Characterization of IS6110 Restriction Fragment Length Polymorphism Patterns and Mechanisms of Antimicrobial Resistance for Multidrug-Resistant Isolates of Mycobacterium tuberculosis from a Major Reference Hospital in Assiut, Egypt

Abstract

We evaluated 25 Mycobacterium tuberculosis isolates from patients at a major Egyptian reference hospital in Assiut, Egypt, who had been treated for at least 1 year for tuberculosis. Typing patterns (IS6110) were diverse, and multidrug resistance was found among 11 (44%) of the isolates. Mutations associated with antimicrobial drug resistance were found in rpoB, katG, rpsL, and embB in the resistant isolates.

Tuberculosis (TB) continues to be a major health problem, particularly in developing countries (10). According to a 1997 report from the Egyptian National Tuberculosis Program, the annual risk of TB infection in this country is 0.32%. This report further revealed that the incidence of smear-positive cases in Egypt is 16 per 100,000 population, with a rate of detection of new smear-positive cases of 70% (3). This report concluded that TB and especially drug-resistant strains of Mycobacterium tuberculosis pose serious public health problems and that multiple drug resistance and low cure rates are the most important problems facing TB control efforts in Egypt.

Despite these findings, there is a paucity of information regarding the distribution of strains and the development of drug resistance, particularly in major population regions outside the immediate vicinity of Cairo. Assiut University Hospital (AUH) is a major Egyptian teaching hospital (1,750 beds) which is located 375 km south of Cairo and which serves more than 3 million people. No data regarding either the rate of incidence of pulmonary TB in the Assiut region or anti-TB drug resistance in this region are available. It is also strongly suspected that many patients are labeled as having pulmonary TB in this region with no bacteriologic confirmation and with inadequate acid-fast microscopy (7). The result of these inadequacies has often been improper, perhaps excessive, treatment regimens. For example, in 1987, 9,460 patients in Egypt were treated for pulmonary TB without confirmation by culturing (3). Inadequate treatment also most likely contributes to both a large number of chronic TB patients often infected with drug-resistant strains and an increased likelihood for community dissemination. However, no database for multidrug-resistant M. tuberculosis (MDRTB) strain types or resistance profiles for strains from AUH exists. In this study, we evaluated the IS6110 fingerprints and resistance patterns of 25 M. tuberculosis isolates in an effort to establish such a database.

Twenty-five M. tuberculosis isolates were obtained from sputum samples from 24 successive and symptomatic patients (there was one pair of isolates from the same patient); these samples had been processed and cultured at AUH. All 24 patients lived in the Assiut region, had undergone at least 1 year of anti-TB therapy, and had been referred to AUH. The doses and the time interval between the cessation of therapy and culturing were unavailable. All patients were symptomatic for pulmonary TB and were found positive for acid-fast bacilli by microscopic smear examination upon admission to AUH. Isolates were sent to the Centers for Disease Control and Prevention from AUH in a coded fashion, with no personally identifying patient information and subsequent to discharge of the patients. In vitro drug susceptibility testing was performed by the modified method of proportions using Middlebrook-Cohn 7H10 agar plates (6).

Crude lysates containing genomic DNAs for use as templates for PCR were prepared from Middlebrook 7H9 broth cultures of bacterial isolates by disruption of cells with siliconized glass beads as previously described (12). Regions of rpoB, katG, rpsL, and embB in which mutations most frequently associated with anti-TB drug resistance have been found were amplified by PCR using previously described conditions and oligonucleotide primers (Table 1) (5, 9, 14) (GenBank accession no. 68081; GenBank accession no. U68480). Amplimers were evaluated for mutations using nonradioactive single-strand conformation polymorphism (SSCP) electrophoresis and automated DNA sequence analysis. Briefly, SSCP was performed by heating a mixture consisting of 5 μl (∼50 ng) of PCR product and 15 μl of deionized formamide at 95°C for 4 min, followed by electrophoresis in 4 to 20% gradient acrylamide gels (Invitrogen Corp., Carlsbad, Calif.) at 300 V for 1.75 h in Tris-borate-EDTA buffer maintained at 13°C (2). Sequencing of both strands of the PCR product was performed with an ABI373 sequencing apparatus according to the protocol supplied by the manufacturer and with the Big Dye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems, Foster City, Calif.).

TABLE 1.

