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Author: Shujauat Hussain Msc Microbiology Doon PG Paramedical College Dehradun UK shujauatdargmail.com DRUG RESISTANCE IN TUBERCLOSIS introDuction:- Correct application of the several powerful drugs in various types of regimens that have evolved from well –planned clinical trials could offer a potentially 100 successful treatment of the disease. Even though such outcome of chemotherapy could not be seen in developing and undeveloped countries owing to several factors many of the developed countries particularly the United States have even entertained hopes of eradicating the disease and have established target dates. Unfortunately however this optimism could not be sustained for long the downward trend of the disease in the United States which continued until the mid 1980’s is now taking an upward turn. Several reasons have been cited for the failure of the TB treatment chief among them is the drug resistance of the tubercle bacilli. Non compliance a notoriously persistent world wide problem magnifies this because many of the patients who fail to take the drugs for the prescribed period end up with drug resistant bacilli. From a microbiologist stand point resistance and susceptibility are relative terms since practically all populations of drug susceptible tubercle bacilli will contain proportions of resistant proportions are very small in wild strains that are not exposed to these drugs 1 in 10 6 for standard drugs and 1 in 10 8 for rifampicin . From a clinical stand point drug resistance can be defined as temporary or permanent capacity of organism and their progeny to remain viable all to multiply in the presence of concentration of drug that would not destroy or inhabit to the growth of cells. According to Mitchison 1961 clinically meaning full drug resistance is indicated by decrease of susceptibility to a drug of a sufficient degree to be reasonably certain that the strain concerned is different from a sample of wild strains of tubercle bacilli that had never came in contact with the drug. Epidemiologically drug resistance in tuberculosis is classified into three types: 1 : Primary drug resistance where previously treated patients are found to have drug resistance organisms presumably because they have been infected from an outside source of resistant bacilli. 2:Acquired drug resistance where the patients who initially have drug susceptible tubercle bacilli later become drug resistance inadequate inappropriate or irregular treatment or more important due to non- compliance in drug taking. 3: Initial drug resistance Mitchison 1968 denotes drug resistance in patients who deny history of previous chemotherapy in reality consists of true primary and an unknown amount of undisclosed acquired resistance. Since it takes elaborate efforts to check conclusively and determine the possibility of previous but unreported treatment initial drug resistance has been gaining practical importance. Various factors favoring developing acquired drug resistance are considered. Historically the problem of drug resistance was recognized in 1946almost immediately following the introduction of streptomycin when Yeoman’s and associates 1946 found that when the drug was given alone there was at first a striking improvement in the patient’s symptoms together with a rapid decrease in the bacilli in the sputum however the number of bacilli soon increased and the condition of the patients deteriorated. These investigators found that the bacilli isolated from the patients were drug resistant that is the bacilli instead of being killed continued to grow in vitro in presence of high concentrations of the same drug. This interesting observation was soon followed by a study by Pyle1947who showed that during treatment with streptomycin alone the

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Table - Natural resistance- frequency of resistantr mutants in a wild strain Drug Concentration of culture medium mcg/ml Natural resistance R 40 1 mutantr in 10 7 bacilli H 02 1 mutantr in 10 4 bacilli EMB 20 1 mutantr in 10 5 bacilli proportion of drug resistant bacilli increased progressively from about in 88750 organisms before the therapy to about 1 in 367 after 15 weeks of treatment. These initial observations were the pioneering studies in the problem of drug resistance in mycobacteria. Subsequent studies have enabled us to recognize that increase in drug resistance following immunotherapy was due to the selection of drug-resistant mutants. A significant landmark soon followed with the discovery by Temple and associates1951that multiple –drug theory was able to cure tuberculosis without development of drug resistance. This pioneering work has been confirmed by studies carried out all over the world and has led to the clinical maxim that tuberculosis patients should be treated with multiple-drug chemotherapy only. The techniques for the detection of drug resistance and the chemotherapeutic and epidemiological importance of the several type of the drug resistance had brought forth a vast literature which was recently summarized by the authorGangadharam1948. Surprisingly not much progress has occurred over the past several years in the theoretical aspects of drug resistance although there had been some improvements in technical aspects particularly in the developed countries where rapid methods have been introduced. No such changes have taken place in developing countries. However the recent epidemics and upsurge in the incidence of drug resistance in many parts of the world are beginning to reawaken interest in this problem. theoretical aspects of Drug resistance A Origin of Drug Resistance All available evidence show that drug resistance in Mycobacterium tuberculosis occurs only by mutation the drug acting merely as a selective agent. It has also been shown that resistance of tuberculin bacilli to isoniazid and streptomycin develops as a single-step mutation unlike the gradual multiple-steps process observed in penicillin resistance Rist1964. Using fluctuation analysis David 1970 has calculated the mutation rates and the proportion of resistant cells in a given bacillary population is greater than mutation rate in addition some mutants do not survive and some may back-mutate to sensitivity. The mutants resistant to one of several anti-tuberculosis drug appears once in every 107 cells whereas the proportion of resistant mutants present in the normally susceptible varies from 1 in 105 to 1 in 109 cells examined MDR-TB and the Mechanisms of Resistance The current mechanism for the emergence of multi-resistant stains which is a major health problem in our days is not due to new mechanisms of resistance but related to mechanisms that we already know for M. tuberculosis i.e. mutations on target genes for the antibiotics. The resistance to isoniazid the most common resistance to first line drugsget the link to chaitanyas is most commonly related to a change on the kat G gene catalase/peroxidase and is also responsible for resistance to ethionamide. Resistance to rifampicin is associated to mutation on rpo B gene and to streptomycin to the genes rps L and rrs. Resistance to rifampicin is most found in association to resistance to isoniazid.

