Survey and Molecular Characterization of Drug- Resistant M. tuberculosis Clinical Isolates from Zunyi, Guizhou Province of China

Tuberculosis (TB) is one of the leading infectious disease killers in the world, especially in developing countries. The increasing frequency of human-to-human transmission of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis poses challenges for effective therapeutic options and infection control. MDR-TB is defined as a form of TB that is resistant to at least isoniazid and rifampin, which are used to treat all TB patients. XDR-TB is a form of TB that is resistant to at least rifampicin and isoniazid in addition to any fluoroquinolone, and at least one of the three injectable secondline anti-TB drugs (amikacin, capreomycin and kanamycin). Based on the WHO Report 2010, China was ranked number one among high burden countries in terms of the estimated number of MDR-TB cases in 2008, and number two in terms of total numbers of TB cases in the world with the incidence rate per capita of 99 per 100,000 populations (WHO, 2011). Guizhou province is one of the highest-incidence-rate areas in China, and its prevalence of drug-resistant TB is higher than most of other provinces of China (Chen et al., 2011). The Affiliated Hospital of Zunyi Medical College is one of the specialized centers in Guizhou certified by the provincial government for the treatment of MDR-TB patients. In 2010, more than 15,000 TB patients were treated at the hospital. In order to determine the molecular characteristics of drug-resistant TB and assist in making informed TB treatment decisions for TB patients in the Zunyi area of Guizhou province, hundreds of M. tuberculosis clinical isolates were collected at the Affiliated Hospital of Zunyi Medical College and used for the systematic surveillance and other studies. We have made some progresses in the detection and molecular characterization of drug-resistant M. tuberculosis clinical isolates using different research methods such as M. tuberculosis culture, spoligotyping, gene sequencing, proteomics and drug susceptibility testing against first-line and second-line antituberculosis drugs.


Introduction
Tuberculosis (TB) is one of the leading infectious disease killers in the world, especially in developing countries.The increasing frequency of human-to-human transmission of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis poses challenges for effective therapeutic options and infection control.MDR-TB is defined as a form of TB that is resistant to at least isoniazid and rifampin, which are used to treat all TB patients.XDR-TB is a form of TB that is resistant to at least rifampicin and isoniazid in addition to any fluoroquinolone, and at least one of the three injectable secondline anti-TB drugs (amikacin, capreomycin and kanamycin).Based on the WHO Report 2010, China was ranked number one among high burden countries in terms of the estimated number of MDR-TB cases in 2008, and number two in terms of total numbers of TB cases in the world with the incidence rate per capita of 99 per 100,000 populations (WHO, 2011).Guizhou province is one of the highest-incidence-rate areas in China, and its prevalence of drug-resistant TB is higher than most of other provinces of China (Chen et al., 2011).The Affiliated Hospital of Zunyi Medical College is one of the specialized centers in Guizhou certified by the provincial government for the treatment of MDR-TB patients.In 2010, more than 15,000 TB patients were treated at the hospital.In order to determine the molecular characteristics of drug-resistant TB and assist in making informed TB treatment decisions for TB patients in the Zunyi area of Guizhou province, hundreds of M. tuberculosis clinical isolates were collected at the Affiliated Hospital of Zunyi Medical College and used for the systematic surveillance and other studies.We have made some progresses in the detection and molecular characterization of drug-resistant M. tuberculosis clinical isolates using different research methods such as M. tuberculosis culture, spoligotyping, gene sequencing, proteomics and drug susceptibility testing against first-line and second-line antituberculosis drugs.

