Mutation and Transmission Profiles of Second-Line Drug Resistance in Clinical Isolates of Drug-Resistant Mycobacterium tuberculosis From Hebei Province, China

The emergence of drug-resistant tuberculosis (TB) is involved in ineffective treatment of TB, especially multidrug resistant/extensively resistant TB (MDR/XDR-TB), leading to acquired resistance and transmission of drug-resistant strains. Second-line drugs (SLD), including both fluoroquinolones and injectable drugs, were commonly proved to be the effective drugs for treatment of drug-resistant TB. The purpose of this study was to investigate the prevalence of SLD-resistant strains and its specific mutations in drug-resistant Mycobacterium tuberculosis clinical isolates, and to acknowledge the transmission pattern of SLD resistance strains in Hebei. The genes gyrA, gyrB, rrs, eis promoter and tlyA of 257 drug-resistant clinical isolates were sequenced to identify mutations that could be responsible for resistance against fluoroquinolones and second-line injectable drugs. Each isolate was genotyped by Spoligotyping and 15-loci MIRU-VNTR. Our results indicated that 48.2% isolates were resistant to at least one of five SLD. Of them, 37.7% isolates were resistant to fluoroquinolones and 24.5% isolates were resistant to second-line injectable drugs. Mutations in genes gyrA, gyrB, rrs, eis promoter and tlyA were detected in 73 (75.3%), 7 (7.2%), 24 (38.1%), 5 (7.9%), and 3 (4.8%) isolates, respectively. The most prevalent mutations were the D94G (23.7%) in gyrA gene and the A1401G (33.3%) in rrs gene. A combination of gyrA, rrs and eis promoter can act as a valuable predicator for predicting XDR phenotype. These results highlight the development of rapid diagnosis are the effective manners for the control of SLD-TB or XDR-TB.


INTRODUCTION
Today, tuberculosis (TB) remains a major threat worldwide than any other single infectious disease. Millions of people continue to fall sick with TB each year. Moreover, the increasing rates of drug-resistant TB (DR-TB) worldwide and the emergence of multidrug/extensively-drug resistant TB (MDR/XDR-TB), leading to a high mortality as well as the financial burden. Regarding cases, estimates of the burden of DR-TB have focused on MDR-TB, there were 457,560 people (range: 396,060-523,980) developed MDR-TB (World Health Organization [WHO], 2018). Among cases of MDR-TB, 8.5% (95%CI: 6.2-11.0) were estimated to have XDR-TB (World Health Organization [WHO], 2018).
MDR-TB patients ordinarily require 18 months of treatment with recommendatory second-line anti-TB drugs (SLD) that primarily include fluoroquinolones (FQ) and second-line injectable drugs (SLID). Similar to other anti-TB agents, the main mechanisms of Mycobacterium tuberculosis (M. tuberculosis) resistant to SLD rely on spontaneous chromosomal mutations. Associated-mutations in the fluroquinolone resistancedetermining region (QRDR) of DNA gyrase-coding genes gyrA and gyrB turned out to be responsible for FQ resistance (FQ r ) (Chien et al., 2016). The genetic determinants of SLID resistance (SLID r ) are more complicated and suggested that being involved in three well-known genes. Cross-resistance between kanamycin (KAN), amikacin (AMK) and capreomycin (CAP) is thought to be associated with mutations in the16S rRNA gene rrs, specifically at position A1401G (Alangaden et al., 1998;Oudghiri et al., 2018). Mutations in promoter region of eis gene, encoding an aminoglycoside acetyltransferase, caused low-level KAN resistance (Bauskenieks et al., 2015;Ngo et al., 2018). CAP resistance has been correlated with mutations in tlyA gene, which encodes a putative 2 -O-methyltransferase (TlyA) (Freihofer et al., 2016;Witek et al., 2017).
Currently, little is known about mutation profiles of SLD resistance (SLD r ) in clinical M. tuberculosis isolates in our area, the objectives of this study were to compare the sequencing data of SLD r associated-genes (FQ for gyrA and gyrB, SLID for rrs region 1400, eis promoter and tlyA) with the phenotypic results by the traditional proportion method in 275 drug-resistant clinical M. tuberculosis isolates collected in Hebei, and to analyze the M. tuberculosis genetic diversity of SLD r -TB (covering FQ r -TB and SLID r -TB), and to elucidate transmission pattern by using Spoligotyping and 15-loci MIRU-VNTR typing.

