Natural Occurrence of Horizontal Transfer of Mycobacterium avium-Specific Insertion Sequence IS1245 to Mycobacterium kansasii

Mycobacterium kansasii carrying IS1245, a highly prevalent insertion sequence among Mycobacterium avium isolates, was detected in a mixed culture of M. avium and M. kansasii. the insertion sequence was stable and able to transpose by a replicative mechanism in M. kansasii. These findings may have significant implications for molecular diagnosis and treatment outcome.Foundation for Research Support of the State of São PauloConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, SP, BrazilInst Adolfo Lutz Registro, Setor Micobacterias, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, SP, BrazilFoundation for Research Support of the State of São Paulo: FAPESP-06/01533-9Web of Scienc

Differential distribution in vitamin D receptor gene variants and expression profile in Northeast Brazil influences upon active pulmonary tuberculosis

Tuberculosis is an infectious disease with variable outcomes. This variability is due to host immune capacity in containing the infection process initiated by the Mycobacterium tuberculosis (MTB). Vitamin D is able to modulate a very specific immune response against MTB infection, and its action relies on vitamin D receptor (VDR) binding. Altered VDR forms may compromise vitamin D pathway and proper immune response after MTB infection. Herein we assessed the relationship of five potentially functional polymorphisms from VDR: rs2228570 FokI, rs11568820 Cdx-2, rs2248098, rs1540339 and rs4760648, with tuberculosis susceptibility. The SNP rs4760648 T/T was associated with differential susceptibility to tuberculosis (OR\u2009=\u20092.50, 95%CI\u2009=\u20091.20-5.36, p\u2009=\u20090.01). The SNP rs1540339 presented association to both T allele (OR\u2009=\u20090.55, 95%CI\u2009=\u20090.35-0.88, p\u2009=\u20090.01) and the T/T genotype (OR\u2009=\u20090.404, 95%CI\u2009=\u20090.20 - 0.78, p\u2009=\u20090.005). The FokI T allele was identified as associated to diminished susceptibility (OR\u2009=\u20090.67, 95% CI\u2009=\u20090.45-0.99, p\u2009=\u20090.04) to active TB, as well as T/T genotype (OR\u2009=\u20090.15, 95%CI\u2009=\u20090.04-0.45, p\u2009=\u20099.58\u2009 7\u200910-5). We also performed the expression analyses and observed a down-regulation of VDR in patients (-10.717 FC, p\u2009=\u20098.42e-12), and according to the presence of associated FokI SNP, we observed that the C/T and T/T genotypes presence increases VDR expression (+\u20091.25 and\u2009+\u20092.35 FC, p\u2009=\u20090.425 and p\u2009=\u20090.506, respectively). This study shows that vitamin D receptor variants can influence upon pulmonary tuberculosis susceptibility and VDR mRNA levels are decreased in those patients

First Description of Natural and Experimental Conjugation between Mycobacteria Mediated by a Linear Plasmid

Background: in a previous study, we detected the presence of a Mycobacterium avium species-specific insertion sequence, IS1245, in Mycobacterium kansasii. Both species were isolated from a mixed M. avium-M. kansasii bone marrow culture from an HIV-positive patient. the transfer mechanism of this insertion sequence to M. kansasii was investigated here.Methodology/Principal Findings: A linear plasmid (pMA100) was identified in all colonies isolated from the M. avium-M. kansasii mixed culture carrying the IS1245 element. the linearity of pMA100 was confirmed. Other analyses suggested that pMA100 contained a covalently bound protein in the terminal regions, a characteristic of invertron linear replicons. Partial sequencing of pMA100 showed that it bears one intact copy of IS1245 inserted in a region rich in transposase-related sequences. These types of sequences have been described in other linear mycobacterial plasmids. Mating experiments were performed to confirm that pMA100 could be transferred in vitro from M. avium to M. kansasii. pMA100 was transferred by in vitro conjugation not only to the M. kansasii strain from the mixed culture, but also to two other unrelated M. kansasii clinical isolates, as well as to Mycobacterium bovis BCG Moreau.Conclusions/Significance: Horizontal gene transfer (HGT) is one of most important mechanisms leading to the evolution and diversity of bacteria. This work provides evidence for the first time on the natural occurrence of HGT between different species of mycobacteria. Gene transfer, mediated by a novel conjugative plasmid, was detected and experimentally reproduced.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Cooperacion Interuniversitaria UAM-Banco Santander con America Latina (CEAL), UAM, SpainConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilLab Nacl Comp Cient, Petropolis, BrazilUniv Autonoma Madrid, Fac Med, Dept Prevent Med, Madrid, SpainInst Adolfo Lutz Registro, Nucleo TB & Micobacterioses, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilFAPESP: FAPESP - 06/01533-9Web of Scienc

