Drug resistant Mycobacterium tuberculosis in Oman: resistance-conferring mutations and lineage diversity

Study population and characteristic of M. tuberculosis isolates

A total of 2,547 unique M. tuberculosis isolates were obtained from pulmonary and extra-pulmonary TB diagnosed Omanis (n = 1, 344) and expatriates (n = 1, 203), between 2009 and 2018 at the Central Public Health Laboratories (CPHL), Ministry of Health, Oman. The laboratory provides TB diagnosis, and in vitro drug susceptibility testing (DST) for the first line anti-TB drugs, to the population of Oman (4.4 million people comprising 2.8 nationals and 1.6 expatriates). The largest proportion of expatriates in Oman are from high TB burden countries such as India, Pakistan and Bangladesh (NCSI, 2020).

Microbiology methods

Standard microbiological assays were carried out by staff at CPHL, for the identification of members of the M. tuberculosis complex. DST was performed on 2,539 isolates (SM, 1.0 µg/ml; INH, 0.1 µg/ml; RIF, 1.0 µg/ml; EMB, 5.0 µg/ml; PZA, 100.0 µg/ml) using the BACTEC MGIT 960 system (Ardito et al., 2001). A sensitive response refers to susceptibility to all first line drugs (SM, INH, RIF, EMB, and PZA), whereas resistance to any drugs refers to growth at or above the lowest drug concentration that inhibits the wild type (susceptible) strain. Different drug resistance profiles were identified including: resistance to a single drug (mono-resistant (MonoR)), resistance to more than one drug but not to both INH and RIF (poly-resistant (PolyR)), resistance to at least INH plus RIF (multi-drug resistant (MDR)),) and MDR plus resistance to a second line anti-TB treatment, fluoroquinolone, and least one of the injectable second-line drugs (extensively-drug resistant (XDR)) (Song et al., 2019).

Ethical approval (SQU-EC/075/18) for the study was granted by the Ethics Committee of The College of Medicine and Health Sciences, Sultan Qaboos University, Oman. Patients’ consent was not obtained as this study examined archived heat killed DNA samples obtained from material cultured as part of the routine diagnosis process by staff of the Central Public Health Laboratories, Ministry of Health, Oman.

Detection of drug resistance-conferring mutations

DNA was extracted from Mycobacterium tuberculosis isolates using the heat-killing method (Warren et al., 2006). PCR and targeted sequencing were used to detect mutations in nine genes implicated in resistance to SM (rpsL and rrs), INH (katG, ahpc, InhA/InhA promoter), RIF (rpoB), PZA (pncA) and EMB (embB and emb C). Primer sequences and PCR conditions are described in Table S1.

Spoligotyping

Spacer oligonucleotide typing (spoligotyping) was successfully completed on 1,295 isolates (629 Omanis and 666 expatriates), as described elsewhere (Al-Mahrouqi et al., 2022; Kamerbeek et al., 1997). Briefly, the direct repeat (DR) region of M. tuberculosis was amplified using a pair of PCR primers. The PCR products were hybridized to a set of 43 oligonucleotide probes corresponding to spacers on the DR region and covalently bound to the membrane. Obtained spoligo-patterns were then compared to the SITVIT-Web database (http://www.pasteur-guadeloupe.fr:8081/SITVIT_ONLINE, http://www.pasteur-guadeloupe.fr:8081/SITVIT2/files/SITVIT-KBBN_report_310313.xls) for identification of spoligo families and spoligotype international types (SIT) (Demay et al., 2012).