M. tuberculosis Isolates
For the present study, we successfully recovered 257 clinical M. tuberculosis isolates. Isolates were obtained from sputum samples provided by confirmed pulmonary patients (167 males and 90 females; age range: 14-83 years; median age: 41 years; 146 new patients and 111 retreated patients) attending eight hospitals in Hebei over 1-year period (From January to December in 2014). One hundred pan-susceptible M. tuberculosis isolates served as negative control. Figure 1 shows the selection process of clinical M. tuberculosis isolates originated from Hebei Province.

DNA Isolation, Amplification, and Sequencing
Genomic DNA extractions were re-suspended a loopful of bacilli in 200 uL of fast lysis buffer (Qiagen, Valencia, CA, United States) and heat inactivated at 80 • C for 10 min, followed by centrifugation at 13,000 g for 5 min. All genes were chosen on the basis of documented association with resistance to SLD and included gyrA and gyrB for FQ and rrs, eis promoter, and tlyA for SLID. Primers and amplicon sizes are presented in the Supplementary Table S1. Each 20 µL PCR mixture was prepared as follows: 10 µL 2× Taq Master Mix (CWBOIO, Beijing, China), 1 µL of the forward and reverse 10 µM primers, 1 µL genomic DNA, and final 7 µL distilled H 2 O complement. PCR program for amplification were 5 min at 94 • C, followed by 35 cycles of 30 s at 94 • C, 30 s at 60 • C, 30 s at 72 • C, and a final extension 72 • C for 10 min.
Sequencing services were provided by Tsingke Biological Technology company (TsingKe, Beijing, China). All the mutations were identified and aligned with the homologous sequences of the reference M. tuberculosis H37Rv strain (GenBank accession number NC_000962) using the BLASTN algorithm 1 .

Genotyping
Spoligotyping was used to identify Beijing family as described previously by Kamerbeek et al. (1997). The direct repeat region was amplified with the primer pairs included DRa (5 -GGTTTTGGGTCTGACGAC-3 ) and DRb (5 -CCGAGAGGGGACGGAAAC-3 ). All PCR products were hybridized to a set of 43 oligonucleotide probes corresponding to each spacer that were covalently bound to a membrane. Spoligotypes in binary formats were matched with Spoldb.4.0 database 2 , and applied the published rules for definition of Beijing family (hybridization to at least three of the spacers 35-43 in direct repeat region and absence of hybridization to spacers 1-34).

Definitions
Based on the following definitions, we utilize these terms throughout the rest of this article. (i) Drug-resistant (DR): defined as resistance to at least one first-or second-line drug;

Data Analysis
To assess the associations between variables (DST, mutations, and genotyping), the χ 2 test, odds ratio (OR) and 95% confidence interval (95%CI) were calculated. And the Fishers' exact was used if any expected counts are less than 5. A P-value of < 0.05 was considered statistically significant. All statistical data analyzed with SPSS version 22.0 (IBM SPSS, Chicago, IL, United States).

Strains Selection and Drug Resistance Patterns
Initially, a total of 316 DR isolates were re-cultured and finally only 257 eligible isolates were included in this study. The selection procedure of strains is shown in Figure 1 (Figure 2A). Among 52

Mutations in gyrA and gyrB
It is known to that the main molecular mechanism of FQ r was caused by mutations in the QRDR of DNA gyrase, which composed of GyrA and GyrB subunits, encoded by gyrA and gyrB genes, respectively. DNA sequencing results of these two genes from FQ r , FQ s and pan-susceptible isolates were summarized in Table 2.

Mutations in rrs, eis Promoter and tlyA
To determine the molecular basis of resistance to SLID, the rrs, eis promoter, and tlyA region were sequenced both in SLID r , SLID s , and pan-susceptible strains. Table 3 showed the mutations in the 1400 region of rrs, eis promoter and tlyA as well as the corresponding resistance phenotypes.
Of 193 SLID s isolates, one isolate each of A1128G, A1138G, C1209T, and C1483T, all occurred in rrs. Furthermore, for eis promoter, a change from G to T at −37 was detected only one isolate.
Among 79 pan-susceptible isolates, none of mutations were tested by us, except that, two clinical isolates with mutations V54G and T53T in tlyA.

