Strains Of Tubercle Bacilli Research Articles

Tuberculosis in the 21st century: an emerging pandemic?

Since the mid-1980ies the world has witnessed a dramatic increase in tuberculosis. Our knowledge of tuberculosis epidemiology was established during the previous epidemic that, at least in Europe, took two hundred years from rise to fall. It is difficult to understand the new epidemic if we assume that the genetics of the host-parasite relationship is unchanged from the previous epidemic.The paper discusses how both host and parasite genetics may have changed. Molecular epidemiology done in Archangel, Russia, where most of the classical reasons for increase in tuberculosis were absent, indicated that strains of Mycobacterium tuberculosis with changed biological properties could be responsible. Strains belonging to the so-called Beijing family were strongly associated with primary drug resistance and caused clusters ten times as big as “traditional” strains.The paper describes how the research consortium takes these observations further to explore the importance of “new” strains of tubercle bacilli in the on-going pandemic. What are the changes in biological properties and what genetic changes do they reflect? The previous epidemic may have changed the genetic susceptibility of the human host by selection, but what has constituted a selective pressure for the bacillary population? The two most dramatic changes in the environment of M. tuberculosis are mass introduction of chemotherapy and BCG vaccination. We explore further the nature of drug resistance in these strains and the possibility that BCG may fail to protect against bacilli of the new pandemic.Lastly the paper points at some action that can be taken instantly and that may have a major impact on transmission, even before the questions mentioned above are answered. The clue here is to shorten the time of transmission by a rapid test to secure early diagnosis and treatment. What is needed, however, is not a diagnostic test for tuberculosis, but a simple screening test with high sensitivity that could tell us whom among the numerous people who have a chronic cough and systemic symptoms who are the true “tuberculosis suspects” eligible for rapid examination of sputum by smear microscopy

Challenges Facing Antitubercular Therapy: New Drugs and Delivery Systems

Tuberculosis (TB) has again emerged as a challenging disease, victimizing a large section of the world population. Long duration of therapy, drug resistance, side effects associated with large doses of number of drugs, and economic reasons have resulted in poor patient compliance and in ineffective anti-TB therapy. In addition, drug-resistant strains of tubercle bacilli have further compounded the problem. In this article challenges facing present therapy, the mode of tuberculosis infection, challenges facing fixed-dose combination (FDC) formulation, new anti-TB molecules from the modification of existing anti-TB drugs for better activity, new molecules acting on the novel targets for reducing bacterial pathogenicity, antibiotics and nonantibiotics exhibiting anti-TB activity, and new drug delivery systems are discussed.

Functional Demonstration of Reverse Transsulfuration in the Mycobacterium tuberculosis Complex Reveals That Methionine Is the Preferred Sulfur Source for Pathogenic Mycobacteria

Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of (35)S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine gamma-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine gamma-synthase (CGS), but demonstration of a gamma-elimination reaction with cystathionine as well as the gamma-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.

A controlled study of rifabutin and an uncontrolled study of ofloxacin in the retreatment of patients with pulmonary tuberculosis resistant to isoniazid, streptomycin and rifampicin

A study of rifabutin in the retreatment of patients with chronic pulmonary tuberculosis whose strains of tubercle bacilli were resistant to all three of the drugs isoniazid, streptomycin, and rifampicin, and usually to others as well, was undertaken in 22 Chinese patients in Hong Kong. They were arranged in 11 pairs such that the resistance pattern of the strains for both patients was the same or closely similar. One of each pair was allocated at random to receive rifabutin (B) daily and the other rifampicin (R) daily. The two members of each pair were also prescribed the same or similar companion drugs, selected on the basis of the results of susceptibility tests and of the history of previous antituberculosis chemotherapy. The bacteriological results showed no evidence of a sustained benefit in any patient. A temporary response was seen on sputum smear examination in 14 patients (7B, 7R), of whom 10 (5B, 5R) had a period of smear-negativity, and on sputum culture examination in 10 patients (5B, 5R), of whom 3 (2B, 1R) had a period of culture-negativity. The duration of response was longer for the B patient in 5 pairs and for the R patient in 5. Two patients (both B) had rifabutin-susceptible strains on admission to the study; their temporary responses were among the best and were associated with the emergence of rifabutin resistance, suggesting that rifabutin may have contributed to their response. A total of 17 patients, including 7 not in the paired comparison, were subsequently retreated with ofloxacin; 10 showed a response, disease becoming and remaining quiescent in 3. The results suggest that rifabutin does not have a useful role in the retreatment of patients with multidrug-resistant pulmonary tuberculosis, which includes rifampicin resistance, except possibly in a small proportion of patients who have rifabutin-susceptible strains. Ofloxacin appears to be a better drug to use, in combination with any companion drugs still available.

