临床常见L型细菌的分布及耐药性变化分析

目的了解临床主要L型致病菌的分布以及对目前常用性变化趋势.方法对2000年7月～2003年7月临床分离的753株药性进行回顾性分析.结果金黄色葡萄球菌、大肠埃希菌、最常见,它们共同对阿米卡星霉素敏感,敏感率达77%以上,共同对罗红霉素、氨苄西林、克林霉素、阿奇霉素耐药,耐药率均在45%以上.结论与我院5年前及10年前L型细菌耐药性对比,抗生素耐药性已发生变迁.掌握L型细菌耐药性的动态,合理使用抗生素.     

Antimicrobial activities of daunorubicin and adriamycin derivatives on bacterial and protoplast type L-form cells of Bacillus subtilis 170, Escherichia coli B, and Proteus mirabilis VI. Structure--activity relationship

The antibacterial activity of ten N-alkylated derivatives of daunorubicin and adriamycin as well as of 5-iminodaunorubicin has been tested by using Bacillus subtilis 170, Escherichia coli B, and Proteus mirabilis VI and their stable protoplast type L-forms in an agar diffusion test. Eight of the substances showed similar activities against B. subtilis and the L-forms of all test organisms, but no activity against the bacterial forms of E. coli and P. mirabilis. The cell wall of these gram-negative bacteria is responsible for this resistance by not allowing the antibiotics to enter the cells. The piperidino compound N-(CH2)5 daunorubicin shows 2-4 times higher activity against B. subtilis and all L-forms in comparison to daunorubicin and the other derivatives. Five of the substances were inactive against all test strains. Their inactivity seems to be associated with the larger substituents at the C-3' position. Relations between molecular structure and activity are discussed considering data about the interaction with DNA and the antitumor activity. Stable protoplast type L-forms and their bacterial forms represent a suitable and effective test system to screen for more effective substances and to get more information about their mode of action.     

The analysis of cell division and cell wall synthesis genes reveals mutationally inactivated ftsQ and mraY in a protoplast-type L-form of Escherichia coli

Cell division and cell wall synthesis are tightly linked cellular processes for bacterial growth. A protoplast-type L-form Escherichia coli, strain LW1655F+, indicated that bacteria can divide without assembling a cell wall. However, the molecular basis of its phenotype remained unknown. To establish a first phenotype-genotype correlation, we analyzed its dcw locus, and other genes involved in division of E. coli. The analysis revealed defective ftsQ and mraY genes, truncated by a nonsense and a frame-shift mutation, respectively. Missense mutations were determined in the ftsA and ftsW products yielding amino-acid replacements at conserved positions. FtsQ and MraY, obviously nonfunctional in the L-form, are essential for cell division and cell wall synthesis, respectively, in all bacteria with a peptidoglycan-based cell wall. LW1655F+ is able to survive their loss-of-functions. This points to compensatory mechanisms for cell division in the absence of murein sacculus formation. Hence, this L-form represents an interesting model to investigate the plasticity of cell division in E. coli, and to demonstrate how concepts fundamental for bacterial life can be bypassed.     

Ellipticines and 9-acridinylamines as inhibitors of D-alanine:D-alanine ligase

D-Alanine:D-alanine ligase (Ddl), an intracellular bacterial enzyme essential for cell wall biosynthesis, is an attractive target for development of novel antimicrobial drugs. This study focused on an extensive evaluation of two families of Ddl inhibitors encountered in our previous research. New members of both families were obtained through similarity search and synthesis. Ellipticines and 9-acridinylamines were both found to possess inhibitory activity against Ddl from Escherichia coli and antimicrobial activity against E. coli and Staphylococcus aureus. Ellipticines with a quaternary methylpyridinium moiety were the most potent among all studied compounds, with MIC values as low as 2 mg/L in strains with intact efflux mechanisms. Antimicrobial activity of the studied compounds was connected to membrane damage, making their development as antibacterial drug candidates unlikely unless analogues devoid of this nonspecific effect can be discovered.     

