Copper, Zinc-Superoxide Dismutase from Clinically Isolated Escherichia coli: Cloning, Analysis of sodC and Its Possible Role in Pathogenicity.

Superoxide dismutase has been discovered within the periplasm of several Gram-negative pathogens. We studied the Cu,Zn-SOD enzyme in Escherichia coli isolated from clinical samples (stool samples) collected from patients suffering from diarrhea. Antibiogram studies of the isolates were carried out to determine the sensitive and resistant strains. The metal co-factor present in the enzyme was confirmed by running samples in native gels and inhibiting with 2 mM potassium cyanide. A 519 bp sodC gene was amplified from resistant and sensitive strains of Escherichia coli. Cloning and sequencing of the sodC gene indicated variation in the protein and amino acid sequences of sensitive and resistant isolates. The presence of sodC in highly resistant Escherichia coli isolates from diarrheal patients indicates that sodC may play role in enhancing the pathogenicity by protecting cells from exogenous sources of superoxide, such as the oxidative burst of phagocytes. The presence of SodC could be one of the factors for bacterial virulence.

Degradation of h-acid by free and immobilized cells of Alcaligenes latus

Alcaligenes latus, isolated from industrial effluent, was able to grow in mineral salts medium with 50 ppm (0.15 mM) of H-acid as a sole source of carbon. Immobilization of Alcaligenes latus in Ca-alginate and polyurethane foam resulted in cells embedded in the matrices. When free cells and immobilized cells were used for biodegradation studies at concentration ranging from 100 ppm (0.3 mM) to 500 ppm (1.15 mM) degradation rate was enhanced with immobilized cells. Cells immobilized in polyurethane foam showed 100 percent degradation up to 350 ppm (1.05 mM) and 57 percent degradation at 500 ppm (1.5 mM). Degradation rate of Ca-alginate immobilized cells was less as compared to that of polyurethane foam immobilized cells. With Ca-alginate immobilized cells 100 percent degradation was recorded up to 200 ppm (0.6 mM) of H-acid and only 33 percent degradation was recorded at 500 ppm (1.5 mM) of H-acid. Spectral analysis of the products after H-acid utilization showed that the spent medium did not contain any aromatic compounds indicating H-acid degradation by A. latus.

Detection, amplification & sequence homology of sodC in clinical isolates of Salmonella sp.

BACKGROUND & OBJECTIVES: Periplasmic copper and zinc superoxide dismutase (Cu,Zn-SOD or SodC) is an important component of the antioxidant shield which protects bacteria from the phagocytic oxidative burst. Cu,Zn-SODs protect Gram-negative bacteria against oxygen damage which have also been shown to contribute to the pathogenicity of these bacterial species. We report the presence of SodC in drug resistant Salmonella sp. isolated from patients suffering from enteric fever. Further sodC was amplified, cloned into Escherichia coli and the nucleotide sequence and amino acid sequence homology were compared with the standard strain salmonella Typhimurium 14028. METHODS: Salmonella enterica serovar Typhi (S. Typhi) and Salmonella enterica serovar Paratyphi (S. Paratyphi) were isolated and identified from blood samples of the patients. The isolates were screened for the presence of Cu, Zn-SOD by PAGE using KCN as inhibitor of Cu,Zn-SOD. The gene (sodC) was amplified by PCR, cloned and sequenced. The nucleotide and amino acid sequences of sodC were compared using CLUSTAL X. RESULTS: SodC was detected in 35 per cent of the Salmonella isolates. Amplification of the genomic DNA of S. Typhi and S. Paratyphi with sodC specific primers resulted in 519 and 515 bp amplicons respectively. Single mutational difference at position 489 was observed between the sodC of S. Typhi and S. Paratyphi while they differed at 6 positions with the sodC of S. Typhimurium 14028. The SodC amino acid sequences of the two isolates were homologous but 3 amino acid difference was observed with that of standard strain S. Typhimurium 14028. INTERPRETATION & CONCLUSIONS: The presence of SodC in pathogenic bacteria could be a novel candidate as phylogenetic marker.

Degradation of h-acid by free and immobilized cells of Alcaligenes latus.

