Abstract

INTRODUCTION:

Mycobacterium tuberculosis (MTB) is a causative agent of tuberculosis (TB) that causes death worldwide.

METHODS:

MTB was subjected to phenotypic drug-susceptibility tests (DST), and drug-resistant genes were sequenced.

RESULTS:

Previously treated patients were more likely to have positive smear results and exhibit drug resistance. New patients were more likely to be mono SM-resistant and less likely to be INH- and RIF-resistant. The most common mutations were katG (S315T), rpoB (S450L), rpsL (K43R), and embB (M306V).

CONCLUSIONS:

The proportion of mono-SM-resistant TB among new patients was higher.

Keywords:
M. tuberculosis; Drug resistance; Gene mutations; Gene sequencing

Tuberculosis (TB), usually caused by Mycobacterium tuberculosis (MTB), remains a major threat to the public health worldwide. Although the directly observed treatment, short-course (DOTS) strategy has significantly reduced the incidence of TB in recent years, the emergence of drug-resistant TB has severely hampered TB prevention and control, especially regarding multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB)¹1. Zhao Y, Xu S, Wang L, Chin DP, Wang S, Jiang G, et al. National survey of drug-resistant tuberculosis in China. N Engl J Med. 2012; 366(23): 2161-70..

TB epidemics are unevenly distributed in China, and there is a high prevalence in rural areas, especially those that are underdeveloped in the northwest and southwest of China¹1. Zhao Y, Xu S, Wang L, Chin DP, Wang S, Jiang G, et al. National survey of drug-resistant tuberculosis in China. N Engl J Med. 2012; 366(23): 2161-70.. Migration, primarily from rural to urban environments, has become common in China in recent decades. Internal migration presents a significant challenge to the national TB control strategies. The latest national survey revealed that the frequency of MDR-TB among pulmonary TB patients in China was 8.32%¹1. Zhao Y, Xu S, Wang L, Chin DP, Wang S, Jiang G, et al. National survey of drug-resistant tuberculosis in China. N Engl J Med. 2012; 366(23): 2161-70.. MDR-TB is a serious clinical and epidemiological problem, incurring substantial economic management costs, as treating patients resistant to isoniazid (INH) and rifampicin (RIF) may be many times more expensive than treating those for whom the main medications are effective. Many studies have investigated the mechanisms of resistance to INH, RIF, streptomycin (SM), and ethambutol (EMB). Mutations in the katG gene are a major cause of INH resistance²2. Unissa AN, Subbian S, Hanna LE, Selvakumar N. Overview on mechanisms of isoniazid action and resistance in Mycobacterium tuberculosis. Infect Genet Evol. 2016; 45: 474-492.. MTB can acquire resistance to RIF through mutations in the rpoB gene, especially in the 81-bp RIF resistance determining region (RRDR)³3. Zhao LL, Chen Y, Liu HC, Xia Q, Wu XC, Sun Q, et al. Molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolates from China. Antimicrob Agents Chemother. 2014; 58(4): 1997-2005.. Mutation-carrying genes, such as rpsL and rrs, which encode the S12 ribosomal protein and 16S rRNA, respectively, are associated with intermediate or high levels of SM resistance⁴4. Hameed HMA, Islam MM, Chhotaray C, Wang C, Liu Y, Tan Y, et al. Molecular Targets Related Drug Resistance Mechanisms in MDR-, XDR-, and TDR-Mycobacterium tuberculosis Strains. Front Cell Infect Microbiol. 2018; 8:114.. Point mutations in embB codon 306, which occur in 30%-69% of clinical EMB-resistant strains, are associated with resistance to EMB⁵5. Ahmad S, Jaber AA, Mokaddas E. Frequency of embB codon 306 mutations in ethambutol-susceptible and -resistant clinical Mycobacterium tuberculosis isolates in Kuwait. Tuberculosis. 2007; 87(2): 123-9.. Thus, the identification of mutations, especially in katG, inhA, rpoB, rpsL, rrs, and embB, is thought to represent a rapid screening method for the detection of first-line drug resistance in clinical isolates.

