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.     

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.     

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.     

Phenotypic and Genotypic Characterization of Pyrazinamide Resistance among Multidrug-Resistant Mycobacterium tuberculosis Isolates in Zhejiang,China

To explore the phenotypic and genotypic characterization of pyrazinamide (PZA) resistance among multidrug-resistant Mycobacterium tuberculosis (MDR-TB) isolates in Zhejiang province, a total of 274 MDR-TB isolates were collected. Drug susceptibility testing and spoligotyping were performed on all clinical isolates. In addition, the mutated features of PZA-resistant loci, including pncA and rpsA, were also analyzed by DNA sequencing. Our results showed that the prevalence of PZA resistance among MDR-TB strains in Zhejiang province was 43.07% and that PZA resistance was associated with concomitant resistance to streptomycin. The majority of PZA-resistant MDR-TB isolates belonged to the Beijing family. Mutations within pncA, not rpsA, constituted the primary mechanism of PZA resistance. Among 118 PZA-resistant isolates, 53 different mutations were observed in pncA, and most of them were point mutations. Compared with the phenotypic data, DNA sequencing of pncA has sensitivity and specificity of 77.97% and 96.79%, respectively. Analysis of pncA provided a robust tool for rapid detection of PZA drug resistance.     

Mutations in gyrA and gyrB among Fluoroquinolone- and Multidrug-Resistant Mycobacterium tuberculosis Isolates

In order to correlate the mutations inside the entire gyrA and gyrB genes with the level of resistance to ofloxacin (OFX) and moxifloxacin (MFX) in isolates of multidrug-resistant Mycobacterium tuberculosis (MDR-TB), a total of 111 isolates were categorized into OFX-susceptible (MIC, ≤2 μg/ml) and low-level (MIC, 4 to 8 μg/ml) and high-level (MIC, ≥16 μg/ml) OFX-resistant isolates and MFX-susceptible (MIC, ≤0.5 μg/ml) and low-level (MIC, 1 to 2 μg/ml) and high-level (MIC, ≥4 μg/ml) MFX-resistant isolates. Resistance-associated mutations inside the gyrA gene were found in 30.2% of OFX-susceptible and 72.5% and 72.2% of low-level and high-level OFX-resistant isolates and in 28.6% of MFX-susceptible and 58.1% and 83.9% of low-level and high-level MFX-resistant isolates. Compared with OFX-susceptible isolates, low-level and high-level OFX-resistant isolates had a significantly higher prevalence of mutations at gyrA codons 88 to 94 (17.0%, 65.0%, and 72.2%, respectively; P < 0.001) and a higher prevalence of the gyrB G512R mutation (0.0%, 2.5%, and 16.7%, respectively; P = 0.006). Similarly, compared with MFX-susceptible isolates, low-level and high-level MFX-resistant isolates had a significantly higher prevalence of mutations at gyrA codons 88 to 94 (14.3%, 51.6%, and 80.6%, respectively; P < 0.001) as well as a higher prevalence of the gyrB G512R mutation (0.0%, 0.0%, and 12.9%, respectively; P = 0.011). D94G and D94N mutations in gyrA and the G512R mutation in gyrB were correlated with high-level MFX resistance, while the D94A mutation was associated with low-level MFX resistance. The prevalence of mutations at gyrA codons 88 to 94 and the gyrB G512R mutation were higher among fluoroquinolone (FQ)-susceptible East Asian (Beijing) and Indo-Oceanic strains than they were among Euro-American strains, implying that molecular techniques to detect FQ resistance may be less specific in areas with a high prevalence of East Asian (Beijing) and Indo-Oceanic strains.     

Analysis of embCAB Mutations Associated with Ethambutol Resistance in Multidrug-Resistant Mycobacterium tuberculosis Isolates from China

Ethambutol (EMB) plays a pivotal role in the chemotherapy of drug-resistant tuberculosis (TB), including multidrug-resistant tuberculosis (MDR-TB). Resistance to EMB is considered to be caused by mutations in the embCAB operon (embC, embA, and embB). In this study, we analyzed the embCAB mutations among 139 MDR-TB isolates from China and found a possible association between embCAB operon mutation and EMB resistance. Our data indicate that 56.8% of MDR-TB isolates are resistant to EMB, and 82.2% of EMB-resistant isolates belong to the Beijing family. Overall, 110 (79.1%) MDR-TB isolates had at least one mutation in the embCAB operon. The majority of mutations were present in the embB gene and the embA upstream region, which also displayed significant correlations with EMB resistance. The most common mutations occurred at codon 306 in embB (embB306), followed by embB406, embA(−16), and embB497. Mutations at embB306 were associated with EMB resistance. DNA sequencing of embB306–497 was the best strategy for detecting EMB resistance, with 89.9% sensitivity, 58.3% specificity, and 76.3% accuracy. Additionally, embB306 had limited value as a candidate predictor for EMB resistance among MDR-TB infections in China.     