Alcaligenes latus, isolated from industrial effluent, was able to grow in mineral salts medium with 50 ppm (0.15 mM) of H-acid as a sole source of carbon. Immobilization of Alcaligenes latus in Ca-alginate and polyurethane foam resulted in cells embedded in the matrices. When free cells and immobilized cells were used for biodegradation studies at concentration ranging from 100 ppm (0.3 mM) to 500 ppm (1.15 mM) degradation rate was enhanced with immobilized cells. Cells immobilized in polyurethane foam showed 100% degradation up to 350 ppm (1.05 mM) and 57% degradation at 500 ppm (1.5 mM). Degradation rate of Ca-alginate immobilized cells was less as compared to that of polyurethane foam immobilized cells. With Ca-alginate immobilized cells 100% degradation was recorded up to 200 ppm (0.6 mM) of H-acid and only 33% degradation was recorded at 500 ppm (1.5 mM) of H-acid. Spectral analysis of the products after H-acid utilization showed that the spent medium did not contain any aromatic compounds indicating H-acid degradation by A. latus.

Faecal carriage of CTX-M-15-producing Klebsiella pneumoniae in patients with acute gastroenteritis.

BACKGROUND & OBJECTIVE: Data on extended-spectrum beta-lactamases (ESBLs) produced by Gram-negative bacteria including Klebsiella pneumoniae especially molecular types of ESBL genes from India are limited. The present study was conducted to investigate the carriage and ESBL contents of multidrug-resistant K. pneumoniae recovered from patients with gastroenteritis in a rural village in southern India. METHODS: Nine K. pneumoniae isolates obtained from 45 stool samples from patients with gastroenteritis from one rural and two urban sites, in southern India were included in the study. Antibiotic susceptibility testing, PCR analysis and sequencing were conducted to characterize the ESBL genes. Clonal relatedness was assessed by pulsed-field gel electrophoresis (PFGE). RESULTS: All the isolates were found to be resistant to at least one of the third generation cephalosporins tested. All the study isolates were confirmed to produce ESBLs. PCR and sequencing revealed the responsible gene to be bla(CTX-M-15). bla(TEM) and bla(SHV) were absent. PFGE indicated that fi ve of seven isolates from villagers were genetically closely related, and in turn were related to isolates from patients in two urban areas in this region. INTERPRETATION & CONCLUSION: Our findings showed that genetically-related isolates of K. pneumoniae producing CTX-M-15 were present in multiple areas in southern India. Larger studies need to be done in various geographical regions of the country to better define the molecular epidemiology of ESBL-producing K. pneumoniae and its clinical implications.

Carriage of capsulated strains of Staphylococcus aureus: a population-based study performed in Gulbarga, South India.

Staphylococcus aureus is a common human pathogen in community- and hospital-acquired infections and its capsule is involved in pathogenesis. We report here the identification of type-5 and type-8 capsular antigens of S. aureus and the prevalence of such strains among volunteers in various age and population groups from different locations in India. S. aureus carriage rates varied between 18 and 50% with the highest values among university students and the lowest in schoolchildren, aged 6-20 years. There was no difference in carriage rates for males vs. females (P=0.415) or in the socioeconomic status of carriers vs. non-carriers or age dependence. Among the carriage isolates 21% were type-5, 52% were type-8 and the remaining 27% were non-typable. Among invasive isolates these percentages were 6, 64 and 30 respectively. This implies that type-5 strains may be less invasive than type-8 strains (P=0.0015).

Effect of trifluoperazine on in vitro ATP synthesis by Mycobacterium leprae.

The effect of trifluoperazine (TFP), a calmodulin antagonist, was investigated on in vitro ATP levels of human derived Mycobacterium leprae. M. leprae were obtained from biopsies from multi-bacillary forms of leprosy and were incubated in a modified Dubos medium system which supports limited in vitro synthesis of M. leprae. This incubation was carried out in the absence and presence of different concentrations of trifluoperazine. Samples for estimation of bacillary ATP levels were taken at day 0 and at 14 days of incubation. TFP inhibited ATP levels in M. leprae and this inhibitory effect was marginal at 2.5 microg ml(-1) (35% inhibition), highly significant at 5 microg ml(-1) (87% inhibition) and almost total at 10 microg ml(-1) (98.5% inhibition). This compound appears to have potential as an anti-leprotic drug and also as a broad spectrum anti-mycobacterial agent in view of its anti-tubercular activity reported earlier.

Viability determination of M.leprae: comparison of normal mouse foot pad and bacillary ATP bioluminescence assay.

