), 10 (8.6%), 55 (47.4%), and 44 (38%) strains, respectively. Fifty-four (46.6%) strains showed the mutation T86I in the gyrA gene, and four (3.4%) strains presented the mutation A2075G in the 23S rRNA gene. The correlation between AST and WGS was 100% for ciprofloxacin, 97.5% for tetracyclines, and 66.7% for erythromycin. The whole genome single nucleotide polymorphism (SNP) tree clustered the C. jejuni strains into two clades comprising strains that were highly related from different sources, places, and years. Conclusion: The high rates of C. jejuni strains resistant to ciprofloxacin and tetracyclines are of concern and may represent a public health problem. WGS has a potential to be a powerful tool for the prediction of resistance of antibiotics used to treat campylobacteriosis. The results obtained by whole genome SNP analysis suggested the potential for transmission between clinical and nonclinical sources and between human and animal sources over the course of 20 years in Brazil.

Correction: Phenotypic and genotypic characterization of Salmonella Typhimurium isolates from humans and foods in Brazil.

[This corrects the article DOI: 10.1371/journal.pone.0237886.].

Phenotypic and genotypic characterization of Salmonella Typhimurium isolates from humans and foods in Brazil.

Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) causes gastroenteritis in many countries. However, in Brazil there are few studies that have conducted a virulence characterization of this serovar. The aim of this study was to evaluate the virulence potential of S. Typhimurium strains isolated in Brazil. Forty S. Typhimurium strains isolated from humans (n = 20) and food (n = 20) from Brazil were studied regarding their invasion and survival in human epithelial cells (Caco-2) and macrophages (U937). Their virulence potential was determined using the Galleria mellonella larvae model combined with the analysis of virulence genes by whole genome sequencing (WGS). A total of 67.5% of the S. Typhimurium studied (32.5% isolated from humans and 35% isolated from food) invaded Caco-2 epithelial cells at levels similar to or greater than the S. Typhimurium SL1344 prototype strain. In addition, 37.5% of the studied strains (25% isolated from humans and 12.5% isolated from food) survived in U937 human macrophages at levels similar to or greater than SL1344. S. Typhimurium strains isolated from humans (40%) and food (25%) showed high or intermediate virulence in G. mellonella larvae after seven days exposure. Approximately, 153 virulence genes of chromosomal and plasmidial origin were detected in the strains studied. In conclusion, the ability of the S. Typhimurium to invade Caco-2 epithelial cells was strain dependent and was not related to the source or the year of isolation. However, S. Typhimurium strains isolated from humans showed greater survival rates in U937 human macrophages, and presented higher proportion of isolates with a virulent profile in G. mellonella in comparison to strains isolated from food suggesting that this difference may be related to the higher frequency of human isolates which contained plasmid genes, such as spvABCDR operon, pefABCD operon, rck and mig-5.

Molecular typing of Campylobacter jejuni strains: comparison among four different techniques.

This study compared the ability of pulsed-field gel electrophoresis (PFGE), flaA small variable region (SVR) sequencing, analysis of the clustered regularly interspaced short palindromic repeats locus by high resolution melting analysis (CRISPR-HRMA), and multilocus sequence typing (MLST) for typing 111 Campylobacter jejuni strains isolated from diverse sources during 20 years in Brazil. For this, we used previous results obtained by PFGE and flaA-SVR sequencing from our research group and performed CRISPR-HRMA and MLST typing for the first time. Furthermore, the discrimination index (DI) of each method was accessed. The DI for PFGE, flaA-SVR sequencing, CRISPR-HRMA, and MLST was 0.980, 0.932, 0.868, and 0.931, respectively. By PFGE and flaA-SVR sequencing, some strains from clinical and non-clinical sources and from humans and animals presented ≥ 80% similarity. Similarly, some strains from different origins presented the same ST and CRISPR-HRMA types. In conclusion, despite the different DI values, all assays provided the same epidemiological information suggesting that a potential transmission may have occurred between C. jejuni from clinical and non-clinical sources and from animals and humans in Brazil. Furthermore it was demonstrated the suitability of PFGE that should be used preferably together with MLST and/or flaA-SVR sequencing for typing C. jejuni strains.

Draft Genome Sequences of 20 Salmonella enterica subsp. enterica Serovar Typhimurium Strains Isolated from Swine in Santa Catarina, Brazil.

