Are Drug Efflux Genes Present among Mycobacterium tuberculosis Isolates from Patients in Lagos, Nigeria?

A major challenge in the treatment of Tuberculosis (TB) is emergence of MultiDrug Resistant Mycobacterium tuberculosis (MDRTB) strains. Efflux genes have been established to be among factors for drug resistance in Mycobacterium tuberculosis (M. tuberculosis) pulmonary infections by conferring bacterial ability to pump-out drugs from intracellular compartment, making it impossible for drugs to attain intracellular concentration lethal to the organ-ism. There is paucity of data on the role of efflux pump in MDRTB in Nigerian strains of M. tuberculosis. Hence, the aim of this study was to detect the carriage, distribution and frequency of efflux pump genes among MDRTB and non-MDRTB isolates from participants with pulmonary tuberculosis in Lagos, Nigeria. This study was carried out on M. tuberculosis isolated from 1020 participants suspected of pulmonary tuberculosis in Lagos State, Nigeria. A total of 78 M. tuberculosis isolates were obtained from the participants suspected of pulmonary tuberculosis. Forty Eight isolates were confirmed as MDRTB and 30 non-MDRTB. Efflux pump genes were investigated in the isolates using the conventional polymerase chain reaction. Statistical analysis was carried out using the Statistical Package for Social Science (SPSS


Introduction
Emergence of drug resistant M. tuberculosis strains is a major challenge in the treatment of tuberculosis (TB). Multi resistant mycobacterial strains (MDRTB) and extensive drug resistance (XDR) have been found to be on the increase and that efflux pump mechanism is an important factor responsible for drug resistance [1]. Efflux pumps are transmembrane proteins which actively take part in transporting wide range of substrates including anti-TB drugs from cytoplasm to exterior of cell, thereby nullifying the inhibitory or lethal activity of the drugs (drug resistance). Efflux pump genes have been reported to have different families and classes and impart resistance to broad range of antibiotics like fluoroquinolone, tetracycline, ofloxacin, and isoniazid [2]. Drug efflux pumps prove to be a major challenge for the treatment of MDRTB; they require special attention to understand their functioning to combat the emerging crisis of MDR and finding a better solution for anti-TB therapy.
The relevance of the efflux mechanism to the resistance of clinical strains of M. tuberculosis is becoming clearer due to reports of experimental introduction of hypothetical efflux genes into M. smegmatis and demonstration of the overexpression of the genes and increases in drug resistance [1] [2]. Other studies [3] showed that the transcription of Rv1258c efflux pump gene in clinical isolates of M. tuberculosis increases upon induction by Rifampicin (RIF) and ofloxacin in a clinical multidrug-resistant M. tuberculosis isolate. This suggests that efflux pumps are involved in MDR in M. tuberculosis. Several mycobacterial efflux pump genes have been characterized using specific markers in laboratory practice [1] [2]. Early studies [4] of drug accumulation in intact cells suggested that drugresistant mycobacteria accumulated fewer drugs within their cells than the susceptible strains and these results were ascribed to the lower cell envelope permeability in resistant mycobacterial cells. However, higher occurrence of drug efflux genes in drug-resistant strains of mycobacterial cell envelope have been reported [1].
Efflux genes have been detected in M. tuberculosis and have also been reported to be responsible for resistance to ant-TB treatment [5]. Bacterial drug efflux pumps have also been classified into five families and these are ATP-binding cassette (ABC) superfamily, major facilitator superfamily (MFS), small multidrug resistance (SMR) family, resistance-nodulation-cell division (RND) family, and the multidrug and toxic compounds extrusion (MATE) family [6]. The proteins of the MFS, SMR, RND and MATE families are secondary transporters in which drug efflux is coupled with proton (H1) influx [5] [6]. These pumps are often referred to as H1-drug antiporters. In contrast, members of the ABC family of multidrug efflux pumps, which are often considered primary transporters, make use of ATP as an energy source [5] [6]. The genome of M. tuberculosis contains genes encoding drug efflux transporters from all these families [1]. Active efflux systems, apart from cell wall permeability, provide resistance by extruding the drug molecules that enter the cell [7] [8]. The intracellular concentration of a given drug depends on the balance between its influx and efflux. The processes of drug influx through porins and drug efflux via drug transporters are among distinct and important processes for drug resistance in mycobacteria [8].
The initial role of these pumps is for intracellular survival and high expression is the major contributing factor for developing MDRTB [7] [8]. Several mycobacterial drug efflux pumps have been identified and characterized experimentally [9]. Studies have suggested that efflux systems are often involved with fundamental cellular physiological processes, suggesting that drug extrusion maybe a secondary function [7] [8] [9]. It has been demonstrated by that efflux pump plays a role in three important processes 1) it extrudes and thus provides resistance to several drugs (including rifampicin, one of the most important frontline TB drugs), 2) it is part of the oxidative stress response, and 3) it is needed to maintain normal growth characteristics both on solid medium and in liquid medium [10]. Studies have shown that if Rifampicin resistant strain of M. tuberculosis were exposed to Rifampicin, it may activate efflux pump genes thereby compromise the efficacy of second-line drugs containing ofloxacin [11]. It has also been shown that Efflux pump inhibitors (verapamil and reserpine) have the potentials to improve the efficacy of anti-tuberculosis drug treatment [11]. S531L. It has also been reported that though these markers are strongly associated with MDR-TB, M. tuberculosis isolates lacking these mutations but eliciting MDR phenotype have been reported by several studies [13] [14]. Studies have also shown that not all rpoB and inhA mutations contribute to multidrug resistance [15]. These two instances have provided the basis for other mechanisms for drug resistance in M. tuberculosis. Apart from its rigid and almost impenetrable cell wall of M. tuberculosis, the efflux gene system of M. tuberculosis has been shown to be an important alternative mechanism of drug resistance [15]. With the knowledge of bioinformatics, it has been predicted that at least 26 complete and 11 incomplete ATP-binding cassette superfamily (ABC) transporters and 16 Major Facilitator Superfamily (MFS) proteins exist in M. tuberculosis [5] and that involvement of several of them in transport of aminoglycosides, fluoroquinolones, chloramphenicol, isoniazid, rifampicin, and tetracyclines has been demonstrated [5]. It has also been shown that the ABC transporters actively pump Rifampicin (RIF) out of the cell thereby lowering the intracellular concentration of RIF to below its binding concentration with the rpoB protein leading to RIF resistance [16]. Expression of 10 different efflux genes in Multi-drug resistant M. tuberculosis has also been reported [16]. It was also reported that the concurrent high expression of some efflux pump genes was associated with resistance to the combination of isoniazid and ethambutol [16]. These antibiotics (isoniazid and ethambutol) plus Streptomycin were identified to group together where efflux-mediated drug resistance appears in M. tuberculosis and that high expression of efflux genes has also been observed with ofloxacin-a second line anti-TB drug [16]. Resazurin Microtitre Assay (REMA) has been described as for testing MIC of M. tuberculosis isolates [16] [17].