Association Between Gene Mutations and Phenotypes
Among the 97 FQ r isolates with gyrA mutations, 63.9% (62/97) were resistant to OFX and 50.5% (49/97) were resistant to LVX. As seen in Figure 2C, strongly evidence showed that gyrA mutations were associated with the cross-resistance of FQ (4.  Table 4).

Genotyping
Three major diverse families were identified among the 124 SLD r isolates, including Beijing, T and H family. Beijing family was the largest sub-lineage with 112 isolates. Nine isolates belonged to the T family, 6 isolates were T1 sublineage, 2 isolates were T2, and 1 isolate was T3. The remaining 3 isolates were identified as H3 sub-lineage.
A total of 124 SLD r isolates were genotyped by 15-loci MIRU-VNTR, revealed that the strains were divided into 113 genotypes, of which 107 were identified as unique patterns and 17 isolates were categorized to cluster patterns (Figure 4). Six small clusters ranging from 2 to 4 isolates were observed in all genotypes. The cumulative clustering rate and recent transmission rate rely on the MIRU-VNTR typing was 13.7% (17/124) and 4.8% (6/124), respectively.
Among six clusters with SLD r strains, two Pre-XDR isolates with a gyrA (A90V or D94G) mutation occurred exclusively in Cluster I. Cluster II contained three isolates, two Pre-XDR isolates presented at gyrA mutation (D94G), one of them accompanied with a mutation at rrs (T1491C), the rest of FQ had no mutation. Cluster III grouped in 4 isolates, the Pre-XDR included 2 isolates with a mutation at gyrA (D94G or D94A), one was FQ and another was SLID without any mutation. Three isolates of cluster IV had three different polymorphisms at either gyrA (A90V or D94N) or rrs (A1401G) locus. Cluster V comprised 3 isolates, the XDR had gyrA (A90V or D94G) and rrs (A1401G) mutations sharing with 2 isolates, whereas one Pre-XDR isolate with gyrA (D94G) and eis promoter C (−14) T.
Cluster VI had one XDR isolate with a mutation at rrs (A1401G) and FQ with a mutation at gyrA (D94G).