Salmonellosis is a disease with a high incidence worldwide, and Salmonella enterica subsp. enterica serovar Typhimurium is one of the most clinically important serovars. We report here the draft genome sequences of 20 S. Typhimurium strains isolated from swine in Santa Catarina, Brazil. These draft genomes will improve our understanding of S. Typhimurium in Brazil.

Draft Genome Sequences of 116 Campylobacter jejuni Strains Isolated from Humans, Animals, Food, and the Environment in Brazil.

Campylobacter jejuni is a major zoonotic pathogen that causes foodborne gastroenteritis worldwide. However, clinical cases of campylobacteriosis have been underreported and underdiagnosed in Brazil. Herein, we describe the draft genome sequences of 116 C. jejuni strains isolated from diverse sources in Brazil.

Pathogenic potential and genotypic diversity of Campylobacter jejuni: a neglected food-borne pathogen in Brazil.

Purpose and methodology.Campylobacter jejuni is a major zoonotic pathogen that causes food-borne gastroenteritis worldwide. However, there are only a few studies available that have molecularly characterized C. jejuni strains isolated in Brazil. The aim of this study was to genotype 111 C. jejuni strains isolated from sick humans (43), monkey faeces (19), chicken faeces (14), chicken meat (33) and sewage (2) between 1996 and 2016 in Brazil using flaA-SVR (short variable region) sequencing and PFGE. Furthermore, the presence of 16 virulence genes was analysed by PCR. RESULTS: Using PFGE and flaA-SVR sequencing, the 111 C. jejuni strains studied were grouped into three and two clusters, respectively, and some strains of different origin presented a similarity of ≥80 %. In total, 35 flaA-SVR alleles were detected. Alleles gt45, gt49 and gt57 were the most prevalent, in contrast with those frequently described in the PubMLST database. All 111 C. jejuni strains contained the genes flaA, flhA, cadF, docA, cdtA, cdtB, cdtC, iamA, ciaB, sodB, dnaJ, pldA, racR and csrA. The wlaN gene was detected in 11 strains (9.9 %), and the virB11 in just one strain (0.9 %). CONCLUSIONS: In conclusion, the pathogenic potential of the C. jejuni strains studied was highlighted by the high frequency of the majority of the virulence genes searched. The flaA-SVR sequencing and PFGE results showed that some of the strains studied presented a high genotypic similarity, suggesting potential for transmission between animal sources and humans in this country. Altogether, the results characterize further C. jejuni isolates from Brazil, an important producer and exporter of chicken meat.

Pesquisa fenotípica de resistência em amostras de Acinetobacter baumannii isoladas em um hospital de emergência

Espécies de Acinetobacter spp. têm emergido como importantes patógenos hospitalares e o A. baumannii tem se mostrado um dos patógenos gram-negativos mais difíceis de serem tratados, especialmente por serem microrganismos multi-resistentes. O A. baumannii tem grande importância clínica devido à tendência de transmissão cruzada, particularmente em UTI, onde numerosos surtos são encontrados. Vários mecanismos de resistência bacteriana são reconhecidos nestas bactérias e uma terapia combinada geralmente é necessária para o tratamento efetivo das infecções por Acinetobacter spp. Essas dificuldades terapêuticas em conjunto com o fato de essas bactérias terem uma capacidade de sobrevivência longa no ambiente hospitalar correspondem a um aumento na transmissão entre os pacientes, através de reservatórios humanos ou de materiais inanimados. O objetivo deste trabalho foi analisar fenotipicamente a presença de ESBL, MβL e AmpC em Acinetobacter baumannii isolados de amostras de urina e sangue de pacientes internados na Unidade de Emergência do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto - USP, no período de 2004 a 2010. De 49 amostras analisadas, 4 (8,16%) foram positivas para ESBL, 16 (32,65%) para MβL e 5 (10,2%) para AmpC. Na detecção de MβL, o EDTA apresentou melhor sensibilidade como agente quelante quando comparado com o MPA. Todas as 16 amostras positivas para MβL foram resistentes a ceftazidima. Os dados obtidos no presente trabalho são importantes para o conhecimento da prevalência de amostras produtoras de enzimas de resistência, bem como para auxiliar na implementação de medidas de controle de infecções hospitalares e no controle da disseminação dos mecanismos de resistência.