Inhibition of efflux by the efflux pump inhibitors reserpine and verapamil
leads to an accumulation of RIF within the cell and concurrent binding of RIF to rpoB, leading to inhibition of transcription and cell death and that the evolution of RIF resistance is a dynamic process involving a cascade of adaptive events which leads to a bacterial growth state where hydrophobic compounds are actively extruded from the cell [17]. This has important ramifications for the treatment of RIF resistant TB and supports the need for the development of anti-TB drugs that target both efflux and ATP synthesis to improve the treatment outcome of MDR-TB and XDR-TB [18]. Efflux genes such as p27 & or p55 gene

Materials and Methods
This study was carried out on MDRTB and non-MDRTB isolated from 1020 Isolation of Mycobacterium tuberculosis on solid medium was done as previously described [23] and on liquid medium using the liquid culture procedure manual. Drug susceptibility testing was carried out using the proportion method procedure. The proportion method was performed as previously described [24].
One hundred microliter of prepared bacterial inoculum was inoculated on one each of separate LJ medium with and without drug for test and as a control. This was followed by incubation at 37˚C for 21 -28 days. Resistance was defined as growth on drug containing tubes greater than 1% of the growth of drug free control medium for INH, RIF, ETM, and 10% for STR [24].
Resazurin Microtitre Assay (REMA) technique was also performed using Middlebrook 7H9-liquid medium. Incubation was done for 14 days and after 7 days  Agarose gel electrophoresis was used for the efflux genes separation and detection after PCR on 2% (w/v) agarose gel pre-stained with ethidium bromide at 0.5 ug/mL. Gels were run in Tris Acetate EDTA (TAE) buffer (40 mMTris, 2 mM sodium acetate 1 mM Na 2 EDTA, pH 8.0) for 1 h. after electrophoresis, separated efflux genes were detected under UV light and photographed using a Thermo Fischer Gel documentation system (Thermo Fisher, USA). The 100 -500 molecular sizes of the amplicon extracted were based on mobility of control DNA ladder (Invitrogen, Life Technologies Ltd., Paisley, UK).
Data obtained were entered into Microsoft excel 2007 version and Epi Info version 6.1. Statistical Package for Social Science (SPSS version 20) were used to obtain descriptive and inferential statistics from the data and summarized as frequency and percentages (%) as well as mean + standard deviation (SD). Chi (X2) square of Fischer Exact (when frequency (n) < 5) test was used to investigate the occurrence and frequency and significance of the targeted efflux genes in the MDR TB and the non-MDRTB isolates. This showed a significant difference between MDRTB and non-MDRTB. On the whole, carriage of 2 or more alleles of efpA efflux gene was associated with multidrug resistance to anti-TB drugs (Table 1, Figure 2). Rv1877 alleles were detected (Table 1, Figure 1      and that Rv2459c (jefA) efflux gene allelic carriage frequency is associated with MDRTB (Table 1, Figure 5).

