Potentiality of a new compound for in vitro differentiation between halophilic and non-halophilic vibrios.

Sensitivity of 21 halophilic vibrios and 16 clinical isolates of non-halophilic vibrios was determined against a new possible antivibrio agent, a pyrimidine analogue, 4, 6-dimethylpyrimidine -2-thiol (4,6-DMPT). It appeared to be a vibriocidal agent, having a mean MIC and MBC of 32 microg/ml for halophilic strains and 64 microg/ml for non-halophilic strains and an LD50 of 300 mg/Kg body weight of mice. Thus, 4,6-DMPT may help an in vitro distinction between halophilic and non-halophilic vibrios. Sensitivity of these strains was also studied with respect to pteridine, crystal violet and Tween 80 hydrolysis as further markers distinguishing between these 2 groups which could also be differentiated by their growth on TCBS or/and CLED media.

Leprosy bacillus--possibly the first chemoautotrophic human pathogen cultivated in vitro and characterised.

Leprosy bacillus (LB) and leprosy derived in vitro culture forms, the chemoautotrophic nocardioform (CAN) bacteria, showed an extremely close homology and identity with each other as regards a chemoautotrophic nutritional pattern, a nocardioform morphology, a weak acid-fastness coupled with Gram and Gomori's stain positivity, an exclusive mycolate and lipid profile, a phenolic glycolipid (PGL-I) and a highly sequestrated DNA characteristic, namely, a unique small size, a low G+C % mole, an exceptionally high gamma and UV radiation resistance, and a high thermal resistance. LB/CAN bacteria (CANb) gave positive signals for 36 kDa protein PCR, as well as, for 65 kDa epitope, and hybridisation with two or more probes and also by RFLP-analysis. Both LB/and CAN bacteria exhibited bacillary multiplication in the mouse footpads (MFP), nerve infiltration and evidences for local pathogenicity associated with pronounced systemic invasion. A highly reproducible mutilation model could be established which enabled a successful application of the postulates of Koch. The proof of their total identity was their anergic reactions in LL cases counterpoised against Mitsuda type strong nodular responses, mirroring the reactions of leprosy bacilli in TT cases, in accordance with the dictum of XIth International Leprosy Congress (1978). Thus, the chemoautotrophic nutritional requirements of LB, entirely unsuspected for a medically important pathogenic bacterium, having dimorphic (both bacillary and mycelial) characters with spores, mycelia and granules and unique pathogenicity of multilation manifested through the virulence factor, the enzyme collagenase, made LB or M leprae the highly enigmatic bacterium for so long.

Impaired function of neutrophils in uraemic patients.

BACKGROUND: Immunodeficiency explains the very high frequency of bacterial infections in patients with chronic renal failure (CRF), which leads to high mortality and morbidity, despite improved therapeutic interventions. Among several factors, the decreased functional capacity of phagocytic leucocytes appears to be responsible for the defective host defence mechanisms against infection in CRF. We evaluated both oxygen-dependent and oxygen-independent microbicidal activity of neutrophils isolated from uraemic patients. METHODS: Forty patients with CRF (20 with mild-to-moderate CRF and 20 with advanced CRF) along with 20 age- and sex-matched healthy controls were studied. The assessment of phagocytic capability, ability to produce superoxide (O2.-) anion and H2O2, myeloperoxidase and granule-specific hydrolytic enzymes such as acid phosphatase, cathepsin D and lysozyme activity of the patient's neutrophils were performed to study their bactericidal activity. RESULTS: The phagocytic index (PI) in the control group was found to be 50.38 (4.58). It was significantly reduced in both mild-to-moderate CRF and advanced CRF, as compared to controls. In mild-to-moderate and advanced CRF patients, O2.- production by resting polymorphonuclear neutrophils (PMN) was low. Also, on stimulation with PMA the O2.- production showed a relative reduction as compared to controls. H2O2 production by resting PMN from CRF patients was unaltered but on stimulation with PMA, the quantum of increase was significantly lower. A marked reduction in the level of intracellular myeloperoxidase activity in PMN was noted in CRF patients. Of the three intracellular lysosomal enzymes assayed, cathepsin D level was increased in the PMN of mild-to-moderate CRF patients; acid phosphatase level was elevated significantly in the PMN of both mild-to-moderate and advanced CRF patients. However, no change in lysozyme levels was observed. CONCLUSION: With increasing severity of uraemia, neutrophils from uraemic patients showed progressive impairment of phagocytic ability. Impairment of oxygen-dependent microbicidal mechanisms was indicated by a decrease in O2.- and H2O2 production. Increased activity of lysosomal enzymes such as cathepsin D and acid phosphatase suggest a state of neutrophil activation in uraemia. It is likely that the immunodeficiency state in uraemics is partly due to reduced bactericidal activity of the neutrophil cell population.

Validity of mechanism of gene transfer in the process called conjugation in bacteria.

