The Department of Microbiology is one of the original and four basic Departments that were established in 1953 when the Institute became operational. Since then, this Department has contributed richly to the understanding of pathogenicity of M tuberculosis and pathogenesis of tuberculosis. Dr. De Monte, Dr. N.P.Gupta and Prof. S.Chandrasekhar are some of the outstanding microbiologists who headed this Department and steered the research activities during the early years of development.

The current research interests of the Department have been expanded further. The spectrum of investigation for mycobacterial diseases has been widened to understand the role of the non-tubercular mycobacterial (NTM) infections which have gained importance as emergent opportunistic pathogens. In the process, presence of plasmids have been Ciemonstrated for the first time from Indian NTM strains by this Department. In addition, this Department has contributed significantly in the study of drug resistance profile of MDR- TB. Studies on pathogenesis and pathogenicity has been ,given a new dimension with our experiments and observations on the nitric oxide mediated mycobactericidal activity of the monocytes and -macrophages cultured from tuberculosis patients and the role of proinflammatory cytokines in it. We demonstrated for the first time that human monocytes/macrophages can be stimulated by appropriate biological response modifiers (BRMS) to release significantly high amount of nitric oxide, to produce mycobactericidal effect in vitro.

During the last decade and a half, the Department has undertaken studies to delineate the profile of aerobic and bacterial pathogens involved, in various respiratory Infections. These studies were carried out by Using transtracheal aspirates, the first ever report of the use of this technique in India. These studies contributed significantly to demonstrate the important role played by the anaerobic bacterial pathogens in respiratory diseases. The Department has also carried out studies on the molecular basis of pathogenicity in respect of Klebsiella pneumoniae, an important causative agent of nosocomial and community acquired pneumonia. Molecular epidemiologic typing of this important pathogen was carried using various techniques e.g., SDS-P AGE of whole cell protein profiling, restriction endonuclease analysis of whole cell DNA and ribotyping.

The role of delayed hypersensitivity (DH) and cellular immunity (CMI) in tuberculosis was studied in collaboration with New Delhi TB Centre. Our studies have shown that DH and CMI are two different processes mediated by different cells and both play a decisive role in tuberculosis.

Studies on isolation and characterization of a new anti-tuberculous antibiotic from a streptomyces strain, plant-scale production, purification, chemical characteristics and toxicity were undertaken. Also a total of 116 new media made from indigenous material were tested as substitute for conventional imported media.

It was shown that tubercle bacilli convert to cell wall deficient forms (CWDF) because of the defence mechanisms of the host. These CWDF when injected into immune deficient guinea pigs revert into acid fast bacilli (AFB). It was suggested that tubercle bacilli convert into filterable forms or CWDF , because of host defence mechanisms or antibiotic treatment. Developed a well defined medium for rapid growth in spheroplast phase of mycobacteria and for their reversion to parent acid fast forms.

Alveolar macrophages from normal and BCG vaccinated guinea pig, the latter being activated, were studied. The acid phosphatase was estimated by histochemical methods. No correlation was observed between macrophage activation and intracellular inhibition of tubercle bacilli. Effect of lysosomal hydrolases of macrophages from immune hosts on the cell wall of acid fast tubercle bacilli was studied. Macrophages from immune hosts were shown to act on the cell walls of mycobacteria which contain lipopolysaccharides to produce cell wall deficient spheroplastic variant.

In vitro studies of the effect of three enzymes namely arabinosidase, mannosidase and n-acetyl glucosaminidase on cultures of M. tuberculosis have been carried out. Acid fast bacilli were shown to revert to non-acid fast and spheroplast forms in the presence of combination of two enzymes. Reversion to parent acid fast forms occurs when grown on medium without the enzymes.

Pulmonary infection caused by Mycobacterium tuberculosis is an unsolved epidemiological problem worldwide. No simple and effective method to determine the source of infection is available till date. We have evaluated the DNA restriction fragment length polymorphism (RFLP) technique for its suitability as an epidemiological tool. Radiolabeled ribosomal RNA prepared from mycobacteria was used as a probe. Unsheared genomic DNA was prepared from one reference strain (M tuberculosis, H37Rv) and thirty native clinical isolates of M. tuberculosis. These were digested with various restriction enzymes, southern blotted and probed with r^32P/ rRNA. The results analysed using the mathematical model showed that restriction enzyme PstI can be used to define a homogenous group of strains (base substitution percentage (P=3% and Percentage DNA homology (H)=94% for all strains) whereas BamHI and EcoRI showed marked variability in P and H values (EcoRI, P-4.3% to 8%, H=84% to 93%; BamHI, P=1.8% to 5%, H=90% to 96.4%). It may be said that a combination of these three restriction enzymes could be a potential tool for molecular fingerprinting of M tuberculosis strains.