Genomic regions examined for mutations

Drug	Gene	Nucleotides	Codons	Primers	Size (bp) of product	Reference and/or source
RIF	rpoB	2316–2571	481–565	BC35 and BC41R	255	9; GenBank accession no. 68081
INH	katG	725–1047	243–349	BC48 and BC51R	321	GenBank accession no. U68480
EMB	embB	7771–8047	270–362	EMB6 and EMB7R	276	14
STR	rpsL	4–310	2–103	ML51 and ML52R	306	5

Isolates were typed by a standard restriction fragment length polymorphism (RFLP) method (15) based upon the strain-specific locations of insertion element IS6110 in the genomes of M. tuberculosis complex organisms. Briefly, genomic DNA was digested with restriction endonuclease PvuII, electrophoresed, blotted onto nylon membranes, and hybridized with a chemiluminescent probe prepared from the 247-bp PCR product of IS6110 (15). Banding patterns on resulting autoradiographs were scanned and analyzed using BioImage whole-band analysis V.3.4 software (Genomic Solutions, Inc., Ann Arbor, Mich.). Statistical evaluation was done by the chi-square test using the SPSS version 9.0 statistical software package. A P value of less than 0.05 was considered statistically significant.

Fourteen (56%) of the 25 isolates from AUH were susceptible to all four drugs tested in vitro, and the remaining 11 (44%) were multidrug resistant (Table 2). Of these resistant isolates, five each were resistant to four drugs and three drugs and one was resistant to two drugs. Eight of 11 rifampin (RIF)-resistant isolates had mutations in an 81-bp region of rpoB. The mutations included S₅₃₁L (four isolates), D₅₁₆V (two isolates), and H₅₂₆C (one isolate), and there was one double mutant (D₅₁₆V-H₅₂₆Y). Eleven isolates that were resistant to 1 μg of isoniazid (INH)/ml were evaluated for mutations in katG codons 243 to 349, and 4 had the same mutation (S₃₁₅T). An additional seven INH-resistant isolates had no mutations in this region. Three of six isolates with high-level resistance to streptomycin (STR) (MIC, >10 μg/ml) had mutations in rpsL codons 2 to 103. The mutations included K₈₈R, T₃₉T(ACC₁₂₀ to ACT), and K₃₄R-T₃₉T. DNA from five single colonies of each double mutant were resequenced in the pertinent regions (rpoB for strain 11 and rpsL for strain 23) (Table 2) and showed the same results, minimizing the possibility of mixed clones each harboring a single mutation. Two isolates with low-level resistance to STR (MIC, 2 to 10 μg/ml) had no mutations in the rpsL region examined. Two of seven ethambutol (EMB)-resistant isolates had mutations in embB codons 270 to 362. The mutations included M₃₀₆I and M₃₀₆V.

TABLE 2.

Genotypic drug resistance analysis of 11 MDRTB isolates from AUH^a

Isolate	RFLP pattern (no of bands)	RIF			INH			STR			EMB
		AP	rpoB	SSCP	AP	katG	SSCP	AP	rpsL	SSCP	AP	embB	SSCP
1	A (10)	R	H526C	+	RH	WT	WT	RL	WT	WT	R	M306I	WT
10	B (14)	R	D516V	+	RH	WT	WT	RH	WT	WT	S	WT	WT
11	C (8)	R	D516V + H526Y	+	RM	WT	WT	RL	WT	WT	S	WT	WT
12	D (10)	R	S531L	+	RH	S315T	+	RH	K88R	WT	R	WT	WT
13	E (12)	R	WT	WT	RH	WT	WT	RH	T39T	+	S	WT	WT
15	F (11)	R	S531L	+	RH	S315T	+	S	WT	WT	R	WT	WT
16	G (8)	R	WT	WT	RH	WT	WT	S	WT	WT	S	WT	WT
18	B (14)	R	D516V	+	RH	WT	WT	RH	WT	WT	R	WT	WT
20	H (11)	R	WT	WT	RH	WT	WT	RH	WT	WT	R	WT	WT
23	D (10)	R	S531L	+	RH	S315T	+	RH	K43R + T39T	+	R	WT	WT
24	I (8)	R	S531L	+	RH	S315T	+	S	WT	WT	R	M306V	+

^aAP results determined by agar proportion (AP) testing: R, resistance; R_L, low-level resistance (MIC, 2 to 10 μg/ml for STR); R_M, moderate resistance (MIC, >1 to ≤5 μg/ml for INH); R_H, high-level resistance (MIC, >10 μg/ml for STR and >5 μg/ml for INH); S, susceptible. Mutations in genes rpoB, katG, rpsL, and embB were determined by autosequence analysis and evaluated by SSCP electrophoresis: WT, wild type; +, mutant. 