Survey of drug-resistant M. tuberculosis clinical isolates
Mycobacteria are aerobes and grow most successfully in tissues with high oxygen content.The suitable growth temperature for mycobacteria is 37°C.Mycobacteria are "acid-fast bacilli" (AFB) because of their lipid-rich cell walls, which are relatively impermeable to various basic dyes unless the dyes are combined with phenol.Once stained, mycobacteria are resistant to decolorization with acidified organic solvents.M. tuberculosis is an intracellular pathogen usually infecting mononuclear phagocytes, and slow-growing with a generation time of 18 to 22 hours.The diagnosis of tuberculosis usually requires the detection of acid-fast bacilli in sputum via the acid-fast staining method (Ziehl-Neelsen method), which uses carbolfuchsin as the stain, acid-alcohol as the destaining solution and methylene blue as the counterstain.The culture confirmation of M. tuberculosis is the gold standard for diagnosis of tuberculosis and the drug susceptibility testing (DST) provides the basis for surveillance of drug-resistant TB and for physicians to adjust chemotherapy.
Even though TB laboratory services formed an essential part of the DOTS (Directly Observed Treatment Short course) strategy for National Tuberculosis Programs, it was often the most neglected component of these programs because of the absence of standardized techniques which complicated the activities of new laboratory services.Based on those considerations, WHO prepared guidelines in 1998 for laboratory services for the framework of National Tuberculosis Programs (WHO, 1998).The guidelines included three detailed manuals, two of which focused on the technical aspects of TB microscopy and culture, and a third one dealt with laboratory management including lab safety and proficiency testing.These manuals were specifically developed for use in low-and middle-income countries with high TB prevalence and incidence rates (WHO, 1998).Most of the methods used in our laboratory at the Affiliated Hospital of Zunyi Medical College and described in this chapter are based on these three manuals.

Collection of sputum specimens from patients
M. tuberculosis may be isolated from various clinical specimens, including respiratory specimens such as sputum, body fluids and body tissues (Goodwin, 2007).TB clinical strains from sputum of patients with active pulmonary tuberculosis were collected at the Affiliated Hospital of Zunyi Medical College.Sputum specimens were collected into a sterile singlecollection Universal container (28ml) with a tightly fitted lid.A good sputum specimen is considered to be recently-discharged materials from the bronchial tree of the patient, with a minimum amount of oral or nasal materials (WHO, 1998).In an ideal situation, a sputum 1. Turn on the thermostat-controlled water-bath and set up the temperature to 56°C. 2. Air dry smear on the slide, flame fix, and transfer it to the water-bath.3. Add carbolfuchsin dye to the heat-fixed smear on slide for 15min, and take out the slide.4. Allow the slide to cool to room temperature, then, decolorize slide by adding the fresh destaining solution, and wait until red color disappears. 5. Add counterstaining solution for 1 min, wash, and dry.6. Examine the slide by a 100× oil immersion objective on a light microscope.

Interpret acid-fast staining results
Smears should be carefully examined with a minimum of 300 fields, and three horizontal sweeps of a smear should be performed (Goodwin, 2007).The positive smear requires the cut-off of at least 5000 bacilli/ml.The overall sensitivity of the acid-fast staining method varies from 20% to 80% (Goodwin, 2007).

Drug susceptibility testing against different drugs
WHO has endorsed commercial liquid culture systems and molecular line-probe assays as gold standards for rapid detection of MDR-TB; however, because of technical complexity, cost and the requirement of sophisticated lab infrastructure, their uses have been limited in many resource-constrained settings (WHO, 2010).Several noncommercial culture and DST methods have been developed for those resource-constrained settings, and assessed by WHO.Based on the testing results, WHO recently recommended MODS (microscopic observation of drug susceptibility) and NRA (nitrate reductase assay) under certain conditions for direct testing of sputum speciments (WHO, 2010).
M. tuberculosis drug susceptibility testing methods using solid media include proportional, resistance ratio and absolute concentration, which are inexpensive and highly standardized for testing susceptibility to many drugs (WHO, 2009).The proportion method is the most commonly used worldwide.Sputum specimens were digested, decontaminated and used to inoculate egg-based media such as L-J slants for DST.The instructions for the preparation of the egg-based media such as L-J, and detailed protocols for performing standardized bacteriological services for detecting infectious cases of pulmonary tuberculosis, monitoring treatment progress and documenting cure at the end of treatment by means of microscopic examination were well described by WHO in Part III (Culture) of the Laboratory Services in Tuberculosis Control (WHO, 1998) and should be followed strictly.

Preparation of culture media
The recommended TB diagnostic laboratory procedures (WHO, 1998;Chinese Antituberculosis Association, 2006) were followed for the preparation of media and suspension, inoculation and mycobacterium culture in a biologic safety cabinet.Löwenstein-Jensen (L-J) solid media were prepared in the laboratory of the Affiliated Hospital of Zunyi Medical College.All the samples were cultured on L-J solid media and grown colonies were identified to the species level using TCH (2-thiophene carboxylic acid) and PNB (paranitrobenzoicacid) selective media or by standard biochemical procedures.

