Eltor型霍乱弧菌L型的诱导及形态学观察

本文主要介绍Eltor型霍乱弧菌在液体和固相的青霉素诱导下形成L型的过程及其菌落和菌体的形态学改变。Eltor型霍乱弧菌在被诱导3—5次即可出现典型的L型菌落。本试验共诱导10次,获得稳定的L型菌株,菌落形态除油煎蛋样外,还可见到双重心样菌落、靶环样菌落、纤维型菌落。菌体的正常状态为徽弧状,经青霉素作用后,微弧状消失,膨大变直,继尔变成杆状和杆状长链。菌落中心多为相互盘绕、交织成网状的丝状体。     

泌尿系细菌L型感染致病性临床研究

目的探讨泌尿系反复感染致病菌的发病特点及细菌L型感染的致病性,为临床诊治提供科学依据。方法将127例清洁中段尿培养确诊的反复发作肾盂肾炎患者,根据致病菌分为细菌型组、细菌伴L型组和细菌L型组,健康体检者为对照组,分析4组患者尿N-乙酰-β-D-氨基葡萄糖苷酶(NAG)、尿视黄醇结合蛋白(RBP)水平变化。结果泌尿系感染各组尿NAG和尿RBP较正常对照组均有不同程度升高,差异有非常显著性(P0.01),单纯细菌L型感染患者较细菌型及细菌伴L型患者稍低,但细菌型、细菌伴L型及细菌L型3组之间差异无显著性(P0.05)。结论细菌L型在泌尿道感染反复发作中占重要地位,泌尿系细菌L型感染具有致病性。     

Molecular detection and beta-glucuronidase expression of gus-marked Bacillus subtilis L-form bacteria in developing Chinese cabbage seedlings

AIM: To detect L-form bacteria in developing Chinese cabbage seedlings. METHODS AND RESULTS: Stable Bacillus subtilis L-forms were genetically modified to express the gus gene (encoding beta-glucuronidase). Germinated seeds of Chinese cabbage were soaked in mannitol based suspensions of the L-form bacteria or with mannitol alone and after washing were grown in aseptic conditions on plant growth medium. Histochemical staining of beta-glucuronidase activity (X-gluc) and Polymerase Chain Reaction (PCR) detection of the gus gene were achieved in the L-form associated seedlings. beta-Glucuronidase was localized in discrete spots, mainly in the roots with staining, and was also observed in the cotyledons and base of stems. Correlation was observed between PCR detection of the gus gene and histochemical staining with detection in similar tissues. Stable L-form bacteria were non-culturable after their association with plant material. CONCLUSIONS: The gus reporter gene system with its associated histological staining for enzyme activity was used successfully for detecting B. subtilis L-form bacteria in plant material. SIGNIFICANCE AND IMPACT OF THE STUDY: These molecular marked L-forms should provide a specific and sensitive technique for detecting L-form bacteria in planta and offer a method for further understanding the L-form/plant association.     

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.     

尿路感染患者尿中细菌L型培养的意义

目的通过对尿路感染患者尿标本中的细菌（普通型和L型）检测,以确定L型菌检测的临床价值。方法采用一般培养法和特殊（L型菌）培养法对临床标本检测。结果364份标本中一般培养法检出细菌112株,阳性率为30．8％,而特殊培养法共培养细菌及L型菌共162株,细菌总检出率为44．5％,其中单独检出L型菌50株。结论细菌在一定条件下可演变成L型菌,增加临床标本中L型菌的培养可明显提高尿路感染的细菌阳性检出率。     

An ELISA for the detection of Bacillus subtilis L-form bacteria confirms their symbiosis in strawberry

AIMS: To develop an ELISA for the detection of antigens derived from stable Bacillus subtilis L-form bacteria and to detect these in plants injected with L-form bacteria. METHODS AND RESULTS: A sandwich ELISA was developed and its specificity was investigated using L-forms and cell-walled forms of B. subtilis, different Bacillus species and a range of bacteria isolated from glasshouse-grown strawberry plants. The detection limits of the ELISA were approximately 10(3) viable cells ml(-1) for L-forms compared with 10(7) viable cells ml(-1) for cell-walled forms. Results showed that L-forms survived and moved within strawberry tissues injected with L-form bacteria. CONCLUSION: An ELISA that selectively detects B. subtilis L-form bacteria was developed and shown to confirm the presence of L-forms in plants. SIGNIFICANCE AND IMPACT OF THE STUDY: This will be a valuable rapid method to further studies on L-form plant interactions.     