We have attempted a new evaluation of the process of conjugation in bacteria, because of some basic dissimilarities observed between this and that of eukaryotes, or plants and animals. Reference donor and recipient strains, widely used to prove conjugation in bacteria, were chosen; addition of DNase during the conjugation process, led to an unexpected but highly reproducible increase in the transconjugant colony counts (TCC; ca. > or = 1 log), when compared with that of the controls without DNase. Transconjugants were also obtained when the same live donors were substituted with the UV-killed ones although the TCC was very low initially. Contrarily, donors treated with DNA-intercalating agents, e.g. acridine orange or ethidium bromide, resulted in a complete failure to produce transconjugants. There was a quantitative relationship between the DNase used on donors and levels of DNA sugars/nucleotides/DNA, which possibly resulted from interaction between the DNase and DNA being present/produced on the donor surface. This may be indicative of what may actually happen in the donor-recipient mixtures in the conjugation test proper, where the recipient DNase may activate a donor DNA production cycle. The evidences presented did not suggest that the donor DNA in the conjugation process is actually vestibuled through any intercellular conjugation passages, and is susceptible to the action of DNase or the intercalating dyes.

Potentiality of tricyclic compound thioridazine as an effective antibacterial and antiplasmid agent.

Thioridazine (Th), which is therapeutically used in psychiatric patients, was found to possess conspicuous antimicrobial activity when tested against 316 strains belonging to a number of Gram positive and Gram negative bacteria. Although Staphylococcus aureus, Vibrio chloerae and V. parahaemolyticus were found to be most sensitive, Th was highly bactericidal against S. aureus and bacteriostatic for vibrios and other Gram negative organisms. In the study of antiplasmid/curing effect of Th on twelve multiply antibiotic and Th resistant bacteria, it was observed that elimination of R plasmids was facilitated by choice of optimal concentration of Th. Significant elimination of single and combined antibiotic resistance occurred in E. coli and Shigella flexneri and not in S. aureus.

Antibacterial activity of the antiinflammatory agent diclofenac sodium.

Antimicrobial property of ten antiinflammatory drugs was tested with eleven sensitive bacteria belonging to both Gram positive and Gram negative types. Since most of the bacteria were moderate to highly sensitive to diclofenac (Dc), this compound was tested in vitro against 397 bacteria, most of which were inhibited by Dc at 50-100 micrograms/ml level. When tested in vivo, Dc at 1.5 and 3.0 micrograms/g body weight of a Swiss strain of white mice, could significantly protect the animals challenged with 50 MLD of Salmonella typhimurium NCTC 74. According to chi 2 test the in vivo data were highly significant (P < 0.001).

Nociception, antinociceptive potency of morphine in streptozotocin induced diabetic rats.

There are controversial reports on the effect of diabetes on the pain threshold. We used male Wistar rats to see the effect of streptozotocin induced diabetes on the tail flick, vocalisation and vocalisation after discharge responses. These represent the spinal, lower brain stem and hypothalamic responses respectively. The effect of morphine in these parameters was studied for both the control and diabetic group. In diabetic rats, the pain threshold was increased. However, this increase was not significant. Morphine produced significant analgesia after thirty minutes for tail flick and vocalisation responses and after fifteen minutes for after discharge in the control group. The antinociceptive effect of morphine was delayed and reduced for all three pain threshold confirming the antagonistic action of glucose on opiate receptors.

Immobilisation stress induced analgesia in diabetic rats.

Stress is known to produce analgesia. The pain threshold is altered in diabetes. We studied the effect of 1 hr of immobilisation stress on pain threshold in male Wistar rats. The same effect was tested in streptozotocin induced diabetic rats. The pain threshold of tail flick, vocalisation and vocalisation after discharge increased in the control group after the stress procedure. Significant analgesia was also obtained in diabetic rats, for flick and after discharge pain threshold. However the vocalisation threshold was not altered, probably due to the antagonistic action of glucose on opiate receptor at the level of brain stem.

Antimicrobial potentiality of a new beta-lactum antibiotic fosfomycin.

Antimicrobial action of penicillin and some of its derivatives including fosfomycin was studied with respect to 225 strains of Gram-positive and Gram negative bacteria. Fosfomycin was found to possess somewhat less activity against Staphylococcus aureus compared with other penicillins; however, it showed powerful activity towards Escherichia coli, Klebsiella spp. and Proteus mirabilis.

In vitro & in vivo experimental susceptibility of Mycobacterium marinum to augmentin.

The effect of augmentin alone and in combination with various beta-lactam antibiotics was studied against a pathogenic Mycobacterium, M. marinum. The in vitro studies did not reveal any additional advantage over that found with augmentin alone and this antibiotic seemed considerably inhibitory to M. marinum at < 1 microgram/ml concentration. In vivo, the effects of augmentin on experimentally produced lesions in the mouse foot pads (MFPs) showed a significant regression of the lesions, which was compatible with an early disappearance of M. marinum from the MFP, in contrast with those of the untreated, control animals.

