Intracellular production of Brucella L forms . II. Induction and survival of Brucella abortus L forms in tissue culture

Hatten, Betty A. (The University of Texas Southwestern Medical School, Dallas), and S. Edward Sulkin. Intracellular production of Brucella L forms. II. Induction and survival of Brucella abortus L forms in tissue culture. J. Bacteriol. 91:14-20. 1966.-Intracellular survival of altered brucellae, possibly L forms, was not greatly affected by penicillin or streptomycin in concentrations ranging from 5.0 to 40 mug/ml, but a combination of these two antibiotics (2.5 to 20 mug/ml each) reduced the number of positive L-form cultures. Tetracycline (2.0 mug/ml) decreased the number of positive L-form cultures at about the same rate as combinations of the higher concentrations of penicillin and streptomycin. Various concentrations of tetracycline (0.1 to 2.0 mug/ml) with 5.0 mug/ml of penicillin or streptomycin significantly reduced the number of positive L-form cultures. L forms were recovered for several days after elimination of bacteria from the cultures by all of the antibiotics tested. L-form production was not dependent upon the presence of antibiotics in the culture medium, but they were recovered in greater numbers when bacteria were still present in the hamster kidney cells. Addition of thallium acetate to infected cells (at varying intervals of time after infection) to control bacterial growth and conversion to the L phase during cellular disintegration decreased the number of positive L-form cultures obtained over a 10-day period. Comparison of the antibiotic sensitivity of bacteria recovered from infected tissue culture cells with the stock strain of Brucella abortus indicated that some resistance to penicillin and tetracycline had developed. A marked resistance to streptomycin was observed in those bacteria recovered from cells maintained in the presence of this antibiotic.     

Ultrastructure of L-forms. II. L-forms of Listeria monocytogenes]

A study was made of ultrathin sections of the stable l-forms of listeria obtained under the action of penicillin in meat-peptone-liver broth. A marked cellular polymorphism was found in the L-form culture: within the same colony cells differed in size, shape and fine structure. It is supposed that polymorphism could be partially explained by a different plasticity and premeability of cytoplasmic membrane in different types of cells of the same L-colony. The three-layer structure of the membrane does not always display the same distinctness in various L-colony cells and also in different areas of the cell surface. Structureless material of low electron density, possibly a defective murein or its precursor, was revealed on the membrane surface. Electrondense inclusion bodies, mesosomes of ring-shaped or more complicated structure and two-contour vesicles were found in the cytoplasm. The cells multiplied by budding, by binary and anomalies division participation of mesosomes in this process was not proved by the L-forms.     

On the pathogenicity of nonstable Brucella L-forms and their revertants.

The pathogenicity of B. abortus 870 L-forms obtained by long-term passaging of virulent culture on media with penicillin and of revertants obtained in vitro and in vivo was studied. L-form cultures stimulated only a mild response of the reticulo-endothelial system of the animal organism, at the same time displaying a certain level of toxicity. In vitro revertants approximated to L-forms, while in vivo revertants stood closer to the initial virulent culture, as regards pathogenicity. This seems to be evidence of a potential danger of brucella L-forms for the human and animal organisms.     

Effect of the composition of reversion medium on change of Staphylococcus aureus lysostaphin protoplasts to coccal forms and L-forms

The experimental conditions under which protoplasts of Staphylococcus aureus strain MS353 (pCp) are converted to the coccal or L-form were investigated. Protoplasts prepared by treating coccal MS353 (pCp) strain with Lysostaphin formed various types of colonies (coccal form, L-form and mixed types) in about 50% yield when they were plated on reversion (R) medium consisting of 2% brain heart infusion, 0.5M sodium succinate, 0.01% bovine serum albumin, 20 mM MgCl2 and 0.6% agar. The L-form type colonies with a typical fried-egg appearance that developed on the R medium at an early stage gradually reverted to the coccal form through a mixed type stage in which a high density area first appeared in the periphery of the colony and then spread throughout the colony. The use of modified R medium without MgCl2 or R medium in which 0.5M sodium succinate as an osmotic stabilizer was replaced by 7.5% NaCl resulted in marked delay in the appearance of reverted cells. R medium without bovine serum albumin yielded atypical L-form type colonies, which contained masses of coccal cells with very irregular margins. On the other hand, R medium without MgCl2 but with penicillin G supported development of L-form type colonies at high rate (13-15%) from the inoculated protoplasts.     

