[Development of a method to determine the minimum inhibitory concentration in reference strains from leptospiras]. / Desarrollo de un método para determinar la concentración mínima inhibitoria en cepas de referencias de leptospiras.

A method to determine the minimum inhibitory concentration for leptospiras was developed, since there is not a standard method to measure it at the international level. Reference strains from the pathogenic complex L. interrogans and L. biflexa were used against penicillin, cyprofloxacine, chloramphenicol, rifampicine and tetracycline. The minimum inhibitory concentration was defined as the lowest concentration of antibiotic where it was observed the inhibition of the bacterial mobility by direct examination in dark field. Ranges for penicillin were from 0.095 to 152 microg/mL, for tetracycline from 0.156 to 3.13 microg/mL, for chloramphenicol, from 0.08 to 12.52 microg/mL, for rifampicine from 0.08 to 1.56 microg/mL, and for cyprofloxacine from 0.15 to 2.4 microg/mL. The antibiotics that showed the lowest values were cyprofloxacine, rifampicine and tetracycline, whereas the most elevated value was obtained against chloramphenicol and penicillin. The strains from the serogroups circulating more frequently in Cuba were used in this research. This study will allow in a near future to determine the antimicrobial susceptibility in autochthonous strains isolated from patients with Leptospirosis at the national level.

Studies on the effect of antibiotic substances on leptospires and their cultivation from material with a high bacterial count.

Leptospira species are difficult to isolate from sperm specimens because rapid growth of the contaminant flora will kill the pathogen. The resistance of 5 Leptospira strains to 5 different antibiotics was examined with a view to an inhibition of such contaminant growth. Neomycin (10, 20, 30 mg/l), vancomycin (5, 8, 10 mg/l), nalidixic acid (50, 75, 100 mg/l), streptomycin (5, 8, 10 mg/l) and chloramphenicol (5, 10, 20 mg/l) were added separately to Korthof's culture medium containing rabbit serum. The comparative growth rates of the leptospires were evaluated. Against the control medium, all 5 antibiotics were found to have an adverse influence on the multiplication phase. In conformity with literature data, vancomycin (10 mg/l) and nalidixic acid (50 mg/l) were found to have the lowest effect. In the cases of streptomycin and chloramphenicol, there was a high reduction of the leptospiral count and even a complete lack of multiplication. A combination of vancomycin (10 mg2l) and nalidixic acid (50 mg/l) was used for the recovery of leptospires from porcine sperm. To inhibit a growth of Ps. aeruginosa, 5000 U/l polymyxin B were added. The strongly inhibitory action of polymyxin B on leptospiral growth could be eliminated by subculturing in a medium free from inhibitory substances after 2 days.

Cultivation of parasitic leptospires: effect of pyruvate.

Sodium pyruvate (100 mug/ml) is a useful addition to the Tween 80-albumin medium for the cultivation of parasitic serotypes. It is most effective in promoting growth from small inocula and growth of the nutritionally fastidious serotypes.

Isolation of the outer sheath of Leptospira and its immunogenic properties in hamsters.

Spherical forms of Leptospira interrogans serotype canicola Hond Utrecht IV were induced with 1 m NaCl. Electron micrographs of these salt-altered cells (SAC) revealed that the outer envelope or sheath had pulled away from the protoplasmic cylinder. Treatment of SAC with 0.02% sodium lauryl sulfate solubilized the sheath and released the protoplasmic cylinder. Further processing of the solubilized sheath yielded a pellet which displayed a membrane structure in electron micrographs. The released protoplasmic cylinder showed loss of intracellular organization and the outer envelope present in normal cells. Immunization of hamsters with whole formalized cells, SAC, or sheath in doses as low as 10 mug/animal protected them from death upon challenge with virulent canicola 27.

Electron microscopy of immune disruption of leptospires: action of complement and lysozyme.

Sequential disruption of the sheath of avirulent leptospires of the serotype canicola with antibody and complement was monitored by electron microscopy. Loosening and separation of the sheath from the protoplasmic cylinder was observed as early as 2 min after exposure to complement. Virulent leptospires of this serotype were morphologically intact after 1 hr of exposure to antibody and complement. Similarly, treatment of leptospires of the serotype patoc with normal serum and complement severely damaged the sheath structure. Removal of the sheath of both serotypes permitted lysozyme to act on the wall of the protoplasmic cylinder. Thus, morphological evidence for the location of the mucopeptide-containing structure of these leptospires was obtained. Viable leptospires with intact sheaths were resistant to lysozyme alone. Sections and negatively stained preparations of sheaths of serotypes canicola and patoc revealed three dense layers with two intermediate light zones and an overall thickness of about 110 A. A periodicity of 40 A was observed in sheath fragments produced by complement. The 70 A wallmembrane complex of leptospires of both serotypes consisted of two dense layers with an intermediate light zone. Structures apparent after removal of the outer sheath included membranous bodies or mesosomes, axial filaments attached to terminal knobs at opposite ends of the cell, and electron-dense intracellular bodies.