The current analysis presents data on the drug resistance profiles of drug-resistant TB as well as gene mutations from a larger MTB DNA sample of the most recent prevalent drug-resistant isolates in rural areas of China. Based on positive sputum culture specimens, MTB isolates were identified, phenotypic drug sensitivity tests (DST) were conducted, and drug-resistant genes were sequenced.

Patient information was registered and verified at the Community Health Care Center. Specimens were collected from specialist TB hospitals in Hangzhou, China, from 2017 to 2019. Sputum samples were collected from patients suspected to have TB. Samples were subjected to acid-fast staining and microscopy tests and were cultured on Lowenstein-Jensen medium according to the national guidelines⁶6. Wang L, Liu J, Chin DP. Progress in tuberculosis control and the evolving public-health system in China. Lancet. 2007; 369(9562): 691-6. (^{Figure 1 in supplemental file} SUPPLEMENTARY MATERIAL Figure 1: Supplemental file. ).

Samples were submitted to the tuberculosis reference laboratory of the Hangzhou Center for Disease Control and Prevention. Phenotypic DSTs for each drug were determined using the proportional method on Lowenstein-Jensen medium. The concentrations of INH (0.2 mg/L), RIF (40 mg/L), SM (4.0 mg/L), and EMB (2.0 mg/L. Sterile deionized water (ddH₂O) and a standard strain of H37Rv (American Type Culture Collection 27294) were used as negative and positive controls, respectively, in all experiments. Drug-resistant MTB genomic DNA was extracted and stored at 20 °C for further use. Genetic fragments associated with drug resistance were amplified using previously reported primers⁷7. Zhao LL, Chen Y, Chen ZN, Liu HC, Hu PL, Sun Q, et al. Prevalence and molecular characteristics of drug-resistant Mycobacterium tuberculosis in Hunan, China. Antimicrob Agents Chemother . 2014; 58(6): 3475-80.. Purified PCR products were sent to Sangon Biotech (Shanghai, China) for sequencing. Sequences were aligned with the published sequences (GenBank accession number NC_000962).

Categorical data were summarized as counts or percentages (%), and the groups were compared using chi-square test or Fisher's exact test. Statistical significance was set at p < 0.05. SPSS 11.0 (IBM SPSS Statistics, United States) software was used for the statistical analyses.

As shown in Table 1, among 2 578 TB patients, the number of male patients was higher than that of female patients in each group (χ² = 23.861, p < 0.001). There were more patients between 76 and 91 years old in the new group (47.80%) than in the previously treated group (27.60%; χ² = 91.690, p < 0.001). The group with no TB treatment history comprised more of clerks (χ² = 48.625, p < 0.001), retirees (χ² = 109.309, p < 0.001), and residents (χ² = 81.078, p < 0.001). Previously treated patients were more likely than new patients to have positive smear results (χ² = 41.079, p < 0.001) and drug resistance (χ² = 196.419, p < 0.001).

Of these, 163 were resistant only to INH, 26 were resistant only to RIF, 168 were resistant only to SM, and 23 were resistant only to EMB (Table 2). Among the 500 drug resistant isolates, 3.45% of isolates (89/2 578) were MDR, 64 were resistant to two drugs, 46 were resistant to three drugs, and 10 were resistant to all drugs.

Sequencing results of katG, inhA, rpoB, rpsL, rrs1, and embB genes showed that 68.68% (193/281) carried a single mutation in the katG gene at codon 315 (Table 3), 9.25% (26/281) carried a single mutation in the inhA gene at codon -15 or -8, and 16.37% (46/281) had mutations in both the katG and inhA genes. The most frequent mutation was S315T (81.14%, 228/281). For RIF-resistant isolates, 90.60% (106/117) carried mutations in the RRDR of rpoB. Seven mutation sites were identified, and the most frequent mutation was at codon 450 (42.74%, 50/117). The second most frequent mutation was found at codon 445 (31.62%, 37/117). In addition, 11.97% (14/117) had mutations at codon 435, while three isolates had synonymous mutations at codons 427, 428 and 430, respectively. Furthermore, 87.55% (204/233) of the SM-resistant isolates harbored mutations in rpsL or rrs1. The most frequent mutation in the rpsL gene was K43R (74.68%, 174/233). A total of 70.91% (39/55) of the EMB-resistant isolates had mutations in the embB gene.

