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.

Keratinocyte-intrinsic MHCII expression controls microbiota-induced Th1 cell responses.

The cross-talk between the microbiota and the immune system plays a fundamental role in the control of host physiology. However, the tissue-specific factors controlling this dialogue remain poorly understood. Here we demonstrate that T cell responses to commensal colonization are associated with the development of organized cellular clusters within the skin epithelium. These organized lymphocyte clusters are surrounded by keratinocytes expressing a discrete program associated with antigen presentation and antimicrobial defense. Notably, IL-22-mediated keratinocyte-intrinsic MHC class II expression was required for the selective accumulation of commensal-induced IFN-γ, but not IL-17A-producing CD4+ T cells within the skin. Taking these data together, this work uncovers an unexpected role for MHC class II expression by keratinocytes in the control of homeostatic type 1 responses to the microbiota. Our findings have important implications for the understanding of the tissue-specific rules governing the dialogue between a host and its microbiota.

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.

Influence of glycemic control on the levels of subgingival periodontal pathogens in patients with generalized chronic periodontitis and type 2 diabetes.

OBJECTIVE: This study evaluated the influence of glycemic control on the levels and frequency of subgingival periodontal pathogens in patients with type 2 diabetes mellitus (DM) and generalized chronic periodontitis (ChP). MATERIAL AND METHODS: Fifty-six patients with generalized ChP and type 2 DM were assigned according to the levels of glycated hemoglobin (HbA1c) into one of the following groups: HbA1c<8% (n=28) or HbA1c≥8% (n=28). Three subgingival biofilm samples from sites with probing depth (PD)<5 mm and three samples from sites with PD≥5 mm were analyzed by quantitative Polymerase Chain Reaction (PCR) for the presence and levels of Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Eubacterium nodatum, Parvimona micra, Fusobacterium nucleatum ssp. and Prevotella intermedia. RESULTS: The mean counts of F. nucleatum ssp. were statistically significantly higher in the sites with PD≥5 mm of the HbA1c≥8% group (p<0.05). Frequencies of detection of T. forsythia, E. nodatum, P. micra and F. nucleatum ssp. were all higher in the sites with PD≥5 mm of the patients with HbA1c≥8%, compared with those of patients with HbA1c<8% (p<0.05). Frequency of detection of P. intermedia was higher in the sites with PD<5 mm of the patients with HbA1c≥8% than those of the patients with HbA1c<8% (p<0.05). CONCLUSIONS: Poor glycemic control, as indicated by HbA1c≥8%, is associated with increased levels and frequencies of periodontal pathogens in the subgingival biofilm of subjects with type 2 DM and ChP.

Influence of glycemic control on the levels of subgingival periodontal pathogens in patients with generalized chronic periodontitis and type 2 diabetes

Abstract Objective This study evaluated the influence of glycemic control on the levels and frequency of subgingival periodontal pathogens in patients with type 2 diabetes mellitus (DM) and generalized chronic periodontitis (ChP). Material and Methods Fifty-six patients with generalized ChP and type 2 DM were assigned according to the levels of glycated hemoglobin (HbA1c) into one of the following groups: HbA1c<8% (n=28) or HbA1c≥8% (n=28). Three subgingival biofilm samples from sites with probing depth (PD)<5 mm and three samples from sites with PD≥5 mm were analyzed by quantitative Polymerase Chain Reaction (PCR) for the presence and levels of Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Eubacterium nodatum, Parvimona micra, Fusobacterium nucleatum ssp. and Prevotella intermedia. Results The mean counts of F. nucleatum ssp. were statistically significantly higher in the sites with PD≥5 mm of the HbA1c≥8% group (p<0.05). Frequencies of detection of T. forsythia, E. nodatum, P. micra and F. nucleatum ssp. were all higher in the sites with PD≥5 mm of the patients with HbA1c≥8%, compared with those of patients with HbA1c<8% (p<0.05). Frequency of detection of P. intermedia was higher in the sites with PD<5 mm of the patients with HbA1c≥8% than those of the patients with HbA1c<8% (p<0.05). Conclusions Poor glycemic control, as indicated by HbA1c≥8%, is associated with increased levels and frequencies of periodontal pathogens in the subgingival biofilm of subjects with type 2 DM and ChP.

Comparison of independent and dependent culture methods for the detection of transient bacteremia in diabetic subjects with chronic periodontitis.

