The focus of the study was to understand mechanisms of rapid amplification of drug resistance by identification of differentially expressed genes in longitudinal clinical isolates. These pairs of isolates were DS at diagnosis and exhibited MDR after five months of compliant DOTS treatment, and had identical DF as determined by spoligotyping and 6 loci MIRU-VNTR. In the current study, it was not feasible to confirm the complete genomic identity of the isolates by whole genome sequencing. In our earlier study, we observed 247 Mtb
isolates from the same population, showed a high discriminatory index of 0.98 on fingerprinting by spoligotyping and MIRU-VNTR (Unpublished data), indicating suitability of using the fingerprint data to ascertain strain identity. The presence of a high proportion of MDR in a DOTS prescribed study population provided the opportunity to detect effects of drug pressures on global gene expression in clinical isolates of Mtb
. We studied three such longitudinal isolate pairs representing the locale predominant spoligotypes: MANU1, CAS and Beijing 
Of the 3 strains, the CAS MDR isolate showed highest and the Beijing MDR the lowest number of differentially expressed genes as compared to their respective DS isolate. Various transcription factors (sig
gene family, rpoB
), cell wall biosynthesis genes (emb
gene familly), protein synthesis genes (rpl
) and several central metabolic pathway genes (ppdK, pknH, pfkB)
were down regulated in the MDR isolates, indicating an overall reduction in metabolism. The reduction in metabolism provided drug tolerance and persistence of Mtb in vivo
, since conventional anti-TB drugs target biosynthetic processes involved in cell growth, including transcription, translation and cell wall biogenesis 
. We also observed various DNA repair and DNA stability genes including rec
, uvr, ruv
genes down regulated. On the other hand, up regulation of various drug efflux pumps, ABC transporters and trans-membrane proteins (some yet to be annotated) was observed in the MDR isolates, indicative of cellular mechanisms which may impart broad spectrum antibiotic resistance. Up regulation of transcriptional genes associated with antimicrobial stress such as whiB
gene familly 
, was observed in the MDR isolates.
The selection of mutations in identified drug target genes, rpoB, inhA, katG, embB
is gradual and sequential and elicits high fitness costs. Thus the acquisition of these mutations in the presence of a multidrug therapy requires ancillary mechanisms, as indicated in our study. For instance, the gyrA
gene has been associated with FQ resistance. These mutations have been reported to have high fitness costs 
. Alternatively efflux pumps have been shown to cause FQ resistance 
. The up regulation of gyr
genes in MDR isolates may therefore indicate compensation for FQ resistance which may be due to up regulation of efflux pumps. These observations highlight a need for drug susceptibility testing of second line drugs even in patients undertaking DOTS.
Efflux pumps have been shown to confer low level resistance to various compounds and anti-TB drugs 
. Additionally slower cellular metabolism and transcription have been associated with drug tolerance in cells without specific drug resistance mutations 
. Thus from our observations, we infer that even in drug compliant patients, over expression of efflux pumps and under expression of metabolic processes may be responsible for low level resistance to multiple drugs without the presence of multiple drug resistance mutations. Such strains may also exhibit a mutator phenotype 
due to reduced DNA repair activity observed in the current study. Further exposure of these isolates to anti-TB drugs may cause “selection and stabilization of spontaneous mutations
”, resulting in clonal expansion of MDR isolates 
, While such mechanisms have been advocated for isoniazid resistance 
, our observations extend these implications to all the first line anti-TB drugs.
Alternate mechanism of rapid amplification of resistance has been hypothesised earlier 
, however it has not been demonstrated in clinical isolates. The three different Mtb
strains: MANU1, CAS and Beijing, used in our study, have different evolutionary history, inducing different mechanisms of drug resistance. The MANU1 genotype is considered endemic in the area 
and has been found to be associated with drug susceptibility 
. Additionally, MDR MANU1 strains have been shown to outcompete drug susceptible CAS and Beijing strains in vitro
. The CAS strains found predominantly in North India 
has been shown to induce greater cavitation 
and consequently result in a more latent disease 
. The Beijing strains are not only associated with MDR, they were also found to produce most fit MDR strains 
. Additionally, the Beijing strains have been shown to able to accumulate MDR mutations when exposed to a different host 
. Thus evidence emphasizes that varied mechanisms may be responsible for drug resistance in these three strains, indicating variations in the gene expression, as seen in our results. Consequently the study may reflect the broad mechanisms of rapid amplification of drug resistance.
Furthermore the observation of upregulation and down regulation of genes involved in the same cellular function including transcription, drug efflux and protein synthesis is indicative of compensation for drug resistance mechanism. For instance the upregulation of Rv1687c, an ABC transporter also known as a drug efflux pump 
, may result in fitness cost due to reduced intake of nutrients. However the down regulation of Rv1686c, a different ABC transporter, may compensate by enhancing selective intake of nutrients. Compensation may also demonstrate the upregulation of sigE
, genes associated with transcription, along with down regulation of whiB
gene, which have been known to play important role in transcriptional regulation 
. Thus compensation (empirical and/or genetic), which results in adaptive evolution leading to drug resistance 
, may have significant role in maintenance of low level drug resistance.
Apart from studying the differential expression of strains with amplified drug resistance, we also examined the differential expression of clinical DS isolates as compared to laboratory standard DS strain H37Rv, initially isolated from a patient in 1905 in the pre-antibiotic era, and which is considered as the neotype of Mtb
. The comparison of DS clinical isolates and H37Rv provides clues for maintenance and increase of pathogenicity of a clinical strain in the presence of drug and immune pressures. This was evinced by the large number of genes differentially expressed in the clinical DS isolate as compare to H37Rv. While these genes were also up regulated in the DS strain with respect to the respective MDR isolate, their over expression when compared to H37Rv is indicative of a human pathogen under drug pressures, which has accumulated functional mutations with least fitness costs enabling it to infect, survive and transmit within human hosts more efficiently.
The observations from the study provide significant candidate genes for studying novel mechanisms of drug resistance, drug tolerance and persistence of Mtb. Efflux pump such as drrA may play a significant role in increasing fitness of low level drug resistant cells and assist in survival of Mtb till acquisition of drug resistant mutations with least fitness costs. Genes like sigD, sigM, gyrA, whiB represent candidates for developing novel markers which predispose Mtb strains to drug resistance.
Limitations of the Study
The small number of clinical isolates (3 pairs) used for GTP, limited by the cost of microarray slides, and scarcity of RNA from clinical samples (reported in earlier studies 
), was a limitation of the study. However while most previous studies performed GTP with laboratory Mtb
strains like H37Rv, H37Ra 
, we performed GTP with 3 pairs (6 isolates) of clinical isolates and H37Rv, which is comparable to earlier reports (1–3 patient isolates) 
. The lack of quantitative validation of the genes is another limitation of the study. Thus the forward path of quantitative real-time PCR on specific genes in relevant biological pathways in increased number of clinical drug susceptible and resistant samples is planned.