Molecular Characterization of Multidrug-Resistant Mycobacterium tuberculosis Isolates from China

To investigate the molecular characterization of multidrug-resistant tuberculosis (MDR-TB) isolates from China and the association of specific mutations conferring drug resistance with strains of different genotypes, we performed spoligotyping and sequenced nine loci (katG, inhA, the oxyR-ahpC intergenic region, rpoB, tlyA, eis, rrs, gyrA, and gyrB) for 128 MDR-TB isolates. Our results showed that 108 isolates (84.4%) were Beijing family strains, 64 (59.3%) of which were identified as modern Beijing strains. Compared with the phenotypic data, the sensitivity and specificity of DNA sequencing were 89.1% and 100.0%, respectively, for isoniazid (INH) resistance, 93.8% and 100.0% for rifampin (RIF) resistance, 60.0% and 99.4% for capreomycin (CAP) resistance, 84.6% and 99.4% for kanamycin (KAN) resistance, and 90.0% and 100.0% for ofloxacin (OFX) resistance. The most prevalent mutations among the MDR-TB isolates were katG315, inhA15, rpoB531, -526, and -516, rrs1401, eis-10, and gyrA94, -90, and -91. Furthermore, there was no association between specific resistance-conferring mutations and the strain genotype. These findings will be helpful for the establishment of rapid molecular diagnostic methods to be implemented in China.     

Molecular Characterization of Multidrug-Resistant Isolates of Mycobacterium tuberculosis from Patients in North India

The World Health Organization has identified India as a major hot-spot region for Mycobacterium tuberculosis infection. We have characterized the sequences of the loci associated with multidrug resistance in 126 clinical isolates of M. tuberculosis from India to identify the respective mutations. The loci selected were rpoB (rifampin), katG and the ribosomal binding site of inhA (isoniazid), gyrA and gyrB (ofloxacin), and rpsL and rrs (streptomycin). We found known as well as novel mutations at these loci. Few of the mutations at the rpoB locus could be correlated with the drug resistance levels exhibited by the M. tuberculosis isolates and occurred with frequencies different from those reported earlier. Missense mutations at codons 526 to 531 seemed to be crucial in conferring a high degree of resistance to rifampin. We identified a common Arg463Leu substitution in the katG locus and certain novel insertions and deletions. Mutations were also mapped in the ribosomal binding site of the inhA gene. A Ser95Thr substitution in the gyrA locus was the most common mutation observed in ofloxacin-resistant isolates. A few isolates showed other mutations in this locus. Seven streptomycin-resistant isolates had a silent mutation at the lysine residue at position 121. While certain mutations are widely present, pointing to the magnitude of the polymorphisms at these loci, others are not common, suggesting diversity in the multidrug-resistant M. tuberculosis strains prevalent in this region. Our results additionally have implications for the development of methods for multidrug resistance detection and are also relevant in the shaping of future clinical treatment regimens and drug design strategies.     

Population Structure Analysis of Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates from Brazil Reveals Predominance of Clonal Complexes 1, 15, and 79

The population structure of 71 carbapenem-resistant Acinetobacter baumannii clinical isolates from several hospitals in Brazil was investigated by ApaI pulsed-field gel electrophoresis, bla_OXA-51-like subtyping, and multilocus sequence typing (Institute Pasteur scheme). In addition to the predominance of strains carrying bla_OXA-23, we detected the presence of bla_OXA-72 and bla_OXA-231. We observed a predominance of clonal complex 1 (CC1), CC15, and CC79 and representative strains of the worldwide-disseminated international clone I.     

Prevalence and Molecular Characterization of Fluoroquinolone-Resistant Mycobacterium tuberculosis Isolates in China

China is one of the countries with the highest burdens of multidrug-resistant (MDR) and fluoroquinolone (FQ)-resistant tuberculosis (TB) globally. Nevertheless, knowledge about the prevalence and molecular characterization of FQ-resistant Mycobacterium tuberculosis isolates from this region remains scant. In this study, 138 M. tuberculosis isolates determined by the agar proportion susceptibility method to be resistant to ofloxacin (OFX) were enrolled from a national drug resistance survey of China. All these strains were tested for susceptibility to ofloxacin, levofloxacin, moxifloxacin, gatifloxacin, and sparfloxacin using liquid Middlebrook 7H9 medium. The entire gyrA and gyrB genes conferring FQ resistance were sequenced, and spoligotyping was performed to distinguish different genotypes. Overall, the prevalence of resistance in China was highest for ofloxacin (3.76%), intermediate for levofloxacin (3.18%) and moxifloxacin (3.12%), and lowest for sparfloxacin (1.91%) and gatifloxacin (1.33%). Mutations in the gyrA gene were observed in 89 (64.5%) out of the 138 OFX-resistant M. tuberculosis strains. Positions 94 and 90 were the most frequent sites of mutation conferring FQ resistance on these strains, accounting for high-level FQ resistance. Furthermore, the Beijing genotype showed no association with high-level FQ resistance or distribution in hot spots in the quinolone resistance-determining region (QRDR) of gyrA. Our findings provide essential implications for the feasibility of genotypic tests relying on detection of mutations in the QRDR of gyrA and the shorter first-line treatment regimens based on FQs in China.     