Cell structure and the pathogenicity of Brucella at different stages of L transformation]

On the basis of changes in the biological properties and morphology of Br. abortus culture under the action of penicillin 3 stages of L-transformation in Brucella were determined. The prevalence of first bacilliform and then typical L-cells and rapid reversion hampering the determination of virulence were characteristic of the initial stage (passages 1-4). Typical L-cells with the wrinkled surface, deep depressions and holes as well as a decrease in virulence and slight pathomorphological changes in the organs of the infected animals were characteristics of the intermediate stage (passages 5-10). Typical L-cells and amorphous masses, a further decrease in virulence, pathomorphological changes of toxic character (only after the injection of L-culture in large doses) were characteristic of the late stage (from passage 11 and further on). At all stages of L-transformation Brucella cultures showed a high reproductive capacity, binary division, the formation of elementary bodies by budding both inside and on the surface of L-cells.     

Physiological characteristics of Salmonella typhimurium treated with penicillin]

Growing bacteria of the two strains of Salmonella typhimurium differing in the sensitivity levels to UV-light formed multinuclear non-septal filaments in the penicillin-containing nutrient medium. The maximum number of the lifefull filaments was formed by the 4th hour of incubation in the beaf-peptone broth at a temperature of 37 degrees C in the presence of 5 gamma/ml of penicillin. The strains exposed to penicillin were less sensitive to UV-light. Exclusion of penicillin from the nutrient medium resulted in a new division of the filamentous cells and reduction of the initial UV-light sensitivity level. It was concluded that the low UV-light sensitivity level of the filaments induced by penicillin was associated with their multinuclear state.     

L-Form Induction, Morphology, and Development in Two Related Strains of Erysipelothrix rhusiopathiae

Two related strains of Erysipelothrix rhusiopathiae, one the parent and the other an L-form revertant, were studied for their propensity or ability to produce L-forms under the influence of penicillin. The parent strain produced L-forms in nutrient solid media in an osmolarity range between 0.85 and 5.0% NaCl concentration whereas the revertant strain did so between 0.5 and 3.0% NaCl concentration. When various hyperosmolar media were tried without penicillin, recovery of L-forms from the revertant strain was optimal at a salt concentration of 2.0%, whereas the parent strain occasionally produced a few L-forms on 3.0% salt medium only. The process of penicillin-induced transformation from bacteria to L-form followed an unusual morphological sequence, beginning with beading of the bacterial body, followed by disintegration into granules from which the L-form colony derived. No large bodies were seen during the initial process of L-form induction, but they evolved later from the original granules and had the potential to reproduce L-type growth. The spontaneous development of L-forms in hyperosmolar media had a different morphological sequence starting with elongation of the bacteria into filaments which later developed polar and central dilatations from which granules and L-type growth developed. The differences in biological behavior between these related bacterial strains suggest that the revertant strain developed new properties, probably of genetic origin. Consequently, the assumption that L-forms revert to the "parent" bacteria may not always be justified. It can be made only after the biological properties of the parent and the revertant organisms have been properly identified.     

Efficiency of procedures for induction and cultivation of Pseudomonas syringae pv. pisi L-form

The L-form of Pseudomonas syringae pv. phaseolicola has been proved to induce resistance to bean halo blight.Various procedures were tested to induce the L-form of Pseudomonas syringae pv. pisi for its potential use as biocontrol agent of pea bacterial blight. Cell-wall deficient cells were induced in a liquid medium with penicillin following a protocol described for P. s. pv. phaseolicola. Cell growth on solid induction medium developed as typical granular and vacuolated structures, and characteristic colonies were observed in the first transfer. However, there was poor growth in subsequent transfers and some reversion to the parental type. To improve the induction procedure, the following new procedures were applied: (1) viability of cells was monitored during induction. The optimum induction time in liquid medium with penicillin was lower for pv. pisi than for pv. phaseolicola. Viability of L-forms in solid induction medium with penicillin was low and decreased in time. (2) the inducer ticarcillin was combined with clavulanic acid, which prevented the reversion to the parental type and (3) a range of concentrations of penicillin and ticarcillin/clavulanic acid was applied by the spiral gradient endpoint method for calculation of minimum inhibitory concentrations (MIC). Based on the results from these tests an induction method for P. s. pv. pisi L-form is proposed and the relevance of L-form is discussed for practice.     

