RESUMO
OBJECTIVES: Elucidating the recent evolutionary history of clinically important antibiotic resistance genes may inform measures to delay the future emergence of additional resistance genes in clinics. This study investigated the recent origin of blaAIM-1, a metallo-ß-lactamase gene found in Pseudomonas aeruginosa, and the possible role of ISCR15 in its mobilisation and transfer into clinical species. METHODS: Comparative genomics were used to identify the recent origin of blaAIM. Mobilisation attempts were performed under different conditions by cloning ISCR15 and the blaAIM-1-like gene in Escherichia coli. RESULTS: Several blaAIM-1 homologues were identified in the Pseudoxanthomonas genus, with conserved synteny of the locus between species and absence of elements associated with mobility. The closest AIM-1 homologue (97.7% amino acid identity) was found in a Pseudoxanthomonas mexicana (P. mexicana) strain. Cloning the blaAIM-like gene in Escherichia coli resulted in high resistance towards carbapenems. While blaAIM-1 is surrounded by ISCR15 elements in clinical strains, in vitro experiments failed to demonstrate their role as mobilising elements. CONCLUSIONS: This study presents evidence that P. mexicana, an environmental species occasionally associated with infections, is the origin of the B3 metallo-ß-lactamase AIM-1. The presence of terIS, a plausible recognition site for ISCR15, in other parts of the P. mexicana genome suggests a more complex and yet not understood mobilisation mechanism.
Assuntos
Proteínas de Bactérias , beta-Lactamases , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/genética , Xanthomonadaceae , beta-Lactamases/metabolismoRESUMO
The emergence of antibiotic resistance in retort to environmental pollutants during wastewater treatment still remains elusive. Here, we first to investigate the emergence of antibiotic resistance in an environmental non-pathogenic bacterium, Pseudoxanthomonas mexicana isolated from a lab-scale bioreactor treating wastewater containing streptomycin. The molecular mechanism of antibiotic resistance development was evaluated in its genomic, transcriptional, and proteomic levels. The streptomycin resistant (SR) strain showed strong resistance to streptomycin (MIC > 600 µg/mL) as well to sulfamethoxazole, ampicillin, and kanamycin (≥250 µg/mL). A 13.4 kb class-1-integron array consisting of a new arrangement of gene cassette (IS6100-sul1-aadA2-catB3-aacA1-2-aadB-int1-IS256-int) linked with Tn5393c transposon was identified in the SR strain, which has only been reported in clinical pathogens so far. iTRAQ-LC-MS/MS proteomics revealed 22 up-regulated proteins in the SR strain growing under 100 mg L-1 streptomycin, involving antibiotic resistance, toxin production, stress response, and ribosomal protein synthesis. At the mRNA level, elevated expressions of ARGs (strA, strB, and aadB) and 30S-ribosomal protein genes (rpsA and rpsU) were observed in the SR strain. The results highlighted the genomic plasticity and multifaceted regulatory mechanism employed by P. mexicana in adaptation to high-level streptomycin during biological wastewater treatment.
Assuntos
Estreptomicina , Águas Residuárias , Antibacterianos/farmacologia , Reatores Biológicos , Cromatografia Líquida , Farmacorresistência Bacteriana Múltipla/genética , Testes de Sensibilidade Microbiana , Proteômica , Estreptomicina/farmacologia , Espectrometria de Massas em Tandem , XanthomonadaceaeRESUMO
BACKGROUND: Hot spring bacteria have unique biological adaptations to survive the extreme conditions of these environments; these bacteria produce thermostable enzymes that can be used in biotechnological and industrial applications. However, sequencing these bacteria is complex, since it is not possible to culture them. As an alternative, genome shotgun sequencing of whole microbial communities can be used. The problem is that the classification of sequences within a metagenomic dataset is very challenging particularly when they include unknown microorganisms since they lack genomic reference. We failed to recover a bacterium genome from a hot spring metagenome using the available software tools, so we develop a new tool that allowed us to recover most of this genome. RESULTS: We present a proteobacteria draft genome reconstructed from a Colombian's Andes hot spring metagenome. The genome seems to be from a new lineage within the family Rhodanobacteraceae of the class Gammaproteobacteria, closely related to the genus Dokdonella. We were able to generate this genome thanks to CLAME. CLAME, from Spanish "CLAsificador MEtagenomico", is a tool to group reads in bins. We show that most reads from each bin belong to a single chromosome. CLAME is very effective recovering most of the reads belonging to the predominant species within a metagenome. CONCLUSIONS: We developed a tool that can be used to extract genomes (or parts of them) from a complex metagenome.