Antimicrobial susceptibility of five subgroups of Mycobacterium fortuitum and Mycobacterium chelonae.

Broth microdilution MICs were determined for 258 clinical isolates of Mycobacterium fortuitum (3 biovariants) and M. chelonae (2 subspecies) with amikacin, tobramycin, cefoxitin, doxycycline, erythromycin, and sulfamethoxazole-trimethoprim and with several new beta-lactams and aminoglycosides and ciprofloxacin. Variations in susceptibility by and within species subgroups confirm the need for susceptibility testing against clinically important strains.     

Emergence and Molecular Characterization of Extensively Drug-Resistant Mycobacterium tuberculosis Clinical Isolates from the Delhi Region in India

We screened 194 Mycobacterium tuberculosis strains isolated from tuberculosis (TB) patients in Delhi and neighboring regions in India to identify the prevalence of extensive drug resistance (XDR) in clinical isolates. Among these, 104 isolates were found to be multidrug resistant (MDR), and 6 were identified as XDR isolates, which was later confirmed by antimicrobial susceptibility testing against the respective drug screening panel. Genotyping was carried out by amplifying and sequencing the following genes: rpoB (rifampin), katG (isoniazid), gyrA (fluoroquinolones), and rrs (amikacin, kanamycin, and capreomycin). Our analyses indicated that mutations at the hot spots of these genes were positively correlated with drug resistance in clinical isolates. The key mutation observed for rpoB was in the codon for amino acid position 531 (S531L), and other mutations were seen in the hot spot, including those encoding Q510P, L511H, D516V, and H526Y mutations. We identified S315T and R463L substitutions encoded in the katG locus. An S95T substitution encoded in the gyrA locus was the most common mutation observed in fluoroquinolone-resistant isolates. In addition, we saw D94G and D94N mutations encoded in the QRDR region. The 16S rRNA (rrs) gene encoded mainly the A1401G mutation and an additional mutation, G1484T, resulting in ribosomal modifications. Taken together, the data in this report clearly establish the presence of phenotypically distinct XDR strains in India by molecular profiling and further identify specific mutational hot spots within key genes of XDR-TB strains.In recent years, the control of tuberculosis (TB) has become a global challenge due to the emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). With 9.2 million new cases and 1.7 million deaths in 2006, TB remains one of the major life-threatening diseases worldwide (22). XDR-TB isolates are resistant to isoniazid and rifampin, to any fluoroquinolone (FQ), and to at least one of the three injectable second-line drugs (amikacin, kanamycin, and capreomycin) (6). As of June 2008, XDR-TB strains have been found in 49 countries, including the United States (6, 22). Furthermore, a recent report points to an alarming increase in the number of tuberculosis patients in the South Asian subcontinent, with India being singled out as having the greatest burden of XDR-TB, with a poor prognosis and high mortality among HIV-infected individuals (4). The risk of XDR-TB spread across country borders has heightened global concern over a potentially untreatable epidemic that may jeopardize recent advances made in global TB control.The prevalence of XDR-TB in India was reported in 2007, but no further efforts have been made to identify its genotypes or geographical spread (9). The present study was undertaken to characterize mutations prevalent in clinical isolates from India with respect to various drug target loci. We examined the drug target genes for rifampin (rpoB), isoniazid (katG), fluoroquinolones (gyrA), and aminoglycosides (rrs), which are commonly prescribed for the treatment of tuberculosis in India. The loci studied were rpoB (RNA polymerase B subunit), katG (catalase-peroxidase), rrs (16S rRNA), and gyrA (DNA gyrase A). Here we report, for the first time, the molecular characterization of XDR-TB isolates from India. This study confirms the presence of XDR-TB in India and simultaneously raises an alarm about its prevalence among TB patients, as many of them may initially have MDR-TB that slowly progresses and mutates to XDR-TB. Furthermore, the fact that some of these patients have HIV infection or the possibility of later coinfection with HIV has the potential to make this global HIV-TB epidemic untreatable with current therapies.     