INTRODUCTION: Oral-derived bacteremia may occur after several dental procedures and routine daily activities. Some conditions of the oral cavity may favor episodes of bacteremia. This would be the case of patients with diabetes mellitus and periodontitis, who exhibit exacerbated gingival inflammation and may be more prone to developing oral-derived bacteremia.  OBJECTIVE: To compare the effectiveness of an independent culture method (quantitative real-time PCR- qCR) and the most commonly used method (BacT-ALERT 3D®) for the diagnosis of bacteremia.  MATERIALS AND METHODS: Blood samples were drawn from subjects with type 2 diabetes mellitus and chronic periodontitis before and after apple chewing. Samples were processed by an automated blood culture system (BacT-ALERT 3D®) monitored for 15 days with suitable subculture of positive cultures. In parallel, whole DNA from blood samples was purified using a commercial kit and screened by qPCR using a universal primer set of16S rDNA for bacteria detection.  RESULTS: Blood cultures taken before apple chewing were shown to be negative by the two diagnostic methods. After chewing, two samples (11%) showed bacterial growth by BacT-ALERT 3D® whereas qPCR did not detect the presence of bacteria in any sample.  CONCLUSIONS: qPCR did not show greater effectiveness than the BacT-ALERT 3D® in the detection of bacteremia of oral origin.

The Current Weight of Evidence of the Microbiologic Profile Associated With Peri-Implantitis: A Systematic Review.

BACKGROUND: There is currently no consensus regarding microorganisms that may be considered true peri-implant pathogens. Therefore, the aim of this systematic review is to determine the weight of evidence for microorganisms related to peri-implantitis based on results of association studies. METHODS: This review was performed following the Preferred Reporting Items for Systematic Reviews and MetaAnalyses (PRISMA). Two independent researchers searched PubMed/Medline, Embase, and Cochrane Library databases up to August 4, 2015, for studies comparing microbiologic outcomes of subgingival biofilm samples from healthy implants and implants with peri-implantitis. RESULTS: A total of 799 titles was identified and 11 studies were included in this review. All data were extracted using a predefined form. Microorganisms found in increased count/abundance/frequency in peri-implantitis belonged to Bacteria domain and viruses, and included a total of six bacterial phyla, 17 bacterial genera, 23 bacterial species, and two genera of viruses. The main bacterial species associated with peri-implantitis are recognized as periodontal pathogens. CONCLUSION: Results of this systematic review suggest moderate evidence supporting association of Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia and some evidence supporting association of Prevotella intermedia and Campylobacter rectus with the etiology of peri-implantitis.

Association of three putative periodontal pathogens with chronic periodontitis in Brazilian subjects.

Objective The aim of this study was to evaluate the association of Porphyromonas endodontalis, Filifactor alocis and Dialister pneumosintes with the occurrence of periodontitis. Material and Methods Thirty subjects with chronic periodontitis (ChP) and 10 with periodontal health (PH) were included in the study. Nine subgingival biofilm samples were collected as follows: i) PH group - from the mesial/buccal aspect of each tooth in two randomly chosen contralateral quadrants; ii) ChP group - from three sites in each of the following probing depth (PD) categories: shallow (≤3 mm), moderate (4-6 mm) and deep (≥7 mm). Checkerboard DNA-DNA hybridization was used to analyze the samples. Results We found the three species evaluated in a higher percentage of sites and at higher levels in the group with ChP than in the PH group (p<0.05, Mann-Whitney test). We also observed these differences when the samples from sites with PD≤4 mm or ≥5 mm of subjects with ChP were compared with those from subjects with PH (p<0.05, Mann-Whitney test). In addition, the prevalence and levels of D. pneumosintes, and especially of F. alocis were very low in healthy subjects (0.12x105 and 0.01x105, respectively). Conclusion F. alocis and D. pneumosintes might be associated with the etiology of ChP, and their role in the onset and progression of this infection should be further investigated. The role of P. endodontalis was less evident, since this species was found in relatively high levels and prevalence in the PH group.

Association of three putative periodontal pathogens with chronic periodontitis in Brazilian subjects

ABSTRACT Objective The aim of this study was to evaluate the association of Porphyromonas endodontalis, Filifactor alocis and Dialister pneumosintes with the occurrence of periodontitis. Material and Methods Thirty subjects with chronic periodontitis (ChP) and 10 with periodontal health (PH) were included in the study. Nine subgingival biofilm samples were collected as follows: i) PH group - from the mesial/buccal aspect of each tooth in two randomly chosen contralateral quadrants; ii) ChP group - from three sites in each of the following probing depth (PD) categories: shallow (≤3 mm), moderate (4-6 mm) and deep (≥7 mm). Checkerboard DNA-DNA hybridization was used to analyze the samples. Results We found the three species evaluated in a higher percentage of sites and at higher levels in the group with ChP than in the PH group (p<0.05, Mann-Whitney test). We also observed these differences when the samples from sites with PD≤4 mm or ≥5 mm of subjects with ChP were compared with those from subjects with PH (p<0.05, Mann-Whitney test). In addition, the prevalence and levels of D. pneumosintes, and especially of F. alocis were very low in healthy subjects (0.12x105 and 0.01x105, respectively). Conclusion F. alocis and D. pneumosintes might be associated with the etiology of ChP, and their role in the onset and progression of this infection should be further investigated. The role of P. endodontalis was less evident, since this species was found in relatively high levels and prevalence in the PH group.

