Molecular Epidemiology and Clinical Characteristics of Drug-Resistant Mycobacterium tuberculosis in a Tuberculosis Referral Hospital in China

Background Despite the large number of drug-resistant tuberculosis (TB) cases in China, few studies have comprehensively analyzed the drug resistance-associated gene mutations and genotypes in relation to the clinical characteristics of M. tuberculosis (Mtb) isolates. Methodology/Principal Findings We thus analyzed the phenotypic and genotypic drug resistance profiles of 115 Mtb clinical isolates recovered from a tuberculosis referral hospital in Beijing, China. We also performed genotyping by 28 loci MIRU-VNTR analysis. Socio-demographic and clinical data were retrieved from medical records and analyzed. In total, 78 types of mutations (including 42 previously reported and 36 newly identified ones) were identified in 115 Mtb clinical isolates. There was significant correlation between phenotypic and genotypic drug resistance rates for first-line anti-TB drugs (P<0.001). Genotyping revealed 101 MIRU-VNTR types, with 20 isolates (17.4%) being clustered and 95 isolates (82.6%) having unique genotypes. Higher proportion of re-treatment cases was observed among patients with clustered isolates than those with unique MIRU-VNTR genotypes (75.0% vs. 41.1%). Moreover, clinical epidemiological links were identified among patients infected by Mtb strains belonging to the same clusters, suggesting a potential of transmission among patients. Conclusions/Significance Our study provided information on novel potential drug resistance-associated mutations in Mtb. In addition, the genotyping data from our study suggested that enforcement of the implementation of genotyping in diagnostic routines would provide important information for better monitor and control of TB transmission.


Introduction
Tuberculosis (TB) remains a major infectious and deadly disease in the world. In addition, multidrug resistance (MDR) and extensively drug-resistance (XDR) pose a more serious problem for TB control. WHO reported that about 8.6 million people developed TB, and 1.3 million died from the disease in 2012. India and China alone accounted for 26% and 12% of global cases, respectively. In addition, the global estimate of the burden of MDR-TB was 300, 000 cases among notified TB patients in 2012. India and China were the two countries estimated to have the largest numbers of TB patients with MDR-TB [1]. Therefore, there is an urgent need to better understand the molecular epidemiology and clinical characteristics of drug-resistant M. tuberculosis (Mtb) isolates, so as to provide knowledge for rapid molecular diagnosis for and better management of drug-resistant TB.
Despite the high TB burden and large number of drug-resistant TB cases in China, relatively few studies have performed comprehensive analysis of drug resistance-associated mutations of clinical Mtb isolates, their genotypes, as well as their association with clinical characteristics. We previously observed high rates of MDR-and XDR-TB among patients from a TB referral hospital in Beijing, China [2]. In addition, we established a TBDReaMDB database-coupled automatic analysis for drug resistance-associated mutations in Mtb isolates [3]. In this study, we further applied this method to perform comprehensive analysis of the drug resistanceassociated mutations (genotypic drug resistance profiles) for a larger sample of Mtb clinical isolates from hospitalized TB patients. Molecular genotyping is a useful tool for analyzing Mtb strain diversity and transmission patterns, especially in high incidence settings such as TB referral hospitals. Genotyping methods based on variable number of tandem repeats (VNTR) of mycobacterial interspersed repetitive units (MIRU) have become popular methods used for TB epidemiological analysis [4,5]. We thus also evaluated the genotypic population structure for the Mtb isolates based on 28 loci MIRU-VNTR genotyping analysis and its correlation with drug resistance associated mutations and clinical characteristics and epidemiology. Our study added to the growing list of potential TB drug resistance-associated mutations, which could be invaluable in applying MDR-and XDR-TB molecular detection tests. In addition, the genotyping data from our study suggested that enforcement of the implementation of genotyping in diagnostic routines would provide important information for better monitor and control of TB transmission.