Assuntos
Algoritmos , Genoma Bacteriano , Metagenômica , Análise de Sequência de DNA/métodos , Xanthomonadaceae/classificação , Xanthomonadaceae/genética , Colômbia , Genes Bacterianos , Marcadores Genéticos , Microbiota , FilogeniaRESUMO
Dipeptidyl aminopeptidase IV (DAP IV or DPP IV) from Pseudoxanthomonas mexicana WO24 (PmDAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position [NH2-P2-P1(Pro/Ala)-P1'-P2' ]. For crystallographic studies, the periplasmic form of PmDAP IV was overproduced in Escherichia coli, purified and crystallized in complex with the tripeptide Lys-Pro-Tyr using the hanging-drop vapour-diffusion method. Kinetic parameters of the purified enzyme against a synthetic substrate were also determined. X-ray diffraction data to 1.90â Å resolution were collected from a triclinic crystal form belonging to space group P1, with unit-cell parameters a = 88.66, b = 104.49, c = 112.84â Å, α = 67.42, ß = 68.83, γ = 65.46°. Initial phases were determined by the molecular-replacement method using Stenotrophomonas maltophilia DPP IV (PDB entry 2ecf) as a template and refinement of the structure is in progress.
Assuntos
Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/isolamento & purificação , Periplasma/enzimologia , Xanthomonadaceae/enzimologia , Cristalização , Cristalografia por Raios X , Dipeptidil Peptidase 4/metabolismo , Cinética , Conformação Proteica , Xanthomonadaceae/classificaçãoRESUMO
The Xanthomonadaceae family consists of species of non-pathogenic and pathogenic γ-proteobacteria that infect different hosts, including humans and plants. In this study, we performed a comparative analysis using 69 fully sequenced genomes belonging to this family, with a focus on identifying proteins enriched in phytopathogens that could explain the lifestyle and the ability to infect plants. Using a computational approach, we identified seven phytopathogen-enriched protein families putatively secreted by type II secretory system: PheA (CM-sec), LipA/LesA, VirK, and four families involved in N-glycan degradation, NixE, NixF, NixL, and FucA1. In silico and phylogenetic analyses of these protein families revealed they all have orthologs in other phytopathogenic or symbiotic bacteria, and are involved in the modulation and evasion of the immune system. As a proof of concept, we performed a biochemical characterization of LipA from Xac306 and verified that the mutant strain lost most of its lipase and esterase activities and displayed reduced virulence in citrus. Since this study includes closely related organisms with distinct lifestyles and highlights proteins directly related to adaptation inside plant tissues, novel approaches might use these proteins as biotechnological targets for disease control, and contribute to our understanding of the coevolution of plant-associated bacteria.
Assuntos
Proteínas de Bactérias/metabolismo , Doenças das Plantas/microbiologia , Xanthomonadaceae/metabolismo , Xanthomonadaceae/patogenicidade , Proteínas de Bactérias/genética , Filogenia , VirulênciaRESUMO
Wohlfahrtiimonas chitiniclastica is an emerging zoonotic bacterium commensally living in larvae of particular flies. It has been associated with human and animal infections but never isolated from food. In the present study, a whole chicken carcass was rinsed in buffered peptone water which was then inoculated into BHI and the growth plated onto selective medium. Species identification was performed by MALDI-TOF MS. Those bacteria identified as W. chitiniclastica were subjected to 16S rRNA sequencing for confirmation and MEGA software was used to obtain their phylogenetic position. The findings of this study raise concerns regarding the abattoir, transport and stock practices of frozen meat carcasses and should be of interest with regard to microbiology, entomology and food production.
Assuntos
Galinhas/microbiologia , Alimentos Congelados/microbiologia , Carne/microbiologia , Xanthomonadaceae/classificação , Xanthomonadaceae/isolamento & purificação , Zoonoses/microbiologia , Animais , Brasil , Doenças Transmissíveis Emergentes/microbiologia , DNA Bacteriano/genética , Dípteros/microbiologia , Manipulação de Alimentos , Microbiologia de Alimentos , Filogenia , RNA Ribossômico 16S/genética , Infecção dos Ferimentos/microbiologia , Xanthomonadaceae/química , Xanthomonadaceae/genéticaRESUMO
Dipeptidyl aminopeptidase BII from Pseudoxanthomonas mexicana WO24 (DAP BII) is able to cleave a variety of dipeptides from the amino-terminus of substrate peptides. For crystallographic studies, DAP BII was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data to 2.3 Å resolution were collected using an orthorhombic crystal form belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 76.55, b = 130.86, c = 170.87 Å. Structural analysis by the multi-wavelength anomalous diffraction method is in progress.