This study analyzed the epidemiology of drug-resistant TB in seven rural areas in Hangzhou (Yuhang, Fuyang, Linan, Tonglu, Jiande, Chunan, and Xiaoshan). Of all included patients, 76.96% (1 984/2578) were Hangzhou residents, and the proportion of previously treated patients was 25.02% (645/2 578), which was significantly lower than the data from other TB hospitals in China⁸8. Cao Z, Lan Y, Chen L, Xiang M, Peng Z, Zhang J, et al. Resistance To First-Line Antituberculosis Drugs And Prevalence Of pncA Mutations In Clinical Isolates Of Mycobacterium tuberculosis From Zunyi, Guizhou Province Of China. Infect Drug Resist. 2019; 12: 3093-3102.. The results indicated a functioning local TB control program in Hangzhou, China. The majority of previously treated patients were male, had positive sputum smear results, and were resistant to RIF (all P < 0.05). A total of 20.25% (522/2 578) isolates were resistant to any of the four first-line anti-TB drugs, and the proportion of MDR-TB was 3.45%, much lower than that reported by Lv et al. (31.1% and 10.1%, respectively)⁹9. Lv XT, Lu XW, Shi XY, Zhou L. Prevalence and risk factors of multi-drug resistant tuberculosis in Dalian, China. J Int Med Res. 2017; 45(6): 1779-1786.. In this study, all TB isolates were collected from specialist TB hospitals in rural areas. We could not obtain all drug-resistant isolates because some patients may seek more highly specialist TB hospitals in urban areas. However, our results revealed, to some extent, the prevalence of drug-resistant TB in the rural areas of Hangzhou.

Drug-resistant TB is often caused by mutations in genes, especially in katG and inhA genes for INH resistance, rpoB for RIF resistance, rpsL and rrs1 for SM resistance, and embB for EMB resistance. Therefore, we analyzed these genes in the current study. The predominance of the S315T substitution in the katG gene in INH resistance has been demonstrated globally, and estimates of this mutation range from < 25% to > 90%, and inhA also confers low-level INH resistance (> 10%)²2. Unissa AN, Subbian S, Hanna LE, Selvakumar N. Overview on mechanisms of isoniazid action and resistance in Mycobacterium tuberculosis. Infect Genet Evol. 2016; 45: 474-492.. We demonstrated that the frequency of the S315T substitution in the katG (81.14%) and inhA (25.62%) genes was higher than that found by Zhao et al., who reported findings (45.3% and 23.3%, respectively) from the neighboring province of Fujian⁷7. Zhao LL, Chen Y, Chen ZN, Liu HC, Hu PL, Sun Q, et al. Prevalence and molecular characteristics of drug-resistant Mycobacterium tuberculosis in Hunan, China. Antimicrob Agents Chemother . 2014; 58(6): 3475-80.. The frequency of the S315T mutation was also higher than that found in studies in Jiangxi Province (67.00%)¹⁰10. Luo D, Chen Q, Xiong G, Peng Y, Liu T, Chen X, et al. Prevalence and molecular characterization of multidrug-resistant M. tuberculosis in Jiangxi province, China. Sci Rep. 2019; 9(1): 7315.. Resistance to RIF is a well-known surrogate marker of MDR-TB, and mutations in the rpoB RRDR remain important RIF resistance markers. In our study, all mutations conferring RIF resistance had mutations within the RRDR, with the most prevalent being S450L (39.32%, 46/117), followed by H445L (11.97%, 14/117). Mutations at codon 445 were the most diverse (H→R/L/D/Y/C/P/N). In agreement with the studies reported previously¹⁰10. Luo D, Chen Q, Xiong G, Peng Y, Liu T, Chen X, et al. Prevalence and molecular characterization of multidrug-resistant M. tuberculosis in Jiangxi province, China. Sci Rep. 2019; 9(1): 7315., 90.60% (106/117) of RIF-resistant isolates had mutations in the RRDR of the rpoB gene, and although they were at different locations, most of them were located at three rpoB codons: 450, 445, and 435.