Ethics statement
The investigation protocols used in this study were approved by the institutional ethics committee of the 309 Hospital, Beijing, China. Written informed consent for Mtb isolates to be collected as well as for their information to be stored in the hospital database for research purposes was provided by participants. Written informed consent was obtained from the next of kin, caretakers, or guardians on the behalf of the minors/children participants involved in this study. Permission for using the information in the medical records of the patients for research purposes was obtained from the 309 Hospital. The Institute ethics committee of the 309 Hospital and the Institute of Microbiology, Chinese Academy of Sciences approved this study.
Mtb isolates, drug susceptibility testing and detection of drug resistance-associated mutations Mtb isolates used in this study were obtained from TB patients being treated in the 309 Hospital, a TB referral hospital located in Beijing, China over a 3-year period (January 1, 2009-December 31, 2011). A total of 3860 culture positive TB patients were diagnosed and treated in the 309 Hospital during 2009-2011. Of these, 150 non-repetitive Mtb isolates were randomly selected to obtain pure cultures for further confirmation by p-nitrobenzoic acid and thiophene carboxylic acid hydrazine resistance tests as well as 16S rDNA sequencing analysis. Thirty-five isolates were excluded as a result of contaminated cultures or ambiguous sequencing results. The remaining 115 isolates were then subjected to DST, sequencing for drug resistance-associated gene mutations, and genotyping analysis. Epidemiologic and clinical data of the patients were extracted from the subjects' medical records. Mtb isolates were cultured using the BACTEC 960 system (Becton Dickinson Diagnostic Systems, Sparks, MD, USA) according to the manufacturer's instructions. The drug susceptibility testing (DST) were conducted according to the WHO guidelines as described previously [2]. Identification of drug resistance-associated mutations of Mtb isolates was conducted as described previously [3]. Written informed consent was obtained from participants.

MIRU-VNTR genotyping of Mtb isolates
Deletion-targeted multiplex PCR (DTM-PCR) method was used initially to identify the Beijing strains [6]. The Mtb isolates were further genotyped by a 28 loci MIRU-VNTR method based on the 24 loci described by Supply et al. [4,7] as well as the four hyper variable loci (1982, 3232, 3820, and 4120) described by Gao et al. [8] and Supply et al. [9]. The amplicons were evaluated on the 2% standard agarose gels by using a 100-bp DNA ladder (Takara), and sizing of the various VNTR alleles was done with the Peak Scanner Software v1.0 (PE Applied Biosystems). The number of repeats at each locus of MIRUs was determined and numerical values were assigned accordingly. Genetic clusters were defined as a group of two or more isolates exhibiting the same MIRU-VNTR pattern. The reference strain H37Rv was run as an additional control.

Phylogenetic analysis
The phylogenetic tree based on the 28 loci MIRU-VNTR data was constructed using PAUP 4.0b software [10] with UPGMA (unweighted pair-group method with arithmetic means) method.

Statistical analyses
All data were analyzed using the SPSS software (15.0 version). Comparisons of categorical variables were performed using the Pearson Chi-square test to compare different groups. A P value of ,0.05 was considered to be statistically significant.

General characteristics of the study subjects
A total of 115 non-repetitive Mtb isolates collected during 2009-2011 period were included in this study. The TB patients from whom the Mtb isolates were obtained included 53 susceptible TB cases, 21 MDR-TB cases, 17 XDR-TB cases, and 24 other types of TB cases (drug-resistant TB cases excluding MDR-and XDR-TB). All cases were HIV negative. The median age was 37 (range 14-86). Sixty-nine (60.0%) patients were male and 46 (40.0%) were female. Sixty-one (53.0%) patients were new cases and 54 (47.0%) were re-treatment cases. More detiled information on socio-demographic and clinical characteristics of the patients is shown in Table S1.

Drug resistance-associated gene mutations in Mtb isolates
We identified 78 types of mutations in drug resistanceassociated genes, including 42 previously reported ones (Table 1) and 36 newly identified ones ( Table 2 and Table 3). More detailed information on the PCR primers used for as well as the mutations identified in drug resistance-associated loci in all Mtb isolates were listed in Table S2 and Table S3, respectively. There was significant correlation between phenotypic and genotypic drug resistance rates for first-line anti-TB drugs (P,0.001 for isoniazid, rifampicin, streptomycin, ethambutol and pyrazinamide by chisquare test). The percentages of genotypic resistant isolates among phenotypic resistant isolates for first-line anti-TB drugs varied between 40.0% to 95.7%. There was also significant correlation between some of the second-line anti-TB drugs (P,0.001 for ofloxacin/levofloxacin and kanamycin by chi-square test). The percentages of genotypic resistant isolates among phenotypic resistant isolates for second-line anti-TB drugs varied between 0% to 41.5% (Table S4)