Assuntos
Cristalografia por Raios X/métodos , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Xanthomonadaceae/enzimologia , Cristalização , Conformação Proteica , Proteínas Recombinantes/químicaRESUMO
We report the first case of fulminant sepsis due to Wohlfahrtiimonas chitiniclastica. This case is also the first one reported in South America. We emphasize the importance of recognizing bacteria that live in the larvae of a parasitic fly as the causative agent of severe infections in homeless patients.
Assuntos
Infecções por Bactérias Gram-Negativas/diagnóstico , Sepse/microbiologia , Xanthomonadaceae/isolamento & purificação , Idoso , Animais , Antibacterianos/farmacologia , Técnicas de Tipagem Bacteriana , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Dípteros/microbiologia , Infecções por Bactérias Gram-Negativas/microbiologia , Pessoas Mal Alojadas , Humanos , Masculino , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , América do Sul , Xanthomonadaceae/classificação , Xanthomonadaceae/genética , Xanthomonadaceae/fisiologiaRESUMO
OBJECTIVE: The aim of this study was to correlate the presence of Enterobacteriaceae, Pseudomonadaceae, Moraxellaceae and Xanthomonadaceae on the posterior dorsum of the human tongue with the presence of tongue coating, gender, age, smoking habit and denture use. MATERIAL AND METHODS: Bacteria were isolated from the posterior tongue dorsum of 100 individuals in MacConkey agar medium and were identified by the API 20E system (Biolab-Mérieux). RESULTS: 43% of the individuals, presented the target microorganisms on the tongue dorsum, with greater prevalence among individuals between 40 and 50 years of age (p = 0.001) and non-smokers (p=0.0485). CONCLUSIONS: A higher prevalence of Enterobacteriaceae and Pseudomonadaceae was observed on the tongue dorsum of the individuals evaluated. There was no correlation between these species and the presence and thickness of tongue coating, gender and presence of dentures.
Assuntos
Enterobacteriaceae/isolamento & purificação , Pseudomonadaceae/isolamento & purificação , Língua/microbiologia , Adulto , Fatores Etários , Contagem de Colônia Microbiana , Prótese Total/microbiologia , Prótese Parcial Fixa/microbiologia , Prótese Parcial Removível/microbiologia , Dentaduras , Enterobacter cloacae/isolamento & purificação , Enterobacteriaceae/classificação , Feminino , Halitose/microbiologia , Humanos , Masculino , Mannheimia haemolytica/isolamento & purificação , Pessoa de Meia-Idade , Moraxellaceae/classificação , Moraxellaceae/isolamento & purificação , Higiene Bucal , Pasteurella pneumotropica/isolamento & purificação , Pseudomonadaceae/classificação , Fumar , Língua/patologia , Xanthomonadaceae/classificação , Xanthomonadaceae/isolamento & purificaçãoRESUMO
OBJECTIVE: The aim of this study was to correlate the presence of Enterobacteriaceae, Pseudomonadaceae, Moraxellaceae and Xanthomonadaceae on the posterior dorsum of the human tongue with the presence of tongue coating, gender, age, smoking habit and denture use. MATERIAL AND METHODS: Bacteria were isolated from the posterior tongue dorsum of 100 individuals in MacConkey agar medium and were identified by the API 20E system (Biolab-Mérieux). RESULTS: 43 percent of the individuals, presented the target microorganisms on the tongue dorsum, with greater prevalence among individuals between 40 and 50 years of age (p = 0.001) and non-smokers (p=0.0485). CONCLUSIONS: A higher prevalence of Enterobacteriaceae and Pseudomonadaceae was observed on the tongue dorsum of the individuals evaluated. There was no correlation between these species and the presence and thickness of tongue coating, gender and presence of dentures.