Procedure for preparation of L-J culture medium (for 1632 ml)
1. Dissolve salts from the following ingredients in order in about 300ml of distilled water by heating (WHO, 1998)

Preparation of drug-containing solid media
Drug stock solutions at recommended concentrations (Table 1) were made in distilled water.To make media containing the end concentration of different drugs (Table 1), 0.4ml of stock solution for each drug was diluted separately with 400ml of L-J mixture mentioned above.
Media containing different drugs were distributed to sterile universal screw-cap centrifuge tubes (7ml medium per tube); tubes were labeled and caps were tightened.The labeled tubes were placed in an oven at a slanted position (30° angle) and baked at 85°C for 50 min.Baked tubes were cooled down to room temperature and stored at 4°C refrigerator until use.
Since the medium was prepared with sterile precautions, this heating process was to solidify the medium instead of sterilizing it.The quality of the egg-based media deteriorates when the baking temperature is too high or the baking time is too long.The L-J medium should be dated and stored in the refrigerator for up to 4 weeks if the caps are tightened to prevent drying of the medium (WHO, 1998).

Digestion and decontamination
M. tuberculosis grows slowly and takes four to eight weeks or longer to give visible colonies.
Cultures are usually made in bottles because of the long incubation time required.The bottles are tightly capped to prevent drying of the cultures.The majority of clinical specimens submitted to the clinical laboratory are contaminated to varying degrees by more rapidly growing normal flora.These would rapidly overgrow the entire surface of the medium before the M. tuberculosis start to grow (Goodwin, 2007).Therefore, specimens must be subjected to digestion and decontamination that liquefies the organic debris and eliminates the unwanted normal flora.All currently available digesting and decontaminating methods are to some extent toxic to M. tuberculosis.Therefore, to ensure the survival of the maximum number of M. tuberculosis in the specimen, the digestion and decontamination procedure must be precisely followed (WHO, 1998).

Drug susceptibility testing
We have collected hundreds of TB culture samples from TB patients, and used them for the DNA extraction and drug-susceptibility testing against seven anti-TB drugs: rifampicin (RIF), isoniazid (INH), streptomycin (STR), ethambutol (EMB), capreomycin (CPM), ciprofloxacin (CIP) and levofloxacin (LVF).The proportion method on L-J media was used to perform the DST against different drugs in most of clinical M. tuberculosis isolates.The following drug concentrations in L-J media were used for the DST: RIF, 40μg/ml; INH, 0.2μg/ml; STR, 4µg/ml; EMB, 2µg/ml; CPM, 40µg/ml; CIP, 2µg/ml; and LVF, 2µg/ml.

Quality control
After coagulation, 5% of the slopes were picked up randomly and continued for incubation for 2 days at 37 °C to check for sterility.If no colony was grown on the solid medium after 48 hours of incubation at 37°C, the whole media batch should be good for DST.

Results criteria
The proportion testing results can be recorded at 28 days and again at 42 days as 3+ for confluent growth; 2+ for more than 100 colonies; and 1-99 colonies for the actual number of colonies.The percentage of drug resistance can be expressed as: Number of colonies on drug-containing medium/Number of colonies on L-J medium × 100%.If the percentage >1, the tested bacterium is considered to be drug resistant.

Results and discussion
We analyzed 316 clinical M. tuberculosis isolates for DST against four drugs (RIF, INH, STR and EMB).Results showed that 51.3% of isolates were resistant to one or more drugs, 19.0% were MDR, 20.9% were resistant to any single drug, 12.0% were resistant to any two drugs, 10.8% were resistant to any three drugs, and 7.6% were resistant to four drugs (Table 2).The prevalence of single drug-resistance was STR>RIF>EMB>INH in combined cases.There were 209 isolates in the first-treated (new TB cases) group, in which 42.1% were resistant to at least one drug, and 23.9% were resistant to any single drug.In addition, there were 107 isolates in the previously treated group, in which 69.2% were resistant to at least one drug, and 15.0% were resistant to any single drug.Our results indicated that the prevalence of drug-resistance in new tuberculosis cases was very high in the Zunyi area of Guizhou province, with 42.1% of the isolates resistant to at least one drug, and there were obvious differences in the drug susceptibility profiles between new and previously treated TB cases.These results also highlight the importance of surveillance of drug-resistant TB in order to improve the treatment outcomes of TB patients.

