Genetic and biochemical characterization of BIM-1, a novel acquired subgroup B1 MBL found in a Pseudomonas sp. strain from the Brazilian Amazon region.

OBJECTIVES: To characterize a novel acquired MBL, BIM-1, in a Pseudomonas #2 (subgroup P. guariconensis) strain isolated from the Aurá river located in the Brazilian Amazon hydrographic basin. METHODS: WGS using an Illumina® MiSeq System was used to characterize the genome of Pseudomonas sp. IEC33019 strain. Southern blotting/hybridization assays were performed to confirm the location of the MBL-encoding gene, blaBIM-1 (Belém Imipenemase). Antimicrobial susceptibility testing, cloning, and biochemical and phenotypic characterization were performed to determine BIM-1 kinetics. RESULTS: The IEC33019 strain showed high resistance rates to ß-lactams, ciprofloxacin and aminoglycosides, being susceptible only to polymyxins and susceptible, increased exposure to aztreonam. WGS analysis revealed a novel acquired MBL-encoding gene, blaBIM-1, found as a gene cassette inserted into a class 1 integron (In1326) that also carried qnrVC1 and aadA11e. In1326 was located in a complex transposon, Tn7122, carried by a 52.7 kb conjugative plasmid (pIEC33019) with a toxin/antitoxin system (vapB/vapC). BIM-1 belongs to the molecular subgroup B1 and shares 70.2% and 64.9% similarity with SIM-1 and IMP-1, respectively. Kinetics analysis of BIM-1 showed hydrolytic activity against all ß-lactams tested. CONCLUSIONS: BIM-1 is a novel acquired MBL encoded by a gene carried by mobile genetic elements, which can be transferred to other Gram-negative bacilli (GNB). Because the IEC33019 strain was recovered from a river impacted by a populous metropolitan region with poor basic sanitation and served by limited potable freshwater, it would be important to establish the role of the BIM-1-producing GNB as nosocomial pathogens and/or as colonizers of the riverside population in this geographical region.

Molecular characterization of bacterial communities of two neotropical tick species (Amblyomma aureolatum and Ornithodoros brasiliensis) using rDNA 16S sequencing.

Ticks are one of the main vectors of pathogens for humans and animals worldwide. However, they harbor non-pathogenic microorganisms that are important for their survival, facilitating both their nutrition and immunity. We investigated the bacterial communities associated with two neotropical tick species of human and veterinary potential health importance from Brazil: Amblyomma aureolatum and Ornithodoros brasiliensis. In A. aureolatum (adult ticks collected from wild canids from Southern Brazil), the predominant bacterial phyla were Proteobacteria (98.68%), Tenericutes (0.70%), Bacteroidetes (0.14%), Actinobacteria (0.13%), and Acidobacteria (0.05%). The predominant genera were Francisella (97.01%), Spiroplasma (0.70%), Wolbachia (0.51%), Candidatus Midichloria (0.25%), and Alkanindiges (0.13%). The predominant phyla in O. brasiliensis (adults, fed and unfed nymphs collected at the environment from Southern Brazil) were Proteobacteria (90.27%), Actinobacteria (7.38%), Firmicutes (0.77%), Bacteroidetes (0.44%), and Planctomycetes (0.22%). The predominant bacterial genera were Coxiella (87.71%), Nocardioides (1.73%), Saccharopolyspora (0.54%), Marmoricola (0.42%), and Staphylococcus (0.40%). Considering the genera with potential importance for human and animal health which can be transmitted by ticks, Coxiella sp. was found in all stages of O. brasiliensis, Francisella sp. in all stages of A. aureolatum and in unfed nymphs of O. brasiliensis, and Rickettsia sp. in females of A. aureolatum from Banhado dos Pachecos (BP) in Viamão municipality, Brazil, and in females and unfed nymphs of O. brasiliensis. These results deepen our understanding of the tick-microbiota relationship in Ixodidae and Argasidae, driving new studies with the focus on the manipulation of tick microbiota to prevent outbreaks of tick-borne diseases in South America.

Emergence of carbapenem-resistant Acinetobacter pittii carrying the blaOXA-72 gene in the Amazon region, Brazil.