Effect of pyrazinamide on nicotinamide adenine dinucleotide glycohydrolase of tuberculous host.

Nicotinamide adenine dinucleotide glycohydrolase (NADase) activity of mice liver was increased after infection with strains of tubercle bacilli either susceptible or resistant to pyrazinamide. Pyrazinamide and nicotinamide, as well as isoniazid, suppressed the increase of NADase associated with tuberculous infection with the bacilli susceptible to these drugs. Pyrazinamide and nicotinamide did not influence the increased NADase produced by bacilli resistant to these drugs. the effects of pyrazinamide and nicotinamide on the host metabolism in tuberculous infection with susceptible bacilli were likely due to their antimycobacterial activities and not their direct action on host metabolism.

Designation of Strain H37Rv as the Neotype of Mycobacterium tuberculosis

Strain H37Rv of Mycobacterium tuberculosis (Zopf) Lehmann and Neumann is typical of the species in growth characteristics, drug susceptibility, and biochemical activity. H37Rv is compared with the hypothetical median strain of M. tuberculosis and agrees with it in 70 of 71 characters. In comparison with other strains of tubercle bacilli, H37Rv has moderate virulence for guinea pigs and mice. This strain agrees well with Koch’s original description of the tubercle bacillus; the few differences that do exist are discussed. The well documented history of H37Rv, coupled with its world-wide distribution and long use by mycobacteriologists, makes it most appropriate as the neotype of the species M. tuberculosis. The strain may be obtained from the Trudeau Institute, Inc., Saranac Lake, N.Y. 12983, as TMC 102 or from the American Type Culture Collection, Rockville, Md. 20852, as 27294.

Infectivity and pathogenicity of drug-resistant strains of tubercle bacilli studied by aerogenic infection of guinea pigs.

Infectivity and pathogenicity of drug-resistant strains of tubercle bacilli studied by aerogenic infection of guinea pigs.

Studies on chemotherapeutics for Mycobacterium tuberculosis. 23. Synthesis and antibacterial activity on Mycobacterium tuberculosis of 3-methoxy-4-phenoxybenzaldehyde and 3-hydroxy-4-phenoxybenzaldehyde derivatives

Forty-two kinds of compounds mainly related to 3-methoxy-4-phenoxybenzaldehyde thiosemicarbazone and 22 kinds of compounds similar to its demethylated compound, 3-hydroxy-4-phenoxybenzaldehyde, were synthesized and their antibacterial action against human tubercle bacilli, H37Rv sensitive strain, was tested, using a Tween-albumin medium. Against the control growth-inhibitory values of 0.0313 μg/ml for isonicotinoyl hydrazide (INH) and 0.625 μg/ml for tibione (TBI), 3-methoxy-4-phenoxybenzaldehyde (p-tolyl) thiosemicarbazone and 4-(o-methoxyphenyl) thiosemicarbazone showed the value of 3.13 μg/ml, and 4-allyl-, 4-propyl-, 4-butyl-, 4-(p-methoxyphenyl)-, 4-(o-ethoxyphenyl)-, and 4-(p-iso-butoxyphenyl)-thiosemicarbazones and amidinohydrazone hydrochloride all showed growth inhibition in a concentration of 6.25 μg/ml. 3-Hydroxy-4-phenoxybenzaldehyde derivatives all failed to show any antibacterial activity and this fact indicated that the presence of a methoxyl group was significant in the appearance of antibacterial activity against this strain of tubercle bacilli. 3-Methoxy-4-phenoxybenzaldehyde isonicotinoylhydrazone inhibited the growth of tubercle bacilli at a concentration of 0.078 μg/ml, but it was found to have cross-resistance with INH.

The isolation of tubercle bacilli from endocervical mucus of infertile women

As infertility is a common and important after-effect of genital tuberculosis in women, an attempt has been made to isolate tubercle bacilli from endocervical mucus of a consecutive and unselected series of 119 women complaining primarily of infertility and clinically unsuspected of genital tuberculosis. Guinea pig tests have demonstrated the presence of tubercle bacilli in endocervical secretions of 7 (5.9 per cent) cases. Culture and microscopic examination gave very poor results. All the strains of tubercle bacilli isolated were found to be of the human type. Three strains showed resistance to streptomycin and P.A.S. Six patients, or 5 per cent, of 119 patients presumably sterile, became pregnant during the period of observation. The figures obtained from the study are too small for statistical treatment, but they are nevertheless informative. As the results of the present findings, it is suggested that bacteriologic examination for tuberculosis of endocervical mucus of all patients with infertility should be a routine procedure.