Molecular characterization of drug-resistant M. tuberculosis
The recommended standard or first-line treatment of tuberculosis includes a combination of four drugs, rifampicin, isoniazid, ethambutol and pyrazinamide, with or without streptomycin.Resistance to the first-line anti-TB drugs has been linked to mutations in at least 13 genes: rpoB for rifampicin resistance, katG, inhA, kasA, ahpC, ndh, furA, and oxyR for isoniazid resistance, embCAB for ethambutol resistance, pncA for pyrazinamide resistance, and rpsL, rrs, and gidB for streptomycin resistance.Resistance to the secondline anti-TB drugs has been linked to mutations in at least 10 genes (Banerjee, et al., 2008).
For the detection of mutations in genes linked to rifampicin and isoniazid resistance, we amplified rpoB and KatG gene fragments from many drug-resistant M. tuberculosis clinical isolations and performed DNA sequencing analyses.We also detected mutations in 5 other genes associated with drug-resistance (inhA, rpsl, rrs, gyrA and gyrB).Nucleotide sequences of primers used for PCR amplification and DNA sequencing were listed in Table 3.

DNA isolation and PCR amplification
The genomic DNA was extracted from M. tuberculosis clinical isolates using a Bacterial DNA Kit (Tiangen, China) following the manufacturer's instruction.DNA fragments for 7 drugrelated genes were amplified by PCR using synthetic oligonucleotide primers listed in Table 3.The following thermocycler parameters were applied with initial denaturation at 94°C for 5 min; 35-42 cycles of denaturation at 94°C for 30 sec; primer annealing at 58-62°C for 30 sec; extension at 72°C for 30-60 sec; and a final extension at 72°C for 7 min.The obtained DNA fragments were analyzed by electrophoresis in 2.0% agarose gel and were visualized under UV light on a transilluminator.

DNA sequencing analysis
In order to determine mutation profiles of 7 drug-related genes (rpoB, katG, inhA, rpsl, rrs, gyrA and gyrB), we purified PCR-amplified gene fragments and sent them to Shanghai Invitrogen for DNA sequencing using primers listed in Table 3.The FinchTV program was downloaded from Geospiza's website and used to view the original sequencing data.Sequence alignment analysis was conducted using the BLAST program at the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to compare our sequencing results with those wild-type M. tuberculosis genes listed in the GenBank.