Comparison of independent and dependent culture methods for the detection of transient bacteremia in diabetic subjects with chronic periodontitis / Comparación entre métodos de cultivo independientes y dependientes para la detección de bacteriemia transitoria en individuos diabéticos con periodontitis crónica

Introduction: Oral-derived bacteremia may occur after several dental procedures and routine daily activities. Some conditions of the oral cavity may favor episodes of bacteremia. This would be the case of patients with diabetes mellitus and periodontitis, who exhibit exacerbated gingival inflammation and may be more prone to developing oral-derived bacteremia. Objective: To compare the effectiveness of an independent culture method (quantitative real-time PCR- qCR) and the most commonly used method (BacT-ALERT 3D ® ) for the diagnosis of bacteremia. Materials and methods: Blood samples were drawn from subjects with type 2 diabetes mellitus and chronic periodontitis before and after apple chewing. Samples were processed by an automated blood culture system (BacT-ALERT 3D ® ) monitored for 15 days with suitable subculture of positive cultures. In parallel, whole DNA from blood samples was purified using a commercial kit and screened by qPCR using a universal primer set of 16S rDNA for bacteria detection. Results: Blood cultures taken before apple chewing were shown to be negative by the two diagnostic methods. After chewing, two samples (11%) showed bacterial growth by BacT-ALERT 3D ® whereas qPCR did not detect the presence of bacteria in any sample. Conclusions: qPCR did not show greater effectiveness than the BacT-ALERT 3D ® in the detection of bacteremia of oral origin.

Introducción. Las bacteriemias de origen oral pueden ocurrir después de procedimientos odontológicos y de otros actos cotidianos. Algunas condiciones de la cavidad oral favorecen las bacteriemias como en el caso de pacientes con diabetes mellitus y periodontitis que presentan inflamación gingival exacerbada. Objetivo. Comparar la eficacia de un método independiente de cultivo (PCR cuantitativa) y otro dependiente (BacT-ALERT 3D ® ) en la detección de la bacteriemia. Materiales y métodos. Se tomaron muestras de sangre de individuos con diabetes mellitus de tipo II y periodontitis, antes y después de la masticación de manzana. Una alícuota se procesó por el sistema automatizado de hemocultivo (BacT-ALERT 3D ® ) y se monitorizó durante 15 días; la otra alícuota fue tratada para la extracción del ADN y procesada por RT-PCR usando un conjunto de cebadores de 16S rDNA exclusivos para bacterias. Resultados. En las muestras tomadas antes de masticar se confirmó la ausencia de bacterias mediante los dos métodos. En las muestras tomadas después de masticar la presencia de bacterias se evidenció únicamente en dos hemocultivos y en ninguna de las muestras se detectó la presencia de bacterias con el método de RT-PCR. Conclusiones. La PCR cuantitativa no mostró mayor eficacia que el BacT-ALERT 3D ® en la detección de la bacteriemia de origen oral.

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.

The effect of systemic antibiotics administered during the active phase of non-surgical periodontal therapy or after the healing phase: a systematic review.

OBJECTIVE: The aim of this systematic review was to compare the clinical effectiveness of systemic antibiotics administered in the active stage of periodontal treatment or after the healing phase. MATERIAL AND METHODS: An electronic search was performed in the databases EMBASE, MEDLINE and Cochrane Central Register of Controlled Trials (CENTRAL), in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement. A manual search of the reference list of selected studies and of review articles was also performed up to November 2013. Randomized Clinical Trials (RCT) that evaluated the systemic administration of antibiotics as adjuvants to scaling and root planning (SRP) at different phases of periodontal treatment were included. Systematic reviews and studies that evaluated subjects with systemic diseases and those that used subantimicrobial doses of antibiotics were excluded. RESULTS: The initial search identified 1,039 articles, of which seven were selected, and only one met the inclusion criteria. This study showed that subjects taking metronidazole and amoxicillin at the initial phase of treatment exhibited statistically significantly greater reduction in pocket depth and gain in clinical attachment level in initially deep sites (PD≥7 mm) than subjects taking antibiotics after healing (p<0.05). This comparison was conducted 2 months after antibiotic intake, at the healing phase. CONCLUSION: To date, only one short-term RCT has directly compared different moments of systemic antibiotics administration, as adjuncts to SRP, in the treatment of periodontitis. Although the results of this study suggested some benefits for antibiotics intake during the active phase of therapy, these findings need to be confirmed by larger placebo-controlled randomized clinical trials with longer follow-up periods.

