The Detection and Sequencing of a Broad-Host-Range Conjugative IncP-1 beta Plasmid in an Epidemic Strain of Mycobacterium abscessus subsp bolletii

Background: An extended outbreak of mycobacterial surgical infections occurred in Brazil during 2004-2008. Most infections were caused by a single strain of Mycobacterium abscessus subsp. bolletii, which was characterized by a specific rpoB sequevar and two highly similar pulsed-field gel electrophoresis (PFGE) patterns differentiated by the presence of a similar to 50 kb band. the nature of this band was investigated.Methodology/Principal Findings: Genomic sequencing of the prototype outbreak isolate INCQS 00594 using the SOLiD platform demonstrated the presence of a 56,264-bp circular plasmid, designated pMAB01. Identity matrices, genetic distances and phylogeny analyses indicated that pMAB01 belongs to the broad-host-range plasmid subgroup IncP-1 beta and is highly related to BRA100, pJP4, pAKD33 and pB10. the presence of pMAB01-derived sequences in 41 M. abscessus subsp. bolletii isolates was evaluated using PCR, PFGE and Southern blot hybridization. Sixteen of the 41 isolates showed the presence of the plasmid. the plasmid was visualized as a similar to 50-kb band using PFGE and Southern blot hybridization in 12 isolates. the remaining 25 isolates did not exhibit any evidence of this plasmid. the plasmid was successfully transferred to Escherichia coli by conjugation and transformation. Lateral transfer of pMAB01 to the high efficient plasmid transformation strain Mycobacterium smegmatis mc(2)155 could not be demonstrated.Conclusions/Significance: the occurrence of a broad-host-range IncP-1 beta plasmid in mycobacteria is reported for the first time. Thus, genetic exchange could result in the emergence of specific strains that might be better adapted to cause human disease.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundao de Amparo a Pesquisa do Estado de São PauloUniversidade Federal de São Paulo, Disciplina Microbiol, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilFed Univ Para, Inst Ciencias Biol, Lab Polimorfismo DNA, BR-66059 Belem, Para, BrazilInst Evandro Chagas, Secao Bacteriol & Micol, Ananindeua, PA, BrazilFed Univ Para, Inst Estudos Costeiros, Braganca, PA, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Disciplina Microbiol, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilFAPESP: FAPESP - 06/01533-9Fundao de Amparo a Pesquisa do Estado de São Paulo: FAPESP - 8/01451-8Web of Scienc

Isolamento, purificação e caracterização de micobacteriófagos ambientais

The first isolation of mycobacteriophages occurred more than 60 years ago. Between the late 40s and early 50s, Gardner and Weiser using M. smegmatis, were the first to note that soil samples could be used as a source for the isolation of these organisms. Since then, mycobacteriophages have been isolated from various sources, such as clinical samples of patients and healthy people, animals, mainly environmental samples, particularly from soil samples. None of the isolates from soil, however, addressed the environmental mycobacteriophages the perspective used in this work. Efforts have been restricted to the isolation of phages, without concern for understanding the dynamics of these viruses in source isolation. Also, the identification of mycobacteria present in the source of isolation of phages remains little explored. Thus, this work presents eight new mycobacteriophages isolates composting material Parque São Paulo Zoo Foundation , referred to herein as Florinda, Girafales, Quico, Nhonho, Barrriga, Pops, Godines and Madruga . These proved incapable of infecting other species of mycobacteria such as M. abscessus, M. tuberculosis and M. bovis. Besides mycobacteriophages, this work identified mycobacteria present in the same material as well as analyzed the behavior of these organisms in the evolution of the composting process. Were identified by the method of PRA-hsp65 species: M. abscessus (58,3% ) , M. fortuitum (29.2 %) , M. peregrinum (9.7%) and a new profile (2,8%). Thus, this paper discusses a new perspective mycobacteriophages and the presence of mycobacteria in the environment.M. smegmatis, foram os primeiros a notar que amostras de solo poderiam ser utilizadas como fonte para o isolamento desses organismos. Desde então, micobacteriófagos têm sido isolados das mais diversas fontes, como amostras clínicas de pacientes e pessoas saudáveis, de animais, de cultivos em laboratórios, mas, principalmente, de amostras de materiais ambientais, em especial de solo. Nenhum dos isolamentos de solo, entretanto, abordou os micobacteriófagos ambientais na perspectiva utilizada neste trabalho. Os esforços têm se restringido ao isolamento dos fagos, sem a preocupação com o entendimento da dinâmica destes vírus na fonte de isolamento. Soma-se a isso o hiato existente na identificação das micobactérias presentes na fonte de isolamento dos fagos. Assim, este trabalho apresenta oito novos micobacteriófagos isolados de material de compostagem da Fundação Parque Zoológico de São Paulo, referidos aqui como: Florinda, Girafales, Quico, Nhonho, Barriga, Pops, Godines e Madruga. Estes se mostraram incapazes de infectar outras espécies de micobactérias como M. abscessus, M. tuberculosis e M. bovis. Além de estudar micobacteriófagos presentes em material de compostagem, este trabalho identificou micobactérias presentes no mesmo material, bem como analisou o comportamento destes organismos na evolução do processo de compostagem. Foram identificadas através do método de PRA-hsp65 as espécies: M. abscessus (58, 3%), M. fortuitum (29,2%), M. peregrinum (9,7%), além de um novo perfil (2,8%). Assim, este trabalho aborda sob uma nova perspectiva a presença de micobacteriófagos e micobactérias no ambiente.Dados abertos - Sucupira - Teses e dissertações (2013 a 2016

Correction: The Detection and Sequencing of a Broad-Host-Range Conjugative IncP-1β Plasmid in an Epidemic Strain of Mycobacterium abscessus subsp. bolletii.