Results and discussion
In 32 rifampicin-resistant strains, we identified 13 different types of missense mutations at codons 509,511,516,522,526,531,533,550 and 572 of rpoB gene, and compared the mutation profile with those of rifampicin-resistant M. Tuberculosis isolates from different geographical regions of the world.Comparison of the results showed that the rpoB gene mutation profile in rifampicin-resistant M. Tuberculosis clinical isolates from Guizhou province differed not only from other provinces of China but also from other countries of Asia, Europe and America (Chen et al., 2010).Two new mutations (Val550Leu and Ser509Arg) were identified and deposited in GenBank (GQ250580 and GQ250581).We concluded that mutation profiles of rifampicin-resistant M. Tuberculosis isolates were variable depending on the geographical locations, and further studies would be needed to determine the molecular basis for such variations (Chen et al., 2010).
In 30 isoniazid-resistant strains, 18 (60%) of them had mutations in katG and/or inhA genes and two newly identified mutations in katG gene (Val230Met and Pro232Gln) were deposited to GenBank (GQ250582 and GQ250583).Results from this study further confirmed that mutations in katG and inhA genes are related to the isoniazid resistance in M. tuberculosis (Chen Y et al., 2010).
To better compare drug resistance and mutation profiles of clinical M. Tuberculosis isolates collected at our hospital, we selected 23 representative isolates and combined the DST results against 4 drugs with mutation profiles in 7 genes related to drug resistance in the same table (Table 4).It is clear that the relationship between drug resistance and mutations in specific genes may be more complex than we expected.For example, no mutations were found in 6 of the 7 drug-related genes for isolate number 2, which was resistant to all 4 drugs tested, suggesting that there must be changes in other regions of the genes or other genes.Further studies will be needed to determine the molecular mechanism underlining the resistance of this kind of isolates to different anti-TB drugs.In another example, two mutations were identified in isolate number 17, one in rpoB gene (TCG531TTG) and another in katG gene (AGC315ACC).However, isolate number 17 was resistant only to rifampicin but sensitive to 3 other drugs.It will be necessary for us to repeat the DST for this isolate to confirm whether this isolate does have a mutation in katG gene but still sensitive to isoniazid.In summary, more studies are need to identify new genes related to MDR-TB.Africa by spoligotyping (Niemann et al., 2000).In addition, Viana-Niero et al. also indicated that the spoligotyping fingerprint of M. Africa was between M. tuberculosis and M. bovis (Viana-Niero et al., 2001).Spoligotyping can be used to distinguish MTC members based on their characteristic spoligotypes (Table 5).Until now, IS6110-RFLP has been the gold standard for genotyping of M. tuberculosis (Majeed et al., 2004).However, a comparison study using IS6110-RFLP and spoligotyping indicated that the genotyping capacity of spoligotyping was better than IS6110-RFLP to strains containing low-copy numbers of IS6110 (Bauer et al., 1999).
To study the genotypic diversity, we choose spoligotyping for molecular typing of 100 clinical M. tuberculosis isolates collected at the Affiliated Hospital of Zunyi Medical College from 2008 to 2009.Bacterial growth and chromosomal DNA isolation were carried out by the method of Soolingen et al. (van Soolinger et al., 1991).The extracted DNA was used as a template for PCR amplification of the DR region of the genome with the biotinylated forward primer, 5'-GGTTTTGGGTCTGACGAC-3' and the reverse primer 5'-CCGAGAGGG GACGGAAAC-3'.
The following thermocycler parameters were applied with initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s; primer annealing at 56°C for 30 s; extension at 72°C for 30 s; and a final extension at 72°C for 8 min.The PCR amplified products were hybridized to a membrane containing a set of 43 immobilized oligonucleotides, each corresponding to one of the unique spacer DNA sequences within the DR locus.DNAs isolated from M. tuberculosis H37Rv and M. bovis BCG were used as controls.The hybridized PCR products were incubated with 1:4000-diluted streptavidin-peroxidase conjugate (Boehringer) for 30min at 42°C.Detection of hybridizing DNA was done by using the chemiluminescent ECL (Amersham) detection system followed by exposure to X-ray film (Hyperfilm ECL; Amersham) in accordance with the manufacturer's instructions.
Spoligotypes in binary format were entered in the SITVIT database (Pasteur Institute of Guadeloupe).Twenty-nine distinct spoligotyping patterns were observed.In total, 20 orphan patterns were identified and the remaining 77 were contained within 9 superfamilies: Beijing, T1, T3, T2, MANU2, Beijing-like, U, H3 and H3-T3.Results showed that almost half of the isolates were clustered to the Beijing lineage, and 13 isolates were clustered to the T1 lineage.
The Beijing genotype family was originally identified in China (Brudey et al., 2006) and defined as strains that presence of at least three spacers 35-43 and absence of spacers 1-34 (van Soolinger et al., 1995).Beijing family strains represent at least 13% of strains worldwide and about half of strains in the East Asia.A total of 2,346 M. tuberculosis isolates from 13 provinces, excluding Guizhou province, in China were genotyped by spoligotyping, and results showed that 74.08% of the isolates belonged to the Beijing family (Dong et al., 2010).
Other studies also examined the relationship of Beijing genotype with drug resistance, however, the association between them was still not clear.The possible reasons for this uncertainty might be: 1) the diversity of treatment programs; 2) compliance to treatment; 3) different quality of anti-TB drugs; and 4) spread of different and not yet distinguished sublineages of the Beijing strains (Parwati et al., 2010).
The "T" families were the most prevalent following Beijing family, belonged to modern TB strains, and ill-defined with more than 600 unclassified STs.The "T" families were further divided into 5 subclades (T1-T5) based on single spacer-differences (Brudey et al. 2006).The numbers of T1 and T2 families were ranked the second and third respectively after Beijing family in the updated SpolDB4.