Correlation between viability assessment by mouse foot pad and ATP bioluminescence was studied in biopsy specimens from multibacillary leprosy cases. Biopsies were processed for inoculation into mouse foot pad and estimation of bacillary ATP levels by bioluminescent assay by earlier established procedures. ATP content as pg/million bacilli was estimated and correlation was assessed with growth in the mouse foot pad. It was observed that when the ATP content was > 36 pg/million bacterial cells, (> 1% probable viables) there was growth in the mouse foot pad from all the specimens. Similar results were observed when the ATP content was in the range of 3.6 to 35.99 pg/million cells (0.1 to 1% probable viables). The positivity rates in the mouse foot pad decreased when the ATP content decreased further. No positive growth in the specimens below 0.04 pg/million bacilli (< 0.001% viable organisms) was observed. These findings show an overall correlation between viability assessed by mouse foot pad and ATP bioluminescence. These observations validate the concept of ATP content of viable unit of M.leprae being in the order of 10(-15) g/live cell which is in the same order of magnitude as a colony forming unit of cultivable mycobacteria.

Detection of M. leprae by gene amplification; combined ethidium-bromide staining and probe hybridization.

Biopsy and skin-scraping specimens from 130 leprosy cases across the disease spectrum (56 TT/BT/I, 73 BB/BL/LL, and 1 neuritic case) and 50 healthy contacts were studied to assess the application of gene amplification. The nucleic acids from these clinical specimens were extracted by an integrated freeze-thawing--optimized lysozyme-/proteinase-k treatment-purification and fractionation procedure. The nucleic acids from cultured organisms were isolated by the stepwise procedure earlier standardized at this laboratory. Gene amplification for a 360-bp fragment of the 18-kDa protein gene was carried out using primer and the procedure described by its developers, and a 360-bp fragment on Southern blot was taken as the yardstick of positivity. The polymerase chain reaction product was analyzed by electrophoresis, ethidium-bromide (EB) staining, and blot (B) hybridization. Overall sensitivity ranged from 71% in specimens with undetectable acid-fast organisms to 100% in specimens with demonstrable acid-fast bacilli. A positivity of 73% in TT/BT/I specimens and 93% in BB/BL/LL specimens was observed. Four combinations were discerned: EB+, B+ (71%); EB-suspicious, B+ (14%); EB-, B+ (3%) and EB-, B- (12%). By combining the blot hybridization with EB staining, the sensitivity could be significantly improved as compared to EB staining alone. The test was found to be absolutely specific by the absence of any false positivity in control specimens as well as with purified DNAs from mycobacterial as well as non-mycobacterial organisms, grown from these specimens. It is recommended that for optimum sensitivity and specificity both EB staining and blot hybridization should be done.

Determination of mycobacterial phylogeny on the basis of immunological relatedness of superoxide dismutases.

Sixteen strains of cultivable mycobacteria were grown in Sauton's medium, and Mycobacterium leprae was purified from armadillo liver. Cell extracts were prepared from log-phase growths of each of the cultivable mycobacterial strains. Superoxide dismutase (SOD) enzyme was purified from all cultivable mycobacterial strains included in the study, and antibodies against purified SOD enzyme were raised in rabbits. Immunological distances (ImDs) between these anti-SOD antibodies and SOD antigens were determined by a previously described immunoprecipitation method and by a recently developed enzyme-linked immunosorbent assay (ELISA) technique. The reciprocal ImDs among mycobacterial strains were constant, reproducible and consistent by these two methods. An evolutionary tree was constructed on the basis of estimated ImDs. Except for M. duvalii and M. terrae, slowly and rapidly growing mycobacterial species appeared to be separately grouped by this analysis. Rapid growers clustered into a group which is near that of some slow-growing mycobacteria. M. avium falls almost in the middle of the evolutionary tree and the position of M. leprae was found to be between those of M. avium and M. bovis BCG. Measurement of immunological relatedness of SODs provides an alternative system with which to study the taxonomical relatedness among mycobacteria.

Development of a SOD ELISA to determine the immunological relatedness among mycobacteria.

This study reports on the standardization of an enzyme-linked immunosorbent assay (ELISA) system for the measurement of immunological distances (ImDs) of the superoxide dismutase (SOD) molecule among the cultivable mycobacteria, namely, Mycobacterium vaccae, M. phlei, M. smegmatis, M. avium, M. scrofulaceum, M. tuberculosis H37Ra, M. tuberculosis H37Rv, and M. bovis BCG, and M. leprae. SODs from cultivable mycobacteria were purified, antibodies were raised against these molecules, and ImDs between these anti-SOD antibodies and antigen (SODs) were determined by an immunoprecipitation technique standardized earlier and by the ELISA technique developed in this study. The ELISA system developed in this study showed higher sensitivity and consistent and reproducible ImDs among various mycobacteria, including pathogens such as M. tuberculosis, M. leprae and M. avium. These values were comparable with the values derived by the immunoprecipitation technique. Our ELISA technique appears to be a sensitive and rapidly reproducible method with the additional advantage of the stability of reagents, and holds promise in the taxonomy as well as in the development of diagnostics for leprosy and other mycobacterial infections.

