Prediction and Analysis in silico of Genomic Islands in Aeromonas hydrophila.

Aeromonas are Gram-negative rods widely distributed in the environment. They can cause severe infections in fish related to financial losses in the fish industry, and are considered opportunistic pathogens of humans causing infections ranging from diarrhea to septicemia. The objective of this study was to determine in silico the contribution of genomic islands to A. hydrophila. The complete genomes of 17 A. hydrophila isolates, which were separated into two phylogenetic groups, were analyzed using a genomic island (GI) predictor. The number of predicted GIs and their characteristics varied among strains. Strains from group 1, which contains mainly fish pathogens, generally have a higher number of predicted GIs, and with larger size, than strains from group 2 constituted by strains recovered from distinct sources. Only a few predicted GIs were shared among them and contained mostly genes from the core genome. Features related to virulence, metabolism, and resistance were found in the predicted GIs, but strains varied in relation to their gene content. In strains from group 1, O Ag biosynthesis clusters OX1 and OX6 were identified, while strains from group 2 each had unique clusters. Metabolic pathways for myo-inositol, L-fucose, sialic acid, and a cluster encoding QueDEC, tgtA5, and proteins related to DNA metabolism were identified in strains of group 1, which share a high number of predicted GIs. No distinctive features of group 2 strains were identified in their predicted GIs, which are more diverse and possibly better represent GIs in this species. However, some strains have several resistance attributes encoded by their predicted GIs. Several predicted GIs encode hypothetical proteins and phage proteins whose functions have not been identified but may contribute to Aeromonas fitness. In summary, features with functions identified on predicted GIs may confer advantages to host colonization and competitiveness in the environment.

In silico comparative analysis of Aeromonas Type VI Secretion System.

Aeromonas are bacteria broadly spread in the environment, particularly in aquatic habitats and can induce human infections. Several virulence factors have been described associated with bacterial pathogenicity, such as the Type VI Secretion System (T6SS). This system translocates effector proteins into target cells through a bacteriophage-like contractile structure encoded by tss genes. Here, a total of 446 Aeromonas genome sequences were screened for T6SS and the proteins subjected to in silico analysis. The T6SS-encoding locus was detected in 243 genomes and its genes are encoded in a cluster containing 13 core and 5 accessory genes, in highly conserved synteny. The amino acid residues identity of T6SS proteins ranges from 78 to 98.8%. In most strains, a pair of tssD and tssI is located upstream the cluster (tssD-2, tssI-2) and another pair was detected distant from the cluster (tssD-1, tssI-1). Significant variability was seen in TssI (VgrG) C-terminal region, which was sorted in four groups based on its sequence length and protein domains. TssI containing ADP-ribosyltransferase domain are associated exclusively with TssI-1, while genes coding proteins carrying DUF4123 (a conserved domain of unknown function) were observed downstream tssI-1 or tssI-2 and escort of possible effector proteins. Genes coding proteins containing DUF1910 and DUF1911 domains were located only downstream tssI-2 and might represent a pair of toxin/immunity proteins. Nearly all strains display downstream tssI-3, that codes for a lysozyme family domain protein. These data reveal that Aeromonas T6SS cluster synteny is conserved and the low identity observed for some genes might be due to species heterogeneity or its niche/functionality.

Virulence characteristics and antimicrobial resistance of Aeromonas veronii biovar sobria 312M, a clinical isolate.

Aeromonas are bacteria widely distributed in the environment, and some species are able to cause infections in humans, of which diarrhea is the most common. The objective of this study was to evaluate the presence of virulence and antimicrobial resistance associated characteristics in A. veronii biovar sobria strain 312M isolated from diarrheal stools. For this, the genome sequencing and phenotypical tests were performed. The draft genome annotation revealed several complete pathways associated with carbon metabolism and a mucin-desulfating sulfatase which may contribute to intestine colonization, and a large number of virulence-associated genes encoding structures associated with adhesion, toxins, and secretion systems. The strain exhibited swimming and swarming motility, biofilm formation, and hemolytic activity. It was resistant to ampicillin, ampicillin/sulbactam, and amoxicillin-clavulanic acid. Although a cphA gene encoding a narrow-spectrum carbapenase was identified in the strain genome, no carbapenemase activity was detected in the antimicrobial susceptibility test. When compared with other A. veronii with complete genomes, the main differences in virulence characteristics are related to lateral flagella and type III and VI secretion systems; the antimicrobial resistance spectrum also varied among strains. The results indicated that A. veronii biovar sobria 312M presents high virulence potential and resistance to limited classes of antimicrobials.

