Associations between Mycobacterium tuberculosis Beijing genotype and drug resistance to four first-line drugs: a survey in China

Abstract

Investigations on the genetic diversity of Mycobacterium tuberculosis in China have shown that Beijing genotype strains play a dominant role. To study the association between the M. tuberculosis Beijing genotype and the drug-resistance phenotype, 1286 M. tuberculosis clinical isolates together with epidemiological and clinical information of patients were collected from the center for tuberculosis (TB) prevention and control or TB hospitals in Beijing municipality and nine provinces or autonomous regions in China. Drug resistance testing was conducted on all the isolates to the four first-line anti-TB drugs (isoniazid, rifampicin, streptomycin, and ethambutol). A total of 585 strains were found to be resistant to at least one of the four anti-TB drugs. The Beijing family strains consisted of 499 (53.20%) drug-sensitive strains and 439 (46.80%) drug-resistant strains, whereas the non-Beijing family strains comprised 202 (58.05%) drug-sensitive strains and 146 (41.95%) drug-resistant strains. No significant difference was observed in prevalence (c²=2.41, P>0.05) between the drug-resistant and drug-sensitive strains among the Beijing family strains. Analysis of monoresistance, multidrug-resistant TB, and geographic distribution of drug resistance did not find any relationships between the M. tuberculosis Beijing genotype and drug-resistance phenotype in China. Results confirmed that the Beijing genotype, the predominant M. tuberculosis genotype in China, was not associated with drug resistance.

Keywords

tuberculosis / drug resistance / genotype / molecular biology

Introduction

Mycobacterium tuberculosis, the agent of tuberculosis (TB), was first identified by Robert Koch in 1882. With the development of modern molecular biology technology, molecular epidemiological studies have identified a genetically related group of M. tuberculosis strains called the Beijing family, which was first described in 1995 by van Soolingen et al. as representing 86% of isolates from Beijing, China [1]. The Beijing genotype originated in East Asia and spread from China [2], where it has established the predominant M. tuberculosis genotype for at least 50 years [3]. Currently, its prevalence is 50% in East Asia and 13% globally. Li et al. reported that 64.9% of M. tuberculosis strains are of the Beijing genotype in China [4]. Recently, we showed that the highest number of Beijing family strains was observed in northern China (85%–95%) with the exception of the Xinjiang Autonomous Region in the northwest (65%), whereas a low percentage was found in southern China (50%–60%). We also uncovered a large proportion of ancestral isolates of the Beijing clade in the Guangxi Autonomous Region, which was in favor of the emergence of this clade in China [5]. Meanwhile, infection by drug-resistant M. tuberculosis (DR-TB) strains is a matter of great concern for TB control programs because no cure is available for multidrug-resistant (MDR-TB) strains of M. tuberculosis, which may spread around the world. Despite the availability of highly efficacious treatment for decades, TB remains a major global health problem.

The M. tuberculosis Beijing genotype has prevailed in China and in other countries in Asia. Both the incidence and prevalence of TB in China have shown a steady decline in recent years. However, the World Health Organization estimates that India, China, and the Russian Federation account for 45% of the combined total of 580 000 new MDR-TB cases and an additional 100 000 people with rifampicin-resistant TB cases [6]. The fifth national TB epidemiological survey in 2010 revealed that the spread of DR-TB, especially MDR-TB, in China, presents a major challenge. The total rate of first-line drug resistance was 36.8%, and the MDR rate was as high as 6.8%. Thus, TB remains a serious burden in China.

In the past, many studies focused on the worldwide spread of Beijing genotype strains. In particular, the association of this genotype with drug resistance is of major interest to a large number of research groups. A high resistance rate has been observed among the Beijing strains, whereas the non-Beijing strains are significantly less resistant to each drug in some countries [7–10]. This phenomenon suggests that the Beijing genotype strains are associated with drug resistance. Therefore, the following hypothesis was proposed to explain the prevalence of the Beijing genotype: the strains of the Beijing family might become drug resistant with high efficiency, implying that drug resistance was responsible for the emergence of this family.

In this study, clinical isolates of M. tuberculosis from the Beijing municipality and nine provinces or autonomous regions in China were used to analyze the relationships between the M. tuberculosis Beijing genotype and drug-resistant phenotypes.

Materials and methods

Standard strain H37Rv

The reference strain H37Rv was cultured, inoculated, conserved, and provided by the National Reference Laboratory of TB, State Key Laboratory for Infectious Diseases Prevention and Control (SKLID)/National Institute for Communicable Disease Control and Prevention (ICDC), Chinese Center for Disease Control and Prevention (China CDC) in Beijing, China.

M. tuberculosis clinical strains and DNA extraction

From 2004 to 2010, for this study, 1286 M. tuberculosis clinical isolates were obtained from 10 different provinces (municipality and autonomous regions) across China, namely, Beijing, Tibet, Jilin, Gansu, Henan, Hunan, Xinjiang, Sichuan, Fujian, and Guangxi. The sputum samples were collected from patients suspected of TB who went to the institutes for TB control and cure or TB hospitals in each province. The bacteria were isolated and inoculated on Löwenstein–Jensen medium [11]. All samples were cultured, and the bacteria were kept in the National Reference Laboratory of TB, SKLID/ICDC, China CDC in Beijing, China. The epidemiological and clinical information of patients was collected using a special epidemiological questionnaire. The strains were genotyped by spoligotyping, and the results were previously reported [5].