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S 40 1 mutantr in 10 4 bacilli ETH 200 1 mutantr in 10³ bacilli Z 250 1 mutantr in 10³ bacilli source: Canetti G. et alli. Bull. WHO 1969 Problem of multi drug resistant tuberculosis MDR-TB One of the classical threats of the tuberculosis epidemic has been the MDR-TB. Drug resistance arises in Mycobacterium tuberculosis MTB generally by mutation of chromosomal genes but despite combined drug therapy the spread of multi-drug-resistant MDR strains is alarmingly increasing. Use and often abuse or misuse of antimicrobial agents has encouraged the evolution of bacteria toward resistance resulting often in therapeutic failure. There are evidences that bacteria have the ability to adapt to this deficit and recover fitness on serial passage1. Resistance to anti-tuberculosis drugs has been a problem since the era of chemotherapy began. After dramatic outbreaks of MDR-TB in the early 1990s resistance became recognized as a global problem. MDR-TB now threatens the inhabitants of countries in Europe Asia Africa and the Americas too. An understanding of the molecular basis of drug resistance may contribute to the development of new drugs. Management of MDR-TB relies on prompt recognition and treatment with at least 3 drugs to which an isolate is susceptible. The roles of drug containing environments and the immunological status of the host and bacterial molecular mechanisms of development of drug resistance to M. tuberculosis have been examined and results are helpful in implementation of modified drug regimens in tuberculosis control programmes. Multidrug resistant strains of M. tuberculosis seriously threaten tuberculosis control and prevention efforts. Molecular studies of the mechanism of action of anti-tubercular drugs have elucidated the genetic basis of drug resistance. Drug resistance in M. tuberculosis has been primarily attributed to the mutations in the drug target genes however the presence of efflux pumps in clinical MDR isolates cannot be ruled out. These mutations lead either to an altered target or to a change in titration of the drug. A diverse array of strategies is already available to assist in rapid detection of drug resistance-associated gene mutations. In spite of remarkable advances in this area much remains to be learned about the molecular genetic basis of drug resistance in M. tuberculosis During the last decade there has been a marked increase in the number and gravity of tuberculosis cases both in developing countries and in industrialized nations. One of the more insidious consequences of this resurgence has been the recent emergence of nosocomial transmission of multi drug resistant strains of M. tuberculosis thus creating untreatable forms of the disease and these strains may become widespread. That the various clinical isolates of M. tuberculosis are geographically partitioned at the global level. MOLECULAR MECHANISM FOR THE EMERGENCE OF DRUG RESISTANCE …… With the last 10 years the mechanism of action of most of the anti-tuberculosis agents has been described and scientists are beginning to understand some of the molecular mechanism whereby Mycobacterium tuberculosis becomes resistant .Ramaswamy s. and J. M. Musser. 1998 Mycobacterium tuberculosis is often acquired early in life with acute infection and with developing immunity granuloma formation and calcification. This is followed by a long latent period which continues until reactivation occurs in a proportion of the individuals. The means that individual strains of Mycobacterium tuberculosis have little opportunity to interact and exchange genetic information with strains compared with for example organism that colonize the nasopharynx or the gastrointestinal tract. In these locations other bacteria may transmit antibiotic resistance transduction or transformation. This option is not available for Mycobacterium tuberculosis so resistance elements such as the insertion sequence IS6110 has been associated with new resistance emerging through the inactivation of critical genes.DaleJ.W.1995 and LemaitureN.W.SoughakoffC. Truffot-pernot and V.