We sought to characterize the genetic context of blaOXA-72 gene in a carbapenem-resistant Acinetobacter pittii strain recovered from a hospitalized patient from Belém, North Brazil, in the Amazon region. We found that the blaOXA-72 gene was carried by a small plasmid, pIEC338SCox, that is 10,498 bp. The gene is flanked by XerC/XerD-like recombinase sites, which suggests that this gene was acquired onto this plasmid by recombination.

Do different probing depths exhibit striking differences in microbial profiles?

AIM: To perform a thorough characterization of the subgingival microbiota of shallow, moderate and deep sites in subjects with chronic periodontitis (ChP). MATERIAL AND METHODS: Subgingival samples were collected from subjects with ChP (n = 3/category of probing depth: ≤3, 4-6 and ≥7 mm) and periodontal health (PH). Individual samples were submitted to 16S rDNA high- throughput sequencing and the analysis was made using mothur and R packages. RESULTS: Nine subjects with ChP and seven with PH were included and 101 samples were evaluated. Thirteen phyla, 118 genera and 211 OTUs were detected. Taxa from Chloroflexi and Spirochaetes phyla were associated with initial stages of disease. Fretibacterium, Eubacterium[XI][G-6], Desulfobulbus, Peptostreptococcaceae[XI][G-1] and [G-3], Bacteroidetes[G-3], Bacteroidaceae[G-1] genera and Filifactor alocis, Fretibacterium fastidiosum, Johnsonella spHOT166, Peptostreptococcaceae[XIII][G-1]HOT113, Porphyromonas endodontalis and Treponema sp. HOT258, which are not conventionally associated with disease, increased with the deepening of the pockets and/or were elevated in ChP; while Streptococcus, Corynebacterium and Bergeyella genera were associated with PH (p < .05). CONCLUSION: Striking differences were observed between the microbiota of shallow and moderate/deep sites, but not between moderate and deep sites in ChP subjects. Differences between shallow sites in PH and ChP were also observed. The characterized microbiota included known oral microorganisms and newly identified periodontal taxa, some of them not-yet-cultured.

Complete Genome Sequence of Staphylococcus aureus FCFHV36, a Methicillin-Resistant Strain Heterogeneously Resistant to Vancomycin.

We report here the sequence of the entire chromosome of Staphylococcus aureus strain FCFHV36, a methicillin-resistant strain heterogeneously intermediate to vancomycin, bearing a type II staphylococcal chromosome cassette mec element (SCCmec), belonging to multilocus sequence type (MLST) 105, and isolated from a vertebra of a patient with osteomyelitis.

Complete Genome Sequence of Linezolid-Susceptible Staphylococcus haemolyticus Sh29/312/L2, a Clonal Derivative of a Linezolid-Resistant Clinical Strain.

We report the whole-genome sequence (WGS) of an in vitro susceptible derivative revertant mutant from a bloodstream isolate involved in a nosocomial outbreak in Brazil. The WGS comprises 2.5 Mb with 2,500 protein-coding sequences, 16rRNA genes, and 60 tRNA genes.

Complete Genome Sequence of Lactococcus lactis Strain AI06, an Endophyte of the Amazonian Açaí Palm.

We report the genome, in a single chromosome, of Lactococcus lactis strain AI06, isolated from the mesocarp of the açaí fruit (Euterpe oleracea) in eastern Amazonia, Brazil. This strain is an endophyte of the açaí palm and also a component of the microbiota of the edible food product.

Draft Genome Sequence of the Brazilian Cyanobium sp. Strain CACIAM 14.

Given the scarcity of data pertaining to whole-genome sequences of cyanobacterial strains isolated in Brazil, we hereby present the draft genome sequence of the Cyanobium sp. strain CACIAM 14, isolated in southeastern Amazonia.

Complete nucleotide sequences of two blaKPC-2-bearing IncN Plasmids isolated from sequence type 442 Klebsiella pneumoniae clinical strains four years apart.

We sequenced the oldest blaKPC-2-bearing plasmid isolated in Brazil and another plasmid also carried by a Klebsiella pneumoniae strain of sequence type 442 (ST442), isolated 52 months later. Both plasmids present an IncN backbone and few acquired regions. Because the 2005 plasmid presented deletions and a truncated gene within Tn4401b compared to the 2009 plasmid, we can thus infer that IncN blaKPC-2-bearing plasmids pFCF1305 and pFCF3SP had a common ancestor circulating in Brazil prior to May 2005.

