Genomic analysis of a cAmpC (CMY-41)-producing Citrobacter freundii ST64 isolated from patient.

Antibiotic resistance in Citrobacter freundii is a public health concern. This study evaluated the closed genome of a C. freundii isolated from the stool of a hospitalized patient initially related to a Salmonella outbreak. Confirmation of the isolate was determined by whole-genome sequencing. Nanopore sequencing was performed using a MinION with a Flongle flow cell. Assembly using SPAdes and Unicycler yielded a closed genome annotated by National Center for Biotechnology Information Prokaryotic Genome Annotation Pipeline. Genomic analyses employed MLST 2.0, ResFinder4.1, PlasmidFinder2.1, and VFanalyzer. Phylogenetic comparison utilized the Center for Food Safety and Applied Nutrition (CFSAN)-single nucleotide polymorphism pipeline and Genetic Algorithm for Rapid Likelihood Inference. Antimicrobial susceptibility was tested by broth microdilution following Clinical and Laboratory Standards Institute criteria. Multi-locus sequence type in silico analysis assigned the C. freundii as sequence type 64 and the blaCMY-41 gene was detected in resistome investigation. The susceptibility to antibiotics, determined using Sensititre® plates, revealed resistance to aztreonam, colistin, cefoxitin, amoxicillin/clavulanic acid, sulfisoxazole, ampicillin, and streptomycin. The genetic relatedness of the C. freundii CFSAN077772 with publicly available C. freundii genomes revealed a close relationship to a C. freundii SRR1186659, isolated in 2009 from human stool in Tanzania. In addition, C. freundii CFSAN077772 is nested in the same cluster with C. freundii clinical strains isolated in Denmark, Mexico, Myanmar, and Canada, suggesting a successful intercontinental spread.

Genomic and phylogenetic analysis of Salmonella enterica serovar Enteritidis strains linked to multiple outbreaks in Brazil.

Salmonella enterica subsp. enterica serovar Enteritidis (SE) has become the prevalent serovar isolated from gastroenteritis cases in Brazil since the 1990s. To better understand the genomic diversity and phylogenetic relationship amongst SE epidemic isolates from Brazil, 30 SE isolates from a variety of implicated foods and case patients of outbreaks between 1999 and 2006 were selected for genome comparison analyses. SE genomes were also compared against publicly available Brazilian SE isolates from pre- and postepidemic period. MLST analysis revealed that all isolates belong to sequence type (ST) 11. A total of seven Salmonella pathogenicity islands (SPIs) (SPI-1, SPI-3-5, SPI-13, SPI14, and C63PI) were identified in the evaluated genomes and all studied SE genomes carried similar prophage profiling. Resistome analysis revealed the presence of resistance genes to aminoglycosides [aac(6')laa, aph(3")-lb, aph(6)-ld], as well as point mutations in gyrA. Phylogenetic analysis demonstrated that certain isolates have circulated in Brazil for years and been involved in distinct outbreaks.

Whole genome sequencing analyses revealed that Salmonella enterica serovar Dublin strains from Brazil belonged to two predominant clades.

Salmonella Dublin is a cattle-associated serovar sporadically causing disease in humans. S. Dublin strains isolated in Brazil and in other countries were analyzed to determine their phylogenetic relationships, the presence of genes, plasmids, genomic regions related to virulence and antimicrobial resistance genes repertoire, using WGS analyses. Illumina was used to sequence the genome of 112 S. Dublin strains isolated in Brazil from humans (n = 82) and animals (n = 30) between 1983 and 2016. Furthermore, 87 strains from other countries were analyzed. WGSNP analysis revealed three different clades, in which the strains from Brazil belonged to two clades, A and C. Most of the genes and genomic regions searched varied among the strains studied. The siderophore genes iroB and iroC were exclusively found in strains from Brazil and pegD gene, related to fimbrial adherence determinants, were positive in 124 strains from clades A and B but absent in all the strains from clade C (n = 71). Eleven plasmid replicons were found in the strains from Brazil, and nine were exclusively found in strains from other countries. The antimicrobial resistance genes mdsA and mdsB, that encode an efflux pump, were found in all the strains studied. The strains from Brazil carried other resistance genes, such as tet(A) (n = 11), tet(B) (n = 4) and tet(C) (n = 4), blaTEM-1 (n = 4), catA1 (n = 1), aadA1 (n = 1), and sul1 (n = 1). In conclusion, S. Dublin strains isolated in Brazil presented some few unique genes not found in strains from other countries and were allocated into two distinct clades with strains of human and animal origin epidemiologically related. This fact stresses the zoonotic potential of S. Dublin circulating in Brazil for more than 30 years.

Genomic investigation of antimicrobial resistance determinants and virulence factors in Salmonella enterica serovars isolated from contaminated food and human stool samples in Brazil.

This study investigated the antimicrobial resistance determinants, virulence factors and identified serovars in 37 Salmonella enterica strains isolated from human stool and contaminated foods linked to outbreaks that occurred in Brazil over 7 years using whole genome sequencing (WGS). Phylogenetic analysis of selected serovars (S. Typhimurium, S. Infantis, S. London, and S. Johannesburg) was performed. Ten distinct serovars were identified and, 51% of the tested strains (n = 19) showed disagreement with the previous conventional serotyping. The antimicrobial resistance (AMR) determinants or plasmids varied among the strains. Resistome analysis revealed the presence of resistance genes to aminoglycosides [aac (6')-laa, aph (3″)-lb, aph (6)-ld, aadA1 and aadA2], sulfonamides (sul1), trimethoprin (dfrA8), fosfomycin (fosA7) and tetracyclines (tetA, tetB, tetC), as well as point mutations in parC (T57S) and gyrA (S83F). Plasmidome showed the presence of IncHI2, IncHI2A, IncFIB (S), IncFII (S), IncI1 and p0111 plasmids. Eight Salmonella pathogenicity islands and up to 102 stress and/or virulence genes were identified in the evaluated genomes. Virulence genes of K88 fimbrial adhesin were first reported in S. enterica (S. Pomona, S. Bredeney and S. Mbandaka strains). pilW gene was first identified in S. Pomona. Phylogenetic analysis showed that some serovars circulated in Brazil for decades, primarily within the poultry production chain. Findings highlighted the virulence and AMR determinants in strains that may lead to recurring food outbreaks.