Assuntos
Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Enterobacteriaceae/isolamento & purificação , Pseudomonadaceae/isolamento & purificação , Língua/microbiologia , Fatores Etários , Contagem de Colônia Microbiana , Dentaduras , Prótese Total/microbiologia , Prótese Parcial Fixa/microbiologia , Prótese Parcial Removível/microbiologia , Enterobacter cloacae/isolamento & purificação , Enterobacteriaceae/classificação , Halitose/microbiologia , Mannheimia haemolytica/isolamento & purificação , Moraxellaceae/classificação , Moraxellaceae/isolamento & purificação , Higiene Bucal , Pasteurella pneumotropica/isolamento & purificação , Pseudomonadaceae/classificação , Fumar , Língua/patologia , Xanthomonadaceae/classificação , Xanthomonadaceae/isolamento & purificaçãoRESUMO
The role of lateral gene transfer (LGT) in prokaryotes has been shown to rapidly change the genome content, providing new gene tools for environmental adaptation. Features related to pathogenesis and resistance to strong selective conditions have been widely shown to be products of gene transfer between bacteria. The genomes of the gamma-proteobacteria from the genus Xanthomonas, composed mainly of phytopathogens, have potential genomic islands that may represent imprints of such evolutionary processes. In this work, the evolution of genes involved in the pathway responsible for arginine biosynthesis in Xanthomonadales was investigated, and several lines of evidence point to the foreign origin of the arg genes clustered within a potential operon. Their presence inside a potential genomic island, bordered by a tRNA gene, the unusual ranking of sequence similarity, and the atypical phylogenies indicate that the metabolic pathway for arginine biosynthesis was acquired through LGT in the Xanthomonadales group. Moreover, although homologues were also found in Bacteroidetes (Flavobacteria group), for many of the genes analyzed close homologues are detected in different life domains (Eukarya and Archaea), indicating that the source of these arg genes may have been outside the Bacteria clade. The possibility of replacement of a complete primary metabolic pathway by LGT events supports the selfish operon hypothesis and may occur only under very special environmental conditions. Such rare events reveal part of the history of these interesting mosaic Xanthomonadales genomes, disclosing the importance of gene transfer modifying primary metabolism pathways and extending the scenario for bacterial genome evolution.
Assuntos
Arginina/biossíntese , Transferência Genética Horizontal , Óperon , Xanthomonadaceae/genética , Arginina/genéticaRESUMO
Three mesophilic bacteria (strains AMX 26B(T), UR374_02 and 12-3(T)) isolated respectively from an anaerobic digester, human urine and urban riverside soil were characterized. Cells were Gram-negative, motile, non-sporulating, straight to curved rods with one polar flagellum and had a strictly respiratory metabolism with O(2) as the preferential terminal electron acceptor. Phylogenetic analysis based on 16S rRNA gene sequences revealed that all strains clustered within the Xanthomonadaceae branch of the Proteobacteria. Isolates AMX 26B(T) and UR374_02 exhibited 100 % 16S rRNA gene sequence similarity and both were related to strain 12-3(T) (99.6 % similarity). The closest relative of all the isolates was Pseudoxanthomonas broegbernensis DSM 12573(T) (similarity 97.1-97.5 %), and they were equidistantly related to Xanthomonas species (95.4-96.6 %), Stenotrophomonas species (95.3-96.1 %) and Pseudoxanthomonas taiwanensis ATCC BAA-4040(T) (95.3-95.4 %). Chemotaxonomic and biochemical data (branched-chain cellular fatty acid pattern without C(13 : 0) iso 3-OH, ubiquinone with eight isoprenoid units, limited range of substrates used, ability to reduce nitrite but not nitrate with the production of N(2)O) supported their affiliation to the genus Pseudoxanthomonas. The results of DNA-DNA hybridization and/or phenotypic analysis allowed them to be differentiated from the two Pseudoxanthomonas species with validly published names and showed that strain 12-3(T) was genomically and phenotypically distinct from the other two isolates. On the basis of these results, two novel species of the genus Pseudoxanthomonas are proposed: Pseudoxanthomonas mexicana sp. nov., consisting of strains AMX 26B(T) (=ATCC 700993(T)=CIP 106674(T)=JCM 11524(T)) (type strain) and UR374_02 (=DSM 15133), and Pseudoxanthomonas japonensis sp. nov., consisting of strain 12-3(T) (=CCUG 48231(T)=CIP 107388(T)=JCM 11525(T)). The report of these two novel species leads to the emendation of the description of the genus Pseudoxanthomonas and the re-evaluation of the phenotype of P. broegbernensis DSM 12573(T) necessitates the emendation of its description.