Electron microscopic characteristics of actinomycetic agents having aetiological association with human leprosy and epizootic ulcerative syndrome of fish.

The transmission electron microscopic (TEM) studies of the human leprosy derived chemoautotrophic nocardio-form (CAN) bacteria and EUS derived CAN bacteria showed presence of double contoured cell-walls consisting of an electron transparent and a dense layer. The fibrillar structures on the surface of these CAN bacterial cells also suggested their similarity to the human tissue derived Mycobacterium leprae cells. These EM studies further revealed mycelial and coccoid bodies in all these bacteria as was observed originally.

Hypotensive effect of intracerebroventricular injection of norepinephrine and its modulation by alpha and beta adrenergic blockers in conscious rabbits.

The present study was designed to investigate the role of central adrenoceptors in the hypotensive effect of intracerebroventricular (ICV) injection of norepinephrine (NE) in conscious rabbits. Experiments were carried out on 19 adult rabbits (oryctolagus cuniculus) of either sex. A dose-dependent hypotensive response to ICV injection of NE was observed with no significant change in heart rate. The hypotensive response of NE was blocked 74.2 +/- 0.7% by yohimbine (alpha-2 adrenergic blocker), and 25.0 +/- 0.5% by metoprolol (beta-1 adrenergic blocker). NE response was not affected either by prazosin or butoxamine (alpha-1 and beta-2 adrenergic blockers respectively). The results suggest that the dose-dependent hypotensive response of ICV administered NE is mediated through alpha-2 and beta-1 central adrenoceptors.

Propagation of Mycobacterium lepraemurium on supplemented minimal medium and its experimental pathogenesis.

The splenic tissue of a mouse experimentally infected with M. lepraemurium (Hawaiian strain, M-65) and developing 'rat leprosy', yielded a pure culture of an acid - fast bacterium having all the characteristics of M. lepraemurium on mineral salt minimal medium supplemented with simple sources of C and N, e.g., NH4 -salts, liquid paraffin, urea, gelatin etc. This could be maintained, by serial passages in vitro with good growth. Its indefinite propagation with tissue - free washed, small inoculum on complex media including Ogawa medium was difficult, and its serial sub-culture was practically impossible. The in vitro isolate from supplemented minimal medium could produce pathological lesions in mice typical of rat leprosy.

Phage-mediated conjugation in staphylococci as a means of transfer of drug-resistance.

Penicillin resistance plasmid was transferred from Staphylococcus aureus B4 (PcrKms, donor) to S. aureus ML351 (PcsKmr, recipient) by co-cultivation of the donor with the recipient in nutrient broth with or without the modifying effects of CaCl2 or sodium dodecyl sulfate. It was found that the transfer of drug-resistance occurred maximally between 6 and 18 hr postinoculation; however, addition of DNase (200 micrograms/ml) could totally prevent such a transfer up to 6 hr and significantly reduce it thereafter. Cell-free filtrate of the donor culture when mixed with the recipient was ineffective in bringing about the transfer of Pcr.

A pathogenicity model for the agents of epizootic ulcerative syndrome (EUS) in fish.

Chemoautotrophic nocardioform (CAN) bacteria had been repeatedly isolated from fish with ulcerative disease syndromes (EUS) from the massive epizootics that had repeatedly occurred since 1988 in eastern India as the major or only pathogenic agent in the background of distinctive environmental and epizootic data. Since these isolates bear significant similarity to the human and rat leprosy bacilli, attempts had been made to demonstrate the pathogenicity of this fish pathogen in the "Swiss" strain of mice as a convenient model. The studies reveal that the fish CAN bacteria could produce pathogenic effects in mice similar to that of the rat leprosy bacillus.

Plasmid-determined degradative metabolism and halophilism of Vibrio parahaemolyticus.

A set of 25 Kanagawa(+) and Kanagawa(-) strains of V. parahaemolyticus was studied for their ability to degrade hydrocarbons in minimal media. All strains gave positive results with respect to crystal violet (CV), methyl violet, liquid paraffin, benzene, naphthalene and phenol. The CV double ring (CVDR) response had earlier appeared to be a significant pathogenic marker [Chakrabarti et al, Indian J Med Res, 85 (1987) 508]. The CVDR response was found also to be a biodegradative marker, and correlates perfectly well with polymyxin resistance and low level of halophilism (4% NaCl). All these markers (characters) were found to be controlled by a single plasmid in the wild type. Elimination of the plasmid, as confirmed by gel electrophoresis studies, resulted in loss of CVDR response, polymyxin resistance, and acquisition of halophilism at a higher level (> 7%). The massive drainage of industrial effluents, rich in hydrocarbons, in the estuarine areas in many countries might have altered the ecosystem in favour of V. parahaemolyticus and its emergence as a new biodegradative and enterotoxigenic pathogen, contaminating fauna and flora in the littoral sea regions, with increased changes of communicability to humans.