Study of the mechanism of horizontal transfer of insertion sequence IS1245, specific of Mycobacterium avium, to Mycobacterium kansasii

Mycobacterium avium and Mycobacterium kansasii are non-tuberculous mycobacteria frequently isolated in infections from HIV positive patients. In a previous study from our laboratory, a mixed culture of M. avium and M. kansasii from a bone marrow isolate of an HIV positive patient was studied. The analysis of isolated colonies allowed the detection of the insertion sequence IS1245, specific from M. avium, in M. kansasii. The presence of this element in M. kansasii was confirmed by PCR-IS1245, RFLP-IS1245 and sequencing. The objective of this study was to investigate the mechanism of transference of the IS1245 to M. kansasii. A band of approximately 100 kb was detected by PFGE in colonies of M. avium and M. kansasii from this mixed culture. Tests of mobility of this DNA band in PFGE gels, in different running conditions, and tests with exonucleases and topoisomerase I demonstrated that this band was a linear plasmid (pMA100) with proteins covalently linked to the 5’ ends. These findings led to the hypothesis that the pMA100 plasmid was responsible for the natural transference of the IS1245 element from M. avium to M. kansasii by conjugation. Experiments performed in vitro reproduced the conjugation event, not only with the M. kansasii strain from the mixed culture, but also with other two unrelated M. kansasii strains. The insertion sequence was stably maintained in the M. kansasii genome after 10 subcultures and its replicative transposition in M. kansasii was also observed. For the first time the natural horizontal gene transfer between different species of mycobacteria has been demonstrated.Mycobacterium avium e Mycobacterium kansasii são micobactérias não tuberculosas frequentemente isoladas em infecções em pacientes HIV positivos. Em um projeto anterior do laboratório foi estudado um cultivo misto de M. avium e M. kansasii isolado da medula óssea de um paciente HIV positivo. Estudando colônias isoladas deste cultivo, foi possível observar que a cepa de M. kansasii possuía a sequência de inserção IS1245, que é especifica de M. avium. A presença deste elemento de inserção em M. kansasii foi confirmada por PCR-IS1245, RFLP-IS1245 e sequenciamento. Este estudo teve como objetivo investigar o mecanismo de transferência da IS1245 para M. kansasii. Uma banda de aproximadamente 100 kb foi detectada por PFGE em colônias de M. avium e de M. kansasii deste cultivo misto. Testes de mobilidade desta banda de DNA em géis de PFGE, em diferentes condições de corrida, e testes com exonucleases e com topoisomerase I comprovaram que esta banda era um plasmídeo linear (pMA100), que continha proteínas covalentemente ligada nos seus terminais 5’. Estes achados levaram à hipótese de que o plasmídeo pMA100 seria responsável pela transferência natural do elemento IS1245 de M. avium para M. kansasii por conjugação. Experimentos in vitro reproduziram o evento de conjugação, tanto com a cepa de M. kansasii isolada do cultivo misto, como com outras duas cepas de M. kansasii não relacionadas. A sequência de inserção se manteve estável no genoma de M. kansasii após 10 subcultivos e foi observada a ocorrência de transposição de modo replicativo em M. kansasii. Pela primeira vez está sendo demonstrada a transferência horizontal de genes natural entre espécies diferentes de micobactérias.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)TEDEBV UNIFESP: Teses e dissertaçõe

Natural Occurrence of Horizontal Transfer of Mycobacterium avium- Specific Insertion Sequence IS1245 to Mycobacterium kansasii▿

Mycobacterium kansasii carrying IS1245, a highly prevalent insertion sequence among Mycobacterium avium isolates, was detected in a mixed culture of M. avium and M. kansasii. The insertion sequence was stable and able to transpose by a replicative mechanism in M. kansasii. These findings may have significant implications for molecular diagnosis and treatment outcome

Diversity of Mycobacteriaceae from aquatic environment at the São Paulo Zoological Park Foundation in Brazil.