Genetic basis for clarithromycin resistance among isolates of Mycobacterium chelonae and Mycobacterium abscessus.

Resistance to clarithromycin among isolates of Mycobacterium chelonae and M. abscessus was observed in 18 of 800 (2.3%) patients tested between 1990 and 1995. Patients whose isolates were resistant had either disseminated disease or chronic lung disease, and the resistant isolates were recovered after clarithromycin monotherapy. Sequencing of the gene coding for the 23S rRNA peptidyltransferase region revealed a point mutation involving adenine at position 2058 (38%) or adenine at position 2059 (62%) in 20 of 20 relapse isolates from the first 13 patients identified. By pulsed-field gel electrophoresis or random amplified polymorphic DNA PCR, initial and relapse isolates were shown to have identical DNA patterns. M. chelonae and M. abscessus isolates were found to have only a single chromosomal copy of the rRNA operon, thus making them susceptible to single-step mutations. Thus, clarithromycin resistance in these species of rapidly growing mycobacteria relates to a point mutation in the gene coding for 23S rRNA and occurs in limited clinical situations, but was identified in almost 5% of isolates tested in 1995.     

pncA Mutations in Pyrazinamide-Resistant Mycobacterium tuberculosis Isolates from Northwestern Russia

Thirty-six pyrazinamide-resistant and eight pyrazinamide-susceptible Mycobacterium tuberculosis isolates from Russia were analyzed for their pncA mutations. Thirty-one (86.1%) of the resistant isolates had a mutation either in pncA or upstream of the gene. Twenty of the 23 different mutations found in this study had not been described earlier. pncA genotype correlated well with pyrazinamidase activity and BACTEC 460 susceptibility test results.     

Molecular Genetics of para-Aminosalicylic Acid Resistance in Clinical Isolates and Spontaneous Mutants of Mycobacterium tuberculosis

