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Spheroplasts of Vibrio choleraetypes Inaba, Hikojima, and Ogawa were produced in serum. An excess of lysozyme was added to expedite transformation, and CaCl2was used as the stabilizing agent. At the optimal time for each strain, when less than 1% classical rod forms were observed microscopically, samples were plated on both conventional and modified L-form media. No growth occurred on conventional media; L-form type colonies appeared on L-form media after 2 to 6 days of incubation. L-form colonies could be subcultured on conventional media and required from four to six passages for complete reversion to classical parent forms. Reaction mixtures of Hikojima and Inaba types were passed through membrane filters (0.45 μ); L-form colonies were grown from both strains after spheroplast transformation. Appropriate controls were negative. It is suggested that the replicative particle may be filtrable.

Because calcium was found to be antagonistic in vitro to the activity of colistin and polymyxin B on Pseudomonas aeruginosa, the effects of calcium and serum on gentamicin and carbenicillin were also examined. Serum was antagonistic to gentamicin in antibiotic tube dilution tests on five strains of P. aeruginosa. Serum was not antagonistic to carbenicillin in tube dilution tests. Physiologic concentrations of calcium antagonized the activity of gentamicin but not carbenicillin. The antagonism observed with gentamicin was less than that previously seen with colistin. The antagonistic effect of calcium and serum was removed by a chelating agent. Gentamicin and carbenicillin may be more active in vivo against P. aeruginosathan colistin or polymyxin B.

Physiological concentrations of calcium in serum antagonize the activities of colistin, polymyxin B, and gentamicin against Pseudomonas aeruginosa. Studies were carried out to determine whether tobramycin, a new aminoglycoside antibiotic, is also antagonized by calcium. The activity of tobramycin in vitro was shown to be antagonized by human serum and by physiological concentrations of calcium. The addition of human serum in broth-dilution tests produced a fourfold rise in the minimal inhibitory concentrations of tobramycin for five strains of Pseudomonas. In disc diffusion tests, the addition of calcium to the agar significantly decreased the size of inhibition zones, and the addition of a chelating agent to the agar increased the zone sizes. In a limited comparative study, tobramycin and gentamicin were tested against both light and heavy bacterial inocula of two strains of Pseudomonas. Tobramycin appeared to be antagonized less by serum than was gentamicin at equal antibiotic concentrations.

While pulmonary infection in cystic fibrosis (CF) is most often initially due to S.aureus, P.aeruginosa often emerges as the major pathogen. The latter may be typed by susceptibility to pyocines, antibacterial substances produced by certain P.aeruginosa and active only against certain other P.aeruginosa. To understand the role of this organism in the bacterial ecology of the respiratory tract, pyocine typing, utilizing NIH indicator strains, was performed on multiple P.aeruginosa isolates from 100 chronic pulmonary disease patients with and without CF, classified according to clinical condition. No specific types were characteristic of non-CF patients, but N-7, N-9, N-ll, N-13, and N-27 were most common to CF patients. Types N-7 and N-ll were found most frequently in those with severe impairment or exacerbation of pulmonary disease, while N-1 and/or N-4 was associated with improvement. Of CF patients in good clinical condition, 33% had N-1 and/or N-4, compared to only 15% of CF patients with severe impairment. Pyocines produced by N-1 and N-4 P.aeruginosa in vitro inhibit N-7 and N-11, but pyocines produced by N-7, N-9, N-11, N-13, and N-27 do not inhibit N-1 and N-4. It is concluded, therefore, that the presence of N-1 and/or N-4 P.aeruginosa may inhibit colonization by P.aeruginosa characteristic of more severe disease as can occur among staphylococci in other clinical conditions.

Human serum inhibits the antibacterial activity of colistin and polymyxin B against Pseudomonas aeruginosa in vitro. By use of a colony-count assay, the inhibitory factor in serum was identified as calcium. The bactericidal activity of colistin was inhibited by either 10−3 m CaCl2, 15% human serum, or 15% heparinized plasma. Colistin was not inhibited by plasma containing chelating agents. Colistin may be less active in vivo than predicted on the basis of in-vitro tests of susceptibility to the antibiotic.