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.     

A Ser315Thr Substitution in KatG Is Predominant in Genetically Heterogeneous Multidrug-Resistant Mycobacterium tuberculosis Isolates Originating from the St. Petersburg Area in Russia

Parts of katG and rpoB from 27 Russian Mycobacterium tuberculosis isolates were sequenced to detect mutations causing resistance to isoniazid (INH) and rifampin (RMP), respectively. All 24 INH-resistant isolates had a mutated katG, and 22 of them (91.7%) carried a mutation coding for a Ser315Thr shift. An rpoB mutation was noted for each of the 21 RMP-resistant isolates, with Ser531Leu being the most prevalent change encoded. Only two isolates had identical IS6110 fingerprints.     

Novel Mutations in ndh in Isoniazid-Resistant Mycobacterium tuberculosis Isolates

Novel mutations in NADH dehydrogenase (ndh) were detected in 8 of 84 (9.5%) isoniazid (INH)-resistant isolates (T110A [n = 1], R268H [n = 7]), but not in 22 INH-susceptible isolates of Mycobacterium tuberculosis. Significantly, all eight isolates with mutations at ndh did not have mutations at katG, kasA, or the promoter regions of inhA or ahpC, except for one isolate. Mutations in ndh appear to be an additional molecular mechanism for isoniazid resistance in M. tuberculosis.     

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.     

Relationship between Pyrazinamide Resistance, Loss of Pyrazinamidase Activity, and Mutations in the pncA Locus in Multidrug-Resistant Clinical Isolates of Mycobacterium tuberculosis

Sixty-two Mycobacterium tuberculosis isolates were tested for pyrazinamidase activity, and their pyrazinamide susceptibility was determined by the radiometric method. Sequencing of pncA genes in the 23 resistant strains revealed mutations in 16 pyrazinamidase-negative strains, 11 of which had not been previously described. Six isolates containing wild-type pncA might possess alternative resistance mechanisms.     

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.     

Meropenem-Clavulanic Acid Has High In Vitro Activity against Multidrug-Resistant Mycobacterium tuberculosis

We investigated the activity of meropenem-clavulanic acid (MEM-CLA) against 68 Mycobacterium tuberculosis isolates. We included predominantly multi- and extensively drug-resistant tuberculosis (MDR/XDR-TB) isolates, since the activity of MEM-CLA for resistant isolates has previously not been studied extensively. Using Middlebrook 7H10 medium, all but four isolates showed an MIC distribution of 0.125 to 2 mg/liter for MEM-CLA, below the non-species-related breakpoint for MEM of 2 mg/liter defined by EUCAST. MEM-CLA is a potential treatment option for MDR/XDR-TB.     

Molecular characterization of ofloxacin-resistant Mycobacterium tuberculosis strains from Russia

In this work, we studied the variation in the gyrA and gyrB genes in ofloxacin- and multidrug-resistant Mycobacterium tuberculosis strains circulating in northwest Russia. Comparison with spoligotyping data suggested that similar to the spread of multidrug-resistant tuberculosis, the spread of fluoroquinolone-resistant tuberculosis in Russia may be due, at least partly, to the prevalence of the Beijing genotype in a local population of M. tuberculosis.     

Sequence Analyses of Just Four Genes To Detect Extensively Drug-Resistant Mycobacterium tuberculosis Strains in Multidrug-Resistant Tuberculosis Patients Undergoing Treatment