The emergence of Mycobacterium tuberculosis resistant to first-line antibiotics has renewed interest in second-line antitubercular agents. Here, we aimed to extend our understanding of the mechanisms underlying para-aminosalicylic acid (PAS) resistance by analysis of six genes of the folate metabolic pathway and biosynthesis of thymine nucleotides (thyA, dfrA, folC, folP1, folP2, and thyX) and three N-acetyltransferase genes [nhoA, aac(1), and aac(2)] among PAS-resistant clinical isolates and spontaneous mutants. Mutations in thyA were identified in only 37% of the clinical isolates and spontaneous mutants. Overall, 24 distinct mutations were identified in the thyA gene and 3 in the dfrA coding region. Based on structural bioinformatics techniques, the altered ThyA proteins were predicted to generate an unfolded or dysfunctional polypeptide. The MIC was determined by Bactec/Alert and dilution assay. Sixty-three percent of the PAS-resistant isolates had no mutations in the nine genes considered in this study, revealing that PAS resistance in M. tuberculosis involves mechanisms or targets other than those pertaining to the biosynthesis of thymine nucleotides. The alternative mechanism(s) or pathway(s) associated with PAS resistance appears to be PAS concentration dependent, in marked contrast to thyA-mutated PAS-resistant isolates.The discovery of the antitubercular activity of para-aminosalicylic acid (PAS) by Lehmann in 1943 (15) was followed by two successful clinical trials conducted in 1944 and 1949 (16, 31). These breakthroughs, combined with the almost simultaneous discovery and introduction of streptomycin (STR), brought much hope in the fight against tuberculosis (TB) (22). The initial success was soon thwarted by the emergence of PAS and STR resistance. This was overcome by coadministering PAS and STR, resulting in the advent of combination therapy (19). In 1951, isoniazid was added to anti-TB regimens until the mid-1960s. Although including PAS combination therapy proved efficacious, side effects attributed to PAS were documented as early as 1951 (6, 25). In addition to PAS-associated gastrointestinal toxicity, elevated and repetitive dosing complicated therapeutic regimens. PAS therapy was discontinued after the introduction of rifampin (rifampicin) and pyrazinamide. PAS was reintroduced in the United States in 1992, following several outbreaks of multidrug-resistant (MDR) isolates (4). Since then, the need for new antibiotics for the treatment of MDR TB has led to the development of novel formulations of PAS, which have proven to be less toxic (5). Today, PAS is used primarily as a second-line drug to treat MDR TB (34).PAS has structural similarities to sulfonamides. Sulfonamides are structural analogues of para-aminobenzoic acid, the substrate of dihydropteroate synthase (encoded by folP1/folP2), and hence function as competitive inhibitors. FolP1 and its putative homologue FolP2 catalyze the condensation of para-aminobenzoic acid and 6-hydroxymethyl-7,8-dihydropterein pyrophosphate to 7,8-dihydropteroate, which is converted to dihydrofolate and reduced to generate the cofactor tetrahydrofolate (THF) by the enzyme dihydrofolate reductase (encoded by dfrA) (Fig. ​(Fig.1).1). Unlike the actions of some sulfonamides or analogues in other pathogens, the PAS-inhibitory activity of folP1 has proven to be unexpectedly poor in vitro (24). More recently, Rengarajan and colleagues (26), using transposon mutagenesis, have shown that PAS resistance is associated with mutations of thymidylate synthase A, encoded by the thyA gene and required for thymine biosynthesis in the folate pathway. This result implies that PAS functions as a folate antagonist, a suggestion supported by the identification of mutations within the thyA coding region in PAS-resistant (PAS^r) clinical isolates (26).Open in a separate windowFIG. 1.The folate pathway and plausible targets for PAS inhibition. The six genes analyzed in this study are underlined.ThyA catalyzes the reductive methylation of dUMP to yield dTMP, required for de novo dTTP synthesis (12). ThyA requires the 5,10-methylene THF cofactor both as a reductant and as a carbon donor in the methylation reaction. The presence of the thyX gene, encoding a functional homologue of thymidylate synthase but with the clear distinction that it utilizes flavin adenine dinucleotide as a cofactor instead of THF, in the Mycobacterium tuberculosis genome is noteworthy (8). Although ThyX utilizes flavin adenine dinucleotide as a cofactor, it still requires 5,10-methylene THF as the methyl donor. It is hypothesized that the bacteriostatic activity of PAS results from perturbation of the folate pathway, although the underlying mechanism has yet to be elucidated (26).Here, we set out to investigate the mutations associated with PAS resistance in a collection of well-characterized M. tuberculosis clinical isolates and PAS^r spontaneous mutants. Five genes, thyA, dfrA, folC, folP1, and folP2, encoding enzymes of the folate pathway; thyX, encoding an alternative thymine-biosynthetic enzyme; and three N-acetyltransferase genes possibly associated with the modification of PAS were analyzed. To better understand PAS resistance, the identified mutations were correlated with MICs and the protein three-dimensional (3D) structure. The structural stability was modeled for all mutants. To our surprise, only 37% of the PAS^r clinical isolates or spontaneous mutants encoded mutations in enzymes of the folate pathway, indicating that other mechanisms associated with PAS resistance have yet to be elucidated.     

Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex

Mycobacterial interspersed repetitive units (MIRU) comprise short tandem repeat structures found at multiple loci throughout the Mycobacterium tuberculosis genome and have been used for typing these pathogens. We have identified MIRU at 18 conserved loci throughout the common portions of the Mycobacterium avium subspecies paratuberculosis (MAP) and M. avium subspecies avium (MAA) genomes. Six of these loci were found to differ between MAA and MAP in the number of tandem repeat motifs occurring at each MIRU locus. Locus specific PCR at 4 of these loci segregated MAP into two major groups, which could be differentiated from ovine-pigmented strains of MAP and the MAP vaccine strain 316F. The same PCR differentiated MAA into five MIRU profiles. PCR at either MIRU locus 1 or MIRU locus 4 distinguished between MAP and all other M. avium complex (MAC) tested. PCR at both loci 1 and 4 also distinguished MAP from Mycobacterium intracellulare. MIRU typing may provide an additional simple and rapid procedure for the differentiation between MAP and other MAC.     

Occurrence of rpoB Mutations in Isoniazid-Resistant but Rifampin-Susceptible Mycobacterium tuberculosis Isolates from Germany

Four out of 143 phenotypically isoniazid-resistant but rifampin-susceptible Mycobacterium tuberculosis strains that were isolated from patients in Germany in 2011 had mutations in the rifampin resistance-determining region of rpoB. After performing drug susceptibility testing (DST) with two methods, the proportion method on Löwenstein-Jensen medium and using the Bactec 960 Mycobacteria Growth Indicator Tube system, we conclude that the two methods are equally reliable for phenotypic DST and MIC determination.     