Data analysis

Associations between categorical variables were tested using Chi-squared and/or Fisher’s exact tests and was applied to the association of DST among isolates from Omanis and expatriates, as well as the association of drug resistance-conferring mutations with their corresponding DST. In addition, chi-squared/Fisher’s exact tests were used to examine the distribution of different resistance profiles (MonoR, PolyR and MDR) and drug resistance-conferring mutations in different M. tuberculosis lineages. Fisher’s exact test was used to compare variables when the observed count was <5 in 20% or more of the cells in 2 × 2 chi-squared table. Logistic regression models were used to estimate the effect of demographic variables (Omanis vs expatriates) on the probability of detecting DST. Odds ratio (OR) and 95% confidence intervals (CI), with their associated log likelihood chi-squared test were presented for logistic regression models. Analyses was conducted in SPSS, version 23 and R program, version 1.4.1717.

Characteristics of study subjects

We examined 2,539 of 2,547 M. tuberculosis strains obtained from Omanis (1,341, 52.8%) and expatriates (1,203, 47.2%). The age of patients ranged between 3 and 100 years, with a mean of 44.2 (20.2 ± SD) among Omanis and 33.7 (10.2 ± SD) for expatriates. Most of the patients were male (n = 1, 549, (61.0%)) with a slightly higher percentage of males among expatriates (51.8%) compared to Omanis (48.2%) (P = 0.218).

Drug susceptibility profile of M. tuberculosis isolates

Of the 2,539 M. tuberculosis isolates examined, 2,198 (86.6%) were sensitive to the first line therapy (INH, RIF, SM, EMB and PZA) (Fig. S1). The remaining 341 (13.4%) isolates fell into four drug resistance profiles: 242 (71%) were MonoR, 40 (11.7%) were PolyR, 49 (14.4%) were MDR, and 9 (2.6%) wereXDR. Of the MonoR strains, 92 (38%) were INH^R, 76 (31.4%) were PZA^R, 65 (26.9%) were SM^R and 9 (3.7%) were RIF^R (Table 1).

Similar rate of resistance to any drug was seen among M. tuberculosis isolated from Omanis and expatriates (P = 0.637). However, variations were noted in INH^R (Log Likelihood ratio = −194.30; χ2, 9.66; P = 0.002), PZA^R (Likelihood ratio = −179.9; χ2,5.01; P = 0.025) and MDR (Log Likelihood ratio = −140.16; χ2,4.275; P = 0.039). INH^R was lower among Omanis (OR = 0.46; 95% CI [0.28–0.75]), contrasting the elevated rates of PZA^R-TB (OR = 1.79; 95% CI [1.07–3.02]) and MDR (OR = 1.90; 95% CI [1.03–3.56]) (Table 2).

Spatial distribution of drug susceptibility in M. tuberculosis

The MonoR forms, SM^R (Log Likelihood ratio = −131.77; χ²,6.19; P = 0.186), INH^R (Log Likelihood ratio = −172.04; χ²,8.51; P = 0.074) and the PolyR cases (Log Likelihood ratio = −104.82; χ²,7.92; P = 0.095) were stable in different regions in Oman. However, variation was seen in RIF^R (Log Likelihood ratio = −31.17; χ2,10.88; P = 0.028), PZA^R (Log Likelihood ratio = −143.94; χ²,13.11; P = 0.011) and MDR (Log Likelihood ratio = −116.82; χ²,23.35; P = 0.000). For example, RIF^R was 14 and 11 times higher in Ad Dakhliyah (OR = 14.53; 95% CI [1.32–324.20]) and Ash Sharqiyah (OR = 11.47; 95% CI [1.05–254.22]), respectively, compared to Muscat. Likewise, in Dhofar, MDR was four time higher (OR = 3.84; 95% CI [1.82–8.38]) and PZA^R was significantly lower compared to Muscat (OR = 0.40; 95% CI [0.15–0.93]) (Fig. 1).