The mean CD4/CD8 ratio at the beginning of the study in the refractory cases (0.69) and the newly diagnosed cases (0.81) were significantly lower than those of the normal control subjects (1.84). After 3 months of chemotherapy all but 3 of the newly-diagnosed cases showed clinical improvement, and all became sputum-negative. Their CD4/CD8 ratio recorded a rise to near normal (1:54). On the contrary, following 3 months of reserve-line regimen, only 7 of the 21 drug resistant cases showed sputum conversion. In all of the drug resistant cases, irrespective of sputum conversion, the CD4/CD8 ratio remained low (1.05).

8. Isolation, identification and molecular characterization of native plasmids from clinical isolates of Mycobacterium avium-intracellulare
Plasmids have been isolated from M. avium intracellulare. One representative plasmid has been characterized for further work to develop a cloning vector for mycobacterial genes. This indigenously developed cloning vector will help in the study of the mycobacterial gene for example the cell wall antigen genes. 


9. In vitro drug sensitivity study of AFB positive isolates from chronic respiratory patient by broth dilution method
A rapid microbroth dilution method has been developed to assess drug susceptibility profile of Mycobacterium avium-intracellulare (isolated from drug failure cases of pulmonary mycobacterial infections) and compared with the conventional plate (proportion) method. The advantages of this microbroth method include the combination of drug susceptibility, and MIC in the same experiment. The materials used are less in quantity and it is a cognately rapid method as results can be read within 7 days. M. avium intracellulare, M. kansasii and M. fortuitum strains were tested with both agar dilution method and broth microdilution method. The test drugs used were isoniazid, streptomycin, ethambutol, rifampicin, kanamycin, amikacin, clofazimine, ciprofloxacin and roxithromycin. For M. avium intracellulare, the agreement between both methods figured around 70% for isoniazid (INH), streptomycin (STR) and ethambutol (EMB). Fot kanamycin (KANA), it was 100% and for rifampicin (RIF), amikacin (AMlKA), clofazimine (CLOFA), ciprofloxacin (CIPRO) and roxithromycin (ROXITH) it was 83.6%.

10. Nitric oxide dependent killing of Mycobacterium tuberculosis by human mononuclear phagocytes from fresh cases of tuberculosis and patients with drug resistant tuberculosis
It was demonstrated in cell culture experiments that nitric oxide, the product of the arginine dependent pathway of human mononuclear phagocytes effectively kills M. tuberculosis in vitro. The release of reactive nitrogen intermediates was triggered by incubation with various proinflammatory cytokines namely IFN-γ,TNF-α and IL-β. We have earlier shown that human mononuclear phagocytes can be induced to release nitric oxide (NO) radicals which can kill tumour cells. We demonstrated by using colony forming assays that human mononuclear phagocytes can effectively kill M tuberculosis by using a NO dependent pathway. Treatment of mononuclear phagocytes with L-arginine resulted in markedly increased killing activity whereas, by using NGMMA, analogue of L-arginine, the cidal activity could be brought down to the basal level. These results clearly suggest that cytokines, particularly IFN-γ, induced NO release and its reactive product with oxygen radical, peroxynitrite, could play an important role in the killing of M. tuberculosis by human mononuclear phagocytes. A significant production of interleukin-4 and interleukin-IO, by the ex-vivo matured, untreated macrophages from the active tuberculosis patients indicate that regulation of cytokine network to encourage in situ/local production of nitric oxide may be useful in management of pulmonary tuberculosis.

11. Molecular epidemiology and typing of strains of Mycobacterium avium intracellulare (MAC)

Analysis of M. avium intracellulare clinical isolates from India by IS-1245 based spoligotyping and restriction fragment length polymorphism (RFLP) of PCR amplified genomic DNA sequence is being undertaken to establish a base data for such strains from India

12. Analysis of respiratory symptomatic patients

Results of direct smear and culture examination of 37,683 sputum samples received from 18,139 patients over a period of ten years (1988-97) have been analyzed. In this study only those patients who submitted more that 2 specimens were included for analysis. In all, 1466 (8..08%) patients were bacteriologically confirmed ( culture positive) for tuberculosis. Among theses, 65.2% (956) were direct smear positive. The remaining 34.7% (509) were cases of smear negative (paucibacillary) tuberculosis. There was a preponderance of male patients in all age groups except in the youngest (<16yrs) where the ratio was reversed. Furthermore, the highest umber of cases belonged to the oldest group of patients (>45yrs), presenting 12.41% of all culture positive cases (males and females) as direct smear negative (paucibacillary). Interestingly, about one third of all culture proven tuberculosis cases among the present group of respiratory symotomatics were smear negative, thus underscoring the actual tuberculosis burden in India.

