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 sSalmonella 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.

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

Background & objectives: Data on extended-spectrum β-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-fi eld 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 confi rmed to produce ESBLs. PCR and sequencing revealed the responsible gene to be blaCTX-M-15. blaTEM and blaSHV were absent. PFGE indicated that fi ve of seven isolates 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 & conclusions: Our fi ndings 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 defi ne the molecular epidemiology of ESBLproducing K. pneumoniae and its clinical implications.

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.

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.

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.

Application of ATP assay for in vitro drug screening testing against human derived M. leprae.

In this study, the ATP content of M. leprae exposed to various antimicrobial agents has been measured to evaluate its usefulness in drug sensitivity screening. Purified M. leprae suspensions from human biopsies have been incubated at 30 degrees C in a modified Dubos medium in the presence of different concentrations of various drugs viz., Rifampicin, Ethionamide, Ethambutol, Cycloserine, Dapsone, Clofazimine, Erythromycin and Tetracycline. ATP levels were estimated at 0, 7 days, 14 days of incubation by the procedures modified and standardised at this laboratory. ATP decay was accelerated by ethionamide, rifampicin, clofazimine, dapsone, erythromycin and to a lesser extent by cycloserine, whereas ethambutol and tetracycline did not have any significant effect. The rate of decay depended on the concentrations of these drugs. ATP assay promises to be a useful system for in vitro drug sensitivity screening against M. leprae isolated from patients.

Metabolic studies on mycobacteria. V. A preliminary report on the ATP synthesis by mycobacteria including M. leprae by using different substrates.

By deletion and addition of various substrates in Sauton's and Dubos media, an experimental system has been standardised in which the role of various nutrients in the energy synthesis of mycobacteria can be determined. By using this system with cultivable mycobacteria it was observed that glycerol and asparagine are the important ingredients for ATP synthesis by mycobacteria. Glucose further enhanced the ATP synthesis and growth of these mycobacteria. In the media containing asparagine or glycerol, there was marginal increase in the ATP in the M. leprae suspensions initially but this was not sustained and there was no progressive increase in biomass or multiplication. When M. leprae was incubated in the media from which both these substrates were deleted, there was progressive decline in ATP levels right from the beginning. From these preliminary results, it appears that asparagine and glycerol may be useful as substrates for ATP synthesis by M. leprae and need to be investigated further. In depth studies are necessary to find out the factors which results in the inability of M. leprae to utilise these and other substrates in a substrained manner for its multiplication and growth in artificial media.

Metabolic studies on mycobacteria. III. Demonstration of key enzymes of TCA cycle in M. leprae.

Cell free extracts of armadillo derived M. leprae, M. phlei, M. smegmatis and normal armadillo liver were analysed for the two key enzymes of TCA cycle. Aconitase activity was assayed in the presence of inhibitor fluorocitrate and it was observed that cell free extracts from cultivable mycobacteria as well as aramadillo derived M. leprae had this enzyme activity and 66-82% of this activity was inhibited by 0.1 mM fluorocitrate. 74% of M. leprae derived enzyme activity was inhibited by fluorocitrate in contrast with armadillo derived enzyme which was only 29% inhibited by fluorocitrate. PAGE separation of cell free extracts and staining for Isocitrate dehydrogenase (ICD) activity showed that an additional bond of ICD activity was demonstrable in the cell free extracts of armadillo derived M. leprae and this was NADP dependent. The mobility (ef) of this band of activity was in the same range as ICD from cultivable mycobacteria and much lower than ICD from normal armadillo liver. From this study and from the previously reported work, it is concluded that like other mycobacteria TCA cycle is operative in M. leprae.

Metabolic studies on mycobacteria. IV. Assay of isocitrate lyase and malate synthase activity in M. leprae.

Cell free extracts from M. tuberculosis H37 Rv, M. smegmatis armadillo derived M. leprae and normal armadillo liver homogenates were assayed for the presence of isocitrate lyase and malate synthase activity. It was observed that significant amount of isocitrate lyase and malate synthase activity was present in M. tuberculosis H37 Rv, M. smegmatis and armadillo derived M. leprae. No such activity was demonstrable in cell free extracts of normal armadillo liver. It is concluded that M. leprae like other mycobacteria has the capability to metabolise via glyoxylate bypass of TCA cycle. These findings may be relevant for understanding the energy metabolism of M. leprae under stress conditions and possibly the 'persister' stage.

Metabolic studies on mycobacteria-I. Demonstration of key enzymes of glycolysis and tricarboxylic acid cycle on polyacrylamide gels.