Antibiotic Resistant Group A Streptococci

A series of Streptococcus pyogenes strains, including strains isolated from patients, mutants which had acquired in vitro resistance to penicillin (Pc), mitomycin C (MC), tetracycline (TC) and chloramphenicol (CM), ultraviolet light induced α hemolytic mutants, as well as β hemolytic mutants (β mutants) derived from α hemolytic mutants (α mutants) were compared as to their antibiotic sensitivity, and physiological, biochemical and serological properties. To obtain β mutants from α mutants the following procedures were employed: (1) serial mouse passage, (2) serial serum-broth transfers, (3) cultivation in heat-killed cultures of parent strains, and (4) cultivation in broth containing bacterial DNA extracted from parent streptococcus cells. From the results obtained these strains could be divided into two major groups, each with two subgroups. Group 1 strains produce soluble hemolysins and are sensitive to Pc. Subgroup 1–1 strains are sensitive to other antibiotics too; subgroup 1–2 are resistant to certain antibiotics other than Pc, bacitracin and MC. Group 2 strains do not produce soluble hemolysins and resistant to Pc. Subgroup 2-1 strains are α hemolytic on horse blood agar and subgroup 2–2 are β hemolytic on the same medium. Pc resistance in group 2 strains was more than 100-fold higher than that of sensitive strains, and was accompanied by MC resistance, but to a lesser degree. Pc resistance in group 2 mutants could be induced by antibiotics other than Pc and also by ultraviolet irradiation. Although group 1 cells retained the characteristics of typical S. pyogenes, group 2 cells, both α and β hemolytic, lost most of the physiological, biochemical and serological properties of this species. The similarity of group 2 strains to group D or group N streptococcal strains in their general properties is discussed.     

Biological properties of promastigotes of Leishmania having undergone lyophilization]

Materials are presented of a comparative study of morphological and cultural properties and virulence of liophylized cultures of Leishmania tropica major after their rehydration or after a long passage on the NNN medium. Results of investigations of two initial strains and three substrains obtained after the recovery of liophylized cultures have shown that liophylization does not reduce essentially the initial strain virulence. The strains undergone liophylization and passed repeatedly on the NNN medium have a tendency to a quicker virulence reduction rate as compared to initial ones. The culture of promastigotes recultivated after liophylization does not differ from the initial one in the ranges of 1-7 passages in its morphology, mobility and ability to grow on the NNN medium.     

Defined Physiological Conditions for the Induction of the L-Form of Neisseria gonorrhoeae

Defined conditions are described which allowed luxuriant growth over continuous subculture of strains of Neisseria gonorrhoeae in broth and on agar. Growth was equal to or surpassed that observed in Mueller-Hinton broth or on Mueller-Hinton blood agar. The final medium adopted consisted of medium 199 and a supplemental mixture of cysteine, glucose, and various salts. Addition of sodium bicarbonate or CO(2) enrichment was not required. For solidification, only agarose allowed growth of all strains; glutamic acid stimulated growth of two strains but was inhibitory for a third. The addition of 8% polyvinylpyrrolidone (PVP), 2% purified albumin, and penicillin resulted in induction of all three strains to the L-form with frequencies up to 0.3%. At present no induction to the L-form has been achieved in the absence of albumin. Various lots of PVP proved toxic in the defined medium, and extensive dialysis was required for good growth and L-form induction. Substitution of PVP with sucrose indicated a sucrose toxicity for the parental gonococcus even on the addition of albumin. L-form induction did occur on sucrose L-medium but at significantly lower frequencies. The colonies appeared 1 week later than those on PVP L-medium but at significantly lower frequencies. The colonies appeared 1 week later than those on PVP L-medium and remained very small and poorly developed.     