MIRU-VNTR genotype profiles of the Mtb isolates
Initially, we applied the DTM PCR method to identify the Beijing genotype strains, and the majority of the studied isolates belonged to Beijing genotype (96.5%, 111/115), which limits the usefulness of spoligotyping. We thus further performed 28 loci MIRU-VNTR analyses to genotype all the isolates. The detailed information on the PCR primers used as well as the 28 loci MIRU-VNTR genotype profiles for each isolates are listed in Table S2 and Table S5, respectively. A total of 101 MIRU-VNTR genotypes were detected. Twenty isolates (17.4%) were clustered and 95 isolates (82.6%) had unique genotypes ( Fig. 1 and Table  S6).

Association between clinical characteristics of the patients and MIRU-VNTR genotype clustering
Comparison of demographic and clinical characteristics of the patients with clustered vs. unique genotype patterns are shown in Table 4. We observed higher proportion of re-treatment cases among patients with clustered isolates than those with unique MIRU-VNTR genotypes (75.0% vs. 41.1%). However, there was no obvious association between the drug resistance as well as several other characteristics of patients (such as gender, age, underlying diseases, and geographic location, etc.) with the clustered MIRU-VNTR genotypes of the corresponding Mtb isolates. By further examining the clinical data of the clustered isolates, we noticed that two groups of clustered isolates had clinical epidemiological links. Specifically, Mtb 1056 (susceptible)

Phylogenetic analysis of Mtb isolates
A total of 38,355 bp covering drug resistance-associated loci in Mtb were used for analysis as described in a previous study [3]. From the UPGMA tree based on the 28 loci MIRU-VNTR data of Mtb isolates, we observed that clustered isolates contained both susceptible and drug-resistant isolates (Fig. 1).