Genome comparison between clinical and environmental strains of Herbaspirillum seropedicae reveals a potential new emerging bacterium adapted to human hosts.

BACKGROUND: Herbaspirillum seropedicae is an environmental ß-proteobacterium that is capable of promoting the growth of economically relevant plants through biological nitrogen fixation and phytohormone production. However, strains of H. seropedicae have been isolated from immunocompromised patients and associated with human infections and deaths. In this work, we sequenced the genomes of two clinical strains of H. seropedicae, AU14040 and AU13965, and compared them with the genomes of strains described as having an environmental origin. RESULTS: Both genomes were closed, indicating a single circular chromosome; however, strain AU13965 also carried a plasmid of 42,977 bp, the first described in the genus Herbaspirillum. Genome comparison revealed that the clinical strains lost the gene sets related to biological nitrogen fixation (nif) and the type 3 secretion system (T3SS), which has been described to be essential for interactions with plants. Comparison of the pan-genomes of clinical and environmental strains revealed different sets of accessorial genes. However, antimicrobial resistance genes were found in the same proportion in all analyzed genomes. The clinical strains also acquired new genes and genomic islands that may be related to host interactions. Among the acquired islands was a cluster of genes related to lipopolysaccharide (LPS) biosynthesis. Although highly conserved in environmental strains, the LPS biosynthesis genes in the two clinical strains presented unique and non-orthologous genes within the genus Herbaspirillum. Furthermore, the AU14040 strain cluster contained the neuABC genes, which are responsible for sialic acid (Neu5Ac) biosynthesis, indicating that this bacterium could add it to its lipopolysaccharide. The Neu5Ac-linked LPS could increase the bacterial resilience in the host aiding in the evasion of the immune system. CONCLUSIONS: Our findings suggest that the lifestyle transition from environment to opportunist led to the loss and acquisition of specific genes allowing adaptations to colonize and survive in new hosts. It is possible that these substitutions may be the starting point for interactions with new hosts.

Genome Analysis of Entomopathogenic Bacillus sp. ABP14 Isolated from a Lignocellulosic Compost.

We report the complete genome sequence of Bacillus sp. strain ABP14 isolated from lignocellulosic compost and selected by its ability in hydrolyzing carboxymethyl cellulose. This strain does not produce a Cry-like protein but showed an insecticidal activity against larvae of Anticarsia gemmatalis (Lepidoptera). Genome-based taxonomic analysis revealed that the ABP14 chromosome is genetically close to Bacillus thuringiensis serovar finitimus YBT020. ABP14 also carries one plasmid which showed no similarity with those from YBT020. Genome analysis of ABP14 identified unique genes related to cell surface structures, cell wall, metabolic competence, and virulence factors that may contribute for its survival and environmental adaptation, as well as its entomopathogenic activity.

Characteristics of an Aeromonas trota strain isolated from cerebrospinal fluid.

Aeromonas are ubiquitous in aquatic habitats. However some species can cause infections in humans, but rarely meningitis. Here we describe the isolation and characterization of an Aeromonas strain from cerebrospinal fluid of a meningitis patient. The isolate, identified as A. trota by biochemical and molecular methods, was susceptible to ampicillin but resistant to cephalothin and cefazolin. Genome sequencing revealed virulence factor genes such as type VI secretion system, aerolysin and lateral flagella. The isolate exhibited swarming motility, hemolytic activity and adhesion and cytotoxicity on HeLa cells. This is the first report of A. trota associated with meningitis and its virulence characteristics.

Utilization of carbon sources by clinical isolates of Aeromonas.