Detection of Ehrlichia canis and Anaplasma platys DNA using multiplex PCR.

We hereby propose a novel sensitive, specific, and cost-efficient method to detect Ehrlichia canis and Anaplasma platys DNA from canine whole blood samples by multiplex PCR. Blood samples from hemoparasited dogs attending the Veterinary Hospital at the Universidade Federal Rural da Amazônia-UFRA, Belém, Brazil, were collected in tubes containing EDTA. Amplification of E. canis and A. platys 16S rDNA by nested (n) PCR was successfully achieved by using primers specific to the Anaplasmataceae in the first round of PCR, followed by a second round of PCR using E. canis-specific primers in conjunction with A. platys-specific primers. The amplicons obtained were cloned and sequenced, yielding sequences of 478 and 473 bp (including primers) pertaining to regions of the 16S rDNA of E. canis and A. platys, respectively. The protocol we here propose may help to measure the prevalence of canine monocytic ehrlichiosis (CME) and canine cyclic thrompocytopenia, not only in northern Brazil, where there is no data available, but also elsewhere.

Diversity and three-dimensional structures of the alpha Mcr of the methanogenic Archaea from the anoxic region of Tucuruí Lake, in Eastern Brazilian Amazonia.

Methanogenic archaeans are organisms of considerable ecological and biotechnological interest that produce methane through a restricted metabolic pathway, which culminates in the reaction catalyzed by the Methyl-coenzyme M reductase (Mcr) enzyme, and results in the release of methane. Using a metagenomic approach, the gene of the α subunit of mcr (mcrα) was isolated from sediment sample from an anoxic zone, rich in decomposing organic material, obtained from the Tucuruí hydroelectric dam reservoir in eastern Brazilian Amazonia. The partial nucleotide sequences obtained were 83 to 95% similar to those available in databases, indicating a low diversity of archaeans in the reservoir. Two orders were identified - the Methanomicrobiales, and a unique Operational Taxonomic Unit (OTU) forming a clade with the Methanosarcinales according to low bootstrap values. Homology modeling was used to determine the three-dimensional (3D) structures, for this the partial nucleotide sequence of the mcrα were isolated and translated on their partial amino acid sequences. The 3D structures of the archaean Mcrα observed in the present study varied little, and presented approximately 70% identity in comparison with the Mcrα of Methanopyrus klanderi. The results demonstrated that the community of methanogenic archaeans of the anoxic C1 region of the Tucurui reservoir is relatively homogeneous.

Diversity and three-dimensional structures of the alpha Mcr of the methanogenic Archaea from the anoxic region of Tucuruí Lake, in Eastern Brazilian Amazonia

Methanogenic archaeans are organisms of considerable ecological and biotechnological interest that produce methane through a restricted metabolic pathway, which culminates in the reaction catalyzed by the Methyl-coenzyme M reductase (Mcr) enzyme, and results in the release of methane. Using a metagenomic approach, the gene of the a subunit of mcr (mcrα) was isolated from sediment sample from an anoxic zone, rich in decomposing organic material, obtained from the Tucuruí hydroelectric dam reservoir in eastern Brazilian Amazonia. The partial nucleotide sequences obtained were 83 to 95 percent similar to those available in databases, indicating a low diversity of archaeans in the reservoir. Two orders were identified -the Methanomicrobiales, and a unique Operational Taxonomic Unit (OTU) forming a clade with the Methanosarcinales according to low bootstrap values. Homology modeling was used to determine the three-dimensional (3D) structures, for this the partial nucleotide sequence of the mcrα were isolated and translated on their partial amino acid sequences. The 3D structures of the archaean mcrα observed in the present study varied little, and presented approximately 70 percent identity in comparison with the mcrα of Methanopyrus klanderi. The results demonstrated that the community of methanogenic archaeans of the anoxic C1 region of the Tucurui reservoir is relatively homogeneous.

Whole genome sequencing of environmental Vibrio cholerae O1 from 10 nanograms of DNA using short reads.

