Natural Occurrence of Horizontal Transfer of Mycobacterium avium - Specific Insertion Sequence IS 1245 to Mycobacterium kansasii

Abstract

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...

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

“…The stability of pMAB01 plasmid was tested using three isolates, INCQS 00594, B52 [1] and IAL 042 [29], according to the protocol described by da Silva Rabello et al [63] with minor modifications. A single colony of each isolate was cultivated in 10 ml of LB broth, at 37°C, in a shaker, at 180 rpm, until the optical density at 600 nm reached 0.6 to 0.8.…”

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

“…Recently, our group confirmed the presence of the IS 1245 element in colonies of Mycobacterium kansasii isolated from a M. avium - M. kansasii mixed culture from a bone marrow specimen of an HIV-positive patient [12]. Fifteen colonies were isolated from the original culture, four of which were identified as M. avium and 11 as M. kansasii .…”

“…Four colonies (88.1. to 88.4) were identified as M. avium and 11 as M. kansasii by phenotypic characteristics (growth rate and pigment production) and by PCR-restriction enzyme analysis of the 16S–23S internal transcribed sequence (PRA-ITS) [47] (Table 2). The 11 M. kansasii colonies showed indistinguishable PFGE-DraI patterns, except for the presence of a band of approximately 100 kb in eight of the 11 isolates and were, in fact, the same strain [12]. Two M. kansasii clinical isolates, IAL 413 and IEC 6805, Mycobacterium smegmatis mc 2 155, Mycobacterium bovis BCG Moreau and Escherichia coli DH5α (Life Technologies Co., Carlsbad, CA) were used in this study (Table 2).…”

Crohn’s Disease May Be Differentiated Into 2 Distinct Biotypes Based on the Detection of Bacterial Genomic Sequences and Virulence Genes Within Submucosal Tissues

“…27 Although gene transfer is generally rare as a result of colonization resistance in the healthy gut that limits direct contact between prospective donor and recipient organisms, 28 altered enterobacterial colonization densities 29 can boost horizontal gene transfer between pathogenic and commensal bacteria. 30,31 It is likely that the eaeA, invA, and yadA invasion/adherence genes detected within submucosal tissues of patients with Crohn's disease have an ancestral origin in Escherichia, Salmonella, and Yersinia spp., respectively, but do not represent recent events. 30,31 It is likely that the eaeA, invA, and yadA invasion/adherence genes detected within submucosal tissues of patients with Crohn's disease have an ancestral origin in Escherichia, Salmonella, and Yersinia spp., respectively, but do not represent recent events.…”