13. Prevalence of drug resistance in a cohort of category I and category II patients of pulmonary tuberculosis
Three consecutive sputum samples from 100 patients of pulmonary tuberculosis attending the Clinical Research Centre at V.P. Chest Institute were examined by Ziel-Neelson staining and cultured on Lowenstain-Jensen medium. The patients included 70 category I and 30 category II cases. Drug susceptibility tests on 35 strains of M. tuberculosis isolated from category I patients showed that 34% strains were resistant to isoniazid. The resistance to rifampicin, ethambutol, and streptomycin was 20%, 23% and 31% respectively. Resistance to both isoniazid and rifampicin was seen in 20% cases. Amongst the 9 strains of M. tuberculosis tested for drug susceptibility from category II patients the resistance to isoniazid and rifampicin was 66% each. Resistance to ethambutol and streptomycin was 33% and 4% respectively. In addition 55% strains were resistance to both isoniazid and rifampicin.

Inter-departmental collaboration

Several research studies were undertaken in collaboration with the Department of Respiratory Medicine, V.P. Chest Institute.

Anaerobes were found to be involved in most of the cases in addition to the pyogenic aerobic bacteria. These findings have a direct bearing on the management and treatment of patients with empyema.

2. The bacterial etiology of acute exacerbations of chronic bronchitis using transtracheal aspirates

This technique was employed for the first time in India for this purpose. Importance of H. influenze and S. pneumoniae as the main etiological agents was demonstrated. The results also proved that anaerobic bacteria have no role to play in the etiology of acute exacerbation.

3. Secondary bacterial flora in cases of pulmonary tuberculosis, using transtracheal aspirates

A number of pyogenic bacteria viz. Klebsiella spp., Pseudomonas spp. and anaerobes (Bacteroides spp.) were found to be causing superimposed infections. Appropriate antibiotics after in vitro susceptibility determination were administered and the patients were found to show excellent response.

4. Usefulness of fibreoptic bronchoscopy in the diagnosis of pulmonary tuberculosis

Suspected cases of pulmonary tuberculosis which were giving consistently negative results by sputum smear examination as well as by culture were selected for the study. Fibreoptic bronchoscopy was found to be very useful in diagnosing a large majority of these cases.

5. Studies on the incidence of plasmid mediated antibiotic resistance in gram negative bacterial clinical isolates

A group of 18 strains, showing high MIC values for various antibiotics were studied for the detection of resistance plasmids by conjugation with a recipient strain of E. coli K12 F^-Nal^r. Seventeen isolates were shown to harbour conjugative plasmids as demonstrated by Anderson's method of direct cross.

6. Studies on association of viral and bacterial infections with acute exacerbation of asthma in smokers and non-smokers

Bacterial infection was detected in 5 cases (10%). S. pneumoniae was recovered in 4 of these and H. influenzae from one case. Interestingly, all the 5 cases had serological evidence of viral infection as well, indicating that bacterial infection was superimposed. All these cases required intervention with antibiotics to control the acute exacerbation. The exacerbation in smoker asthmatics tended to last longer than in non-smokers. 

With klebocin technique only 39 (~ 70%) out of 50 strains could be typed while pp A had 100% typeability. Only 8 strains could be ascribed to unique klebocin types while by protein profiling as many as 31 strains were shown to have unique protein profiles, thereby demonstrating its higher power of strain discrimination. Thus, protein profiling in comparison to klebocin typing was shown to have 100% typeability, 100% reproducibility and a much higher level of strain discrimination capability

8. Bacterial etiological profile of adult community acquired pneumonia

Gram stained smear examination of a properly expectorated sputum specimen (having less than 10 squamous epithelial cells and > 25 polymorphonuclear cells) was found to have an excellent correlation with identification of the pathogen by sputum and bronchial aspirate culture. Bacterial pathogens could be isolated in 18 of the 30 cases; S. pneumoniae, the predominant pathogen from ~37%, K. pneumoniae from ~ 17%, S. aureus from 4% and S. faecalis from 4% of the cases. Bacteremia accompanying pneumonia was detected in 20% of the patients; S. pneumoniae accounting for 10% of these and K. pneumoniae for the rest of the cases.


