Lack of Correlation between embB Mutation and Ethambutol MIC in Mycobacterium tuberculosis Clinical Isolates from China

Abstract

Seventy-four Mycobacterium tuberculosis clinical isolates from China were subjected to drug susceptibility testing using ethambutol, isoniazid, rifampin, and ofloxacin. The results revealed that the presence of embB mutations did not correlate with ethambutol resistance but was associated with multiple-drug resistance, especially resistance to both ethambutol and rifampin.

Ethambutol (EMB), often used in combination with isoniazid, rifampin, and pyrazinamide, is a key drug of first-line antituberculosis treatment. EMB seems to exert its toxic effect by inhibiting the embABC-encoded proteins, and mutations in the embB gene appear to play a major role in the development of EMB resistance in Mycobacterium tuberculosis (13). The marked clinical association between embB codon 306 mutations and EMB resistance in M. tuberculosis at one time led to its proposal as a marker for EMB resistance in diagnostic tests (6, 8, 11, 15). However, discrepancies between the results of genotypic and phenotypic EMB resistance testing have raised questions about the accuracy of molecular assays based on the detection of point mutations in embB codon 306 for prediction of EMB resistance (3, 4, 7, 14). Hazbon et al. (3) has reported that for embB codon 306 mutations, there is “a novel association with broad drug resistance and IS6110 clustering rather than ethambutol resistance.”

For DNA sequencing of the embB gene (primer set includes forward, 5′-CGGCATGCGCCGGCTGATTC, and reverse, 5′-TCCACAGACTGGCGTCGCTG) from 141 EMB-resistant and 40 EMB-sensitive clinical isolates from Henan Province, China, ABI Prism Big Dye terminator sequencing kits were used. We found that 45.2% of EMB-resistant isolates harbored embB codon 306 mutations (ATG to ATA, GTG, ATT, CTG, or ATC [five types]) (12). We also found that 15% (6/40) of EMB-susceptible isolates had embB gene mutations (the breakpoint concentration of EMB is 2 μg/ml in L-J medium) in 2000. After a previous analysis by denaturing high-pressure liquid chromatography (DHPLC) of drug resistance genes in M. tuberculosis in our laboratory (9, 10), we went back to test these isolates and found that the DHPLC results for the embB gene were completely consistent with those of DNA sequencing. Figure 1 shows the DHPLC and DNA sequencing results for the six isolates that were EMB sensitive but harbored embB mutations. Results similar to those we obtained for streptomycin resistance were obtained (R. Shi, J. Zhang, C. Li, Y. Kazumi, and I. Sugawara, unpublished data). We also found that 22% (16/72 EMB-sensitive clinical isolates from an affiliated hospital of the Beijing Tuberculosis and Lung Tumor Research Institute) possessed embB mutations in 2006. We tested the EMB MICs for these 16 isolates, and all were less than 2 μg/ml, but the MIC for four isolates was 0.125 μg/ml (data not shown). We also tested 100 clinical isolates of M. tuberculosis from Henan Province, China (9 were isoniazid monoresistant, and all others were pansusceptible), and 200 clinical isolates from Fukujuji Hospital, Tokyo, Japan (7 were isoniazid monoresistant, 1 was rifampin monoresistant, and all others were pansusceptible), by the DHPLC method to screen the embB gene, but no mutation was found (DHPLC data not shown).

FIG. 1.

embB DHPLC pattern of six EMB-susceptible isolates from Henan Province, China. I, isoniazid; R, rifampin; S, streptomycin; E, EMB; +, resistant; −, susceptible.

In the present study, 74 clinical isolates from the Beijing Tuberculosis and Lung Tumor Research Institute were subjected to testing for drug susceptibility (in L-J medium) to EMB, isoniazid, rifampin, and ofloxacin, and analysis of their respective resistance genes, embB, katG, rpoB, and gyrA, was done by DHPLC and DNA sequencing (9, 10, 11). Variable-number tandem repeat analysis was also performed using 12 standard loci of mycobacterial interspersed repetitive units reported by Mazars et al. (5). Among the 74 isolates, 14 were pansusceptible and no embB mutations were found. When a MIC of more than 2 μg/ml was taken as a standard for EMB resistance (and the World Health Organization suggests that 2 μg/ml in L-J medium is the MIC), 17 isolates were found to be resistant, of which 65% (11/17) showed embB mutations, while 56% of EMB-susceptible isolates (24/43) revealed embB mutations. Of these latter 24 isolates, 21 were multidrug resistant, and 3 were monoresistant to rifampin at a high level. Three isoniazid-sensitive isolates were found to possess embB mutations, while seven isolates showing a high level of isoniazid resistance had no embB mutations. None of the rifampin-sensitive isolates were found to have embB mutations, while most of the isolates showing high rifampin resistance harbored embB mutations. The variable-number tandem repeat analysis results revealed that the embB mutations were clustered (data not shown). Table 1 shows the correlation of an embB mutation and resistance to four drugs at different concentrations for the 74 clinical isolates. DHPLC data and DNA sequencing results for the katG, rpoB, and gyrA genes are omitted. As shown in Fig. 2, embB mutations were distributed among isolates with susceptibility or resistance to EMBat concentrations ranging from 0.25 to 20 μg/ml, but the isolates exhibited multidrug resistance, indicating that the presence of mutations in embB codon 306 is not applicable for prediction of EMB resistance in clinical M. tuberculosis isolates. A chi-square test revealed that the presence of an embB mutation was strongly correlated with rifampin resistance and an increased frequency of resistance to other drugs, whereas it showed no correlation with isoniazid resistance (Table 2).