Comparative characteristics of antigenic peculiarities and several other properties of stable long-term cultures of salmonella L-forms]

Enzymatic properties, sensitivity to antibiotics and peculiarities of the antigenic structure of stable L-forms of S. paratyphi B (L-115), S. typhimurium (L-71a-16) and S. typhi (L-5761) were studied. In difference from the initial strains, the L-form under study possessed a high penicillin and ampicillin sensitivity, and also a marked sensitivity to polymyxin and detergents; this characterized them as L-forms of protoplastic type. All the L-variants differed considerably by the enzymatic properties from the initial strains, and, in the majority of cases, retained the antigenic components and the species-specificity characteristic of the initial parental cultures. Despite the loss of the cell wall the synthesis of O-antigen in the L-forms under study was undisturbed. The 6-forms contained an antigenic component not found in the initial cultures and apparently causing the neutralizing activity of the L-antiser.     

The immunochemical characteristics of the lipopolysaccharides of Pseudomonas syringae (pathovars atrofaciens and phaseolicola) and P. holci (serogroup VI)]

Lipopolysaccharides (LPS) of the representatives of strains of serogroup VI Pseudomonas syringae (P. syringae pv. atrofaciens 2399, pv. phaseolicola 120a, 7842 and P. holci 8299) possessing virulence and confinement to the host-plant are characterized by high serological activity in direct and cross reactions of the binary diffusion in agar, immunoelectrophoresis, passive hemagglutination and inhibition of passive hemagglutination. A supernatant and a sediment obtained after ultracentrifugation of LPS preparations possessed O-antigenic activity. O-specific polysaccharide (PS) is serologically less active than the LPS preparations. Problems of the intergroup and intragroup serological affinity in connection with the structure of O-specific PS. It is proved that the basic chain of O-specific polysaccharide (D-rhamnane) plays definite (but not a single) part in displaying antigenic properties of the whole LPS macromolecule.     

Stable L-forms of Clostridium perfringens and their growth on glass surfaces.

L-forms of Clostridium perfringens were induced in brain heart infusion broth containing 10% sucrose and 2 units of penicillin. After a few hours of growth, spheroplasts, granules, and elongated bacilli were apparent. At 24-h intervals, serial subcultures were made in the above medium which resulted in a culture composed entirely of spheroplasts (or protoplasts) and granules. Upon the withdrawal of penicillin these L-form cultures grew well and, after 100 passages, there was no reversion to the bacillary form. Sucrose could also be withdrawn from the medium. The effects of centrifugation, osmotic stabilizer, ultraviolet light, temperature, pH, and lyophilization upon stable L-forms were examined. L-forms were found to attach to the walls of culture tubes during trowth and sheets of L-form growth were obtained on cover slips in Leighton tubes and on the sides of medicine bottles.     

Reduction in virulence of Babesia bovis due to rapid passage in splenectomized cattle.

A marked loss of virulence of Babesia bovis for normal cattle was observed during its rapid serial, blood passage in splenectomized calves. In 2 strains studied closely, responses to infection in cattle inoculated with parasites collected from the 11th passages were minimal, although the splenectomized donors were severely affected. The change was reversed by passaging in intact hosts, and in one experiment the parasites had become very virulent at passage 5. The finding has proved useful in the preparation of safe, living vaccines to control babesiosis. Selection either for immunogenic antigens, against immunosuppressive ones, or a combination of these effects may cause the decrease in virulence.     