Multiple Displacement Amplification (MDA) of DNA using φ29 (phi29) DNA polymerase amplifies DNA several billion-fold, which has proved to be potentially very useful for evaluating genome information in a culture-independent manner. Whole genome sequencing using DNA from a single prokaryotic genome copy amplified by MDA has not yet been achieved due to the formation of chimeras and skewed amplification of genomic regions during the MDA step, which then precludes genome assembly. We have hereby addressed the issue by using 10 ng of genomic Vibrio cholerae DNA extracted within an agarose plug to ensure circularity as a starting point for MDA and then sequencing the amplified yield using the SOLiD platform. We successfully managed to assemble the entire genome of V. cholerae strain LMA3984-4 (environmental O1 strain isolated in urban Amazonia) using a hybrid de novo assembly strategy. Using our method, only 178 out of 16,713 (1%) of contigs were not able to be inserted into either chromosome scaffold, and out of these 178, only 3 appeared to be chimeras. The other contigs seem to be the result of template-independent non-specific amplification during MDA, yielding spurious reads. Extraction of genomic DNA within an agarose plug in order to ensure circularity of the extracted genome might be key to minimizing amplification bias by MDA for WGS.

Rapid hybrid de novo assembly of a microbial genome using only short reads: Corynebacterium pseudotuberculosis I19 as a case study.

Due to the advent of the so-called Next-Generation Sequencing (NGS) technologies the amount of monetary and temporal resources for whole-genome sequencing has been reduced by several orders of magnitude. Sequence reads can be assembled either by anchoring them directly onto an available reference genome (classical reference assembly), or can be concatenated by overlap (de novo assembly). The latter strategy is preferable because it tends to maintain the architecture of the genome sequence the however, depending on the NGS platform used, the shortness of read lengths cause tremendous problems the in the subsequent genome assembly phase, impeding closing of the entire genome sequence. To address the problem, we developed a multi-pronged hybrid de novo strategy combining De Bruijn graph and Overlap-Layout-Consensus methods, which was used to assemble from short reads the entire genome of Corynebacterium pseudotuberculosis strain I19, a bacterium with immense importance in veterinary medicine that causes Caseous Lymphadenitis in ruminants, principally ovines and caprines. Briefly, contigs were assembled de novo from the short reads and were only oriented using a reference genome by anchoring. Remaining gaps were closed using iterative anchoring of short reads by craning to gap flanks. Finally, we compare the genome sequence assembled using our hybrid strategy to a classical reference assembly using the same data as input and show that with the availability of a reference genome, it pays off to use the hybrid de novo strategy, rather than a classical reference assembly, because more genome sequences are preserved using the former.

Type IV SCCmec found in decade old Brazilian MRSA isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) commonly causes infection in hospitalized patients. Since its appearance in the 1960s, the SCCmec has evolved throughout the years into 5 different types (I-V), each bearing a different set of genes. Infection with MRSA SCCmec types I, II or III is almost exclusively restricted to hospitalised patients. However, recently, community acquired MRSA (CA-MRSA) infections have been reported with increasing frequency, usually caused by a type IV SCCmec MRSA in nosocomial settings. We studied the prevalence of SCCmec types in 50 nosocomial strains collected from 1995 to 1999. The SCCmec complex type and presence of Panton-Valentine leukocidin (PVL) were determined by PCR. Strains had been previously typed by PFGE and were now typed by MLST. We found that 3 of the isolates studied bore a type IVc SCCmec all having different PFGE and MLST profiles (ST3, ST5 and ST88). All strains bearing a type III SCCmec belonged to MLST ST239 (Brazilian/Iberian clone). Only the strain which presented the ST5 profile bore the pvl gene. The type IVc SCCmec strains presented relatively lower levels of resistance to oxacillin in comparison to the type III SCCmec strains. The pattern of dissemination of the type IV SCCmec remains to be elucidated. The finding of strains carrying a type IV SCCmec in the present study among strains isolated at least 7 years ago indicates that clones bearing a type IV SCCmec have been present in Brazil for quite some time, and must have gone by undetected.