DISCUSSION
To our best knowledge, the present study is the first to provide a description of mutations associated with SLD r in M. tuberculosis strains from Hebei. Nevertheless, we examined the phylogenetic diversity in SLD r strains through genotyping profiles. The present findings revealed that 48.2% (124/257) were resistant to at least one five SLD. Previously, several studies reported the resistance rate of SLD strains has been estimated to range from 25.7 to 51.9% (Bakuła et al., 2016;Brossier et al., 2017;Hu et al., 2017). According to a recently published World Health Organization (WHO) report, among cases of MDR-TB in 2017, 8.5% (95%CI, 6.2-11.0%) were estimated to have XDR-TB (World Health Organization [WHO], 2018). We reported the frequency of XDR-TB is 10.9%. FQ r isolates is primarily attributed to mutations in the QRDR of gyrA. According to a systematic review, gyrA mutations were reported in codons 88-94 appeared to account for roughly 60.0-90.0% of FQ r globally (Avalos et al., 2015). In our present study, mutations at gyrA codon 89, 90, 91, and 94 were observed in 75.3% of resistant isolates. The frequency of mutations conferring FQ r is similar to Shanghai (76.0%), although it is lower than those in Russia (83.0%), India (81.0%) and Thailand (92.3%), and higher than those in Morocco (30.0%) and New York (67.0%) (Sullivan et al., 1895;Mokrousov et al., 2008;Zhu et al., 2012; Disratthakit et al., 2016;Singhal et al., 2016;Chaoui et al., 2018), indicating that mutations in gyrA tend to differ by geographic region. In line with previous studies (Singhal et al., 2016;Pang et al., 2017;Chawla et al., 2018), substitutions at codon 94 were the most frequent mutation among FQ r isolates. This phenomenon may be explained by the fact that codon 94, which aims at the water-magnesium ion bridge with a conserved C3/C4 keto acid moiety of quinolones, plays an important role in stabilizing the quinolone molecule in the quinolone binding pocket, an amino acid substitution at this position will exaggerate the deleterious effect of the binding between most quinolones and DNA gyrase (Aldred et al., 2016;Blower et al., 2016;Disratthakit et al., 2016).
Interestingly, significant evidence has demonstrated a link between gyrA mutations and FQ, MDR, Pre-XDR and XDR, suggesting that mutations at gyrA might be act as a candidate diagnostic marker for FQ, MDR and a possible indicator of Pre-XDR-TB or XDR-TB. In Mycobacterium, the interactive effect between rifampicin-and FQ-resistant mutations was influenced by epistasis and produced a varying degree of loss in fitness (Borrell et al., 2013). We hypothesized that the progression of MDR to Pre-XDR or XDR may be caused by the positive epistasis between gyrA mutations and mutations in drug resistant gene conferring rifampicin. Certainly, further research is required to confirm this hypothesis.
Unlike the high frequency of gyrA mutations, mutations in gyrB gene are seldom commonly associated with FQ r in M. tuberculosis isolates. A gyrB mutation (L442L, S447F, N499T, A504V, or A508A) was identified in 7 FQ r isolates. As far as we known, a L442L, S447F, N499T, A504V mutation has not been previously reported. Due to the small numbers of isolates with gyrB mutations, no such mutation was significant independently associated with FQ r . Additionally, we found that both FQ r and FQ s strains exhibited an A508A mutation in gyrB, T53T 1 a KAN, kanamycin; AMK, amikacin; CAP, capreomycin; R, resistance; S, sensitivity. b WT, wild-type.   making it difficult to acknowledge the contribution to phenotypic resistance. Several publications reported double mutations in gyrA, gyrB or both gyrA and gyrB, which ranged from 1.0 to 3.0% (Avalos et al., 2015;Chien et al., 2016;Kateete et al., 2019). Double mutations in both gyrA and gyrB were found in FQ r isolates but the absence of susceptible isolates, suggesting that although rare, it can be as the highly specific predictor of FQ r . Previous reports indicated the A1401G in rrs effectively identify the phenotypic resistance to KAN, AMK and CAP (Malinga et al., 2016;Varghese and Al-Hajoj, 2017). In our study, 36.5, 63.2, and 50.0% of sensitivities with an A1401G mutation in rrs were resistant to KAN, AMK, and CAP, respectively (Supplementary Tables S6-S8). Compared to DST test, it seems that a mutation at A1401G provided a better maker for AMK and CAP than for KAN. However, the canonical A1401G rrs mutation explains only around 56.0% of KAN r , while the mutations G10A and C14T in the promoter of eis, explain another 33.0% of low-level KAN r as estimated by one review (Georghiou et al., 2012). Our study supports these finding, as 5 KAN r isolates were found to have mutations in eis promoter, including three of G (−10) A and two of C (−14) T. Mutations in tlyA is also known to responsible for resistance to CAP. In fact, we observed three isolates were resistance to CAP alone, but KANsensitive and AMK-sensitive isolates had no mutations in tlyA gene. Consequently, tlyA should be included in the molecular analysis of CAP-resistance.
In our study, rrs mutations were significantly associated with MDR-TB (5.1 OR, 95%CI [2.0, 13.0], P < 0.001), and 95%CI [9.4,62.2], P < 0.001), suggesting the detection of rrs mutations provide the valuable information to support the initiation of effective treatment regimens for MDR-TB or even XDR-TB cases. Specifically, as previously reported (Zhang et al., 2014;Ogari et al., 2019), we observed a significant cross-resistance between KAN, AMK and CAP associated with rrs gene. Therefore, rrs might serve as a marker to predict the cross-resistance among SLID agents.
The traditional phenotypic DSTs lead serious delays to the detection of resistance just because of the extremely slow growth of M. tuberculosis. However, several commercial diagnostic tests such as GeneXpert MTB/RIF, Genotype MTBDRplus/MTBDRsl assays, have been developed to rapidly detect both first-and second-line drug resistance in M. tuberculosis by scanning the associated mutations (Xie et al., 2017;Kim et al., 2019). Our performance showed that analysis of the associated mutations was recommended to provide a good sensitivity for rapid verification of FQ r (79.4%), SLID r (50.8%), Pre-XDR (80.4%), and XDR (92.9%). In addition, sensitivities and specificities for predicting phenotypic resistance to five SLD in M. tuberculosis isolates as shown in Supplementary Tables S4-S8.
Spoligotyping result showed that the predominance of Beijing family (SIT1), which accounted for the largest cluster (71.8%) among SLD r strains in this study. No significant associations were found between the genotypes and specific types of resistance. But a high frequency of Beijing family strains among FQ r isolates was previously reported from Vietnam and Russia (Mokrousov et al., 2008;Duong et al., 2009). One possibility is that the Beijing family strains have a high level of intrinsic resistance to FQ, providing an opportunity to tolerate low level of FQ and subsequently generate gyrA mutations, or that these mutations confer an advantage under the absence of antibiotics pressure (Mokrousov et al., 2008). An observation revealed that eis mutations were considered to be associated with the Beijing clades (Casali et al., 2014); however, this is not applicable for all geographical settings. Generally, we found no significant correlation between any mutation and a spoligotype family, which is accordance with reports from China (Zhang et al., 2013(Zhang et al., , 2014. In this study, an UPGMA-tree was generated from 15-loci MIRU-VNTR among 124 SLD r isolates, which was slightly similar with our previous study on the acquired resistance of MDR-TB in this region (Li et al., 2019). The highly different genotypic patterns and drug resistance profiles suggest that acquisition of resistance is also an important cause for the emergence of SLD r in Hebei Province. MIRU-VNTR data revealed a moderate level of genotypic diversity, which implicated composite mechanisms of resistance, including transmitted and acquired resistance, as a potential cause for the emergence of SLD r strains. Transmission of clustered SLD r strains with identical mutations may indicated the acquisition of drug resistance typically confers a reduction in fitness cost, and mutations may further contribute to the spreading of Pre-XDR-TB or XDR-TB. Therefore, the strict DOTS is the necessary principle to reduce the incidence of acquired strains. In addition, 28.6% of XDR strains were clustered, and it can be inferred that there exists cloning transmission between a small number of XDR strains.
The main limitation of this study was only ability to perform at critical concentrations for DST but not established MICs. This discrepancy of the critical-concentration method used for DST, such that up to 5% of wild-type M. tuberculosis strains are classified as drug resistant. While our study is also limited by the number of strains, further studies continue to collect larger additional strains to depict acquired resistance in the community and household and build transmission chain.