Of 17 mutations identified by sequence analysis, 14 were detected by nonradioactive SSCP electrophoresis (Table 2). Four pairs of isolates each shared unique IS6110 RFLP fingerprints (patterns B, D, L, and Q) (Fig. 1 and 2). Two of these patterns (B and D) were found among MDRTB isolates from four patients, and each matching pair also had identical or similar resistance genotypes (Table 2). The remaining 17 isolates, including 7 MDRTB isolates, had distinct IS6110 fingerprints. Analysis of the dendrogram (Fig. 2) revealed that patterns D and H were >90% related (both being found in MDRTB isolates with 9 matching bands but unrelated resistance genotypes) and that patterns E and P were ∼85% related (each having 12 bands with minor differences in mobilities). No more than 73% relatedness was observed among the remaining patterns.

FIG. 1.

IS6110 RFLP patterns found among 25 M. tuberculosis isolates from 24 patients undergoing prolonged anti-TB therapy at AUH. Numbers of bands and associated antimicrobial drug data are shown in Table 2. The size standard (lane std) was a prepared series of clones each bearing one or more copies of a segment of insertion element IS6110. Panels A and B show the results of different experimental runs.

FIG. 2.

Dendrogram produced using BioImage whole-band analysis V.3.4 software and showing the relationship of 21 RFLP patterns identified for 25 M. tuberculosis isolates from AUH. Std, standard. RFLP patterns are shown in Figure 1.

Although the prevalence of multidrug resistance among new isolates of M. tuberculosis in Egypt was reported to be 3.2% in the 1997 Egyptian National Tuberculosis Program report (3), we found 44% of a sample of 25 consecutive M. tuberculosis isolates at AUH to be MDRTB. This high prevalence was most likely influenced by the criteria for isolate selection, which particularly limited the sample to isolates from patients undergoing prolonged anti-TB therapy. The treatment regimen included RIF, INH, EMB, and most often STR (occasionally pyrazinamide) for 2 months followed by RIF and INH for an additional 7 months. Fujiwara et al. (4) reported that the percentage of multidrug-resistant isolates among previously treated patients in New York City in 1994 was 19.1%, but no Egyptian data were available for comparison.

The problem of M. tuberculosis drug resistance in Egypt and particularly in the Assiut region is further complicated by the absence of in vitro drug susceptibility testing and also by patient noncompliance to prescribed treatment regimens. It appears more likely that drug-resistant strains of M. tuberculosis would be isolated from patients who have been subjected to extensive and apparently unsuccessful treatment courses (16). We found, however, that the majority of isolates (56%) in this study were susceptible to all drugs tested, and an explanation for the failure to cure patients infected with susceptible isolates unfortunately is unknown; both the time interval between the cessation of therapy and positive culturing and the extent of treatment noncompliance were unavailable. While directly observed therapy programs for TB control have become established in various regions of Egypt, they have only recently been introduced in Assiut. These directly observed therapy programs may alleviate problems associated with incomplete treatment regimens among patients in the Assiut region (16).

The mutations found among the MDRTB isolates in this study have been previously reported (11). The percentages of isolates with mutations in the regions examined were, however, unexpectedly low. For example, we found that only 8 of 11 RIF-resistant isolates (73%) had mutations in rpoB (codons 481 to 565), compared to >90% of isolates with such mutations in previous studies (P = 0.314). With the exception of one isolate with a rare double mutation, the rpoB mutations that we found are common among RIF-resistant isolates. Likewise, only 4 of 10 high-level INH-resistant isolates had S₃₁₅T mutations in katG; this rate was lower than what has been found in other studies (8, 13). It is likely, however, that mutations in other gene regions that we did not evaluate (e.g., inhA and other katG regions) (12) were responsible for the INH resistance in some of our AUH isolates. The prevalence of rpsL mutations in high-level STR-resistant isolates and of embB mutations in EMB-resistant isolates was also lower than what has been found in previous studies (1, 14). Although EMB resistance has been previously ascribed to the embB mutations found among our isolates (14), only two of the three rpsL mutations found could be associated with phenotypic STR resistance, since one mutation was silent (T₃₉T); i.e., no amino acid was substituted. Although the prevalence of known resistance mutations that were identified by the SSCP method was low, this technique may be a rapid and relatively inexpensive screening method to use in conjunction with conventional drug susceptibility testing for larger collections of isolates under study in settings such as AUH, where DNA sequencing instruments are not yet available.

We are currently expanding this database by implementing genotypic testing of M. tuberculosis in the AUH laboratory. In addition to RFLP analyses, rapid screening of isolates for drug-resistant mutations may be a useful adjunct to other programs oriented toward controlling anti-TB drug resistance in the Assiut region.