Intracellular Production of Brucella L Forms I. Recovery of L Forms from Tissue Culture Cells Infected with Brucella abortus

Hatten, Betty A. (The University of Texas Southwestern Medical School, Dallas), and S. Edward Sulkin. Intracellular production of Brucella L forms. I. Recovery of L forms from tissue culture cells infected with Brucella abortus. J. Bacteriol. 91:285-296. 1966.-Infectivity of virulent Brucella abortus strain 3183 was less for hamster macrophages after a 2-hr adsorption period than for an attenuated strain (S19) and its tissue culture variant (30). Both strains S19 and 30 were very toxic for the cells, but 3183 was not toxic. Two types of L forms were recovered from a large percentage of hamster kidney cell cultures when disintegration of infected cells was accelerated by tissue culture medium of high pH. One type grew in finely granular microcolonies, was isolated from cells infected for short periods of time, and often reverted to the bacterial form. The other type occurred in small irregularly shaped forms which later developed into round bodies. Both stained specifically with fluorescein-conjugated B. abortus antiserum. Semisolid media containing 0.7% agar provided optimal subsurface L-form growth. L forms also grew well in Thioglycollate Medium but grew poorly in other liquid media. Surface L-form growth was supported by several agar media, but CO(2) was required for optimal growth. Monolayers infected with strain 3183 and examined immediately after adsorption contained occasional small, round bodies. Bizarre forms increased in number with time and, after 24 to 72 hr, large pink-staining inclusions were often present which persisted for several days. Also appearing at about the same time were smaller, dark-staining forms which were first seen in clusters but later dispersed and finally occurred in chainlike configurations. Direct fluorescent-antibody stains of infected cells established that the intracellular forms were related to the infecting strain of B. abortus.     

Ultrastructure of Brucella abortus L-forms induced by penicillin in a liquid and in a semisolid medium

Brucella abortus L-forms were induced by 5.0 or 10.0 mug of penicillin/ml in a broth medium containing 0.3 m sucrose, and in a semisolid medium containing 10% calf serum and 20.0, 40.0, or 60.0 mug of penicillin/ml. After 96 hr of incubation, L-forms of various sizes and shapes were observed. Basic structures of the L-forms were similar whether induced in liquid or semisolid medium. L-forms had two "unit" membranes, each consisting of two outer dense layers separated by a lucent layer. A few large, irregularly shaped organisms in penicillin-treated broth cultures had additional surface material and were referred to as "transitional" forms. In contrast with L-forms, the bacterial cells were fairly uniform in size and shape, were smaller, and had a more complex cell wall structure. Small bodies limited by a "unit" membrane were present within and around numerous L-forms from liquid and semisolid medium cultures. Other internal membranous structures were also seen in some L-forms. Most Brucella L-forms described in this paper reverted to bacteria in the absence of penicillin and were structurally characteristic of unstable L-forms.     

Study of the L-transforming action on Brucella cells of tetracycline, streptomycin, rifampicin and their combinations with penicillin in in vitro experiments]

The in vitro study of the possible formation of L-forms of Brucella under the effect of tetracycline, streptomycin, rifampicin or their combinations with penicillin showed that passages of various Brucella species on media containing increasing concentrations of the antibiotics resulted in insignificant morphological changes in the cells. Passages of the same cultures on media containing combinations of the above antibiotics with penicillin resulted in more significant changes in the cell morphology, up to formation of spheroplasts. No L-transformation was observed under the experimental conditions.     

Susceptibility of Cheese and Yoghurt Starter Bacteria to Antibiotics

Eight single-strain lactic streptococci, three commercial cheese starters, and six lactic acid bacteria isolated from yoghurt were examined for their susceptibility to penicillin, cloxacillin, tetracycline-hydrochloride and streptomycin. The ranges of the antibiotics causing 50% inhibition of the bacteria were (mug/ml): penicillin, 0.009 to 0.20; cloxacillin, 0.24 to 2.50; tetracycline, 0.09 to 0.60; and streptomycin, 0.35 to 13.0. The average concentrations required to cause 50 and 100% inhibition of the cheese starters were (mug/ml): penicillin, 0.12 and 0.26; cloxacillin, 1.91 and 3.9; tetracycline-hydrochloride, 0.13 and 0.36; and streptomycin, 0.59 and 2.06. All the cocci were about equally susceptible to tetracycline, and all organisms were more resistant to cloxacillin than penicillin. The yoghurt isolates were more resistant to streptomycin and more susceptible to penicillin than the cheese starters. The 2, 3, 5-triphenyltetrazolium chloride test, using Streptococcus thermophilus BC as assay organism, does not detect low levels of streptomycin in milk. However, it is useful in detecting cloxacillin residues.     