EXPERIMENTAL STUDIES ON THE SURVIVAL OF THE TUBERCLE BACILLUS OF THE GUINEA PIG CADAVERS AFTER INTERMENT

According to the recent governmental statistics, about 50,000 death are attributed to tuberculosis. Tuberculosis death occurs mostly in the domestic dwellings like other deaths, because of limited medical facilities. Cadavers are usually disposed of by a conventional method of interment. Handling of cadavers is for the most cases performed by close relatives since the funeral system of western standards is lacking. Although sanitary regulations require cremation or interment with strict provision of the cadavers resulted from acute communicable diseases, sanitary control of those resulted from chronic diseases like tuberculosis is not strongly enforced. Furthermore, current land planning programs often necessitate removal of buried cadavers, thus generating risk of unhygienic handling of infected cadavers. This experiment was designed to observe how long the bacilli can survive in the cadavers after interment. Two strains of tubercle bacilli, a H37RV standard strain, and a resistant strain called H531 were used for the study. The H531 strain was isolated from a tuberculosis patient and found to have developed resistance to INH(10r/ML) and Streptomycin (10r/ML). The bacterial suspension was inoculated into 300 guinea pigs weighing 300 gms on the average by way of intracardiac, intraperitoneal and subcutaneous routes. Morbid or dead animals were sacrificed and isolation of the bacilli was made of the lungs, liver, spleen and mesenteric lymph nodes, using 3% KH2PO4 Ogawa media. Simultaneously these necropsied animals were buried in the ground with depth of 1.5 meters from the surface. After internment for different lengths of time periods, the reisolation of the bacilli was made of specimens taken from the decaying cadavers. The experiment was carried out from December 1963 to the end of 1965. The following can be summarized: 1. The recovery rate of tubercle bacilli, the H37RV strain, from the cadavers was 100% after interment for 30 days of the Winter period, December-February. The rate, however, decreased to 29.4% after 60 days. 2. When buried in Spring month, March, bacilli, the H37RV strain, were recovered with 100% after 30 day internment and 14.3% after 60 days. 3. When buried during Summer months, June-July, the bacilli, the H37RV strain were recovered with 100% after 30 days, 33,3% after 60 days and nil after 70 days. 4. The recovery rate of the bacilli of the resistant strain, from the cadavers, was 100% after 30days of interment in Spring season, March-May and decreased to 33.3% after 60 days. 5. When buried during Summer months, June-July, the bacilli were recovered with 100% after 30 days and 22.5% after 60days. 6. No difference on the survival rate of the vacilli was observed between two strains. 7. The recovery rates of the bacilli, H37RV strain, from the cadavers framed with different conditions are 100% in all cases after 30 days internment. The rates, however, differed after 60 days interment according to the condition of framing, wiz the rate for a wooden box was 53.3%, that for a vinyl sac was 33.3% and those buried with the naked condition showed nil. 8. No survival of the bacilli, either the strain of H37RV or that of H534 was seen after 70 days internment during any month of he year. 9. It was confirmed that the drug resistance of the bacilli by no means differs between groups before and after interment. 10. It was observed through animal experimentation, that the resistant strain, H543, produced a definite sign of progressive tuberculosis though possessed low virulence.

EXPERIMENTAL RE-EVALUATION STUDIES ON ISONICOTINIC ACID HYDRAZIDE lN VITRO AND IN VIVO Ⅰ. In vitro Inhibitory Effects of Isonicotinic Acid Hydrazide (INH) Against Tubercle Bacilli

