Molecular analysis of kanamycin and viomycin resistance in Mycobacterium smegmatis by use of the conjugation system

Taniguchi, Hatsumi; Chang, Bin; Abe, Chiyoji; Nikaido, Yoshihiko; Mizuguchi, Yasuo; Yoshida, Shin Ichi

doi:10.1128/jb.179.15.4795-4801.1997

Cited by 68 publications

(68 citation statements)

References 28 publications

(50 reference statements)

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“…We anticipate that this conjugal system will improve the genetic versatility of M. smegmatis, and mc 2 155 in particular, and facilitate the characterization of the mycobacterial genome. In fact, the utility of this system was demonstrated by the ability to transfer auxotrophic markers between strains (Mizuguchi and Tokunaga, 1971) and, more recently, by the ability to mobilize resistance markers between strains as shown here and demonstrated by Taniguchi et al (1997).…”

Section: Chromosomal Transfer Is Extensivementioning

confidence: 74%

Conjugal transfer of chromosomal DNA in Mycobacterium smegmatis

Parsons

Jankowski

Derbyshire

1998

Molecular Microbiology

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SummaryThe genus Mycobacterium includes the major human pathogens Mycobacterium tuberculosis and Mycobacterium leprae. The development of rational drug treatments for the diseases caused by these and other mycobacteria requires the establishment of basic molecular techniques to determine the genetic basis of pathogenesis and drug resistance. To date, the ability to manipulate and move DNA between mycobacterial strains has relied on the processes of transformation and transduction. Here, we describe a naturally occurring conjugation system present in Mycobacterium smegmatis, which we anticipate will further facilitate the ability to manipulate the mycobacterial genome. Our data rule out transduction and transformation as possible mechanisms of gene transfer in this system and are most consistent with conjugal transfer. We show that recombinants are not the result of cell fusion and that transfer occurs from a distinct donor to a recipient. One of the donor strains is mc 2 155, a highly transformable derivative that is considered the prototype laboratory strain for mycobacterial genetics; the demonstration that it is conjugative should increase its genetic manipulability dramatically. During conjugation, extensive regions of chromosomal DNA are transferred into the recipient and then integrated into the recipient chromosome by multiple recombination events. We propose that DNA transfer is occurring by a mechanism similar to Hfr conjugation in Escherichia coli.

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Section: Chromosomal Transfer Is Extensivementioning

confidence: 74%

Conjugal transfer of chromosomal DNA in Mycobacterium smegmatis

Parsons

Jankowski

Derbyshire

1998

Molecular Microbiology

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“…In several research studies, low-level KAN R strains differ from high-level KAN R strains by both lacking rrs mutations and cross-resistance to other ribosome binding drugs, including AMK (6)(7)(8)(9)(10). The prevalence of mutations in the eis promoter in 79% of the low-level KAN R clinical isolates studied and absence from the KAN S isolates studied suggests that these mutations account for much of the observed low-level KAN resistance.…”

Section: Discussionmentioning

confidence: 99%

“…Mutations in the 16S rRNA gene, rrs, can cause high-level resistance to KAN [minimum inhibitory concentration (MIC) Ն80 g/mL], and some mutations can confer crossresistance to other second-line drugs, including amikacin (AMK) and capreomycin (CAP) (6). However, up to 80% of KAN-resistant (KAN R ) clinical isolates display low-level resistance to KAN (5 g/mL Ͻ MIC Ͻ 80 g/mL), do not contain rrs mutations, and do not exhibit cross-resistance (7)(8)(9)(10). The molecular basis of this low-level resistance is unclear.…”