Preliminary results of a bacteriologic study of the blood and urine of children with nephritis]

Single examination of the urine and blood by the method described in this paper was carried out in 14 children suffering from glomerulonephritis. L-form cultures were isolated in 11 cases. The cultures were isolated both at the active phase and during the remission. Three L-form cultures reverted into streptococci spontaneously. The revertants showed a sharp difference from the L-forms by the cell morphology and also by the colour and morphology of the colonies.     

细菌L型的厌氧诱导和培养

厌氧条件下以羧卡青霉素诱导金黄色葡萄球菌、大肠杆菌和蜡样芽胞杆菌形成Ｌ型,观察细菌Ｌ型在厌氧条件下的形成、形态、生长及时渗透压的敏感性等特性。结果表明：蜡样芽胞杆菌在厌氧条件下不能形成Ｌ型或其Ｌ型在厌氧条件下亦不能返祖。金黄色葡萄球菌和大肠杆菌在厌氧条件下虽能诱生Ｌ型,但形成丝状体的构成Ｌ型菌落难以传代培养,厌氧培养未见Ｌ型圆球体和典型Ｌ型油煎蛋样菌落。金黄色葡萄球菌Ｌ型在含１％～１０％ＮａＣｌ的Ｌ型培养基上可生长形成Ｌ型菌落或非菌落形式存在的Ｌ型巨形体;大肠杆菌和蜡样芽胞杆菌的Ｌ型在含２％～６％ＮａＣｌ的Ｌ型培养基上可生长形成Ｌ型菌落或非菌落形式存在的Ｌ型巨形体。涂片染色或返祖试验证实细菌Ｌ型在含０．５％ＮａＣｌ的Ｌ型培养基或常规细菌学培养基上亦可生存。非菌落性Ｌ型巨形体和丝形体是细菌Ｌ型在琼脂培养基上广泛的存在形式。     

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.     

Unique biological properties of Mycobacterium tuberculosis L-form variants: impact for survival under stress

Bacteria can, under certain conditions, enter into a cell-less state known as L-form conversion. This phenomenon is universal, but also recognized with difficultly by microbiologists. The current study addresses several aspects concerning the ability of tubercle bacilli to use L-form conversion as a unique adaptive strategy to survive and reproduce under unfavorable conditions. Nutrient starvation of M. tuberculosis in vitro followed by passages in Middlebrook 7H9 semisolid medium was used for stress induction and the selective isolation of mycobacterial L-form variants. Light and electron microscopy images evidence the peculiar characteristics of mycobacterial L-forms. For example, mycobacterial L-forms were observed to lose their acid-fastness and change their morphology. In addition, wide morphological variability, the presence of large and elementary bodies, coccoids and small granular forms, as well as the appearance of unusual modes of irregular cell division were observed. Unlike classical tubercle bacilli, L-form variants grew and developed typical "fried-egg" colonies faster. L-forms were verified as M. tuberculosis by spoligotyping. The results provide insights into the nature of L-form phenomena in M. tuberculosis and link them to the mechanisms allowing mycobacterial survival under stress.     

假结核耶氏菌在实验条件下细菌L型的观察

细菌L型广泛分布在生物体内外,并为细菌生命周期的一相。本文应用假结核耶氏菌(Yersinia pseudotuberculosis)作为研究材料,在L型培养基中无抗生素存在情况下连续传代,观察到:正常型(杆状)假结核菌出现圆球体、丝状体,继续传代又返祖为正常型。实验表明,细菌L型是细菌生命周期中的一相。     

15年前后细菌L型临床耐药性的变迁

目的检测15年前后细菌L型临床耐药性的变迁,分析影响因素,指导临床用药。方法对新乡市中心医院15年来临床检出的常见细菌L型菌株逐个进行药敏监测,逐年进行耐药性变化分析研究。结果1990年至1999年细菌L型常见菌株耐药性逐年上升,特别是青霉素类大部耐药;2000年至2005年,耐药性趋势变缓,青霉素类耐药率有所下降。结论合理应用抗生素,不滥用抗生素是细菌L型耐药性变迁的重要原因,药物敏感试验是良好的监测方法。     

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.     

Serum inhibits penicillin-induced L-form growth in Staphylococcus aureus: a note of caution on the use of serum in cultivation of bacterial L-forms.