InhA and katG genes were the most clinically relevant and determined resistance in most clinical isolates, and this was the main reason we included only inhA and katG in our study. We found that 5.69% of INH-resistant isolates were not associated with any genotypic mutations in inhA or katG, which was a much lower result than that reported by Hazbon et al. previously¹¹11. Hazbón MH, Brimacombe M, Bobadilla del Valle M, Cavatore M, Guerrero MI, Varma-Basil M, et al. Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis. Antimicrob Agents Chemother . 2006; 50(8): 2640-9.. Furthermore, we reported the frequency of the S315N mutation to be 3.91%, which was much lower than that reported in Taiwan¹²12. Tseng ST, Tai CH, Li CR, Lin CF, Shi ZY. The mutations of katG and inhA genes of isoniazid-resistant Mycobacterium tuberculosis isolates in Taiwan. J Microbiol Immunol Infect. 2015; 48(3): 249-55.. To date, the S315N mutation has rarely been reported in Hangzhou, China.

Regarding the two other first-line anti-TB drugs, 87.55% (204/233) of SM-resistant isolates and 70.91% (39/55) of EMB-resistant clinical isolates were identified using molecular techniques. The findings were similar to those reported by Zhao et al. (82.9% and 70.6%, respectively)⁷7. Zhao LL, Chen Y, Chen ZN, Liu HC, Hu PL, Sun Q, et al. Prevalence and molecular characteristics of drug-resistant Mycobacterium tuberculosis in Hunan, China. Antimicrob Agents Chemother . 2014; 58(6): 3475-80.. Our results showed that 70.91% of EMB-resistant isolates carried mutations at codon 306, which was higher than that reported previously (58.00%)¹³13. Al-Mutairi NM, Ahmad S, Mokaddas E. Molecular Screening Versus Phenotypic Susceptibility Testing of Multidrug-Resistant Mycobacterium Tuberculosis Isolates for Streptomycin and Ethambutol. Microb Drug Resist. 2018; 24(7): 923-931.. Of the SM-resistant isolates, 12.45% had mutations in neither rpsL nor rrs1, and 29.09% of EMB-resistant isolates had no mutations in the embB gene, so other related genes may be involved¹⁴14. Islam MM, Tan Y, Hameed HMA, Chhotaray C, Liu Z, Liu Y, et al. Phenotypic and Genotypic Characterization of Streptomycin-Resistant Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates in Southern China. Microb Drug Resist . 2020; 26(7): 766-775.. GidB mutations have been found in both resistant and susceptible clinical drug-resistant MTB isolates¹⁵15. Nhu NT, Lan NT, Phuong NT, Chau Nv, Farrar J, Caws M. Association of streptomycin resistance mutations with level of drug resistance and Mycobacterium tuberculosis genotypes. Int J Tuberc Lung Dis. 2012; 16(4): 527-31.; therefore, we did not include the gidB gene in the analysis we used to detect SM resistance. We sequenced the associated gene fragments of 30 all-drug susceptible MTB isolates simultaneously; however, we found none of the mutations mentioned above.

Our study showed that the overall prevalence of the first-line drug-resistant TB in the rural areas of Hangzhou, China was low. However, the proportion of INH and SM resistance were higher. The most prevalent genetic mutations associated with INH, RIF, SM, and EMB resistance were katG (S315T, 81.14%), rpoB (S450L, 39.32%), rpsL (K43R, 74.68%), and embB (M306V, 49.09%), respectively. Additionally, we identified a rare substitution mutation of S450P in the RRDR of the rpoB gene. Furthermore, we found that new TB patients were more likely to be resistant only to SM and less likely to be resistant to both INH and RIF than previously treated patients. Our findings could be helpful in the development of rapid molecular diagnostic methods and may improve our understanding of drug resistance in Hangzhou, aiding the development of precision medicine for TB and the disturbance of drug-resistant TB transmission.

ACKNOWLEDGMENTS

We offer our deepest thanks to the institutions that provided technical support for the development and implementation of this study.

REFERENCES

SUPPLEMENTARY MATERIAL

Figure 1:
Supplemental file.

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