CONCLUSION
In summary, this study showed that 48.2% of isolates were resistance to at least one of five SLD, and most of which were resistant to OFX, LVX, and KAN. The majority of FQ r and SLID r strains were associated with gyrA mutation at D94G and rrs mutation at A1401G, respectively. Mutations in gyrA, rrs, and eis promoter seem to be the causative biomarker for the screening of resistance to SLD, even XDR-TB itself. No correlation was found between any mutation and a spoligotype family. Furthermore, acquired resistance is one of the critical factors driving the SLD r strains in Hebei Province. These results highlight the use of appropriate treatment regimens and the development of early rapid diagnosis are the effective manners for the control of SLD-TB or XDR-TB patients.

DATA AVAILABILITY
All the data analyzed throughout this research are included in this published article.

ETHICS STATEMENT
Approval for this study was obtained from the Medical Ethics Committee of Scientific Research Project of the Fifth Hospital of Shijiazhuang. Written informed consent was obtained from each participant according to the Federal and Institutional Guidelines.

AUTHOR CONTRIBUTIONS
QL performed the DST and DNA extraction, analyzed the data, and wrote the first draft of the manuscript. HG, ZZ, and YT performed the DST and spoligotyping. TL performed the MIRU-VNTR typing. YW collected the samples and supported the study. JL and YL contributed to the conception and design of the study. ED designed the study, collected the samples, and revised the manuscript. All authors read and approved the final manuscript.