Results
Rv1002c efflux gene alleles were detected in the isolates with MDR M. tuberculosis isolates accounting for 62.5% of the alleles and 40.0% (p = 0.017).   This showed significant difference in the MDRTB and non-MDRTB isolates and that carriage of Rv1002c efflux gene alleles was associated with MDR TB (Table 1, Figure 6). Rv0342 efflux gene alleles were detected in the isolates with MDR M. tuberculosis isolates accounting for 66.7% of the alleles and non-MDRTB accounting for 33.3% (p = 0.024). This showed that carriage of Rv0342 was associated with multidrug resistance (  Figure 4. It also showed that was significant difference in allelic abundance in the carriage of drrC efflux genes between MDRTB and non-MDRTB isolates and is therefore associated with multidrug resistance.

Discussion
Efflux gene types responsible for extrusion of isoniazid (INH), ethambutol (ETM) and Rifampicin (RIF) were detected in the MDRTB and non-MDRTB isolates in this study. This finding is similar to those previously reported in mycobacterial isolates [19] [20] [22]. There was significant difference in allelic abundance of mutation determinants in MDRTB and non-MDRTB (60.6% -79.7% in MDRTB vs. 20.3% -39.4% in susceptible M. tuberculosis).
MDR-TB rate of 19.4% and 15.8% by proportion method and Genotype MTBDR Plus assay respectively were found in this study, suggesting that the difference may be due to efflux genes as another mechanism of resistance. Other efflux pump genes in MDRTB genome detected were Rv2486c (efpA), Rv1877, Rv1002c, Rv2686, drrC and Rv2459c (jefA). They were found to be significantly associated with multidrug resistance (P < 0.05). This finding is similar to the study conducted by [5] and [16] where it was reported that at least 26 complete The danger in exposing rifampicin-resistant strains of M. tuberculosis to rifampicin therapy had been reported [11]. Rifampicin-resistant strains of M. tuberculosis exposed to Rifampicin, caused a reduction in susceptibility to ofloxacin (a second-line anti TB drug) to above the critical concentration of 2μg/ml which is used to define resistance to the drug [11]. The lesson from this is that all rifampicin resistant patients must be screened for the presence of efflux pump genes by molecular methods before exposing the patients to rifampicin therapy to avoid creating resistance to even the second-line anti TB drugs. It is therefore important to determine resistance to rifampicin either by detection of mutation markers of rifampicin resistance or by detection of efflux genes targeted at rifampicin in M. tuberculosis causing infection in a particular patient before the T. Y. Raheem et al.
commencement of anti-TB regimens. This is to avert raising strains that will be resistant to second line anti-TB drug such as ofloxacin [16] [17].
More than 8 efflux gene of different families, classes and functions were observed in this study. This finding is similar to the study conducted by [5] and [16] where it was reported that at least 26 complete and 11 incomplete ATPbinding cassette superfamily (ABC) transporters and 16 major facilitator superfamily (MFS) proteins exist in M. tuberculosis and reported expression of 10 different efflux genes in M. tuberculosis respectively. However, there is need to demonstrate loss of resistance by MDRTB using efflux gene inhibitors reported by [5] and [16] to confirm or refute reports that specific inhibitors of efflux pumps (verapamil and reserpine), inhibit efflux systems in M. tuberculosis and produced an increase in susceptibility to antibiotics [20].
Results of this study also showed that the level of streptomycin, isoniazid, rifampicin and ethambutol resistance varies independently of the mutation in the rpo B gene. This also suggested that other biological mechanisms such as efflux pumps were responsible for resistance even where mutation of the rpoB gene did not occur in the M. tuberculosis. It has been shown that Efflux pump inhibitors (verapamil and reserpine have the potentials to improve the efficacy of anti-tuberculosis drug treatment [11]. It had also earlier been reported that specific inhibitors of efflux pumps, verapamil and reserpine, exposure of bacteria to substances that inhibit efflux systems produces an increase in susceptibility to antibiotics [19]. The findings in this study further confirm what bioinformatics knowledge has shown that there is expression of over 10 different efflux genes in Multi-drug resistant M. tuberculosis [16] and at least 26 complete and 11 incomplete ATP-binding cassette superfamily (ABC) transporters and 16 major facilitator superfamily proteins exist in M. tuberculosis [16] [17]. It was also reported that the simultaneous over-expression of some efflux pump genes was associated with resistance to the combination of isoniazid and ethambutol [17]. These antibiotics (isoniazid and ethambutol) plus Streptomycin were identified to group together where efflux-mediated drug resistance appears to be important in M. tuberculosis. Over expression of efflux genes has also been observed with ofloxacin-a second line anti-TB drug [17].

Conclusion
Different efflux gene types and functions were detected in the MDR Mycobacterium tuberculosis isolates from this study. The multiplicity of efflux gene alleles was seen more in MDRTB isolates than in non-MDRTB. Further studies to investigate the possible inhibition of the detected efflux genes using beta blockers such as verapamil and reserpine are recommended.