TABLE 1.

Correlation of embB mutation and resistance to four drugs for 74 clinical isolates from Beijing, China

EMB MIC (μg/ml)	No. of isolates with:			No. of resistant isolates at indicated drug concn (μg/ml)a									Total no. of isolates with embB mutation and resistance to other drug(s) (n = 74)
				INH			RIF			OFX
	No embB mutation	embB mutation in codon 306	embB mutation at another position	≤1	1 to 10	≥10	≤50	50 to 400	≥400	≤4	4 to 16	≥16
0.25	9	6	1	14	2	0	9	4	3	12	3	1	16
0.5	14	6	0	14	5	1	9	6	5	17	1	2	20
1	9	9	0	14	3	1	7	7	4	12	4	2	18
2	1	2	0	1	2	0	1	0	2	2	1	0	3
5	2	3	0	2	1	2	4	1	0	3	1	1	5
10	2	3	0	2	1	2	2	3	0	4	1	0	5
20	2	5	0	1	0	6	1	2	4	4	1	2	7

^aINH, isoniazid; RIF, rifampin; OFX, ofloxacin.

FIG. 2.

Distribution of embB mutation rates among isolates at different EMB concentrations. White bars indicate the embB mutation rates, and shaded bars show the multidrug resistance rates among isolates for each EMB MIC. Numbers inside the bars represent numbers of embB-mutated (or multidrug-resistant) isolates relative to the total number of isolates at each MIC.

TABLE 2.

Relationship between embB mutation and resistance to isoniazid, rifampin, and ofloxacin and to increasing concentrations of drugs

Drug resistancea	Total no. of isolates	No. (%) of isolates with embB mutation:		P (chi-square test)
		Present	Absent
Resistant to
INH alone	54	31 (57)	23 (43)	0.10
>1 μg/ml	13	6 (46)	7 (54)	0.71
>10 μg/ml
RIF alone	52	35 (67)	17 (33)	1.27 × 10−5
>50 μg/ml	35	26 (74)	9 (26)	1.46 × 10−8
>250 μg/ml
OFX alone	52	32 (62)	20 (38)	0.007
>2 μg/ml	21	14 (67)	7 (33)	0.008
>8 μg/ml
INH and RIF	47	32 (70)	15 (30)	1.137 × 10−5
Any single drug	4	1 (25)	3 (75)	0.25
Any two drugs	15	4 (27)	11 (73)	0.03
All three drugs	42	30 (71)	12 (29)	2.03 × 10−5
Pansusceptible	14	0 (0)	14 (100)

^aINH, isoniazid; RIF, rifampin; OFX, ofloxacin.

In summary, our data reveal no evidence of embB mutations in pansusceptible clinical isolates. An emb mutation was restricted to EMB-susceptible strains that were already resistant to other antituberculosis drugs. There was no strong relationship between the presence of an embB mutation and the EMB MIC. Our results support the findings of Hazbon et al. (3) that an embB mutation is strongly associated with resistance to an increased concentration of drugs. It is speculated that the development of embB mutations may predispose an isolate to the development of resistance to multiple antibiotics and may increase the ability of these multiple-drug-resistant clinical isolates to be transmitted between subjects. Our data also suggest that an embB mutation has a strong relationship to rifampin resistance but no relationship to isoniazid resistance. Although there was also a significant correlation with ofloxacin resistance, it is entirely possible that this may have been an indirect association, as ofloxacin is a second-line antituberculosis drug and in China it is usually used in place of rifampin when rifampin resistance becomes evident. The precise mode of action of EMB and the molecular basis of resistance are not fully understood. The effects of EMB are pleiotropic, and several hypotheses have been proposed for its mode of action (2). Inhibition of cell wall biosynthesis may not play an important role, and inhibition of RNA metabolism may be partly responsible (1, 2). The present study did not provide firm evidence to allow a conclusion to be drawn as to whether there is a close relationship between an embB gene mutation and rifampin resistance because of the limited number of samples and the assay system used. However, our findings suggest that studies of interrelationships among mechanisms of antituberculosis drugs and drug resistance genes would be a fruitful area of research.