Temporal distribution of drug susceptibility in M. tuberculosis

Overall, resistant M. tuberculosis isolates remained stable over the study period, with the exception of INH^R (Table S2). The PolyR and MDR M. tuberculosis strains remained stable over the study period (P = 0.158 and 0.248 respectively) (Figs. 2A & 2B). However, the MonoR strains increased steadily, with 2 times higher prevalence in 2018 compared to 2009 (Log Likelihood = −791.78; χ2,19.98; P = 0.025) (OR 2.1; 95% CI [1.13–3.76]) (Fig. 2C), driven by PZA^R (Log Likelihood = −161.4; χ²,42.00; P = 0.000) (Fig. 2D). In contrast, INH^R decreased steadily (Log Likelihood = −189.7; χ²,19.00; P = 0.025) (Fig. 2E) while SM^R (P = 0.113) and RIF^R (P = 0.210) remained unchanged (Figs. 2F & 2G).

Diversity of drug resistant M. tuberculosis isolates

Spoligotyping was completed for 242/341 (70.9%) drug-resistant M. tuberculosis isolates (106 Omanis and 136 expatriates) (Fig. S1). Of these, 187 (76.4%) exhibited known spoligotype patterns (SIT), while 55 (22.7%) were ‘orphans’. The isolates with known spoligotypes comprised 8 lineages, EAI (66, 35.3%), CAS (50, 26.7%), Beijing (28, 15.0%), T (21, 11.2%), LAM (10, 5.3%), H (5, 2.7%), X (2, 0.5%), Ural-1 (2, 0.5%), Cameroon (2, 0.5%) and UK (1, 0.5%). Of these, 137 (73.26%) isolates existed in clusters of 33 spoligo-profiles (with 2 to 28 isolates), and 50 (26.74%) were singletons, each with a distinct profile. Of the above clusters, 16/33 (48.48%) included M. tuberculosis isolates from both Omanis and expatriates (Table 3), suggesting close genetic relatedness and possible transmission of drug-resistant lineages between the two groups.

The patterns of resistance (MonoR, PolyR and MDR) were found in most of the spoligotype-defined lineages (Table 4). However, MonoR and MDR were significantly associated with strain lineage (p = 0.000) for both types of resistance patterns (Table 3). MonoR was disproportionately higher in the EAI and CAS lineages, whereas MDR was over-represented in the Beijing, CAS, and T lineages. The variation in MonoR seen in the different linages was linked to SM^R (p > 0.001) and PZA^R (p > 0.001) (Table 4).

Mutations in M. tuberculosis drug resistance genes

Full length sequences for nine genes implicated in resistance to SM (rpsL and rrs), INH (katG, ahpc, InhA/InhA promoter), RIF (rpoB), PZA (pnc A) and EMB (emb B and emb C) were obtained for 356 of the M. tuberculosis isolates (211 (58.8%) sensitive and 154 (42.2%) resistant to any drug) (Fig. S1). The resistant isolates were obtained from 58 (37.7%) Omanis and 96 (62.3%) expatriates (Table 5).

Drug resistance-conferring mutations among Omanis and expatriates

The rpsL K43R, katG S315T and rpoB S450L mutations associated with SM^R, INH^R and RIF^R, respectively, existed at similar frequencies among Omanis and expatriates (Fig. 3). However, the C-15T mutation in the inhA promoter linked to INH^R existed at a significantly higher prevalence among expatriates compared to Omanis (Fig. 3).

Drug resistance-conferring mutations in different M. tuberculosis lineages

We examined whether the distribution of the drug resistance-conferring mutations were influenced by M. tuberculosis genetic background. The rpsL K43R, katG S 315T and rpoB S450L mutations associated with SM^R, INH^R and RIF^R, respectively (Table 6), existed at different frequencies in different lineages (Table 6). The rpsL K43R, katG S315T and ropB S450L mutations were disproportionately higher in isolates of the Beijing lineage, whereas katG S315T and inhA C-15T mutations were over-represented in the EAI lineage. In contrast, isolates from the CAS lineage harboured all of the above mutations (Table 6). Interestingly, the orphan isolates which most likely represent locally evolved strains, had high rates of the evolutionary mutations, embB E378A, embC T270I, embC N394D and katG R463L (Table 6).