Heat-inactivated horse serum inhibited penicillin-induced L-form colony formation in Staphylococcus aureus when included in an osmotically stabilized culture medium. Most, perhaps all, L-form colonies that appeared with low frequencies on the serum-supplemented medium were of the penicillin-independent, stable type. This relationship must be taken into account when use of serum is considered for L-form cultivation.     

Survival of Escherichia coli under lethal heat stress by L-form conversion

Transition of bacteria to cell wall deficient L-forms in response to stress factors has been assumed as a potential mechanism for survival of microbes under unfavorable conditions. In this article, we provide evidence of paradoxal survival through L-form conversion of E. coli high cell density population after lethal treatments (boiling or autoclaving). Light and transmission electron microscopy demonstrated conversion from classical rod to polymorphic L-form shape morphology and atypical growths of E. coli. Microcrystal formations observed at this stage were interpreted as being closely linked to the processes of L-form conversion and probably involved in the general phenomenon of protection against lethal environment. Identity of the morphologically modified L-forms as E. coli was verified by species specific DNA-based test. Our study might contribute to a better understanding of the L-form phenomenon and its importance for bacterial survival, as well as provoke reexamination of the traditional view of killing strategies against bacteria.     

The symbiosis of Bacillus subtilis L-forms with Chinese cabbage seedlings inhibits conidial germination of Botrytis cinerea

AIMS: To establish whether germination of Botrytis cinerea was affected by the symbiosis of Bacillus subtilis L-form bacteria with Chinese cabbage. METHODS AND RESULTS: Germinating seeds of Chinese cabbage were co-cultivated with either L-forms of Bacillus subtilis or 5% (w/v) mannitol by soaking for 3 h. Seeds were then washed in sterile water, sown on a minimal medium and incubated in controlled conditions. L-form symbiosis was detected over a time course by ELISA. Conidial germination of Botrytis cinerea was significantly reduced on cotyledonous leaves of L-form-treated plants compared with controls. CONCLUSIONS: Symbiosis of B. subtilis L-form bacteria during seed germination of Chinese cabbage inhibits conidial germination in plants on subsequent exposure to Botrytis cinerea. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first account of plant symbiosis with L-form bacteria showing antagonism to a fungal plant pathogen. This has promising implications for the use of this L-form as a biocontrol agent.     

Induction of L-Phase Variant from Protoplast of Staphylococcus aureus

Protoplasts of Staphylococcus aureus 209P and Cowan 1 were induced by treatment with lysostaphin. These protoplasts were sensitive to detergent, a low concentration of sodium chloride and low temperature. Almost all protoplast cells spread on CLYS agar medium (casein hydrolysate, yeast extract, Na-lactate, and NaCl) formed typical L-form colonies. Horse serum (0.25%) and Mg²⁺ (109 mm) are essential factors for formation of the L-form colonies of 209P. In the case of Cowan 1, Mg²⁺ was not required. The active factor(s) in horse serum was heat-resistant and protein in nature.     

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.     

Regeneration of protoplasts of Bacillus subtilis 168 and closely related strains

Regeneration of protoplasts to bacilli was attempted in several strains of Bacillus closely related to Bacillus subtilis 168. On DM3 and similar media using succinate as osmotic support, only B. subtilis 168 and Bacillus natto ATCC 15245 were able to regenerate. Media containing mannitol as osmotic support, and agar as gelling agent gave rise to L-form colonies with Bacillus licheniformis NCTC 6346. Many of the L-form colonies were able to regenerate to the bacillary form when plated on the mannitol medium solidified with gelatin. All of the Bacillus species tested were able to regenerate on the latter medium at rates sufficient to allow protoplast transformation and fusion experiments.     

Filterability of Streptococcal L-Forms

The filterability of the broth-grown stable L-form derived from Streptococcus faecium F24 was tested by filtration under the influence of varying amounts of applied pressure. A decrease in the pore size of the filter resulted in a corresponding decrease in viable count, but no major effect was noted due to the different pressures applied. Serial filtration of a deoxyribonuclease-treated L-form culture in the mid-logarithmic phase of growth resulted in recovery of viable L-forms from the 0.45-mum filtrate but not from the 0.22-mum filtrate. It is possible that disruption of the L-form bodies with release of small viable elements had occurred. Protoplasts, diluted in an osmotic stabilizer, were filtered similarly; L-forms could be grown from the filtrate passing through the filters of 0.45 mum or greater. Filtration of the parent streptococci gave rise to streptococcal colonies from the 1.2-mum filtrate only.