Cell fusion between L-forms and protoplasts of Staphylococcus aureus

Mixtures of various combinations of Lysostaphin protoplasts and stable L-forms of Staphylococcus aureus, which have different markers for drug resistance, were treated with polyethylene glycol (PEG) to examine the development of doubly resistant fusion products (fusants). To recover doubly resistant colonies as L-forms, they were incubated in 4.5% NaCl-brain heart infusion (BHI) broth containing penicillin G (PCG) for enrichment culture and cultured in PCG-4.5% NaCl-BHI agar medium (method 1), while to recover doubly resistant fusants as L-forms and coccal forms, they were grown on reversion medium (R medium) which causes reversion of protoplasts or fusants to parent type cells, and then cultured on assay media, i.e., R medium, BHI agar medium or PCG-4.5% NaCl-BHI agar medium (method 2). Under both experimental conditions, doubly resistant fusants developed as L-form cells by PEG treatment of pairs of protoplasts carrying the chloramphenicol (CP)-resistance plasmid and L-forms having chromosomal resistance to streptomycin (SM). In the reverse combinations, i.e., protoplasts showing chromosomal SM-resistance and L-form cells carrying the CP-resistance plasmid, the first method gave no doubly resistant colonies. By the second method, without enrichment culture on R medium, the latter combination gave doubly resistant fusants as L-form, coccal-type and mixed-type colonial forms, while when the PEG-treated mixture was enriched on R medium, fusants were obtained exclusively as the coccal type on either R medium or BHI agar assay medium. Neither of the methods yielded colonies of doubly resistant fusants on PEG-treatment of pairs of protoplasts and L-forms both of which were chromosomal, but with different drug resistances. These results show that PEG-induced cell fusion between protoplasts and L-forms of S. aureus, unlike the fusion between protoplasts or between L-forms, resulted in transfer of the drug resistance controlled by the plasmid to the fusion products. The fusants obtained were L-forms in method 1, and coccal type in the method 2.     

Identification with the aid of various serological reactions of L forms of streptococci isolated from the live organism]

Growth inhibition, agglutination, precipitation, and passive hemagglutination tests were used for the identification of the L-forms of streptococci isolated from the organism of experimental rabbits both after the infection with the L-forms of streptococci and with the streptococci of group A. The tests were positive not only with the antiserum of homologous, but also of heterologous strains of the L-form of streptococcus, group A. The L-form cultures isolated from the experimental animals failed to differ from the laboratory strain of the L-forms of streptococcus, group A, by serological properties.     

Differentiation of mycoplasmatales from bacterial protoplast L-forms by assay for penicillin binding proteins

Membrane proteins with the specific ability for binding penicillin with high affinity (penicillin binding proteins) were found to be present in two strains of the cell wall-less protoplast L-form of P. MIrabilis and were absent from different species of Mycoplasma and from Acholeplasma laidlawii. Thus, the assay for penicillin binding proteins appeared to be suitable for the differentiation of the cell wall-less procaryotes. The absence of penicillin binding proteins from the mycoplasmatales further confirmed the unrelatedness of this group to the bacteria.Non-Standard Abbreviation PBP penicillin binding protein Dedicated to Professor Dr. Otto Kandler on the occasion of his 60th birthday     

Effect of Ca2+ and K+ on the intracellular pH of an Escherichia coli L-form.

The L-form NC7, derived from Escherichia coli K12, grew in a complex medium containing 0.2 M-CaCl2 as osmotic stabilizer, but not at pH values above 7.8. The cessation of growth at alkaline pH was not due to cell death. In complex media containing K+ or Na+, the L-form grew ove a wide pH range. Growth at alkaline pH was inhibited by 1 mM-amiloride, indicating that Na+/H+ antiport activity was required for growth at alkaline pH. The internal pH (pHi) of the L-form in media containing K+, Na+ or Ca2+ was constant at about 7.8 to 8.0 at external pH (pHo) values of 7.2 and 8.2. The rates of O2 consumption by intact cells, lactate oxidation by membrane vesicles from cells grown in Ca(2+)-containing medium, and cell division were all strongly repressed under alkaline conditions.     

Division and death rates of Salmonella typhimurium inside macrophages: use of penicillin as a probe.

In mouse peritoneal macrophages infected in vitro with Salmonella typhimurium the number of viable bacteria and the number of stainable bacteria detected by light microscopy both increased at similar rates with a doubling time of more than 1 h. Antibiotics were not present; instead extracellular bacteria were removed by frequently rinsing the cells. The bacterial doubling time in the same medium in the absence of macrophages was about 20 min. Penicillin added to macrophage monolayers rapidly entered the macrophages, reaching a diffusion equilibrium. The penicillin-induced bacterial death rate appeared to depend on the bacterial division rate as well as on the penicillin concentration. These properties of penicillin were used to monitor intracellular bacterial division and death rates. The results indicated that intracellular killing, with the disappearance of stainable bacteria, did not contribute to the extended doubling time in macrophages. It was concluded that the intracellular environment of the bacteria was probably growth inhibitory but not bactericidal.