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jarlier. 1999 MUTATION… In prokaryotic genomes mutations are constantly occurring due to base changes caused by exogenous agents DNA polymerase errors deletions insertions and duplications for prokaryotes there is a constant rate of spontaneous mutation of 0.0033 mutations/DNA replication that is uniform for a diverse spectrum of organism DrakeJW.1999. The mutation rate for individual genes varies significantly between and within genes. The reasons for these variations are uncertain but are thought to be under the influence of the local DNA sequence. For example significant differences between the evolutionary rates of heat shock protein genes within the Mycobacterium genus have been detected. The nonsynonymous sites of the GroEL gene evolved twice as fast as those of the HSP65HughesAL.1993 gene. The antibiotic resistance genes encoding fundamental replication functions of the organism such as rpoB and gyrA are typically highly conservedDriclaj.w.1999. and TelentiA.P.ImbodenF.MarchesiD.LowerieS.ColeM.J.ColstonL.Matter K.Schopferand T.Bodmer.1993. GENETIC BASIS OF RESISTANCE…… Talenti and colleagues were the first to determine the site of mutation that resulted in rifampicin resistance in Mycobacterium tuberculosis . They used the evidence that E.coli became resistant to rifampicin through mutation in the beta subunit of the rpoB gene and sequenced this gene from a series of epidemiology unrelated strainsTelentiA.P.ImbodenF.MarchesiD.LowerieS.ColeM.J.ColstonL.Matter K.Schopferand T.Bodmer.1993. They showed that almost all rifampin-resistant isolates had mutations in a small region of rpoB . Subsequently further clinical studies indicated that mutation are found in his region up to 95 of resistant isolates.RamswamyS. and J.M.Musser 1998. A similar approach has been adopted to detect to mutations conferring resistance to another antibiotics. The genetic basis of resistance for some anti-tuberculosis agents is not fully known. For example streptomycin resistance emerges through mutation in rrs and rpsl that produce alteration in the streptomycin binding site but these changes are identified in just over one-half of the strains studied till date jindaniA.V.R.Aber EA.Edwwards and D.A.Mitchison1980. Thus there is considerable amount of research into the mechanism of resistance that is still required. It should be noted that in many cases mutations found in association with drug resistance organism may cause different levels of resistance and may not be directly related to the mechanism of resistance. Isoniazid-resistance is a case in a point. Modification of KatG partial or total deletions point mutations or insertions leads to the abolition or diminution of Catalase activity and high level resistance to isoniazid HeymB.P.AlzariN.H Honore and N.Lehn2000 and Zhang Y T Garbe and D. young. Catalase activity is essential in activating isoniazid to the active hydrazine derivatives. A deficiency in enzyme activity produces high-level resistance and is found in more then 80 of isoniazid resistant strains. Alternatively low-level resistance caused by point mutations in the regulatory region of inhA operon resulting in over expression of inhA . Strains with this mutation have normal mycolic acid synthesis but low level resistance to isoniazid. Point mutations in the regulatory region of ahpC have also been demonstrated these are a compensation for the effects of absent or reduced Catalase katG function and do not directly result in resistance. Most pyrazinamide-resistant organism have mutations in the pyrazinamidase gene although the gene may also be inactivated through the insertion of IS6110 Lemaiture N. W. SougakoffC.Truff-pernot 1999. Pyrazinamide is essential producing the active derivative and mutants are unable to produce an active drug. In addition to this some resistant strains have no defined mutation Davies A.P. O J. BillingtonT.D.McHughD.A Mitchison and S H Gillespie 2000. MUTATION RATE: The rate at which resistance emerges differs for all of the anti-tuberculosis agents being highest for ethambutol and lowest for rifampin and quinolones. The risks of mutation for most of the antibiotics used in tuberculosis treatment have been defined previously David H.L1970for rifampin isoniazid streptomycin and ethambutol

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they are 3.32x10 9 2.56x10 8 2.29x10 8 and 1.0x10 7 mutations per bacterium per cell division respectively. The mutation rate rather than the mutation frequency is most reliable measure it records the risk of mutation per cell division rather than the proportions of mutant cells. Mutation frequency is significantly affected by “jackpot” mutation occurring early in the culture. There are several different calculation methods used to determine the mutation rate accurately and the mathematics behind calculation is beyond the scope of this review. Readers seeking further information can find a detailed description of these methods in one of several review articles JonesM.E.Thomson and A. Rogers.1994. These methods have been applied to anti-tuberculosis drugs to calculate the estimated rates of mutation to resistance to the major anti-tuberculosis drugsDavid H.L 1970. It has been assumed that the risk that an organism will develop resistance to two agents is the product of the risks of developing resistance to each separately. For example the risk of resistance for a