Bacteria in the genus Aeromonas are primarily aquatic organisms; however, some species can cause diseases in humans, ranging from wound infections to septicemia, of which diarrhea is the most common condition. The ability to use a variety of carbon substrates is advantageous for pathogenic bacteria. Therefore, we used Biolog GN2 microplates to analyze the ability of 103 clinical, predominantly diarrheal, isolates of Aeromonas to use various carbon sources, and we verified whether, among the substrates metabolized by these strains, there were some endogenous to the human intestine. The results indicate that Aeromonas present great diversity in the utilization of carbon sources, and that they preferentially use carbohydrates and amino acids as carbon sources. Among the carbon sources metabolized by Aeromonas in vitro, some were found to be components of intestinal mucin, including aspartic acid, glutamic acid, l-serine, galactose, N-acetyl-glucosamine, and glucose, which were used by all strains tested. Additionally, mannose, d-serine, proline, threonine, and N-acetyl-galactosamine were used by several strains. The potential to metabolize substrates endogenous to the intestine may contribute to Aeromonas' capacity to grow in and colonize the intestine. We speculate that this may help explain the ability of Aeromonas to cause diarrhea.

Qualidade físico-química e atividades biológicas de caseinofosfopeptídeos obtidos por hidrólise tríptica de caseinato de sódio sob diferentes condições / Physico-chemical quality and biological activities of Casein phosphopeptides obtained by sodium caseinate tryptic hydrolysis under different conditions

Muitos métodos têm sido empregados na produção de peptídeos bioativos para a promoção da saúde. O objetivo deste estudo foi produzir caseinofosfopeptídeos por hidrólise tríptica do caseinato de sódio usando diferentes temperaturas (37 e 50 °C) e tempos de reação (2 e 4 h), caracterizá-los e analisar sua influência nas atividades citotóxica, antimicrobiana e antioxidante. Caseinofosfopeptídeos foram caracterizados através da composição centesimal, eletroforese em gel de poliacrilamida, Espectrometria de Massas e Cromatografia Líquida de Alta Eficiência. Toxicidade para leucócitos humanos, atividade antimicrobiana utilizando o teste de microdiluição em caldo e determinação da capacidade antioxidante pelo método de espécies reativas ao ácido tiobarbitúrico foram os ensaios biológicos realizados. Os resultados mostraram que as quatro frações peptídicas obtidas apresentaram-se com baixo peso molecular e elevados teores proteico e mineral; quanto ao perfil aminoacídico, apresentaram elevadas e diferenciadas quantidades de ácido glutâmico e serina, que pouco variaram de acordo com o processo de obtenção; não se mostraram tóxicos para leucócitos humanos; demonstraram atividade antimicrobiana para Escherichia coli e Salmonella Enteritidis e elevada capacidade antioxidante. Os resultados físico-químicos das frações de caseinofosfopeptídeos demonstraram elevada composição nutricional em termos de proteína e, principalmente, cálcio. O conjunto de dados indicou que alterações no tempo e na temperatura de reação para a obtenção dos hidrolisados não interferem nas suas qualidades biológicas, mostrando serem seguros para a promoção da saúde e para a aplicação em situações especiais, que envolvem pacientes desnutridos, imunossuprimidos, com comprometimento ósseo ou gastrintestinal decorrentes de inflamações e infecções.

Several methods have been employed for the production of bioactive peptides for health promotion. The aim of this study was to produce and characterize Casein phosphopeptides obtained by sodium caseinate tryptic hydrolysis under different temperatures (37 and 50 °C) and reaction times (2 and 4 h), and evaluate their biological capabilities. They have been characterized by assessing their centesimal composition, by polyacrylamide gel electrophoresis, Mass Spectrometry, and High-Performance Liquid Chromatography. The biological activities tested included toxicity for human leukocytes, antimicrobial assay using the microdilution test, and determination of the antioxidant capacity by the thiobarbituric acid reactive species method. The results showed that the four fractions obtained were of low molecular weight with high protein and mineral contents; their amino acid profile showed high and differentiated amounts of glutamic acid and serine independent of the methodological procedures. The results also showed no toxicity for human peripheral leukocytes, demonstrated antimicrobial activity against Escherichia coli and Salmonella Enteritidis as well as high antioxidant capacity. The results of the physicochemical Casein phosphopeptides' fractions showed high nutritional composition in terms of protein and, particularly, calcium. The biological assays indicated that time and temperature changes in the process for obtaining casein hydrolysates have not interfered with their biological qualities. In addition, they have proven safe in promoting health in special conditions involving malnourished and/or, immunocompromised patients or those with bone and/or gastrointestinal impairment due to inflammations and infections.