Of course, it is importanat to discover new antituberculous drugs, but it is also very important to know how present anti-tuberculous drugs should be used for tuberculosis control. It is well known that isonicotinic acid hydrazide(INH) is the most widely used effective drug in chemotherapy of tuberculosis. It seems that a new drug more effective than INH will not be discovered in the near future. From this view point, many investigators are still continuing their efforts to know the best way of using INH, part icularly in optimal dose and mode of administration. In Korea, much of the treatment of pulmonary tuberculosis depends on the use of INH alone, therefore suitable therapeutic methods using INH for control of tuberculosis in this country are urgently required. Under the above mentioned circumstances the authors have made a series of experimental studies on re-evaluation of INH in vitro and in vivo. Two human strains, H<sub>37</sub>Rv and Kurono, and one bovine strain, Ravenel, were used. Ten day old cultures from Ogawa media were used as inocula throughout the experiments. Proskauer-Beck medium with the addition of 10 percent horse serum was used for determination of inhibitory effects of INH against the above three strains. The following conclusions were reached: 1. Non-contaminated INH solution was obtained by using ten fold dilution or twice dilution methods with sterilized distilled water or Proskauer-Beck medium without autoclaving or Seitz-filtration. 2. Minimum completely inhibitory concentration o INH to human H<sub>37</sub>Rv strain and Kurono strain was between 0.02mcg per ml. and 0.04 mcg per ml. but to bovine Ravenel strain was around 0.04 mcg per ml. 3. Slide culture method with petroleum ether suspension of the tubercle bacilli is a rapid quantitative method for measuring inhibitory effect of INH against the tubercle bacilli. 4. Under the constant conditions, minimum completely inhibitory concentration of INH against the same strain of tubercle bacilli is always constant. The deep cultures should be observed at least within one week. 5. Microscopic examination of stained slides by slide culture method should be made at three or four days cultivation, because INH activity begins to decrease.

CLASSIFICATION OF THE TUBERCLE BACILLI ISOLATED IN CHONNAM DISTRICT 1. Classification by Spot Test

The authors have isolated, during 1962. 10-1964. 2, using the Lowenstein-Jensen media, 130 strains of tubercle bacilli from the patients, diagnosed already as open lung tuberculosis or intestinal tuberculosis, called on Chonnam T.B. Association, Kwangju Grahms Hospital, Chonnam University Hospital, Mokpo Red Cross Hospital and Hwasu Tangkwang Hospital and also the T.B. patients have being taken to their beds in each home of Kwangju and Kangjin area. The 4 strains among 130 rains of T.B. bacilli show stain positive and culture-negative phenomena. And also, the spot test of 128 strains was positive and the remained 2 strains were negative. The most favorable pH for spot test was pH7.0-8.0.

Drug-resistant tubercle bacilli.

In a recently published communication, Cohen 1 calls attention to the disturbing problem of drug-resistant tubercle bacilli. This report and similar ones from widespread areas merit serious consideration but require critical analysis. The problem of drug-resistant tubercle bacilli may be examined from 2 aspects: (1) from the point of view of the reduction in therapeutic effectiveness of the drugs in an individual patient and (2) in terms of the epidemiological implications of the spread of drug-resistant organisms within a community. From the therapeutic point of view, there is general agreement that the demonstration of the emergence of streptomycin resistance in vitro correlates closely with the loss of drug effectiveness in the patient. This is probably true, also, for the secondary antituberculous drugs such as aminosalicylic acid, pyrazinamide, cycloserine, viomycin, kanamycin, and ethionamide. There are considerable differences of opinion concerning the clinical significance of isolating isoniazid-resistant strains of tubercle bacilli from

Interaction of lysates of histiocytes and tubercle bacilli.

SummaryCell lysates derived from peritoneal histiocytes of normal and BCG-immunized rabbits or guinea pigs had no demonstrable direct effect on the morphology, neutral red binding capacity, viability or cytotoxic activity of virulent and attenuated strains of Mycobacterium tuberculosis. Histiocytic lysate in combination with Tween 80 was found to be bactericidal for strains of tubercle bacilli but not for a strain of Escherichia coli or a strain of Staphylococcus aureus. The mechanism of this activity appeared to be due to the enzymatic degradation of Tween 80 by histiocytic lipases with the formation of oleic acid. A similar antimycobacterial activity was manifested in mixtures of histiocytic lysates with crude lipid extracts of normal rabbit serum and with lipemic rabbit serum. No difference in activity was found between cell lysates prepared from histiocytes of normal and BCG-immunized rabbits or guinea pigs.

The consistency of the susceptibility to hydrogen peroxide of tubercle bacilli isolated from South Indian patients before treatment

Summary Isoniazid-sensitive strains of tubercle bacilli cultured from pairs of sputum specimens obtained pretreatment from 7 South Indian patients were exposed to 0·02 per cent hydrogen peroxide for 60 and 90 min., and the numbers of viable organisms were counted. The variation in the percentage of surviving organisms from patient to patient was greater than the variation from culture to culture obtained from the same patient. Thus, strains from different Indian patients have characteristic degrees of susceptibility to hydrogen peroxide.