combination of rifampin streptomycin and isoniazid is 10 25 /bacterium/generation. The risk of mutants emerging in a patient depends partly on this and the size of bacterial population within compartments. Therefore risk of resistance may be more accurately calculated using the formula P1-1-r n where P is the probability of the drug resistance emerging r is the mutation rate and n is the number of bacilli in a lesion usually calculated to be 10 6 per lesionsShimai T 1987. I f single drug therapy with a risk of resistance emerging is 100. If two drugs with a combined mutation rate pf 10 12 are used then the risk is 0.01 however if the bacterial population in a lesion is 10 10 and the mutation rate is 10 12 then there is a 1 risk of resistance emerging. If mycobacteria are found in different compartments or in different physiology states it is likely that this equation is an oversimplification. It is likely that even if a patient is receiving optimum chemotherapy there are populations of mycobacterial cells that are effectively receiving monotherapy or dual therapy. This means that the above equation is an optimistic estimate of the risk of resistance. This is in accord with clinical experience which suggests that a relatively small deviation from the standard regimen may lead to the emergence of resistance. Factors responsible for the Development of Drug resistance….. Primary drug resistance due to the infection of patients with drug-resistant bacilli generated somewhere else could be a serious epidemiological problem. Increase in such resistance is due to the increase in resistant cases in the community and the chances of exogenous infectious. Acquired drug resistance which develops in patient who had originally drug-susceptible organisms but whose bacilli become resistant can be due to several reasons discussed below Davidson 1987 Biological:- a Initial bacterial population b Local factors inside the host favorable for multiplication of drug-resistant bacilli. c Presence of drug in insufficient concentrations d Patient’s drug inactivation status. Clinical:- a Treatment with single drugs b Inadequate dosage of the drugs c Insufficient duration of treatment d Adding a single drug to failing regimen e Interference by occult or quack medicine f Interference by any other indigenous systems of medicine Pharmaceutical and pharmalogical:- a Insufficient concentration of pure drug.

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b Inadequate standardization of the bioavailability of the drug c Improper storage conditions of the preparations d Improper bioavailability of combined tablet preparations e Confusion created by trade names of various preparations of combined tablets f Improper or incorrect dispensing of the drug Administrative a Insufficient supplies of the drug b Bureaueatic influence in the ordering and supply of the drugs c Substandard drugs purchased because of cost considerations and government regulations d Administrative delays in the release of the drugs from ports of entry e Administrative controls of the drug dispensing Sociological patient’s cooperation a Noncompliance b irregularity in drug intake c Premature discontinuation in drug intake d Avoidance of other exogenous infections with drug-resistant bacilli Table 2 factors responsible for influence the chance for drug resistance .. INCREASED CHANCE:- 1. Previous treatment of tuberculosis a Greatest risk if treatment fails or disease relapses while patient is still on drugs b Significant risk if treatment is inadequate c Treatment received while living in a high-tuberculosis-incidence area 2. Birth and/or recent residence in a high-tuberculosis-incidence area a Particularly Asia south and central America and Africa b Certain localized areas without development countries c The younger the age the greater the risk 3. Recent exposure to known case of drug-resistant disease 4. Infection with mycobacteria other then Mycobacterium tuberculosis SUMMARY:- Drug resistant tuberculosis posses a significant threat to human health and it is important to understand how the resistance emerges if were to reverse the upward trend. Treatment with internationally approved regimens results in a very high cure rate with a few relapse and without the emergence of resistance. These regimens are effective in preventing the emergence of resistance because combination chemotherapy makes it highly unlikely that there will be a spontaneous mutant resistant a of the components chemotherapy. Patients with uncomplicated tuberculosis who receive inadequate treatment provide a selection advantage for resistant mutants because bacteria may be exposed to monotherapy preventing the emergence of resistance to single agents and then to multiple agents as the protection combination is eroded. That Mycobacterium tuberculosis cells within the body