The rapid detection of Mycobacterium tuberculosis isolates resistant to second-line drugs is crucial for the institution of appropriate treatment regimens as early as possible. Although molecular methods have successfully been used for the rapid detection of resistance to first-line drugs, there are limited data on mutations that confer resistance to second-line drugs. To address this question, we analyzed Mycobacterium tuberculosis strains resistant to ofloxacin (n = 26) and to capreomycin and/or amikacin (n = 48) from Uzbekistan for variations in target genes (gyrA, gyrB, rrs, and tlyA). Strains susceptible to ofloxacin (n = 49) and capreomycin and/or amikacin (n = 39) were included as controls. Mutations in gyrA or gyrB were found in 96% (25/26 strains) of the ofloxacin-resistant strains, while none of the susceptible strains displayed mutations in those two genes. The most common mutation occurred in gyrA at codon 94 (17/26 strains [65.4%]), followed by mutations at codons 90 and 91. Two strains showed a mutation in gyrB, at codons 485 and 543, respectively; both mutations have not been reported previously. The most frequent mutation in strains resistant to both amikacin and capreomycin was A1401G in rrs (34/40 strains [85.0%]). Three strains had mutations in tlyA, of which two (at codons 18 and 118) were associated with resistance to capreomycin alone. Overall, none of the 10 resistant strains (5 amikacin-resistant and capreomycin-susceptible strains) and none of the 39 susceptible control strains had mutations in the genes investigated. Our results clearly demonstrate the potential of sequence analyses of short regions of relatively few target genes for the rapid detection of resistance to second-line drugs among strains isolated from patients undergoing treatment for multidrug-resistant tuberculosis. The mechanisms that confer amikacin resistance in this setting remain unclear.With 9.2 million new cases and 1.7 million deaths in 2006, tuberculosis (TB) remains one of the most serious infectious diseases worldwide (28). In many settings, the emergence and transmission of drug-resistant Mycobacterium tuberculosis strains further threaten TB control efforts. TB fails to respond to treatment with the standard first-line drugs isoniazid (INH) and rifampin (RIF; rifampicin) in patients infected with so-called multidrug-resistant (MDR) Mycobacterium tuberculosis strains. MDR-TB is particularly difficult to treat because the second-line drugs used are less potent and more expensive (12).Disease caused by extensively drug-resistant (XDR) M. tuberculosis isolates, defined as MDR with resistance to any fluoroquinolone and at least one of three injectable second-line drugs (amikacin [AMK], capreomycin [CM], or kanamycin), is associated with very poor treatment outcomes (9).The rapid detection of microbial resistance to second-line drugs prior to and during treatment is therefore of essential importance for the implementation of increased infection control measures that may well limit the spread of TB to other patients.Compared to conventional liquid medium-based drug susceptibility testing, which still takes about 7 to 10 days, beginning from the time that a positive culture is obtained (18), the detection of genetic variants which mediate resistance to certain antimicrobial agents represents a more rapid alternative. Tests for the detection of MDR M. tuberculosis that detect mutations in the rpoB and katG/inhA regions, which confer resistance to RIF and INH, respectively, are now commercially available (3, 8, 13).Resistance to fluoroquinolones, such as ofloxacin (OFX), commonly used to treat MDR-TB is thought to be mediated by mutations (single nucleotide polymorphisms [SNPs]) in the target genes gyrA and, less frequently, gyrB, which encode the respective subunits of the DNA topoisomerase gyrase (24). Most mutations conferring resistance to quinolones are known to accumulate in a short discrete region of the gyrA and gyrB genes termed the quinolone resistance-determining region (QRDR) (2).Resistance to the aminoglycoside AMK and to CM are associated with SNPs in the 16S rRNA gene (rrs), especially in the region between nucleotides 1400 and 1500 (1, 23). Resistance to CM is thought to be additionally mediated by mutations located anywhere in the tlyA gene, which encodes a 2′-O-methyltransferase (10, 15).In the study described here, we aimed to determine if molecular analyses of target genes could be used for the rapid, specific, and sensitive detection of resistance to second-line drugs among new cases of TB and among previously treated patients in a high-incidence setting.Sequence analyses of the genes associated with fluoroquinolone resistance (gyrA and gyrB) and aminoglycoside or CM resistance (rrs and tlyA) were carried out with a panel of MDR- and XDR-TB strains arising from an MDR-TB treatment program in Karakalpakstan, a region in Uzbekistan with a high incidence of anti-TB drug resistance. Genetic data were then correlated with the results of phenotypic resistance testing.     

Selection of Mutations To Detect Multidrug-Resistant Mycobacterium tuberculosis Strains in Shanghai,China