The effect of systemic antibiotics administered during the active phase of non-surgical periodontal therapy or after the healing phase: a systematic review

Objective The aim of this systematic review was to compare the clinical effectiveness of systemic antibiotics administered in the active stage of periodontal treatment or after the healing phase. Material and Methods An electronic search was performed in the databases EMBASE, MEDLINE and Cochrane Central Register of Controlled Trials (CENTRAL), in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) statement. A manual search of the reference list of selected studies and of review articles was also performed up to November 2013. Randomized Clinical Trials (RCT) that evaluated the systemic administration of antibiotics as adjuvants to scaling and root planning (SRP) at different phases of periodontal treatment were included. Systematic reviews and studies that evaluated subjects with systemic diseases and those that used subantimicrobial doses of antibiotics were excluded. Results The initial search identified 1,039 articles, of which seven were selected, and only one met the inclusion criteria. This study showed that subjects taking metronidazole and amoxicillin at the initial phase of treatment exhibited statistically significantly greater reduction in pocket depth and gain in clinical attachment level in initially deep sites (PD≥7 mm) than subjects taking antibiotics after healing (p<0.05). This comparison was conducted 2 months after antibiotic intake, at the healing phase. Conclusion To date, only one short-term RCT has directly compared different moments of systemic antibiotics administration, as adjuncts to SRP, in the treatment of periodontitis. Although the results of this study suggested some benefits for antibiotics intake during the active phase of therapy, these findings need to be confirmed by larger placebo-controlled randomized clinical trials with longer follow-up periods. .

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.

Microbiological diversity of peri-implantitis biofilms.

The open-ended microbial diagnostic approaches such as the complete or partial sequencing of the 16S ribosomal gene by Sanger sequencing or by pyrosequencing have provided new insights into the diversity of the oral microbiota. These techniques have recently been used to evaluate the microbiota associated with osseointegrated implants and these results have expanded the knowledge on the diversity of the microbial communities associated with peri-implantitis. Taken together, the results of these studies suggest that the diversity of the microbial community of peri-implantitis and periodontitis might not be as similar as previously thought. Although certain known periodontal pathogens may also be associated with the etiology of peri-implantitis, apparently there were many differences between these two clinical conditions, involving distinct microorganisms. Further investigations on the diversity of peri-implant microbiota would be essential in order to define effective preventive and therapeutic strategies for peri-implantitis. It is also important to standardize laboratory protocols to make the results of the open-ended diagnostic techniques based on PCR amplification more comparable throughout the different research groups.

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.

Metronidazole and amoxicillin as adjuncts to scaling and root planing for the treatment of type 2 diabetic subjects with periodontitis: 1-year outcomes of a randomized placebo-controlled clinical trial.

AIM: To evaluate the clinical and microbiological effects of the use of metronidazole (MTZ) + amoxicillin (AMX) as adjuncts to scaling and root planing (SRP) for the treatment of chronic periodontitis (ChP) in type 2 diabetic subjects. MATERIAL AND METHODS: Fifty-eight type 2 diabetic subjects (n = 29/group) with generalized ChP were randomly assigned to receive SRP alone or with MTZ [400 mg/thrice a day (TID)]+AMX (500 mg/TID) for 14 days. Subgingival biofilm samples were analyzed by qPCR for the presence of seven periodontal pathogens. Subjects were monitored at baseline, 3, 6 and 12 months post-therapies. RESULTS: The group receiving SRP+MTZ+AMX presented greater mean probing depth (PD) reduction and clinical attachment gain, a lower number of sites with PD ≥5 mm (primary outcome variable) and a reduced number of subjects with ≥9 of these residual pockets than the control group at 1-year post-therapy (p < 0.05). The antibiotic-treated group also presented reduced levels and greater decreases of the three red complex species, Eubacterium nodatum and Prevotella intermedia, compared to the control group at 1 year (p < 0.05). CONCLUSIONS: The adjunctive use of MTZ+AMX significantly improved the clinical and microbiological outcomes of SRP in the treatment of type 2 diabetic subjects with ChP.

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.

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.