Studies on microbial aerobic flora of skin in leprosy patients.

This study reports the isolation and identification of aerobic organisms from biopsies/slit-skin smears/scrapings from 129 leprosy patients and 50 healthy controls. These include 56 paucibacillary (PB) and 73 multibacillary (MB) cases. Thirty-six isolates from the specimens from 21 patients and 15 healthy controls were grown. The non-mycobacterial isolates from clinically PB leprosy (TT/BT/I) patients were: (1) Gram-positive cocci: Staphylococcus aureus(1), Staphylococcus albus(1); (b) Gram-positive bacilli: Bacillus subtilis(1), Corynebacterium xerosis(1); (c) Gram-negative bacilli: Escherichia coli(1), Proteus mirabilis(2), Klebsiella pneumoniae(1) and Pseudomonas aeruginosa(1). The isolates from clinically MB leprosy (BB/BL/LL) patients were: (a) Gram-positive cocci: Micrococci(1), Staphylococcus aureus(1) and Staphylococcus albus(1); (b) Gram-positive bacilli: Corynebacterium xerosis(1); Corynebacterium hofmanni(1) and Bacillus cereus(1). (c) Gram-negative bacilli: Escherichia coli(2), Klebsiella pneumoniae(1) and Proteus mirabilis(2). The specimens from healthy controls yielded similar organisms. These were (a) Gram-positive cocci: Staphylococcus albus(2), Staphylococcus aureus(2) and Micrococci(2); (b) Gram-positive bacilli: Corynebacterium xerosis(1), Bacillus subtilis(2), Corynebacterium hofmanni(1) and Bacillus cereus(1); (c) Gram-negative bacilli: Escherichia coli(3), Proteus vulgaris(1) and Proteus mirabilis(1). While these results show no significant differences in the species types of non-mycobacterial aerobic organisms isolated from healthy skin and PB/MB types of leprosy, these isolates need to be characterized by immunological/molecular methods to find out subtypes if any.

Isolation and characterization of cultivable mycobacteria from leprosy skin.

Attempts were made to isolate cultivable mycobacteria from 129 biopsies/slit-skin scrapings from the skin of leprosy patients (73 multibacillary-BB/BL/LL and 56 paucibacillary-TT/BT/I) as well as 50 healthy controls. Among the 19 isolates obtained, 17 were from specimens from leprosy cases whereas two were from healthy controls. 14 of the 17 isolates were from multibacillary cases and three were from paucibacillary patients. The mycobacteria isolated were: M.scrofulaceum (4 = all LL cases); M.avium (3 = 2 from LL cases and 1 from healthy control); M.avium-intracellulare complex (1 LL); M.gordonae (2 = 1 from BT and BB each); M.flavescens (1 BL); M.smegmatis (2 = both LL); M.phlei (4 = 1 LL, 1 BL, 1 BT and 1 healthy control); M.fortuitum (1 BL); and M.chelonei (1 BT relapse). The results of this study suggest a preferential colonization of skin of lepromatous leprosy cases by M.scrofulaceum and M.avium. As such isolates have been reported by the investigators from other parts of the world, independent confirmation of such trends in Indian patients is significant and casual relationship (if any) between such colonization and development of lepromatous disease merits further investigation.

Treatment of bacilliferous BL/LL cases with combined chemotherapy and immunotherapy.

Thirty-six, untreated borderline lepromatous/lepromatous (BL/LL) leprosy patients with an initial bacterial index (BI) of 4+ to 6+ were serially allocated to three treatment groups. Group I patients received a slightly modified WHO regimen (rifampin once a month, clofazimine and dapsone daily) and BCG intradermally (i.d.) (0.1 mg/per dose). Group II patients were administered the same MDT and Mycobacterium w (2 x 10(8)) killed bacilli/dose i.d., and Group III received the same MDT with 0.1 ml of distilled water i.d. Vaccination was repeated every 6 months. Biopsies were taken from the local site of vaccination and from a distant site, i.e., the back. The progress was monitored periodically by clinical, histopathological and bacterial (BI, mouse foot pad, ATP) parameters. Twenty-five patients had completed a follow up of more than 2 years. These included: 7 in Group I, 10 in Group II, and 8 in Group III. One patient of the MDT + BCG group who was progressing well dropped out after 28 months. In cases on combined chemotherapy and immunotherapy, no viable bacilli were demonstrable by mouse foot pad and ATP measurement after 6 months (at 12 months or afterward). However, in come of the control cases on MDT alone, viable bacilli could be detected even up to 18 months (by mouse foot pad) and 2 years (by ATP estimation). With 36 months of treatment, the mean BI decreased from 4.64+ to 1.66+ in the group on MDT alone (controls), 4.9+ to 0.08+ in the MDT + BCG group, and 4.75+ to 0 in the MDT+Mycobacterium w group. Compared with the MDT and MDT + BCG groups, the fall in the BI was significantly more in the MDT + Mycobacterium w group at 12, 18, and 24 months. While all of the cases in the Mycobacterium w groups became smear negative by 36 months, it took 42 months for all of the BCG group to achieve negativity. Immunotherapy appears to have a significant effect on the killing and clearance of bacilli and should be considered as an adjunct to chemotherapy, especially in bacilliferous lepromatous cases.

