An outbreak of infections affecting 311 patients who had undergone different invasive procedures occurred in 2004and 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.Rapidly growing mycobacteria (RGM) are widely distributed in the environment, especially in water (rivers, lakes, potable water), and can contaminate reagents and medical equipment. Most RGM infections in humans are caused by species belonging to the Mycobacterium fortuitum, Mycobacterium chelonae-Mycobacterium abscessus, and Mycobacterium smegmatis groups (6).The M. chelonae-M. abscessus group comprises two genomospecies, M. chelonae and M. abscessus, which have been differentiated on the basis of Ͻ70% genomic homology by DNA-DNA hybridization (16, 18). These species have been isolated from sporadic cases of chronic lung disease associated with bronchiectasis and cystic fibrosis, disseminated cutaneous infections, and postsurgical wound infections. They have also been implicated in outbreaks in cardiac, ophthalmologic, and plastic surgeries and pseudo-outbreaks related to contaminated bronchoscopes and contaminated laboratory reagents (6,12,20,25,26). Mycobacterium immunogenum was included in this group in 2001. It was isolated from metalworking fluids and was associated with cases of hypersensitivity pneumonitis in factory workers. This species has also been detected in cutaneous, catheter-related, articular, and lung infections; in an outbreak related to ophthalmologic surgeries; and in a pseudo-outbreak related to bronchoalveolar lavage procedures (19,27). Mycobacterium massiliense was vali...

BackgroundIn 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 FindingsA 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/SignificanceHorizontal 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.

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.Mycobacterium avium and Mycobacterium kansasii are major human mycobacterial pathogens, and both can cause pulmonary infections in immunocompetent individuals, as well as disseminated infections in the immunocompromised (6). Coinfection with M. avium and M. kansasii has also been documented in HIV-positive patients (8,15). Current treatment of M. avium and M. kansasii infections is based on long-term antibiotic therapy and relies on different drug combinations, justifying all efforts directed to the correct identification of clinical isolates. Insertion sequences (IS) are mobile genetic elements within mycobacteria that are often species specific, a property that can be exploited for diagnosis and epidemiological studies (4). The host range of the IS1245 element was considered to be limited to M. avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. silvaticum (3,9,(11)(12)(13)17).Detection of IS1245 by PCR was used for confirmation of the presence of M. avium in a mixed culture from a bone marrow specimen from an HIV-positive patient. Fifteen slowgrowing colonies were recovered from the bone marrow primary culture by plating dilutions on Middlebrook 7H10 solid medium supplemented with oleic acid, albumin, catalase, and dextrose (7H10-OADC). Four colonies (88.1 to 88.4) were nonpigmented, slow-growing mycobacteria identified as M. avium by PCR-restriction enzyme analysis (PRA) using the 16S-23S rRNA internal transcribed spacer (ITS) sequence as the target (14). Eleven colonies (88.5 to 88.15) produced yellow pigment after exposure to light and were identified as M. kansasii by PRA-ITS (Fig. 1A). For further characterization of these colonies, a 427-bp fragment from IS1245 was amplified by PCR (3). Unexpectedly, IS1245 amplicons were detected not only in the nonpigmented M. avium colonies but also in 8 out of 11 M. kansasii colonies (Fig. 1B). Amplicons generated with M. kansasii DNA were sequenced and showed 100% identity with the deposited IS1245 sequence (accession number L33879) (data not shown). Final identification of the 15 colonies to the species level was obtained by sequencing a 440-bp fragment of the 5Ј 16S rRNA gene (Escherichia coli positions 54 to 510) (16). Colonies 88.1 through 88.4 showed 100% sequence similarity to the corresponding sequence of M. avium type strain ATCC 25291 (accession number EF521895). Colonies 88.5 through 88.15 showed 100% similarity to the corresponding sequence of M. kansasii type strain CIP 104589 (accession number AF547940). The two sequences differed at 12 positions (97.3% similarity).To confirm the presence of IS1245 copies in the eight colonies of M. kansasii, restriction fragment length pol...