Polyacrylamide gel electrophoresis (PAGE) technique was standardised to demonstrate some key enzymes of glycolysis, hexose mono phosphate (HMP) pathway and tricarboxylic acid cycle in slow growing mycobacteria (M. avium. M. gastri) as well as in fast growing mycobacteria (M. vaccae, M. phlei). The enzymes studied were lactate dehydrogenase (LDH) glucose-6-phosphate dehydrogenase (G6PD), aconitase, isocitrate dehydrogenase (ICD), succinic dehydrogenase (SDH), fumerase and malate dehydrogenase (MDH). All the three pathways were found to be operative in slow as well as fast growing mycobacteria. Using this technique M. leprae specific MDH activity was demonstrated in the cell free extract of M. leprae. It's (MDH) electrophoretic mobility on gels lies in the range shown by other mycobacterial species studied and was distinct from that of host MDH. It appears that PAGE offers a useful tool for metabolic characterization of M. leprae using infected tissues.

Metabolic studies on mycobacteria--II. Glyoxylate by-pass (TCA cycle) enzymes of slow and fast growing mycobacteria.

Glyoxylate by-pass of tricarboxylic acid cycle (TCA) comes into prominence during survival of microorganisms under oxygen limitations and study of these enzymes may contribute to understanding of physiology of 'persisters' in various mycobacterial diseases. The enzymes of glyoxylate by-pass have been assayed in the extracts of various mycobacterial species, namely, M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. flavescens, M. vaccae, M. smegmatis and Mycobacteria strain w (M.w.). M.w. has been included because of its close antigenic resemblance to M. leprae. It has been found that all of the above investigated species possess isocitrate lyase and malate synthetase, the key enzymes of glyoxylate by-pass. The presence of the enzymes is being reported for the first time in M. flavescens, M. vaccae and M.w. whereas these were earlier shown to be present in M. tuberculosis and M. smegmatis. It was also demonstrated in M.w. where acetate alone could not serve as sole source of carbon, but in the presence of glycerol stimulates the activity of glyoxylate pathway enzymes. The importance of these findings has been discussed.

Isoenzymes of mycobacteria. II. Relevance of LDH zymograms in taxonomy and identification.

The cell free extracts of mycobacteria namely M. kansasii M. avium, M. tuberculosis, BCG (Glaxo), M. gastri, M. phlei, M. smegmatis, M. vaccae, M. strain w., M. scrofulaceum, M. gordonae, M. nonchromogenicum E. coli, Staph, aureus, and M. leprae infected skin have been electrophoresed and stained for LDH activity. Normal skin tissue was also taken as control. It was found that all the organisms tested showed distinct species specific LDH isoenzyme patterns. There was no extra band but an aberrant zone of LDH activity was seen in M. leprae infected human skin in comparison to LDH isoenzymes from normal skin. No strain variations was found among the different strains of species investigated. Results described in the present paper indicate that LDH isoenzyme patterns of mycobacteria could be of identification value at species level.

Isoenzyme patterns of mycobacteria. I. Factors influencing LDH isoenzymes of mycobacteria.

Cell free extracts of a fast growing mycobacterium (M. phlei) and a slow growing mycobacterium (M. tuberculosis H37Ra) were analysed for lactate dehydrogenase (LDH) isoenzymes under different experimental conditions. It was observed that growth of M. phlei when taken from Lowenstein Jensen (LJ) as well as Sauton's medium showed identical band but for (M. tuberculosis H37Ra the number of bands observed were less when grown on LJ-medium. There was no difference in LDH isoenzyme patterns when the mycobacteria were incubated at 30 degrees C and 37 degrees C and under different pH conditions (6.2-8.2). Actively growing cultures of both the species showed distinct LDH isoenzyme patterns whereas the activity and bands became indistinct in old cultures. The LDH bands from lyophilized growth studied resembled to those of fresh growth. The treatment of growth with 1M NaOH for one hour resulted in marked diminution of LDH activity. Sonication with wet growth weight of 0.5 gm per ml of distilled water was found to give clearer bands as compared to phosphate buffer. No loss of LDH isoenzymes activity was noticed after storing the extracts at -80 degrees C for one month, treating to 58 degrees C for one hour or freezing and thawing for 2 times whereas these isoenzymes were quite unstable at other storage temperatures. Increasing the staining time was found helpful in getting clearer bands when activity was low. It is concluded that the factors studied have important bearing on LDH isoenzyme patterns of mycobacteria and must be kept in mind while studying the LDH zymograms for any taxonomic identification of mycobacteria or for studying the metabolic role. These are important both for sensitivity and reproducibility of LDH zymograms.