9. Incidence of virulence factors in clinical isolates of Klebsiella pneumoniae
As many as 53% of the 225 strains were found to be serum resistant. 1:1 Urinary and respiratory isolates had the highest incidence (~ 68%) of serum resistance. Aerobactin production was detected in 26% of the strains. Of these, highest frequency of aerobactin production was observed in the respiratory isolates. As many as 92% of the isolates were shown to secrete enterochelin, while only ~ 20% of the isolates were positive for mucoidy. Only 3% of the total isolates were found to possess multiple virulence factors viz: serum resistance, aerobactin, mucoidy and enterochelin production.

The assay developed by us was compared with the conventional viable count method. There was an excellent agreement between the two methods as the number of strains categorised as sensitive, intermediate sensitive and resistant by the two methods was almost identical. In comparison to the conventional technique which is labour intensive, cumbersome and time consuming the rapid assay developed by us is simple, easy and rapid and thus can serve as an excellent alternative for determining the bacterial serum susceptibility.

11. Studies on the incidence of 3rd generation cephalosporins (3GC) resistance in Klebsiella pneumoniae

As many as 70% of the strains of K. pneumoniae were resistant to ceftazidime, and of these 40% were also resistant to cefuroxime. Gentamicin resistance was also detected in 50% and ciprofloxacin resistance in 11% of the ceftazidime resistant strains. Of the ceftazidime resistant strains as many as 70% had minimum inhibitory concentrations (MIC) ranging between 64 mg/ml to 256 mg/ml.

12. Detection of extended spectrum β- lactamases in clinical isolates of Klebsiella pneumoniae

By minimum inhibitory concentration (MIC) reduction test it was possible to detect extended spectrum β-lactamase (ESBL) production in 50 of the 68 strains screened. By another technique viz: double disk synergy test (DDST) it was shown that 70% the strains produced extended spectrum β lactamases. The two techniques showed good agreement in detecting ESBL production in K. pneumoniae clinical isolates tested. Of the 50 strains which were positive for ESBL production, as many as 10 strains had been categorised as sensitive to ceftazidime by the standard disk diffusion test interpreted according to the NCCLS standards. This observation has serious implications since, the strains categorised as sensitive by the routine in-vitro test could be ESBL producers and, hence, therapeutic failure with ceftazidime treatment could result in such cases.

13. Identification, isolation and characterisation of plasm ids encoding ESb lactamases in clinical isolates of K. pneumoniae

It was possible to demonstrate transfer of ceftazidime resistance by conjugation to a recipient strain of E. coli K12 L^- F^- Nal^r. In 3 transconjugants the minimum inhibitory concentration (MIC) of ceftazidime was as high as that of the donor K. pneumoniae strains. However, the MIC of ceftazidime in 5 other transconjugants was much less than that of the corresponding K. pneumoniae donor strains. Co-transfer of resistance to gentamicin as well as co-transfer of resistance to gentamicin, cefuroxime, ciprofloxacin along with ceftazidime resistance could be detected. The plasmid encoding ceftazidime resistance was identified and was characterised as a ~ 180 kb conjugative plasmid.

14. Development of a simple and rapid procedure for isolation of plasmid DNA from mucoid gram negative bacteria

A modified rapid method for extraction of plasmids from mucoid isolates was developed, standardised and tested. It proved to be extremely useful in detecting plasmid especially large ones in mucoid isolates of K. pneumoniae. This protocol eliminated the polysaccharide contamination and yielded plasmid DNA samples with high degree of purity (A₂₆₀/A₂₈₀ ratio 1.8 to 1.9). The plasmid bands were sharp, intense, with no background smearing. The modified protocol proved to be an excellent alternative to the conventional Kado and Liu's method especially for the detection and isolation of large sized (> 100 kb) plasmids in K. pneumoniae.

15. Plasmid encoded serum resistance in K. pneumoniae

Serum resistance encoding plasmids could be transferred from serum resistant K. pneumoniae to a highly serum sensitive recipient strain of E. coli. Plasmids of various sizes ranging from ~140 to ~183 kb were found to be associated with this phenotype. Serum resistance of transconjugants was found to be of a lower degree than that of the donor strains. This observation indicates that the serum resistance is mediated by more than one factor. Many of these plasmids were characterized as R-plasmids. To the best. of our knowledge this study is the first of its kind in the world in which serum resistance phenotype has been demonstrated to be plasmid encoded in Klebsiella pneumoniae.

