L-Forms of Pseudomonas aeruginosa

Abstract

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 wallactive 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%o of that produced by the bacterial form. The role of L-forms in infectious diseases in general and in urinary tract infections in particular has not been clarified. However, there is inferential evidence that these wall-deficient variants may be significant, not only as primary etiologic agents, but particularly may account for relapse after apparent cure (23). Postulated mechanism for production in vivo is based on the in vitro demonstration that wall-deficient variants can be induced by serum action on the parent form (1, 37). L-forms have been isolated from urine of patients with urinary tract infection (14) and inferential evidence has been obtained by treatment of experimental animals with antibiotics (15). Pseudomonas infections present an important problem, particularly in urinary tract infections and burn patients, because antibiotics in general use are not active against this organism. The semisynthetic penicillin, carbenicillin (disodium a-carboxybenzylpenicillin; BRL 2064), has been demonstrated to possess significant activity against strains of Pseudomonas aeruginosa, both in vitro (40) and in vivo (21, 39, 44). In view of this activity, it was felt that carbenicillin provided an opportunity to determine whether L-forms could be produced in vitro from P. aeruginosa and to study some biological properties of the L-forms. This study details initial production of the Lform of P. aeruginosa and the achievement of nonreversion in the absence of antibiotic. Comparisons of morphology,...

Tension-activated channels in the mechanism of osmotic fitness in Pseudomonas aeruginosa

“…We attempted the same procedure on PA only to find that the PA14 strain is completely insensitive to cephalexin. However, following earlier work by Hubert (Hubert et al, 1971), we were able to effectively induce filamentous growth of PA in the presence of 0.2-0.3 mg/ml carbenicillin (Fig. 3).…”

Increased Antibiotic Sensitivity in Pseudomonas aeruginosa Following Passage in Carbenicillin‐containing Media

“…After several passages on carbenicillin-free medium there was no loss of such antibiotic sensitivity. This evidence confirms that of Hubert et al (1971) for the development of stable L-forms by certain strains of P. aeruginosa L-formation in vitro in P. aeruginosa would appear to be a variable phenomenon and from our data concerning the onset of increased antibiotic sensitivity there is an indication that in certain strains the development of L-form type variants occurs after only a few passages in the presence of the cell-wall inhibitor, carbenicillin. The prolonged therapeutic usage of this penicillin against P. aeruginosa might, thus, be expected to induce L-formation in vivo with specific strains if the antibiotic failed to kill all of the infecting cells.…”

“…Our results are somewhat at variance with those obtained by Hubert et al (1971) for P. aeruginosa B10490 with regard to specific antibiotics potentiated against the L-forms. In particular while colistin (colomycin) sulphate and gentamicin, in contrast to the inactivity recorded for other aminoglycoside antibiotics, were found by these workers to be more effective against the bacterial rather than the L-form, the reverse effect was found with our variants.…”

“…Although the L-form appeared more sensitive than the bacterial form to several antibiotics in a disc sensitivity test an opposite effect was found with colistin (colomycin) sulphate, gentamicin and polymyxin B. Hubert et al (1971) could not suggest any explanation for this latter phenomenon which disagrees with results previously obtained for both Gram-positive and Gram-negative bacteria Teuber, 1969). The current quantitative investigation was undertaken to determine whether such results are characteristic for clinicallyisolated strains of P. aeruginosa.…”

“…Reports of L-form production in P. aeruginosa appear limited to that of Hubert et al (1971) in which a single strain, B 10490, was shown to undergo L-formation following serial subculture in osmotically-stabilized medium containing carbenicillin. Although the L-form appeared more sensitive than the bacterial form to several antibiotics in a disc sensitivity test an opposite effect was found with colistin (colomycin) sulphate, gentamicin and polymyxin B.…”