Antibiotic sensitivity of S. typhi cultures that have reverted from L forms isolated from the blood of patients]

L-forms of bacteria were isolated in 18 out of 300 fever patients with diagnoses of typhoid-paratyphoid fever, grippe, virus respiration disease and others in the Diagnostic Department of an Infection Hospital during bacteriological tests of the blood. Among the cultures tested 13 were instable and reversed to the bacterial form. The type identification showed that only 9 revertants possessed properties characteristic of the typhoid fever microbes and belonged to S. typhi. Sensitivity of the typhoid fever revertants to levomycetin, sintomycin, streptomycin, pencillin and tetracycline was studied. The studies showed that the typhoid fever revertants from the L-forms isolated from the patients were sufficiently sensitive to levomycetin, sintomycin, penicillin and tetracycline. The minimum bactericidal concentrations of the above antibiotics ranged within 12.5--100 gamma/ml.     

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.     

In Vitro Susceptibility of Brucella to Various Antibiotics

A series of 27 strains of six species of Brucella was tested for susceptibility in vitro to a representative cross section of antibiotics in current use. The activity against each species was plotted, with the cumulative per cent of strains inhibited indicated for each concentration. As a class, the tetracycline antibiotics were the most effective. Erythromycin, gentamicin, streptomycin, and kanamycin, as well as rifampin, were quite active. The penicillin-cephalosporin group, with the exception of ampicillin, was comparatively ineffective, as were the polypeptides and the miscellaneous group of chloramphenicol, lincomycin, cycloserine, and sulfadiazine. Species differences were noticeable, with some strains of B. canis being considerably more resistant to streptomycin and the tetracyclines than B. suis and B. abortus. B. melitensis, B. ovis, and B. neotomae were intermediate in antibiotic susceptibility.     

In Vitro Effect of Rifampin on Mycobacteria

Rifampin inhibited 20 strains of Mycobacterium tuberculosis in concentrations of 0.005 to 0.02 mug/ml in 7H-9 broth with Tween 80 and killed all or nearly all of the inoculum in four to eight times greater concentrations. In the same medium without Tween 80, as well as on 7H-10 agar, about 16 to 64 times these amounts were required to produce the same effect. Rifampin was also active against M. kansasii and some of the nonchromogenic mycobacteria. The incidence of mycobacterial cells resistant to rifampin within the cultures studied was in the range of one to four per 10(8) to 10(9) colony-forming units with concentrations of 4 to 125 mug of rifampin per ml. Only one of the Battey cultures and that of M. fortuitum yielded cells resistant to rifampin at 125 mug/ml but not at 500 mug/ml. The same strains yielded more than double that number of organisms resistant to streptomycin and up to 100 times more organisms resistant to isoniazid. All three drugs stopped the growth or reduced the mycobacterial population in growing cultures after contact for 24 to 48 hr. Complete inhibition of growth was produced by rifampin at 1.0 mug/ml in an average of 6 days and by streptomycin at 5.0 mug/ml in 3 days. After an average contact of 10.7 days with rifampin, five of seven strains resumed growth and all strains began regrowth after exposure to streptomycin for 9.4 days. The marked susceptibility of M. tuberculosis and of atypical mycobacteria to rifampin in vitro and the relatively low incidence of resistant mutants suggests that this agent may have clinical usefulness in the treatment of tuberculosis and some other mycobacterioses.     

Effect of Penicillin-Streptomycin and Other Antibiotics on Melanogenic Parameters in Cultured B16/F10 Melanoma Cells

Penicillin and streptomycin, the most widely used antibiotics in mammalian cell cultures, caused a moderate stimulation in dopa oxidase and tyrosine hydroxylase activities, but a slight inactivation in the dopachrome tautomerase activity of B16/F10 melanoma cells at the routine concentration (100 units/ml penicillin and 100 μg/ml streptomycin) used for preventing bacterial growth in cultured animal cells. At these concentrations, tyrosinase activities and melanin content augmented with time during the first 24–48 hr. The opposite effect acted on cell viability. After withdrawal of the antibiotics from the culture medium, the recovery of melanogenic parameters to normal values was fully reached after few hours (around 10), and it was already noticeable as soon as 4 hr after removal. Other antibiotics used in cell culture, like kanamycin, gentamicin, and the antimicotic nystatin, exerted similar low effects at the recommended concentrations, always lower than two-fold and thus lower than those reported for amphotericin B. Taking into account these relatively low effects, and the high risk of contamination of mammalian cells culture without antibiotics, penicillin and streptomycin may still be routinely used in experiments leading to explore the melanogenic activity of malignant melanocytes in culture, unless very precise studies and strict conditions were needed.     