Proteomics and its application in tuberculosis research
Proteomics is defined as the large-scale study of proteins, their post-translational modifications, and their structures and functions underlying different biological processes.Proteomics enables the qualitative and quantitative analyses of proteins in complex biological systems, such as cells, tissues, and body fluids under specific conditions or in response to different stimuli (Graham et al., 2011;Lim & Elenitoba-Johnson, 2004;List et al., 2008;Wu & Liu, 2009).Proteomics is an effective means to rapidly identify new proteins as diagnostic or prognostic markers and as therapeutic targets for various diseases including cancers, genetic diseases, and infectious diseases such as tuberculosis (Chung et al., 2007;Kavallaris & Marshall, 2005;Drake et al., 2005;Stulik & Butaye, 2011).
Comparative proteome analyses of culture supernatant proteins from virulent M. tuberculosis H37Rv and attenuated M. bovis BCG (Mattow et al., 2003) or H37Ra (He et al., 2003) were performed to identify new virulent factors.Proteomics was also used for genome-wide analysis of the host intracellular network regulating survival of M. tuberculosis (Kumar et al., 2010).It was demonstrated that protein expression by a Beijing strain was different from that of another clinical isolate and M. tuberculosis H37Rv (Pheiffer et al., 2005).
The major membrane protein of virulent M. tuberculosis was characterized using proteomics technology (Lee et al., 1999).Xie et al. used proteomics to compare proteins of MDR-TB isolate with drug-sensitive isolate (Xie et al. 2009).Additionally, proteomics has been successfully used for vaccine design to improve protection against tuberculosis (Mollenkopf et al., 2004).
The development of proteomic profiles for M. tuberculosis is needed to identify proteins that are differentially expressed in clinical strains with different drug susceptibility profiles such as MDR-TB and XDR-TB comparing to the standard strain, which can provide insights into the mechanisms by which the mycobacteria resistant to anti-TB drugs and the selection of suitable biomarkers for new diagnostics, new virulent factors and/targets for the development of potential therapeutics and new vaccines.
The most commonly used method for proteomic analysis is the two-dimensional polyacrylamide gel electrophoresis (2-DE), which allows the separation and display of thousands of proteins from a complex mixture by their charges (pI) and relative molecular mass (Mr).Gel-separated proteins can be identified rapidly by mass spectrometry (MS), and such analyses permit the systematic identification of the proteome if genomic information is available.In this preliminary study, we used 2-DE and mass spectrometry to compare protein expression profiles among different clinical M. tuberculosis strains such as MDR-TB, XDR-TB and the reference strain H37Rv, and identified some up-regulated, down-regulated genes which might be associated with MDR or XDR.

Sample preparation for MDR/XDR-TB isolates
The sensitive strain used in this study was the standard M. tuberculosis strain H37Rv, which was obtained as a gift from the Chinese Centre for Disease Control and Prevention.Both MDR-TB and XDR-TB strains were clinical isolates collected at the Affiliated Hospital of Zunyi Medical College.Five to six inoculating loops of M. tuberculosis colonies were scraped from improved L-J culture media and transferred to 4 ml of purified water in a screw-top centrifuge tube.The mycobacterium suspension was heated at 80°C for 30 min in a water bath to inactivate M. tuberculosis, and centrifuged at 4000g for 10min for removing the supernatant.The precipitation was resuspended in the phosphate-buffered saline (pH7.4), and centrifuged at 4000g for 10min to remove the supernatant.The inactivated M. tuberculosis sample was resuspended in 0.3ml of lysis buffer, and 10l of proteinase inhibitors were added to the suspension followed by incubation at 4°C for 20 min.The suspension was then sonicated (200W, 1min×30 times), and centrifuged at 4000g for 30min at 4°C.Four-fold volume of cold dimethyl ketone were added to the suspension and the mixture was kept at -20°C overnight for precipitating proteins.The protein precipitates were centrifuged at 10000g for 5min at 4°C, and resuspended in 7M carbamide and 2M sulfourea solution to dissolve proteins followed by centrifugation at 4000g for 30min at 4°C.Concentration of protein samples was measured by the NanoDrop-1000 (Thermo, Germany) and protein samples were stored at -20°C until use.

Two-dimensional electrophoresis (2-DE)
Protein samples (200-300g) were resuspended in 480l of rehydration buffer and applied to pH 4-7 IPG strips (Amersham Biosciences) for rehydration.When IPG strips were rehydrated with the protein samples, isoelectric focusing (IEF) was performed in the following voltage mode: 0V 1h, 50V 10h fast voltage, 500V The pI and Mr gradient of the 2-DE gels were determined using an iterative calibration method after 2-DE gel images transferred to computer by means of the image scanner.Spot detection and image analysis were performed and compared using the program Progenesis SameSpots (Nonlinear Dynamics, UK) for 2-DE database construction.Images of MDR-TB and XDR-TB were compared to the gel image of the standard strain H37Rv, and protein spots showing differential expression levels of more than two-fold were analyzed.