Effects of ortho-phthalaldehyde,glutaraldehyde and chlorhexidine diacetate on Mycobacterium chelonae and Mycobacterium abscessus strains with modified permeability

The mechanisms of the mycobactericidal action of ortho-phthalaldehyde (OPA), glutaraldehyde (GTA) and chlorhexidine diacetate (CHA) were investigated using mycobacterial spheroplasts of two reference strains, Mycobacterium chelonae NCTC 946, Mycobacterium abscessus NCTC 10882 and two GTA-resistant strains, M. chelonae Epping and M. chelonae Harefield. Transmission electron microscopy of the spheroplasts revealed an altered cell wall structure compared with the parent cells. Structural alterations resulting from the spheroplasting process were in part correlated to a loss of lipid content. Low concentrations of CHA induced protein coagulation in M. chelonae NCTC 946 spheroplasts, which also exhibited the highest loss of free non-polar lipids. Higher concentrations of CHA were required to produce similar results to the other spheroplasts investigated in which there was a less substantial decrease in lipid content. OPA (0.5% w/v) readily penetrated the residual cell wall and cytoplasmic membrane, producing significant protein coagulation in M. chelonae NCTC 946. GTA (0.5% v/v) induced a similar effect but to a lesser extent. Pre-treatment of the spheroplasts with OPA and GTA and their subsequent suspension in water demonstrated that GTA was a more potent cross-linking agent. This protective effect of GTA results from extensive cross-linking of amino and/or sulphydryl side-chain groups of proteins. The rapid mycobactericidal effect of OPA probably arises from its more efficient penetration across biological membranes. Mycobacterial spheroplasts represented a useful cellular model with an altered cell wall permeability. This study also showed the importance of the mycobacterial cell wall in conferring intrinsic resistance to CHA.     

Mycobacterium chelonae bloodstream infection induced by osteomyelitis of toe: A case report

Mycobacterium chelonae is a rapidly growing mycobacterium that has the potential to cause refractory infections in humans. Mycobacteremia resulting from the organism is extremely rare, and its clinical features are yet to be uncovered. We herein present a case of M. chelonae bloodstream infection involving an immunocompromised older patient. A 79-year-old woman, on a long-term treatment with prednisolone plus tacrolimus for rheumatoid arthritis, visited our outpatient department complaining of deteriorating pain and swelling at her right 1st toe. Laboratory parameters showed elevated C-reactive protein and leukocytosis, and magnetic resonance imaging indicated osteomyelitis at the proximal phalanx of her right 1st toe. Considering the refractory course, the infected toe was immediately amputated. M. chelonae was isolated from bacterial cultures of the resected tissue and blood (BD BACTEC™ FX blood culture system, Becton Dickinson, Sparks, MD, USA), leading to a diagnosis of disseminated M. chelonae infection. We treated the patient with an antibiotic combination of clarithromycin, minocycline, and imipenem (2 weeks), which was converted to oral therapy of clarithromycin, doxycycline, and levofloxacin. This case highlighted the potential pathogenesis of M. chelonae to cause mycobacteremia in an immunocompromised patient.     

In vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium avium, Mycobacterium fortuitum, and Mycobacterium chelonae to ticarcillin in combination with clavulanic acid.

The in vitro susceptibility of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium avium, Mycobacterium fortuitum, and Mycobacterium chelonae (M. chelonei) to ticarcillin in combination with calvulanic acid (CA) was studied by the agar dilution method. All the M. tuberculosis, M. bovis, and M. africanum strains were inhibited at a ticarcillin concentration of 32 micrograms/ml or lower in combination with 5 micrograms of CA. M. chelonae and M. avium strains proved resistant to more than 128 micrograms of ticarcillin plus 5 micrograms of CA per ml. M. fortuitum strains needed 128 micrograms of ticarcillin plus 5 micrograms of CA to inhibit approximately 30% of the isolates.     

Efficacy of Nitazoxanide against Clinical Isolates of Mycobacterium tuberculosis

Nitazoxanide (NTZ) has bactericidal activity against the H37Rv laboratory strain of Mycobacterium tuberculosis with a MIC of 16 μg/ml. However, its efficacy against clinical isolates of M. tuberculosis has not been determined. We found that NTZ''s MIC against 50 clinical isolates ranged from 12 to 28 μg/ml with a median of 16 μg/ml and was unaffected by resistance to first- or second-line antituberculosis drugs or a diversity of spoligotypes.