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.

Successive exposure to Mentha piperita L. essential oil affects the culturability and induces membrane repair in a persister epidemic Salmonella Typhimurium PT4.

This study had as aims to evaluate the effects of successive exposures to Mentha piperita L. essential oil (MPEO) on culturability and physiological functions of Salmonella Typhimurium PT4. S. Typhimurium PT4 cells (108 log CFU/mL) were exposed to the same (1.25 µL/mL) or increasing MPEO concentrations (1.25-80 µL/mL) during 252 h. At each 36-h interval, the viable cell counts, and distinct cell functions were assessed using plate counting and flow cytometry, respectively. As the exposure time to the same MPEO concentration increased, the population of S. Typhimurium PT4 cells with damaged, permeabilized and depolarized membrane, and compromised efflux activity decreased. Otherwise, S. Typhimurium PT4 cells with damaged membrane physiological functions increased over the exposure to increasing concentrations of MPEO. Genomic analyses showed that the strain carries 17 genes associated with stress responses and the persistence of the tested strain among sources associated with poultry spanning more than 16 years and its virulence for humans. Therefore, successive exposure to a sublethal concentration of MPEO induced S. Typhimurium PT4 cells capable of maintaining the membrane integrity and its functions despite their non-culturable state.

Draft Genome Sequences of 30 Salmonella enterica Serovar Enteritidis Isolates Associated with Multiple Outbreaks in Brazil.

Salmonella enterica subsp. enterica serovar Enteritidis has been the prevalent serovar isolated from gastroenteritis cases in Brazil since the 1990s. Here, we report the draft genomes of 30 S Enteritidis isolates originating from a variety of patients and implicated foods during outbreaks between 1999 and 2006 in Brazil.

Draft Genome Sequences of 26 Salmonella Isolates Associated with Multiple Outbreaks in Brazil.

Salmonella enterica subsp. enterica isolates are the leading cause of foodborne illness worldwide. Here, we report the draft genomes of 26 Salmonella isolates of food and clinical origin, belonging to four serovars, associated with outbreaks from 1999 to 2006 in the south of Brazil.

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.

Changing of the Genomic Pattern of Salmonella Enteritidis Strains Isolated in Brazil Over a 48 year-period revealed by Whole Genome SNP Analyses.

Salmonella Enteritidis became the main serovar isolated from gastroenteritis cases in Brazil after the 90's. In this study we used whole genome sequence analysis to determine the phylogenetic relationships among a collection of strains isolated in Brazil to identify possible genomic differences between the strains isolated in the pre and post-epidemic period. Also, we compared our data from strains isolated in Brazil to strains available in the public domain from other South American countries. Illumina technology was used to sequence the genome of 256 Salmonella Enteritidis strains isolated over a 48 year-period in Brazil, comprising the pre- and post-epidemic period. Phylogenetic analyses revealed distinct lineages for strains isolated before and after 1994. Moreover, the phage region SE20 that may be related to the emergence of Salmonella Enteritidis worldwide was present only in strains of the post-epidemic cluster. In conclusion, our results showed that the genomic profile of Salmonella Enteritidis strains isolated in Brazil shifted after 1994, replaced by a global epidemic group of strains. It may be hypothesized that the presence of the prophage SE20 might have conferred to these strains a better ability to colonize chicken and consequently to infect and cause disease in humans, which might better explain the increase in the number of S. Enteritidis cases in Brazil and other South American countries. However, to verify this hypothesis further studies are needed.

Draft Genome Sequences of 112 Salmonella enterica Serovar Dublin Strains Isolated from Humans and Animals in Brazil.

Salmonella enterica serovar Dublin is a strongly adapted serovar that causes enteritis and/or systemic disease in cattle and results in high rates of mortality. Here, we report the draft genome sequences of 112 S. Dublin strains isolated from humans and animals in Brazil. These draft genome sequences will help enhance our understanding of this serovar in Brazil.

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.

Draft Genome Sequences of Yersinia frederiksenii, Yersinia intermedia, and Yersinia kristensenii Strains from Brazil.

Yersinia enterocolitica-like strains are usually understudied. In this work, we reported the draft genome sequences of two Yersinia frederiksenii, two Yersinia intermedia, and two Yersinia kristensenii strains isolated from humans, animals, food, and the environment in Brazil. These draft genomes will provide better molecular characterizations of these species.

Draft Genome Sequences of 256 Salmonella enterica subsp. enterica Serovar Enteritidis Strains Isolated from Humans, Food, Chickens, and Farm Environments in Brazil.

Salmonella enterica subsp. enterica serovar Enteritidis emerged in the late 1980s as the most isolated Salmonella serovar worldwide. Here, we report the draft genomes of 256 S Enteritidis strains isolated from humans, food, chickens, and farm environments in Brazil. These draft genomes will help enhance our understanding of this serovar in Brazil.