16. To study the effect of acrylamide concentration on the SDS-PAGE protein patterns of K. pneumoniae

Polyacrylamide gels with different concentrations of acrylamide i.e. 10%, l2%, 13% and 15% were constituted. Protein extracts of a set of six clinical isolates of Klebsiella pneumoniae were run on each of these gels. The protein patterns obtained were subjected to computerised numerical analysis. The 12% acrylamide gel gave the best results as the protein bands were most sharply resolved and well separated, maximum number of bands (28-34) were resolved and there was maximum percentage similarity between the different protein patterns. It was thus concluded that 12% acrylamide gel is the most suitable for undertaking epidemiologic typing of K. pneumoniae isolates by whole-cell protein profile analysis.

17. To study the effect of storage of bacterial cultures on the SDS-PAGE protein patterns

Whole-cell protein patterns of six freshly isolated clinical isolates were obtained by SDS-PAGE. The protein patterns of these strains were obtained after storage as nutrient agar slants and peptone agar stabs for different periods of time viz. 2 months, 4 months and 6 months. It was found that bacterial culture storage (tested up to 6 months) did not affect the protein patterns of the isolates. Moreover, the nature of the culture medium (nutrient agar/peptone agar) used for maintaining the stock cultures had no effect on the protein patterns of K. pneumoniae isolates.

18. Epidemiologic typing of K. pneumoniae isolates by SDS-PAGE of whole-cell proteins

A total of 55 clinical isolates of Klebsiella pneumoniae collected from two hospitals [Sir Ganga Ram Hospital (SGRH) and Safdarjang Hospital (SH)] was subjected to typing-by SDS-PAGE of whole-cell proteins. The protein patterns of these isolates were subjected to computerised numerical analysis. The 29 isolates of SGRH could be divided into 14 protein types while 26 SH isolates could be ascribed to 21 protein types. Apart from a few small clusters, most of these isolates could be assigned unique protein types confirming that these isolates were epidemiologically unrelated. The SDS-P AGE technique had a discriminatory index of 0.97.

19. Characterization of Klebsiella pneumoniae isolated from nosocomial outbreaks of neonatal septicemia using SDS-PAGE whole-cell protein profile analysis

A total of 44 isolates of K. pneumoniae; 15 from the neonatal nursery ward of Sir Ganga Ram Hospital (SGRH), and 29 from the neonatal nursery ward of Safdarjang Hospital (SH) were typed by whole-cell protein profiling. The protein patterns were subjected to computerised numerical analysis. The protein patterns of all the 15 isolates of SGRH were 100% similar, indicating that only a single clone was responsible for the neonatal septicemia outbreak. The 29 isolates of SH could be divided into 4 protein types. Twenty-six isolates belonged to a single type while each of the remaining 3 isolates had a unique protein type. In SH also a single clone was responsible for the nosocomial outbreak though sporadic cases caused by other clones were also occurring. SDS-P AGE whole-cell protein profiling Was thus found to be an excellent epidemiologic typing technique for investigation of nosocomial outbreaks.

20. Evaluation of the effect of different concentrations of acrylamide on the DNA patterns

The whole-cell DNA patterns of clinical isolates of K. pneumoniae were resolved in polyacrylamide gels constituted with 5%, 7.5% and 10% acrylamide. The 5% acrylamide concentration was found to be the most optimum since the DNA bands were sharp and clearly resolved; DNA patterns had the maximum percentage similarity and results could be interpreted with the minimum number of DNA bands.

21. Epidemiologic typing of K. pneumoniae by SF-REA of whole-cell DNA

Fifty-five clinical isolates of Klebsiella pneumoniae, collected from two hospitals (SGRH and SH) over a period of 3 years, were typed by small fragment- restriction endonuclease analysis (SF-REA) of whole-cell DNA. The DNA patterns of these isolates were subjected to computerised numerical analysis for an objective comparison. The 29 isolates of SGRH were divided into. 25 DNA types giving a discriminatory index of 0.98. The 26 isolates of SH could be ascribed to 26 DNA types giving a discriminatory index of 1.0. The SF-REA of whole cell DNA was thus demonstrated to be an excellent typing technique with a very high power of strain discrimination.

22. Characterization of nosocomial isolates of Klebsiella pneumoniae using SF-REA of whole-cell DNA

Thirty six blood isolates of K. pneumoniae obtained from neonatal nursery wards of SGRH and SH were characterized, by small fragment-restriction endonuclease analysis (SF-REA) of whole-cell DNA. The DNA patterns were subjected to computerized numerical analysis. All the 15 GR. Isolates belonged to a single DNA type indicating that a single epidemic strain was responsible for these fifteen cases of neonatal septicemia. Similarly, the 21 SH isolates were of a single DNA type thereby confirming that all the neonatal septicemia cases were caused by a single clone. The epidemic strains of the two hospitals were however, different and did not resemble each other.