The consistency of the virulence in the guinea-pig of tubercle bacilli isolated on different occasions from South India patients before treatment

Summary Isoniazid- and streptomycin-sensitive cultures of tubercle bacilli, isolated pretreatment from 12 South Indian patients when they first attended the Centre, and at 7 and 42 days thereafter, were tested for their virulence in the guinea-pig. The variation in virulence between patients was greater than the variation between cultures from the same patient. Thus, consistent differences exist between the virulence of strains of tubercle bacilli isolated from different Indian patients. No systematic alteration in virulence occurred during the 42-day period, during which no patient was receiving anti-tuberculosis chemotherapy. The variation in virulence between cultures obtained from the same patient was no greater than could be attributed to natural variation in the response of the guinea-pigs.

VACCINATION AGAINST TUBERCULOSIS

WHILE DEATHS from tuberculosis have decreased sharply in this country, the number of new cases has not, so that there is still leeway for improvement in the control of this disease. Safe, effective immunizing agents have contributed immeasurably toward the control of other communicable diseases. What is the situation in 1959 with respect to vaccination against tuberculosis? This Commentary and the paper by Barclay in this issue of Pediatrics consider this question. Already in Koch's day the search was on for a vaccine against tuberculosis. Tuberculin, as well as tubercle bacilli killed or attenuated in various ways and strains of tubercle bacilli derived from cold-blooded animals, proved disappointing. Calmette and Guérin, working with a virulent bovine strain of tubercle bacillus isolated in 1902, found that it lost virulence when sub-cultured at frequent intervals on special media. By 1922, the "BCG" strain appeared stable and was administered orally to infants. Soon afterwards, despite pleas from prominent French clinicians of the day, mass vaccination was undertaken without adequate controls, and the vaccine fell into disrepute.

PROCEEDINGS

THE PURPOSE of this presentation is to review very briefly some of the history of the controversial BCG vaccine. This is a living, attenuated, bovine strain of tubercle bacilli. This strain was first isolated in Paris as a very virulent organism for animals, and it was attenuated by serial passage in artificial media for a period of 13 years. By 1921, Calmette and Guérin were satisfied in their own minds that they had produced an attenuated bacillus, but they delayed the announcement and the distribution of cultures to other laboratories until about 1924. From 1921 to 1928, large numbers of experiments were conducted to try and revert this attenuated bacillus to its original virulent characteristic. All experimental attempts in this direction failed. In 1930 the vaccine received a setback from which it has never fully recovered, when 240 children received it as an orally administered vaccine at Lubec and 72 of them died. 2 A full-scale investigation subsequently revealed that the BCG strain was not the cause of the disaster. A contaminating strain of bacilli in the culture administered was positively identified by growth and color-producing characteristics. I think it is improper to speak of "a BCG vaccine." Just as the original vaccine was able to mutate from a fully virulent strain of organisms to an attenuated form, mutations continue to go on producing relative degrees of infectivity or invasiveness. There is no such thing as absolute virulence and absolute avirulence. Rather, there is a continuous spectrum of invasiveness which an organism may exhibit, and an organism may change its characteristics of invasiveness from one culture to another. Therefore, one may read in the literature that a BCG vaccine given in Scandinavia has produced a certain amount of immunity and a certain amount of hypersensitivity, but one cannot expect to exactly reduplicate the same result here; because you just cannot get the same Scandinavian vaccine here and reduplicate that experience. In this country there are several sources of BCG vaccine that a laboratory may acquire or a physician may use.

In vitro inhibition of growth of M. tuberculosis by certain 11-oxygenated steroids.

SummaryHydrocortisone and corticosterone in a concentration of 100 μg/ml markedly inhibit growth of isoniazid sensitive and resistant strains of tubercle bacilli grown in a simple synthetic culture medium. Ten and 20 μg/ml produce slight slowing of growth in some strains.

Formation of protoplasts from mycobacteria by mycobacteriophage.

SummaryVarious mycobacterial cells were converted to protoplasts by treatment with a mycobacteriophage concentrate. The technic involved production of sufficient adapted phage by growth either in the 607 or H37Ra strains of tubercle bacilli. Further concentration was necessary in order to obtain a sufficiently high phage bacteria ratio. This was accomplished by lyophilization. The significance of the reported findings is discussed.