Correlation between inhibitory effect of quinolones and mycolic acid metabolism in mycobacteria.

Mycolic acids are important components having a significant role in maintaining the rigidity of mycobacterial cell wall. They could also be the barrier for penetration of certain drugs into the bacterial cell. A novel in vitro model system was established for assessing the effect of Ciproflaxacin on mycolic acid metabolism in pathogenic mycobacteria M. Kansasii (which has similar mycolic acid pattern to that from M. leprae) and the effect of norfloxacin in M. intracellulare. These test mycobacteria were exposed in their midlogarithmic phase of growth to 0.5, 1, 2, 3, 4, 5 and 6 micrograms ml of ciprofloxacin and norfloxacin respectively for 1, 2 and 24 hours. Ciprofloxacin completely inhibited the synthesis of mycolates in M. kansasii at 3, 4 and 5 micrograms/ml; whereas norfloxacin exhibited its maximum inhibitory action on mycolic acids in M. intracellulare at 6 micrograms/ml for all the durations of exposure. Inhibition of mycolates directly correlated with bacterial viability which was estimated by colony forming units. The effect of quinolones on mycolic acid metabolism appears to be direct and not secondary to DNA gyrase. The results obtained from this study and our previous findings show that mycolic acid metabolism is affected by various groups of drugs, whose primary sites of activity may be different. The findings of the present study may have significant therapeutic implications in leprosy and other mycobacterial diseases.

Rapid characterization of Mycobacterium fortuitum-chelonei complex by restriction fragment length polymorphism of ribosomal RNA genes.

Using labelled, gamma-32P rRNA of mycobacteria as a probe restriction fragment length polymorphism (RFLP) of rRNA genes of strains belonging to the Mycobacterium fortuitum-chelonei complex was analysed. Each DNA sample was cleaved with EcoRI restriction endonuclease, the fragments were separated by agarose gel electrophoresis and transferred to nitrocellulose membrane. Fragments of DNA containing rRNA genes were identified by hybridization with gamma-32P-labelled rRNA. Patterns were found to be species specific and both the species were distinguishable from each other. Results indicate that this approach can be used for rapid genomic characterization of the Mycobacterium fortuitum-chelonei complex.

Effect of chemotherapy on viability of Mycobacterium leprae as determined by ATP content, morphological index and FDA-EB fluorescent staining.

Viable bacterial populations were estimated in bacilli purified from 105 biopsies from 40 untreated and 65 multibacillary leprosy patients treated with multidrug therapy (MDT) for varying periods. The bacilli were purified and viability was determined by ATP content, morphological index (MI), and fluorescein diacetate-ethidium bromide (FDA-EB) staining. Viable populations were calculated, taking 3.58 x 10(-15) g/solid bacillus as the mean ATP content of a viable unit of Mycobacterium leprae. The proportion of viable bacilli was also estimated in the same specimens using solid-staining (MI) and green-staining bacilli by the FDA-EB method. In the untreated cases, the positive viability by ATP assay was 100%, 92% by MI, and 100% by FDA-EB. ATP content per solid bacillus was relatively constant, which was not the case with ATP content per green-staining bacillus. While the MI was zero in all cases, viable bacilli could still be detected by ATP estimations in 5 of the 32 (16%) patients after 2 years of MDT and in 1 of the 20 (5%) patients after 3 years of MDT. No viable bacilli could be detected even by this method beyond 3 years of MDT. On the other hand, green-staining bacilli were demonstrable in 7/32 (22%) of cases after 2 years of MDT, 2/20 (10%) after 3 years of MDT, and 1/13 (8%) after more than 3 years of treatment, indicating that the FDA-EB staining and ATP assay did not detect the same populations. A determination of the ATP content of M. leprae could be used as a reliable and sensitive tool for determining viability of the bacilli.