Pyelonephritis XI. Effect of Growth Phase of Streptococcus faecalis on Serum Susceptibility and Virulence.

Rapidly growing Streptococcus faecalis was found to be markedly susceptible to the bactericidal activity of normal rat and rabbit sera, whereas stationary-phase organisms were resistant. In contrast, human serum killed neither phase organism. Further, human serum was found to interfere with killing by rabbit serum. The serosusceptible, rapidly growing organisms were less virulent than the seroresistant, stationary-phase organisms. This was tested by production of pyelonephritis in the rat after intravenous inoculation of the organism. Renal bacterial content over a period of 4 weeks was significantly less in those animals which had received rapidly growing organisms.

Osmotic stability and sodium and potassium content of L-forms of Streptococcus faecalis.

A stable L-form of Streptococcus faecalis (T(53)) was transferred in media containing decreasing concentrations of sucrose until it grew in medium without added osmotic stabilizer. This L-form (designated T(53I)) was compared with T(53) from which it was derived. The survival of these two L-forms suspended at different osmolalities showed that maximal survival for T(53I) was 350 to 400 milliosmolal and for T(53) was 900 to 1,000 milliosmolal. Both peaks were at the osmolality of their growth media. Measurement of intracellular potassium and sodium showed that the concentration of these ions was reduced in T(53I).

Pyelonephritis. XIV. Effect of immunization on experimental Escherichia coli pyelonephritis.

Mice immunized subcutaneously with heat-killed Escherichia coli were protected from pyelonephritis produced by the intravenous route, but there was little or no protection from ascending infection. No significant protection from ascending or hematogenous pyelonephritis was demonstrated when immunization was accomplished by injecting heat-killed E. coli into the bladder. Heat-killed E. coli injected either subcutaneously or into the bladder protected mice from early endotoxic death after intravenous or bladder challenge.

Activity of methacycline, related tetracyclines, and other antibiotics against various L-forms and their parent bacteria in vitro.

The activity of methacycline against microbial L-forms and their parent bacteria was compared with that of oxytetracycline, chlortetracycline, tetracycline, and demethylchlortetracycline, as well as with that of 22 other antibiotics which included examples of major groups of antibiotics. The L-forms and bacteria used were Streptococcus faecalis, S. faecium, S. faecalis var. zymogenes, Staphylococcus aureus (three strains), Proteus mirabilis (two strains), Pseudomonas aeruginosa, Escherichia coli (two strains), Sarcina flava, Serratia marcescens, and Klebsiella pneumoniae. The five tetracyclines had similar activities and were more active against L-forms than bacterial forms, except that the bacterial form of S. flava was more susceptible than the L-form. In general, other antibiotics (except the penicillins) were more active against L-forms than bacterial forms. There were certain exceptions where the bacterial form was more susceptible than the L-form. These included the effect of polymyxin B and colistin on P. aeruginosa, E. coli, and P. mirabilis, and the effect of gentamicin on P. aeruginosa, E. coli, S. flava, and S. marcescens.

Defects in prodigiosin formation by L-forms of Serratia marcescens.

An L-form of Serratia marcescens has previously been shown incapable of producing the red pigment, prodigiosin, characteristic of the parent bacteria. Mutants of S. marcesens, unable to form one or the other of the two prodigiosin precursors, 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde or 2-methyl-3-n-amylpyrrole, were used to test the nature of the L-form defect. The L-forms failed to form sufficient amounts of either precursor to be detected by the appropriate mutant, and, when furnished the precursors, failed to couple them to form prodigiosin.

L-forms of Pseudomonas aeruginosa.

L-forms of a strain of Pseudomonas aeruginosa were produced by serial subculture of the bacterial form on agar medium containing sucrose as an osmotic stabilizer and carbenicillin. L-forms eventually became stable, i.e., would not revert in the absence of antibiotic, and were adapted to grow well in broth with the osmotic stabilizer. Gross morphology and light microscopic colony morphology were typical of an L-form. L-form cells were approximately spherical and bounded in part by a plasma membrane; they lacked the triple-layer cell wall structure and coarse, electron-dense nucleoidal granules of the parent bacterial form. The L-form, but not the bacterial form, contained cores, organelles previously reported only in group D streptococci. Antibiotic disc-sensitivity studies showed the stable L-form to be as sensitive as, or more sensitive than, the bacterial form to most antibiotics. Exceptions were polymyxin B, colimycin sulfate, and gentamicin, which were more active against the bacterial form. The remainder of the aminoglycosides and cell wall-active antibiotics showed no inhibition of either form. The L-form was more susceptible to cidal activity of normal human serum than the parent form. The L-form exhibited fewer biochemical activities than the parent bacteria or bacterial forms derived by reversion at a time when the L-form was still unstable. L-form colonies appeared colorless, and chemical analysis demonstrated that, if the L-form produces pigment at all, which was not demonstrated, it could not have been more than 3.6% of that produced by the bacterial form.

Differential action of a streptococcal bacteriocin on mycoplasmas and microbial L-forms.

Bacteriocin activity of Streptococcus faecalis var. zymogenes was tested against a variety of bacteria, L-forms, and mycoplasmas. Both a partially purified liquid preparation and a colony overlay technique were used. Other S. faecalis strains were the only bacteria whose growth was inhibited. The liquid preparation inhibited growth of all but three of the tested L-forms (whether derived from gram-positive or gram-negative bacteria), and two of these exceptional organisms were inhibited when the colony overlay technique was employed. On the other hand, the L-form of Streptobacillus moniliformis and all 33 tested mycoplasmas grew readily in the presence of the bacteriocin when either method was employed. It is suggested that the presence of cholesterol in the Streptobacillus and mycoplasmal membranes, unique in this regard among procaryotic cells, may be responsible for the differential pattern of growth inhibition.