MALDI-MS and database
Twenty protein spots of interest (5 spots from H37Rv, 10 spots from XDR-TB, and 5 upregulated protein spots from MDR-TB) were excised from Coomassie Brilliant Blue G-250 stained two dimensional gels (2-DE) and digested in gel using trypsin for 18h at 37°C.Masses of the peptides extracted from gel slices were applied to the sample plate of a matrix-assisted laser desorption ionization-time-of-flight MS (MALDI-TOF-MS).Peptide

Comparison of XDR and MDR patterns
The comparison was repeated at least three times, and only those differences confirmed in all comparisons were accepted as strain specific.This study compared the proteome of XDR-TB clinical isolate to those of MDR-TB clinical isolate and the standard strain H37Rv, and demonstrated that the 2-DE protein expression patterns of XDR-TB and MDR-TB clinical isolates were highly correlated, but there were some visible differences between drug-resistant strains and the reference H37Rv strain.Results also showed that more basic proteins were expressed in drug-resistant M. tuberculosis isolates than in the standard H37Rv stain.Figure 1 shows differences among three different mycobacterium strains.There were 19 proteins specifically overexpressed in XDR-TB (Table 6), 3 proteins upregulated (Table 7), 13 proteins down-regulated (  7. Expression levels of 3 up-regulated proteins in XDR-TB (protein expression level=number in the table×10 4 ) Out of these forty-five protein spots, twenty were selected for the mass spectrometry analysis, and only three proteins were identified by the MALDI-TOF-MS (Table 9).The differential expression of the ribosomal protein S3 and 19 kda major membrane protein in the XDR-TB strain was validated by the real-time RT-PCR (data not shown).It is not clear how differential expression of these three proteins contributes to the drug resistance of the XDR-TB.Further function analysis of these three proteins will be conducted in our laboratory to determine their relationship with drug resistance of XDR-TB.

Conclusion
In this chapter, we have provided the detailed description about our current progresses in the field of survey and molecular characterization of drug-resistant M. tuberculosis clinical isolates using different research methods such as M. tuberculosis culture, spoligotyping, gene sequencing, proteomics and drug susceptibility testing against first-line and second-line anti-tuberculosis drugs.We hope this chapter will be useful for researchers, scientists and physicians in academic institutions, clinical laboratories, pharmaceutical companies and research hospitals who have interest in the research related to drug-resistant M. tuberculosis, especially MDR/XDR-TB.

Fig. 1 .
Fig. 1.Protein expression patterns of XDR-TB and MDR-TB clinical isolates compared to the standard strain H37Rv in two-dimensional electrophoresis

Table 2 .
Drug susceptibility profiles of 316 clinical M. tuberculosis isolates.

Table 3 .
Primers used in this study for PCR amplification and DNA sequencing

Table 4 .
Drug susceptibility profiles and mutational patterns of 23 representative clinical isolates

Table 5
. Characterization of M. tuberculosis complex by Spoligotyping MTC includes M. tuberculosis, M. bovis, M. voles and Africa mycobacterium.M. tuberculosis is one of the leading pathogens to human and animal, followed by M. bovis.M. tuberculosis and M. bovis can be distinguished from MTC by using L-J agar slants, PNB and TCH.M. bovis can be quickly identified by spoligotyping.Considering the difficulties in determining M. vole and Africa mycobacterium by traditional methods of bacteriology, Soolingen et al. developed the spoligotyping method to distinguish the M. vole from MTC (van Soolinger et al. 1998).Results from Niemann et al. proved that M. bovis could be differentiated from M.
Survey and Molecular Characterization of Drug-Resistant M. tuberculosis Clinical Isolates from Zunyi, Guizhou Province of China 447 www.intechopen.com Survey and Molecular Characterization of Drug-Resistant M. tuberculosis Clinical Isolates from Zunyi, Guizhou Province of China 449 mass fingerprint data were searched using the Mascot search (www.matrixscience.com) of the National Center for Biotechnology Information (NCBI) database. www.intechopen.com

Table 6 .
Expression levels of 19 proteins specifically overexpressed in the XDR-TB (protein expression level=number in the table×10 4 ) Table 8) and 10 proteins disappeared in the XDR-TB clinical isolate compared to the reference H37Rv strain.

Table 8 .
Expression levels of 13 down-regulated proteins in XDR-TB (protein expression quantity=number in the table×10 4 ).

Table 9 .
Description of three proteins identified by MALDI-TOF-MS