Type IV SCCmec found in decade old Brazilian MRSA isolates

Methicillin-resistant Staphylococcus aureus (MRSA) commonly causes infection in hospitalized patients. Since its appearance in the 1960s, the SCCmec has evolved throughout the years into 5 different types (I-V), each bearing a different set of genes. Infection with MRSA SCCmec types I, II or III is almost exclusively restricted to hospitalised patients. However, recently, community acquired MRSA (CA-MRSA) infections have been reported with increasing frequency, usually caused by a type IV SCCmec MRSA in nosocomial settings. We studied the prevalence of SCCmec types in 50 nosocomial strains collected from 1995 to 1999. The SCCmec complex type and presence of Panton-Valentine leukocidin (PVL) were determined by PCR. Strains had been previously typed by PFGE and were now typed by MLST. We found that 3 of the isolates studied bore a type IVc SCCmec all having different PFGE and MLST profiles (ST3, ST5 and ST88). All strains bearing a type III SCCmec belonged to MLST ST239 (Brazilian/Iberian clone). Only the strain which presented the ST5 profile bore the pvl gene. The type IVc SCCmec strains presented relatively lower levels of resistance to oxacillin in comparison to the type III SCCmec strains. The pattern of dissemination of the type IV SCCmec remains to be elucidated. The finding of strains carrying a type IV SCCmec in the present study among strains isolated at least 7 years ago indicates that clones bearing a type IV SCCmec have been present in Brazil for quite some time, and must have gone by undetected.

Avaliação da funcionalidade do locus accessory gene regulator (agr) em cepas de Staphylococcus aureus brasileiras com sensibilidade reduzida aos glicopeptídeos / Evaluation of the functionality of the accessory gene regulator (agr) in Brazilian Staphylococcus aureus strains with reduced susceptibility to vancomycin

O tratamento de infecções por Staphylococcus aureus tem sido problemático devido ao surgimento de cepas resistentes a múltiplios antibióticos. O antibiótico de escolha para o tratamento de infecções por S. aureus resistente a oxacilina é o glicopeptídeo vancomicina. Desde o primeiro isolamento de cepas com sensibilidade reduzida a vancomicina (VISA) em 1997, tem havido crescente preocupação com a disseminação da resistência a este antibiótico. Os mecanismos moleculares que levam à resistência de baixo nível a vancomicina ainda não foram elucidados. A detecção deste fenótipo na rotina de laboratório clínico é laboriosa, pois as técnicas disponíveis são de difícil execução e interpretação. Até agora, não há relato de transmissão horizontal de infecção por VISA, e todas as cepas com este fenótipo foram isoladas de pacientes que faziam o uso prolongado de vancomicina. Uma deficiência no locus regulador de genes acessórios (agr) foi postulado como fator de risco para a aquisição do fenótipo VISA por uma cepa sensível a este antibiótico. Para este estudo, foram selecionadas 47 cepas de S. aureus, com sensibilidades variadas a vancomicina, inclusive 5 cepas VISA isoladas no Brasil. Determinou-se nas cepas as concentrações inibitórias mínimas de vancomicina e oxacilina, a atividade hemolítica em ágar sangue de carneiro e de coelho, a capacidade de aderir ao poliestireno e o polimorfismo do locus agr. Determinou-se a integridade do locus agr por PCR-RFLP e sequenciamento de bases em 13 cepas representativas das 47 estudadas. A integridade do locus regulador acessório sarA também foi avaliada por sequenciamento de bases nestas 13 cepas. Foram escolhidas 18 cepas sensíveis a vancomicina com variadas características fenotípicas e estas foram submetidas à indução de resistência a vancomicina através da passagem seriada em concentrações crescentes deste antibiótico...

First isolation of metallo-beta-lactamase-producing multiresistant Klebsiella pneumoniae from a patient in Brazil.

A multiresistant Klebsiella pneumoniae isolate was taken from the blood of a 75-year-old patient with nosocomial pneumonia who developed septic shock and failed therapy with imipenem. The isolate presented an MIC of imipenem of 128 microg/ml, and the production of a metallo-beta-lactamase was confirmed by phenotypic and genotypic techniques. We here report, for the first time, the detection of a metalloenzyme (IMP-1)-producing K. pneumoniae clinical strain in Latin America. The gene responsible for this phenotype was found to be bla(IMP-1), carried in a class 1 integron.