In vitro activities of antimicrobials against Brucella abortus isolates from cattle in Korea during 1998-2006

In vitro activities of 13 antibiotics were assessed against 85 Brucella abortus isolates from naturally infected cattle in the Republic of Korea during 1998-2006, using broth microdilution test. Tetracyclines showed the most excellent activity against B. abortus, displaying MIC values of 0.5 μg/ml or below. In particular, minocycline showed the lowest MIC??/?? values (0.125/0.125 μg/ml) in this study. Among four fluoroquinolones tested, ciprofloxacin (MIC??/??, 0.5/1 μg/ml) and norfloxacin (MIC??/??, 8/8 μg/ml) had the most and the least activities, respectively. Gentamicin (MIC??/??, 1/1 μg/ml) was more effective than streptomycin, erythromycin, rifampin, and chloramphenicol (MIC??/??, 2/2 μg/ml).     

Detection of Brucella , abortus , in the cervical mucus of nulliparous heifers

Sixteen nulliparous Holstein heifers were exposed artificially to Brucella , abortus , biotype 1 strain 2308. Attempts were made to recover the organism from blood, udder secretions and cervical mucus. Blood cultures 2 to 4 wk postexposure were positive. B . abortus , was recovered from one or more udder quarters in 11 heifers. B . abortus , was recovered from cervical mucus of one heifer on Day 18 postexposure. All heifers were serologically positive within 5 wk. The presence of B . abortus , in the nongravid uterus is transitory and associated with the bacteremic phase. It is limited or prevented in most heifers due to the effect of estrus. Nulliparous heifers are suitable candidates for use as donors of Brucella -free embryos, even where infection is known to exist in the herd.     

Antibiotic Susceptibility of Anaerobic Ruminal Bacteria

This study demonstrated that 15 species of ruminal bacteria with no previous history of contact with antibiotics are susceptible to bacitracin, chloramphenicol, chlortetracycline, erythromycin, novobiocin, oleandomycin, oxytetracycline, penicillin, tetracycline, tylosin, and vancomycin. A number of the species were not inhibited by kanamycin, neomycin, polymyxin, and streptomycin. The data suggest that antibiotic-resistant cells occur within susceptible cultures of these species. Streptococcus bovis FD-10 and a nonruminal anaerobe, Bacteroides melaninogenicus BE-1, showed similar antibiotic susceptibilities.     

布鲁氏菌2308ery基因启动子缺失株的构建及鉴定

目的:构建布鲁氏菌2308株ery基因启动子缺失株。方法:用PCR方法从亲本株2308上扩增ery基因启动子侧翼序列,将该片段与pMD19-T连接,亚克隆为自杀载体pGEM-7zf-Δery-sacB。将自杀载体电转化布鲁氏菌感受态细胞中经同源重组后,分别用100 mg/L氨苄和7%蔗糖筛选。对获得的基因缺失株进行RT-PCR鉴定和遗传稳定性检测。结果:成功获得ery基因启动子缺失株,2308Δery基因启动子缺失株未扩增出eryA基因。并且该缺失株在10代以内未发生回复突变。结论:成功构建2308Δery基因启动子缺失株,为研究布鲁氏菌的毒力基因及其流产机制奠定基础。     

Completion of the genome sequence of Brucella abortus and comparison to the highly similar genomes of Brucella melitensis and Brucella suis

Brucellosis is a worldwide disease of humans and livestock that is caused by a number of very closely related classical Brucella species in the alpha-2 subdivision of the Proteobacteria. We report the complete genome sequence of Brucella abortus field isolate 9-941 and compare it to those of Brucella suis 1330 and Brucella melitensis 16 M. The genomes of these Brucella species are strikingly similar, with nearly identical genetic content and gene organization. However, a number of insertion-deletion events and several polymorphic regions encoding putative outer membrane proteins were identified among the genomes. Several fragments previously identified as unique to either B. suis or B. melitensis were present in the B. abortus genome. Even though several fragments were shared between only B. abortus and B. suis, B. abortus shared more fragments and had fewer nucleotide polymorphisms with B. melitensis than B. suis. The complete genomic sequence of B. abortus provides an important resource for further investigations into determinants of the pathogenicity and virulence phenotypes of these bacteria.     

Streptomycin resistance in chloramphenicol-producing strains of Streptomyces species 3022a.