[This corrects the article on p. e60746 in vol. 8.]

Predicted coding sequences on plasmid pMAB01 and the putative functions.

*<p>: Certain CDSs showed more than one best hit, although the first one was selected to represent the homologous sequence.</p

Plasmid pMAB01 purified from INCQS 00594 and from <i>E.coli</i> transconjugants and transformants.

<p>Plasmid from <i>M. abscessus</i> subsp. <i>bolletii</i> INCQS 00594 was extracted with QIAGEN Plasmid Maxi Kit and plasmids from <i>E. coli</i> with QIAGEN Plasmid Mini Kit. M, <i>E. coli</i> NCTC39 R861 (used as marker), 1, <i>M. abscessus</i> subsp. <i>bolletii</i> INCQS 00594; 2, <i>E. coli</i> BL21(DE3); 3, <i>E. coli</i> BL21(DE3) transformed with pMAB01; 4, transconjugant <i>E. coli</i> BL21(DE3); 5, <i>E. coli</i> C600 Nal^R; and 6, transconjugant <i>E. coli</i> C600 Nal^R</p

Phylogenetic tree of the genes <i>trfA</i>, <i>ssb</i>, <i>korB</i> and <i>klcA</i> from IncP-1 plasmids.

<p>The eighteen IncP-1 plasmid sequences corresponding to sub-groups α, β, γ and ε were obtained from GenBank, and the pMAB01 sequences were obtained in this study. The posterior credibility values are represented for each node. Plasmid QKH54 was used as outgroup. Plasmid pMAB01 is indicated with a black triangle. The scale bar corresponds to the nucleotide substitution rate. The vertical distance is provided for illustrative purposes only.</p

Characterization of mycobacteria and mycobacteriophages isolated from compost at the São Paulo Zoo Park Foundation in Brazil and creation of the new mycobacteriophage Cluster U

Abstract Background A large collection of sequenced mycobacteriophages capable of infecting a single host strain of Mycobacterium smegmatis shows considerable genomic diversity with dozens of distinctive types (clusters) and extensive variation within those sharing evident nucleotide sequence similarity. Here we profiled the mycobacterial components of a large composting system at the São Paulo zoo. Results We isolated and sequenced eight mycobacteriophages using Mycobacterium smegmatis mc2155 as a host. None of these eight phages infected any of mycobacterial strains isolated from the same materials. The phage isolates span considerable genomic diversity, including two phages (Barriga, Nhonho) related to Subcluster A1 phages, two Cluster B phages (Pops, Subcluster B1; Godines, Subcluster B2), three Subcluster F1 phages (Florinda, Girafales, and Quico), and Madruga, a relative of phage Patience with which it constitutes the new Cluster U. Interestingly, the two Subcluster A1 phages and the three Subcluster F1 phages have genomic relationships indicating relatively recent evolution within a geographically isolated niche in the composting system. Conclusions We predict that composting systems such as those used to obtain these mycobacteriophages will be a rich source for the isolation of additional phages that will expand our view of bacteriophage diversity and evolution

Genetic map of the IncP-1β plasmid pMAB01.

<p>The coding regions are indicated with arrows showing the direction of transcription. The inverted repeats (IRs) of the transposons and integrons are identified. The origins of vegetative replication (<i>oriV</i>) and plasmid transfer (<i>oriT</i>) are indicated with black boxes. The functional modules of the plasmid backbone are differentiated by colors in the inner circle: Tra1 (<i>tra</i>) and Tra2 (<i>trb</i>) (green); replication (rep) module (<i>trfA-ssb</i>) (grey); central control (ctl) region encoding regulatory and stability functions (<i>kfrA – relE</i>) (light brown). One genetic load region (load 1) contains the following: a Tn<i>501</i>-like mercury-resistance (<i>mer</i>) transposon (dark blue); a truncated Tn<i>5393c</i> streptomycin-resistance transposon (yellow); and a copy of the insertion element IS<i>1071</i> (orange). The second genetic load region (load 2) contains a class 1 integron with an integrase (<i>intI</i>) and the integron-specific segment <i>qacEdelta1-sul1-orf5</i> (light blue), with two cassettes, encoding an aminoglycoside 6'-N-acetyltransferase (<i>aac(6’)-Ib</i>) and a glyoxalase-like domain protein (pMAB01-024) (brown). A detailed description of the accessory regions is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060746#pone-0060746-g002" target="_blank">Figure 2</a>.</p

Pulsed field gel electrophoresis (PFGE) and Southern blot hybridization results.

<p>(<b>A)</b> PFGE-<i>Dra</i>I and (<b>B)</b> Southern blot hybridization using the <i>trfA</i>-derived probe of selected isolates showing the PFGE patterns of the epidemic strain: 1, the sequenced isolate INCQS 00594, showing the ∼50 bp PFGE-<i>Dra</i>I band that hybridized with the <i>trfA</i> derived probe; 2, isolate showing a band with faster migration in PFGE-<i>Dra</i>I and Southern blot hybridization; 3, no plasmid band detected in PFGE-<i>Dra</i>I and a hybridization band visible near gel origin; 4, two plasmid bands with different migration; 5, no evidence of the presence of pMAB01 either using PFGE-<i>Dra</i>I or hybridization; and 6, cured INCQS 00594 colony. The asterisks indicate the plasmid bands.</p