Novel tools are urgently needed for the rapid, reliable detection of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis. To develop such tools, we need information about the frequency and distribution of the mycobacterial mutations and genotypes that are associated with phenotypic drug resistance. In a population-based study, we sequenced specific genes of M. tuberculosis that were associated with resistance to rifampin and isoniazid in 242 phenotypically MDR isolates and 50 phenotypically pan-susceptible isolates from tuberculosis (TB) cases in Shanghai, China. We estimated the sensitivity and specificity of the mutations, using the results of conventional, culture-based phenotypic drug susceptibility testing as the standard. We detected mutations within the 81-bp core region of rpoB in 96.3% of phenotypically MDR isolates. Mutations in two structural genes (katG and inhA) and two regulatory regions (the promoter of mabA-inhA and the intergenic region of oxyR-ahpC) were found in 89.3% of the MDR isolates. In total, 88.0% (213/242 strains) of the phenotypic MDR strains were confirmed by mutations in the sequenced regions. Mutations in embB306 were also considered a marker for MDR and significantly increased the sensitivity of the approach. Based on our findings, an approach that prospectively screens for mutations in 11 sites of the M. tuberculosis genome (rpoB531, rpoB526, rpoB516, rpoB533, and rpoB513, katG315, inhA-15, ahpC-10, ahpC-6, and ahpC-12, and embB306) could detect 86.8% of MDR strains in Shanghai. This study lays the foundation for the development of a rapid, reliable molecular genetic test to detect MDR strains of M. tuberculosis in China.Multidrug-resistant (MDR) tuberculosis (TB), defined as resistance to at least rifampin (RIF) and isoniazid (INH), and extensively drug-resistant (XDR) TB, defined as additional resistance to any fluoroquinolone and one injectable second-line drug, are among the most serious health threats of the 21st century. The epidemic of MDR TB is especially severe in China, a nation with the world''s second largest number of TB cases and the largest number of MDR TB cases (39). A recent study reported that 9.3% of all TB cases in China are MDR, almost twice the worldwide MDR prevalence (4.8%) (14). While a lot of attention has been focused on acquired drug resistance among TB patients who receive an inadequate treatment regimen or who cannot adhere to their treatment regimen, several studies also showed that a large number of MDR TB cases are likely caused by transmission of MDR strains of Mycobacterium tuberculosis (2, 19). Therefore, there is an urgent need for new tools and approaches that will provide a rapid, reliable, and cost-effective diagnosis of MDR TB, particularly in resource-limited settings. This will help to prevent transmission of MDR strains and to optimize treatment regimens for MDR cases.Drug susceptibility testing by the conventional solid medium culture method is highly sensitive and specific but extremely slow, due to the slow growth of M. tuberculosis. Liquid culture methods can reduce the turnaround time but require specialized instrumentation and reagents and are not feasible in most resource-limited settings. New molecular diagnostic methods represent a potentially rapid and sensitive alternative to conventional diagnostics. The molecular basis for phenotypic rifampin resistance is linked to mutations in the 81-bp core region of rpoB. Phenotypic isoniazid resistance has been associated with mutations in katG, particularly at codon 315, as well as with mutations in inhA, the promoter of mabA-inhA, and the intergenic region of oxyR-ahpC (3, 33, 34, 37, 43). Recently, a database of tuberculosis drug resistance mutations (TBDReaMDB) was established (27), and several genotypic diagnostic methods based on specific drug resistance-conferring mutations were developed. Two line probe assays, the INNO-LiPARif.TB assay (Innogenetics, Belgium) and the GenoType MTBDR Plus assay (Hain Lifescience, Nehren, Germany), have been approved by the World Health Organization (WHO) as tools for the rapid diagnosis of MDR TB (11, 20, 22). These tools are rapid and reproducible, but performance varies by geographic location, depending on the prevalent strains of M. tuberculosis and the type and frequency of drug resistance-conferring mutations in the population being tested (20, 22). Therefore, a thorough understanding of the diversity of the mycobacterial genetic mutations will form the foundation for new diagnostic methods.Despite the large number of MDR TB cases in China, relatively few studies have determined the prevalence of different drug resistance-conferring mutations among MDR clinical isolates. In this study, we investigated the type and frequency of drug resistance-conferring mutations that occurred among M. tuberculosis clinical isolates that were phenotypically MDR. Our goal was to identify and select a limited, parsimonious number of mutation sites that can be used to prospectively and rapidly screen isolates to detect MDR TB in Shanghai.     

Identification and Characterization of Multidrug-Resistant Salmonella enterica Serotype Albert Isolates in the United States

Salmonella enterica is one of the most common causes of bacterial foodborne illness in the United States. Although most Salmonella infections are self-limiting, antimicrobial treatment of invasive salmonellosis is critical. The primary antimicrobial treatment options include fluoroquinolones or extended-spectrum cephalosporins, and resistance to these antimicrobial drugs may complicate treatment. At present, S. enterica is composed of more than 2,600 unique serotypes, which vary greatly in geographic prevalence, ecological niche, and the ability to cause human disease, and it is important to understand and mitigate the source of human infection, particularly when antimicrobial resistance is found. In this study, we identified and characterized 19 S. enterica serotype Albert isolates collected from food animals, retail meat, and humans in the United States during 2005 to 2013. All five isolates from nonhuman sources were obtained from turkeys or ground turkey, and epidemiologic data suggest poultry consumption or live-poultry exposure as the probable source of infection. S. enterica serotype Albert also appears to be geographically localized to the midwestern United States. All 19 isolates displayed multidrug resistance, including decreased susceptibility to fluoroquinolones and resistance to extended-spectrum cephalosporins. Turkeys are a likely source of multidrug-resistant S. enterica serotype Albert, and circulation of resistance plasmids, as opposed to the expansion of a single resistant strain, is playing a role. More work is needed to understand why these resistance plasmids spread and how their presence and the serotype they reside in contribute to human disease.