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are susceptible to different components of antituberculosis chemotherapy means that the risk of resistant emerging is higher then would be expected if the whole population of bacterial cells should be counted together. Clinical complications such as empyema and extensive caviation permit a large population to develop in a compartment into which drugs may not penetrate there is an increased likelihood of resistance emerging. A similar situation may develop in patients with extensive disease or poor immunity. We have learned that some physiological conditions may induce a hypermutable state making multiple resistance more likely. The assumption that resistant organisms are less fit then wild type strains may not be correct as the initial fitness deficits may be attenuated by adaption by multiple passage. Instances of isoniazid resistance where attenuated virulence is common may occur because the molecular mechanism of resistance directly affects a system required by the organism for intracellular survival. The importance lesson these clinical and molecular studies teach us that resistant organisms over time will be fully virulent and that if we ware to prevent an epidemic of multiple-drug –resistant tuberculosis we must take steps to ensure that all patients are diagnosed and effectively so that strains are not created and transmitted in the community. Hypothetical View about the Spread Of Drug Resistance……… Drug resistant tuberculosis can occur due to different resistant in different places such as-- In developing countries such as India drug resistance may be due to following reasons- 1. Irregular dose 2. If proper or complete dose is not taken when first time attacked by Mycobacterium tuberculosis. 3. Unawareness among illiterate people. 4. Because of substandard drugs. 5. Monotherapy. 6. Use of drugs such as rifampin for other nonmycobacterial disease. 7. Unavailability of latest facilities 8. Economically not efforting the cost of drugs 9. Due to unavailability of laboratories for detecting the drug resistant organism 10. One of the most is HIV infection among tuberculosis patients Drug resistance in developed countries is very rarely caused by above reasons the main reasons for drug resistance in developed countries may be as:- 1. Drug addiction 2. Alcoholic 3. HIV infection among tuberculosis is common every where and has a very much contribution in drug resistance While these factors contribute mostly to the spread of primary drug resistance the bulk of primary drug resistance is shown to be due to immigrants from high-prevalence countries. Some of these immigrants who had undiagnosed tuberculosis or drug resistance would soon develop resistance to rifampin as well. Thus in the absence of facilities to obtain rapid information on drug resistance the patient receiving both isoniazid and rifampin will in essence receive a single drug rifampin since he has undiagnosed isoniazid resistance. Research Needs- Hypothetical concepts………….. To make treatment against the drug resistant mycobacteria following things should be done as soon as possible

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these may be helpful- 1. In case streptomycin resistant patient if therapy of streptomycin is not stopped the resistant bacteria replicates and removes the susceptible one in case it is stopped as soon as possible the susceptible bacteria are removed automatically. To control it the patient sputum should be examined for drug resistance after every month. 2. Sulfatides are responsible for prevention of mycobacteria from engulfment of bacteria by macrophages by preventing the phagosome lysosome fusion. How to prevent the synthesis of these sulfatides or sulfated aryl trehalose it may by use of some drugs. 3. Mutation is one of the cause of drug resistance mutation of genes. Mutagens can be used to mutate the most important gene responsible for the virulence or drug resistance of mycobacteria. REFRENCES:- 1. Ananthanarayan and paniker book of microbiology 8 th edition. 2. A text book of microbiology by john l. ingraham university of California Davis 3. A text book of microbiology by Prescott edition 7 th . 4. Encyclopedia of infectious disease book of microbiology 5. A book on tuberculosis by Riechman 103-116EDWARD A. NARDELL Harvard Medical College The Cambridge Hospital Cambridge Masachusetts department of public health Boston. 6. Desert medicine research centre pall road Jhodpur India 7. Department of Microbiology Dr. S.N. medical college jhodpur 8. Royal free and University college Medical college University London London NW3 2PF United kingdom 9. www.ncbi.com 10. Gangadharm book of tuberculosis by LEE Riechman university of lllinosis college of medicine at Chicago vol. 8 293-322. 11. Portability distribution of drug-resistant mutants in unselected populations of mycobacterium tuberculosis Applied microbiology by David H.L. 12. FOX W. and Mitchison short course chemotherapy of pulmonary tuberculosis 13. Heifets L.B. drug susceptibility in the chemotherapy of mycobacterial infections and synergistic effects of rifampin streptomycin ethionamide and ethambutol on mycobacteria 14. Bottger E. C. B. Springer M. Pletschette and P. Sander. 1998. Fitness of antibiotic-resistant microorganisms and compensatory mutations. Nat. Med. 4:1343–1344. CrossRef Medline 15. Breathnach A. S. A. de Ruiter G. M. Holdsworth N. T. Bateman D. G. O’Sullivan P. J. Rees D. Snashall H. J. Milburn B. S. Peters J. Watson F. A. Drobniewski and G. L. French. 1998. An outbreak of multi-drug-resistant tuberculosis in a London teaching hospital. J. Hosp. Infect. 39:111–117. CrossRef Medline 16. Cambau E. W. Sougakoff M. Besson C. Truffot-Pernot J. Grosset and V. Jarlier. 1994. Selection of a gyrA mutant of ycobacterium tuberculosis resistant to fluoroquinolones during treatment with ofloxacin. J. Infect. Dis. 170:479–483. Medline 17. Canetti G. 1955. The tubercle bacillus in the pulmonary lesion of man. 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