Draft Genome Sequence of Aeromonas caviae 8LM, Isolated from Stool Culture of a Child with Diarrhea.

Aeromonas spp. are Gram-negative rods ubiquitous in aquatic environments; however, some species are able to cause a variety of infections in humans. Here, we report the draft genome sequence of Aeromonas caviae 8LM isolated from stool culture from a child with diarrhea in southern Brazil.

Biochemical characteristics, adhesion, and cytotoxicity of environmental and clinical isolates of Herbaspirillum spp.

Herbaspirillum bacteria are best known as plant growth-promoting rhizobacteria but have also been recovered from clinical samples. Here, biochemical tests, matrix-assisted laser deionization-time of flight (MALDI-TOF) mass spectrometry, adherence, and cytotoxicity to eukaryotic cells were used to compare clinical and environmental isolates of Herbaspirillum spp. Discrete biochemical differences were observed between human and environmental strains. All strains adhered to HeLa cells at low densities, and cytotoxic effects were discrete, supporting the view that Herbaspirillum bacteria are opportunists with low virulence potential.

Impact of Aeromonas and diarrheagenic Escherichia coli screening in patients with diarrhea in Paraná, southern Brazil.

INTRODUCTION: A wide diversity of bacterial agents may cause diarrhea, presenting challenges to clinical laboratories to define a diagnosis. Considering that most stool cultures are negative, we screened stool samples from patients with diarrhea for the presence of 14 bacterial enteropathogens, aiming to establish which of them should be included in routine stool analysis. METHODOLOGY: Stool samples from 400 patients with diarrhea were analyzed for the presence of Salmonella, Shigella, Campylobacter, Aeromonas, Plesiomonas shigelloides, Vibrio, Yersinia enterocolitica, and diarrheagenic Escherichia coli using conventional microbiological methods and PCR. Two distinct samples were studied; one included predominantly patients involved in outbreaks, and the other patients of low socioeconomic status presenting sporadic cases of diarrhea. RESULTS: In total, 86 cultures (21.5%) were positive. Mixed infections were found in five patients, leading to recovery of 91 strains of enteropathogenic bacteria: Salmonella Enteritidis (9.2%), Aeromonas (7.2%), diarrheagenic E. coli (5.2%), and C. jejuni (1%). However, Salmonella predominated, with 11.5% frequency in diarrhea outbreaks, while Aeromonas predominated among patients of low socioeconomic status, with 14.6% frequency. CONCLUSION: Aeromonas and diarrheagenic E. coli, which are not routinely screened for, deserve to be included in laboratory screening panels.

Detection of diarrheagenic Escherichia coli using a two-system multiplex-PCR protocol.

BACKGROUND: Diarrheagenic Escherichia coli (DEC) strains are important causes of diarrhea. However, they cannot be distinguished from E. coli of the intestinal microbiota by conventional microbiological tests. METHODS: This work presents a two-system multiplex PCR for detection of DEC. Primers for 16S rRNA gene were added as internal amplification control to validate negative reactions. The multiplex-PCR system 1 contains primers for detection of Shiga toxin producing E. coli (STEC; stx1, stx2), enteropathogenic E. coli (EPEC; eae, bfpA), atypical enteropathogenic E. coli (aEPEc; eae), enteroinvasive E. coli (ETEC; lt, st), enteroinvasive E. coli (EIEC; ial), and the internal amplification control 16S rRNA. The system 2 contains primers for EIEC (ipaH), enteroaggregative E. coli (CVD432), diffusely adherent E. coli (daaE), and 16S rRNA. The protocol was tested with E. coli reference strains, and also with cultures of fecal specimens of people with diarrhea and healthy controls. RESULTS: The protocol correctly identified the DEC reference strains. No DEC marker was amplified for negative controls; these results were validated by the amplification of a fragment of the 16S rRNA gene. The frequency of DEC was 7.6% for both patients and healthy controls; two Shigella sonnei strains were detected in the group with diarrhea. The identity of the amplicons was confirmed by DNA sequencing. CONCLUSION: The protocol is specific for DEC Shigella and is suitable for clinical laboratories.