We investigated the species diversity of Mycobacteriaceae in surface water samples from six environments at the zoological park in São Paulo, Brazil. Three hundred and eighty isolates were cultivated and identified by phenotypic characteristics (growth rate and pigmentation) and sequencing of hsp65, rpoB and 16S rRNA genes. The results revealed that almost 48% of the isolates could be identified at the species level; about 50% were classified at the genus level, and only less than 2% of the isolates showed an inconclusive identification. The isolates classified at the genus level and not identified were then evaluated by phylogenetic analyses using the same three concatenated target genes. The results allowed us to identify at the genus level some isolates that previously had inconclusive identification, and they also suggested the presence of putative candidate species within the sample, demonstrating that this zoological park is an important source of diversity

Molecular Characterization of Mycobacterium massiliense and Mycobacterium bolletii in Isolates Collected from Outbreaks of Infections after Laparoscopic Surgeries and Cosmetic Procedures▿

An outbreak of infections affecting 311 patients who had undergone different invasive procedures occurred in 2004 and 2005 in the city of Belém, in the northern region of Brazil. Sixty-seven isolates were studied; 58 were from patients who had undergone laparoscopic surgeries, 1 was from a patient with a postinjection abscess, and 8 were from patients who had undergone mesotherapy. All isolates were rapidly growing nonpigmented mycobacteria and presented a pattern by PCR-restriction enzyme analysis of the hsp65 gene with BstEII of bands of 235 and 210 bp and with HaeIII of bands of 200, 70, 60, and 50 bp, which is common to Mycobacterium abscessus type 2, Mycobacterium bolletii, and Mycobacterium massiliense. hsp65 and rpoB gene sequencing of a subset of 20 isolates was used to discriminate between these three species. hsp65 and rpoB sequences chosen at random from 11 of the 58 isolates from surgical patients and the postinjection abscess isolate presented the highest degrees of similarity with the corresponding sequences of M. massiliense. In the same way, the eight mesotherapy isolates were identified as M. bolletii. Molecular typing by pulsed-field gel electrophoresis (PFGE) grouped all 58 surgical isolates, while the mesotherapy isolates presented three different PFGE patterns and the postinjection abscess isolate showed a unique PFGE pattern. In conclusion, molecular techniques for identification and typing were essential for the discrimination of two concomitant outbreaks and one case, the postinjection abscess, not related to either outbreak, all of which were originally attributed to a single strain of M. abscessus

Conjugation experiments using <i>M. avium</i> 88.3 as the donor strain.

<p>*three experiments with 15, 20 and 25 days incubations, respectively.</p><p>**three experiments with 2, 4 and 10 days incubations, respectively.</p><p>§pigmented colonies after exposure to light.</p><p>£PCR-IS<i>6110</i> positive.</p><p>¶grown on LB agar.</p

PFGE of DNA genomic preparations.

<p>(A) PFGE with undigested DNAs from <i>M. avium</i> 88.3 (1) and <i>M. kansasii</i> 88.8 (2) under different switch times, indicated below each figure; (B) pMA100 extracted from PFGE gels and treated with exonuclease III (3) or exonuclease lambda (4); (C) pMA100 extracted from PFGE gels and treated (+) or not (-) with topoisomerase I; (D) DNA prepared with (+) or without (-) adding proteinase K to the lysis buffer; (E) same as in (D) in PFGE gels and running buffer prepared with 0.2% SDS. λ: DNA concatemers of the bacteriophage λ genome.</p

Analysis of transconjugants isolated in mating experiments using <i>M. avium</i> 88.3 as donor strain.

<p>(A) PFGE with undigested DNA; (B) Southern blot of PFGE gels with undigested DNA and hybridization with IS<i>1245</i>-derived probe; (C) PFGE-DraI; (D) Southern blot of PFGE-DraI gels and hybridization with pMA100-derived probe. Open arrows indicate the linear plasmid pMA100. 1: <i>M. avium</i> 88.3; 2: <i>M. kansasii</i> 88.6; 3: <i>M. kansasii</i> IAL 413; 4: <i>M. kansasii</i> IEC 6805; 5: <i>M. bovis</i> BCG Moreau; W: wild-type colony; T: transconjugant colony.</p