Drug sensitivity test

The proportion method and the absolute concentration method were used to test the susceptibility of the isolates to the four first-line anti-TB drugs, namely, isoniazid (INH), rifampicin (RFP), streptomycin (SM), and ethambutol (EMB) [11].

Statistical analysis

Chi-square test and Fisher exact test were used to determine the association of variables with the Beijing genotype and to estimate the odds ratio (OR) and the 95% confidence interval (CI) of OR. A P value of 0.05 was used as the cutoff level for significance.

The main bioinformatics analysis tools used were NCBI (http://www.ncbi.nlm.nih.gov/), Gel Compar 4.0 (Applied Maths, Kortrijk, Belgium), BioNumerics (Version 5.10), and SPSS for Windows 21.0 (SPSS Inc., Chicago, IL, USA).

Results

Role of drug resistance in the dissemination of the Beijing family of M. tuberculosis

In 1286 M. tuberculosis clinical isolates that were genotyped by spoligotyping, 701 were tested for susceptibility to the four first-line anti-TB drugs (i.e., INH, RFP, SM, and EMB) by the absolute concentration method and proportion method. In Beijing family isolates, 499 (53.20%) drug-sensitive strains and 439 (46.80%) drug-resistant strains were noted. No significant statistical difference (c²=2.41, P>0.05) was found between the drug-resistant and drug-sensitive isolates. By comparison with the relative percentages of resistant and susceptible strains in the non-Beijing families, drug resistance was not frequent in Beijing family strains (Table 1).

The results of single-drug resistance to the four drugs showed that the highest single-drug resistance rates among these isolates were observed for INH and RFP (32.89% and 29.32%, respectively). Isolates of the Beijing genotype were found in both drug-susceptible and drug-resistant isolates for each drug. A comparison of the relationship between the Beijing genotype and single-drug resistance to four drugs revealed that resistance to INH (OR=1.08; 95% CI, 0.83–1.42), RFP (OR=1.19; 95% CI, 0.90–1.58), EMB (OR=0.86; 95% CI, 0.59–1.24), and SM (OR=1.26; 95% CI, 0.94–1.69) was not associated with infection by strains of the Beijing genotype (Table 2).

We also found that the prevalence of single-drug resistance and multidrug resistance in 10 provinces was 12.69% and 34.12% in Beijing family strains and 9.77% and 32.18% in non-Beijing family strains, respectively. The difference was not significant. In the 10 selected regions, drug susceptibility, whether single-drug resistance or multidrug resistance, was not statistically different in the Beijing genotype and non-Beijing genotype isolates (Table 3). These results indicated no associations between resistance to different anti-TB drugs and the Beijing genotype.

Discussion

With the development of molecular techniques, exploring the contagious and pathogenic mechanism of TB at the molecular level is possible. To date, many studies proved that M. tuberculosis population is organized in different clades characterized by particular molecular signatures [12,13]. One of the most interesting discoveries was that of the M. tuberculosis Beijing family by Dick van Soolingen and his colleagues in 1995 [1]. Although this family was first recognized as highly prevalent in East Asia, molecular epidemiological data collected in several countries in the past decade have revealed that the genotype is widespread around the world [14–16].

However, where and how this genotype has emerged remains unknown, although several arguments are in favor of a Chinese origin [5]. In particular, the association of the M. tuberculosis Beijing genotype with drug resistance is of major interest to a large number of research groups. The large epidemiological study we performed in China may help shed some light on these problems.

As the strains of the Beijing family could become drug resistant with high efficiency possibly through the existence of mutator genes, drug resistance might be a factor that enhances the emergence of this family [17]. Many outbreaks of MDR-TB, with poor response to treatment and high disease and death rates, have been reported. For example, in the United States, large outbreaks of MDR-TB were caused by strains of the W family, forming a minor subgroup of the M. tuberculosis Beijing family [14,18,19]. Some results showed that the drug-resistant prevalence of Beijing family strains was higher than that of non-Beijing family strains [10,20], whereas others reported no significant differences between the two genotypes [21,22]. Chan et al. reported that the drug-resistant prevalence of Beijing family strains is lower than that of non-Beijing family strains [23].

In the present study, no statistical correlation between single-drug and multidrug resistance and the Beijing genotype was observed. On the basis of the geographical distribution, all P values did not meet the level for significance. No association was found between mono-drug and multidrug resistance and the Beijing genotype in 10 different provinces. Therefore, our results demonstrated that the Beijing family and drug resistance in China were not correlated.

In summary, this work is the largest M. tuberculosis group of isolates used to analyze the association between the M. tuberculosis Beijing genotype and drug resistance. We observed no evidence showing that drug resistance was associated with the transmission of M. tuberculosis Beijing genotype strains in China.

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