Purpose To determine clinical safety and cardiovascular, cardiac autonomic and inflammatory responses to a single session of inspiratory muscle training (IMT) in obstructive sleep apnea (OSA) subjects. Methods In a randomized controlled trial individuals of both sexes, aged between 30 and 70 years old with diagnosis of moderate to severe OSA were enrolled. Volunteers with OSA (n = 40) performed an IMT session with three sets of 30 repetitions with a 1-min interval between them. The IMT group (n = 20) used a load of 70% of the maximum inspiratory pressure (MIP), and the placebo group (n = 20) performed the IMT without load. Measurements of systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), heart rate variability (HRV), and inflammatory markers were performed pre, post-immediate and 1 h after the IMT session. Results No differences were shown in SBP, DBP, HRV, or inflammatory markers at any of the intervals analyzed. However, HR in the IMT group was lower 1 h after the IMT session compared to the pre-session values (p = 0002). HR was higher in the placebo group when comparing pre × post-immediate (p < 0.001). HR decreased after the first hour in relation to the pre (p < 0.001) and post-immediate (p < 0.001) values. Conclusion IMT sessions promote discreet hemodynamic, cardiac autonomic and inflammatory responses. Therefore, IMT is considered clinically safe and can be performed at home, guided but unsupervised, with lower cost and greater adherence to exercise program for subjects with OSA.

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 = 2.50, 95%CI = 1.20-5.36, p = 0.01). The SNP rs1540339 presented association to both T allele (OR = 0.55, 95%CI = 0.35-0.88, p = 0.01) and the T/T genotype (OR = 0.404, 95%CI = 0.20 -0.78, p = 0.005). The FokI T allele was identified as associated to diminished susceptibility (OR = 0.67, 95% CI = 0.45-0.99, p = 0.04) to active TB, as well as T/T genotype (OR = 0.15, 95%CI = 0.04-0.45, p = 9.58 × 10 -5 ). We also performed the expression analyses and observed a down-regulation of VDR in patients (-10.717 FC, p = 8.42e −12 ), and according to the presence of associated FokI SNP, we observed that the C/T and T/T genotypes presence increases VDR expression (+ 1.25 and + 2.35 FC, p = 0.425 and p = 0.506, respectively). This study shows that vitamin D receptor variants can influence upon pulmonary tuberculosis susceptibility and VDR mRNA levels are decreased in those patients.

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.

BACKGROUND Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the number of new cases of multidrug resistant TB (MDR-TB), pre extensively drug-resistant TB (pre-XDR-TB) and extensively drugresistant TB (XDR-TB) has increased considerably worldwide.OBJECTIVES Herein, using 156 M. tuberculosis isolates from 106 patients previously classified as MDR or pre-XDR or XDR isolates, we investigated the genetic mutation profiles associated with phenotypic resistances in patients with MDR-TB, pre-XDR-TB and XDR-TB, treatment outcomes and resistance evolution.METHODS Molecular analyses were performed by partial sequencing of the rpoB, katG, gyrA, gyrB, rrs genes and analysis of the fabG-inhA promoter region. Clinical, epidemiologic and demographic data were obtained from the TB Notification database system of São Paulo (TB-WEB) and the Information System for Special Tuberculosis Treatments (SITE-TB).FINDINGS Drug resistance was attributed to previously known mutations and a novel Asp449Val mutation in gyrB was observed in four isolates from the same patient. Ten patients had more than one isolate evaluated and eight of these patients displayed resistance progression. MAIN CONCLUSIONSThe present study is the first to report the frequency of mutations related to second-line drug resistance in MDR-TB, pre-XDR-TB and XDR-TB isolates. The results could lead to the improvement of available technologies for the rapid detection of drug resistant TB.

No abstract