Discussion
Early detection of drug resistance in Mtb is important for the successful treatment of TB. New tools for rapid and cost-effective diagnosis of drug resistance in Mtb isolates are urgently needed to control drug-resistant TB, especially the more dangerous MDRand XDR-TB epidemics. Molecular diagnostics could potentially fill this need but require comprehensive information on the type and frequency of specific drug resistance-associated mutations. A recent study by Zhang et al. reported that through sequencing 161 Mtb isolates with a range of drug resistance profiles, they discovered 72 new genes, 28 intergenic regions, 11 nonsynonymous SNPs and 10 intergenic region SNPs with strong and consistent associations with drug resistance in Mtb [11]. In this study, we further identified 36 unreported mutations in drug resistance-associated genes in clinical Mtb isolates. Our study adds to the growing body of knowledge on potential drug resistanceassociated mutations in Mtb, which could lead to the development of rapid and more comprehensive molecular diagnostic methods for drug-resistant TB.
Some mutations we identified were seen in both susceptible and resistant isolates. For example, Rv1592c T70 (del) (INH), gidB S100F (SM), embC R927 (EMB), ethA Q360 H (ETH), gidB E92D (SM), gyrA (S95T) and gyrA E21Q (OFX, LVX) etc. were seen in identical or similar prevalence between susceptible and resistant isolates, thus those mutations should not be associated with drug resistance. Instead, they could be considered as reference specific mutations or phylogenetic informative mutations. The relatively low correlation between the phenotypic and genotypic drug resistance profiles for some drugs were consistent with our previous study and also some other studies [3,12]. Consistent with several previous studies, our observations showed that certain mutations were more frequently identified in resistant isolates vs. susceptible isolates and thus could serve as useful marker for rapid detection of resistance in the clinical Mtb isolates. For example, S315T and R463L in katG (for isoniazid resistance); S531L in rpoB; K43R and K88R in rpsL (for streptomycin resistance); M306V in embB (for ethambutol resistance); D94G and A90V in gyrA (for fluoroquinolone resistance); and G1332A and A1401G in rrs (for kanamycin resistance), etc. [13][14][15]. Except for those known drug resistance mutations, our studies also revealed a few more newly identified potential drug resistance mutations. For example, A1081 (del) in rpoB, A908C in rrs, A119D, A193 and A205 in gidB, A28 (del) in pncA, and A1789 (del) in rpoB. We then performed genetic studies by creating point mutation in the susceptible reference strain H37Rv using the pJV53K system [16] for those newly identified potential drug resistance-associated mutations, but failed to confirm their roles in causing drug resistance (data not shown). Our study further confirmed the notion that the genetic basis of drug resistance is more complex than previously anticipated, and a single point mutation could possibly contribute to but is not efficient for drug resistance [17,18].
Analysis of VNTR profiles specific to endemic strains in certain settings may be useful to identify the sources of outbreaks and transmission pathways. For example, genotyping analysis of MDR-TB isolates revealed that active transmission of MDRand XDR-TB is taking place in Portugal, and that the high prevalence of observed XDR-TB is due to the continued transmission of particular genetic clusters [19,20]. But another recent study from Mexico showed that almost all MDR cases studied were epidemiologically unrelated, indicating that the genetic variations observed among those strains are suggestive of emergence of acquired drug-resistance during the course of treatment [21]. A large proportion of the analyzed cases in our study were primary MDR-or XDR-TB. In addition, the clustered MDR-, and XDR-TB cases among the new cases strongly indicated that those patients had been primarily infected with those highly drug-resistant Mtb strains. The observation that clustered isolates contained both susceptible and drug-resistant isolates in the UPGMA tree suggested that the drug resistance traits of Mtb strains are not conserved within defined strain clusters but rather that individual isolates within each cluster have evolved unique characteristics during long-term antibiotic treatment and complicated microbe-host interaction process. This observation further confirmed the previous notion that drugresistant Mtb isolates have evolved and acquired different mutations independently [3]. We also observed higher proportion of re-treatment cases among patients with clustered isolates than those with unique MIRU-VNTR genotypes. This indicates that those re-treatment cases could be linked with reactivation or recent transmission of new strains. We did notice that several groups of isolates among clustered isolates had clinical epidemiological links, but since those epidemiologically linked clustered isolates had similar but not identical phenotypic and genotypic drug resistance profiles, thus those data could not support the incidence of patient-to-patient transmission during their hospitalization. A previous study indicated the limitations of using MIRU-VNTR typing and epidemiological investigations to detect transmission of Mtb in high burden settings and suggested that whole-genome sequencing (WGS) could provide better solution for tracing the transmission of Mtb in such settings [22]. Thus WGS could be used in the future to better discriminate the possibility of direct transmission events defined by the MIRU-VNTR typing in this study. Nevertheless, since the clustered isolates have a potential of transmission among patients as suggested by several previous studies [19,20,23], thus early detection of those isolates and strengthening of control measures to prevent transmission of them are warranted. A limitation of this study is a possible sampling bias, which could be caused by the following factors. First, the sampling of the participants was not completely randomized but was based on voluntary participation of the patients and availability of the pure cultures of the isolates. Second, since the sample size is relatively small and some mutations were detected at a relatively low frequency, thus the statistical analysis used to identify potential drug resistance mutations in this study might not be suitable for some mutations. For example, D516V in rpoB was previously considered to be a drug resistance mutation [24], while the P value for this mutation was higher than 0.05. By contrast, a couple of mutations that were known not associated with drug resistance, such as gidB E92D [25] and gyrA S95T [26], were found with a P value lower than 0.05. Third, the sampling was not continuously done, which could lead to reduced clustering of cases. Nevertheless, this study provided important baseline data that may be used to understand and monitor the molecular epidemiology of Mtb isolates in China. Further studies are warranted to better understand the detailed molecular mechanisms in MDR-and XDR-Mtb isolates.

Author Contributions
Conceived and designed the experiments: CHL. Performed the experiments: QW CHL. Analyzed the data: QW SKPL FL PCYW CHL. Contributed reagents/materials/analysis tools: YZ HML PCYW CHL. Contributed to the writing of the manuscript: CHL QW SKPL BXL FL YZ HML YLH PCYW.