The plasma logarithm of the triglyceride/HDL-cholesterol ratio is a predictor of low risk gestational diabetes in early pregnancy.

BACKGROUND: The plasma lipid profile changes atherogenically during normal pregnancy. Gestational diabetes mellitus (GDM) can exacerbate the changes in metabolism. The logarithm of the ratio triglycerides/HDL-cholesterol is an atherogenic index of the plasma (AIP) and can be used as a marker for plasma atherogenicity. METHODS: Serum of 576 unrelated Euro-Brazilian pregnant women was collected and the subjects were classified as healthy pregnant women (control, n=288) and gestational diabetic patients (GDM, n=288) according to the ADA 2010 criteria. Both studied groups were sub classified in 4 gestational periods: (i) 12-23, (ii) 24-28, (iii) 29-32 and (iv) >32 weeks of gestation. RESULTS: Except for the AIP, the other parameters showed low discrimination between control and GDM groups (ROC curves). When analyzed by ROC curves the AIP of subjects in the early period of gestation showed sensitivity and specificity of 82.6% and 83.4%, respectively, with a cut-off point of 0.099 (AUC 0.886, P<0.0001). CONCLUSIONS: The AIP is a valuable index to identify pregnant women with low risk of gestational diabetes before 24 weeks of gestation.

Morphological and molecular identification of filamentous fungi isolated from cosmetic powders

Seven fungi were isolated from 50 samples of cosmetic powders. Morphological analyses and ribosomal DNA Internal Transcribed Spacers sequencing were performed which allowed the discrimination of the isolated fungi as Aspergillus fumigatus, Penicillium sp., and Cladosporium sp. which could have, among their species, potentially pathogenic microorganisms.

A prospective study on Aeromonas in outpatients with diarrhea in the central region of Rio Grande do Sul State

Aeromonas spp. were identified in five (2,7%) of 182 diarrheal stool cultures, A. caviae was predominant, resistant mainly to ampicillin and cephalotin. This is the first study showing the presence of Aeromonas spp. in diarrheal stools of outpatients in the central region of Rio Grande do Sul State, Brazil.

A prospective study on Aeromonas in outpatients with diarrhea in the central region of Rio Grande do Sul State.

Aeromonas spp. were identified in five (2,7%) of 182 diarrheal stool cultures, A. caviae was predominant, resistant mainly to ampicillin and cephalotin. This is the first study showing the presence of Aeromonas spp. in diarrheal stools of outpatients in the central region of Rio Grande do Sul State, Brazil.

Biochemical properties, enterohaemolysin production and plasmid carriage of Shiga toxin-producing Escherichia coli strains.

Thirty-eight strains of Shiga toxin-producing Escherichia coli (STEC) were characterised in terms of biochemical properties, enterohaemolysin production and plasmid carriage. A wide variation in the biochemical properties was observed among the STEC, with 14 distinct biotypes identified. Biotype 1 was the most common, found in 29% of the strains. Enterohaemolysin production was detected in 29% of the strains. Most of the bacterial strains (95%) carried one or more plasmids and considerable heterogeneity in size and combinations was observed. Seven distinct plasmid profiles were identified. The most common profile, characterised by the presence of a single plasmid of ~90 kb, was found in 50% of these strains. These data indicate extensive diversity among STEC strains. No correlation was found among biotype, serotype, enterohaemolysin production and plasmid profile.

Biochemical properties, enterohaemolysin production and plasmid carriage of Shiga toxin-producing Escherichia coli strains

Thirty-eight strains of Shiga toxin-producing Escherichia coli (STEC) were characterised in terms of biochemical properties, enterohaemolysin production and plasmid carriage. A wide variation in the biochemical properties was observed among the STEC, with 14 distinct biotypes identified. Biotype 1 was the most common, found in 29 percent of the strains. Enterohaemolysin production was detected in 29 percent of the strains. Most of the bacterial strains (95 percent) carried one or more plasmids and considerable heterogeneity in size and combinations was observed. Seven distinct plasmid profiles were identified. The most common profile, characterised by the presence of a single plasmid of ~90 kb, was found in 50 percent of these strains. These data indicate extensive diversity among STEC strains. No correlation was found among biotype, serotype, enterohaemolysin production and plasmid profile.