mentioning

confidence: 99%

Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis

Zaunbrecher

Sikes²,

Metchock³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

300

311

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The emergence of multidrug-resistant (MDR) tuberculosis (TB) highlights the urgent need to understand the mechanisms of resistance to the drugs used to treat this disease. The aminoglycosides kanamycin and amikacin are important bactericidal drugs used to treat MDR TB, and resistance to one or both of these drugs is a defining characteristic of extensively drug-resistant TB. We identified mutations in the ؊10 and ؊35 promoter region of the eis gene, which encodes a previously uncharacterized aminoglycoside acetyltransferase. These mutations led to a 20 -180-fold increase in the amount of eis leaderless mRNA transcript, with a corresponding increase in protein expression. Importantly, these promoter mutations conferred resistance to kanamycin [5 g/mL < minimum inhibitory concentration (MIC) <40 g/mL] but not to amikacin (MIC <4 g/mL). Additionally, 80% of clinical isolates examined in this study that exhibited low-level kanamycin resistance harbored eis promoter mutations. These results have important clinical implications in that clinical isolates determined to be resistant to kanamycin may not be cross-resistant to amikacin, as is often assumed. Molecular detection of eis mutations should distinguish strains resistant to kanamycin and those resistant to kanamycin and amikacin. This may help avoid excluding a potentially effective drug from a treatment regimen for drug-resistant TB.T he World Health Organization estimates that 9.2 million new cases of tuberculosis (TB) occur each year (1). Despite intensive efforts to ensure proper drug dosages and patient compliance with drug regimens, multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis have emerged (2). These strains cause extensive mortality in immunocompromised individuals (3) and hinder the control and prevention of the disease. XDR and MDR TB infections cannot be adequately treated with the first-line anti-TB drugs and require expensive, prolonged treatment with second-line anti-TB drugs. The rapid determination of the resistance profile of an isolate can facilitate selection of an appropriate drug regimen and preclude development of additional drug resistances. Rapid detection of resistances is best achieved with molecular diagnostic approaches, particularly in developing countries where access to culture facilities is limited. Such strategies require a detailed understanding of the molecular basis for drug resistance. Although the mechanisms of resistance to first-line drugs such as isoniazid and rifampin are well characterized, much less is known about such mechanisms for the second-line drugs (4).An important second-line anti-TB drug is the aminoglycoside kanamycin (KAN), which binds to the 16S rRNA in the 30S ribosomal subunit and inhibits protein synthesis (5). In other bacteria, characterized mechanisms of KAN resistance include altered efflux or influx of the drug, inactivation of the drug by enzymatic modification, and mutation or methylation of rRNA, which disrupts binding of the drug to the riboso...

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“…This rRNA methyltransferase modifies nucleotide C1409 in helix 44 of 16S rRNA and nucleotide C1920 in helix 69 of 23S rRNA [119]. Additionally, mutations in the rrs gene that encodes the 16S rRNA are also associated with resistance to VM and CPM, specifically a G→T nucleotide changes at codon 1484 [120,121]. Cross-resistance can be found between KM, AMK, CPM and VM [6].…”

Section: Second Line Drugsmentioning

confidence: 99%

Review: The Molecular Basis of Resistance in <i>Mycobaterium tuberculosis</i>

Santos¹

2012

OJMM

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Tuberculosis is a serious global public health problem and its high prevalence is strongly associated with the increase of drug resistance. This steady increase in the frequency of M. tuberculosis strains resistant to one or more agents commonly used to treat tuberculosis has drawn worldwide attention to understanding the molecular basis of resistance in M. tuberculosis. TB resistance is a great concern in the antibiotic resistance pandemic due to the high risk of death, as patients can remain infected for months or years and also because of the difficulty of the treatment. A molecular understanding of the series of events that render M. tuberculosis multi-drug resistant is very important in order to find a fast and appropriated diagnosis as well as a new target for new drugs.

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Molecular analysis of kanamycin and viomycin resistance in Mycobacterium smegmatis by use of the conjugation system

Cited by 68 publications

References 28 publications

Conjugal transfer of chromosomal DNA in Mycobacterium smegmatis

Conjugal transfer of chromosomal DNA in Mycobacterium smegmatis

Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis

Review: The Molecular Basis of Resistance in <i>Mycobaterium tuberculosis</i>

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Molecular analysis of kanamycin and viomycin resistance in Mycobacterium smegmatis by use of the conjugation system

Cited by 68 publications

References 28 publications

Conjugal transfer of chromosomal DNA in Mycobacterium smegmatis

Conjugal transfer of chromosomal DNA in Mycobacterium smegmatis

Overexpression of the chromosomally encoded aminoglycoside acetyltransferase eis confers kanamycin resistance in Mycobacterium tuberculosis

Review: The Molecular Basis of Resistance in &lt;i&gt;Mycobaterium tuberculosis&lt;/i&gt;

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Review: The Molecular Basis of Resistance in <i>Mycobaterium tuberculosis</i>