Resistance to low (5 mug/ml) concentrations of streptomycin in agar media was not inherited by all of the surviving population. Outgrowth of cultures in liquid media supplemented with the antibiotic depended upon inoculum size. Antibiotic titers in the supplemented cultures decreased during incubation, and an inactive radioactive product was detected when [14C] streptomycin was used. This low-level resistance is, therefore, attributed to enzymic inactivation of the antibiotic. Growth 10 mug/ml or higher concentrations of streptomycin on agar media was due to selection of resistant variants present in the parent strain. A range of such variants existed, decreasing in frequency as their degree of resistance increased. Examination of one that was resistant to moderate concentrations of streptomycin, (25 mug/ml) and a second that was resistant to high (100 mug/ml) concentrations of streptomycin suggested that both possed ribosomes which had lower affinity for the antibiotic than those of the parent strain, and that tolerance to high levels of streptomycin was due to a resistant ribosomal system for protein biosynthesis.     

Development of a novel technique for axenic isolation and culture of thraustochytrids from New Zealand marine environments

Aims: To maintain axenic cultures of commercially important thraustochytrids, a novel procedure was developed for the isolation of zoospores and sporangium from heterotrophic seawater samples and axenic culture on solid media. Methods and Results: Thraustochytrid cultures were isolated from Whangapoua Harbour in North East New Zealand and subjected to two antibiotic and antifungal treatment regimes designed to eliminate bacteria and fungi. Antibiotic trial 1 was designed to determine the appropriate combination of antibiotics (including streptomycin/penicillin, ampicillin, rifampicin, nalidixic acid, tetracycline, gentamicin and the antifungal agent nystatin). Antibiotic trial 2 determined the optimal dosing frequency and concentration of the antibiotics, and antifungal found to be the most promising in trial 1. Axenic cultures were then spread plated onto nutrient agar containing the optimal antibiotic cocktail, and pure thraustochytrid colonies were purified on solid media using standard microbiological techniques. Conclusions: Removal of bacteria and fungi was best accomplished using a mixture of three antibiotics and one antifungal; rifampicin (300 mg l^?1), streptomycin/penicillin (25 mg l^?1) and nystatin (10 mg l^?1) were incorporated in seawater samples and incorporated into cultures every 24 h for a minimum of 2 days. Significance and Impact of the Study: The axenic isolation and culture of marine thraustochytrids from a marine habitat in New Zealand have significant implications for the biotechnological development of these potentially valuable protists. This method has global significance as it is reasonable to assume it could be used throughout the world to obtain axenic thraustochytrid cultures.     

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.     

Transformation of Clostridium perfringens L forms with shuttle plasmid DNA.

L-form (L-phase) cultures of Clostridium perfringens were tested for their transformability with plasmid DNA. Three L-form strains were transformable, but one, strain L-13, was superior to the others. This strain was easily and reproducibly transformed with previously described shuttle vectors which were derived from either C. perfringens or Escherichia coli. Strain L-13 was transformable by a variety of methods, and a new micromethod worked well under both aerobic and anaerobic conditions. The maximal number of transformants was attained after strain L-13 was exposed for 4 h to the transforming DNA and polyethylene glycol. Viable counts determined in tubes of semisolid brain heart infusion medium containing 10% sucrose, with or without 2 micrograms of tetracycline per ml, showed a transformation rate of 3.9 X 10(-5) (transformants per viable cells).     

Phage Typing Reactions on Brucella Species

The nature of the phage typing reactions on Brucella species was determined by rates of adsorption and infection, one-step growth experiments, and susceptibility to lysis from without. The highest rates of adsorption and infection were obtained on smooth B. abortus cultures, and large clear plaques were produced. One or a few phage particles per B. neotomae cell killed about one-half of the cells, but some went through an infective cycle and released mature phage that resulted in production of small clear plaques. With B. suis, more phage particles per cell were required to kill, replication did not occur, and plaques were not observed. Still greater numbers of phage particles were required to cause some inhibition of growth of B. melitensis lawns. Rough Brucella cultures and species, such as B. ovis and B. canis, were not affected by the highest concentrations of phage. B. abortus cultures of intermediate colonial morphology adsorbed phage, but only a few infected cells (after a delayed latent period) released mature phage. An infected culture or colony appeared normal until spontaneous phage mutants appeared which could penetrate the cell wall more effectively than the parent phage. The mutant phage multiplied more rapidly, and the colony changed to a sticky white form.     

The ultrastructural organization of Brucella L forms and revertant cultures

The submicroscopic organization of Brucella cells in the process of L-transformation and reversion has been studied. As revealed in this study, at its initial stages L-transformation is accompanied by the loss of cell-wall peptidoglycan and by considerable polymorphism of Brucella cells. Further stages are characterized by the presence of a great number of closed annular membrane structures both in the cytoplasm and outside the cells. At late stages of L-transformation the destruction of the cytoplasm and the cells has been found to occur. In revertant cultures the restoration of the cell wall has been noted.