Isolation,structural and functional characterization of Staphylococcus aureus protoplasts obtained using lysoamidase

The action of the lysoamidase bacteriolytic complex on Staphylococcus aureus VKM B-209P cells has been studied to obtain protoplasts. The cells in the midlogarithmic phase were the most sensitive to lysoamidase action. It led to local destruction of cell wall due to hydrolysis of the peptidoglycan. Protoplast formation occurred in two steps in the presence of 1 M sucrose. First, osmotically fragile spheroplasts were formed. Then, the protoplasts were released from the destructed cell wall. The protoplast yield was about 80%. The protoplasts preserved the intact ultrastructure and were able to synthesize peptidoglycan fibrillae. Mainly the spheroplasts that maintained the cell-wall residues reversed into bacterial forms. The protoplasts had respiratory activity similar to cells. Respiration of cells and protoplasts was stimulated by various substrates. High rates of oxygen consumption were observed with -glycerophosphate and ethanol as substrates.     

Nature of the IR spectra of intact cells of Gram-negative microorganisms

The character of changes in the infrared spectra of E. coli in the process of their mechanical disintegration has been studied. The destruction of E. coli cell structures has been shown to produce no changes in the optical density of the main analytical absorption bands in infrared spectra. This fact suggests that the infrared absorption spectra of E. coli are the sum of the spectra of all chemical components of the cell, which is confirmed by the infrared spectral study of E. coli cell-wall preparations. Similar results have been obtained in the study of the preparations of B. pertussis cell walls, protoplasts and intact cells.     

STREPTOCOCCAL L-FORMS IV. : Comparison of the Metabolic Rates of a Streptococcus and Derived L-Form.

Panos, Charles (University of Illinois College of Medicine, Chicago, and Albert Einstein Medical Center, Philadelphia, Pa.). Streptococcal L-forms. IV. Comparison of the metabolic rates of a Streptococcus and derived L-form. J. Bacteriol. 84:921-928. 1962.-Glycolytic rates of hexoses, amino sugars, pentoses, two-carbon compounds, and certain intermediates of glycolysis and the adaptive response to glucose of a group A Streptococcus and its derived L-form were compared. It was found that removal of the streptococcal cell wall did not result in the loss of the homolactic characteristic of the parent coccus or in a marked increase in the metabolism of certain glycolytic intermediates by the L-form. It was shown that (i) a major difference exists between the coccus and its L-form in the metabolism of glucosamine and N-acetylglucosamine; (ii) apparently, a loss of selectivity and internal control occurred in the transformation to the L-form; and (iii) this form, unlike the parent coccus, displayed an adaptive response to glucose. These data were not the result of an internal loss of essential cofactors or enzymes by diffusion from within the L-form. Nor could they be accounted for by dry-weight differences due to loss of the streptococcal cell wall. Finally, it was observed that the sonically disintegrated L-form in 0.5 m NaCl was capable of a glycolytic activity of 46% of that of the total intact culture. These data suggest that the conversion of a streptococcus to the L-form is accompanied by an alteration in carbohydrate metabolism as well as the loss of the cell wall. Previously reported data are in agreement with these findings and support the conclusion that the resulting form is not merely a bacterial cell without a rigid cell wall.     

Alpha-helical cationic antimicrobial peptides: relationships of structure and function

Antimicrobial peptides (AMPs), with their extraordinary properties, such as broad-spectrum activity, rapid action and difficult development of resistance, have become promising molecules as new antibiotics. Despite their various mechanisms of action, the interaction of AMPs with the bacterial cell membrane is the key step for their mode of action. Moreover, it is generally accepted that the membrane is the primary target of most AMPs, and the interaction between AMPs and eukaryotic cell membranes (causing toxicity to host cells) limits their clinical application. Therefore, researchers are engaged in reforming or de novo designing AMPs as a ‘single-edged sword’ that contains high antimicrobial activity yet low cytotoxicity against eukaryotic cells. To improve the antimicrobial activity of AMPs, the relationship between the structure and function of AMPs has been rigorously pursued. In this review, we focus on the current knowledge of α-helical cationic antimicrobial peptides, one of the most common types of AMPs in nature.     

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.     

Comparison of freezing tolerance in cultured plant cells and their respective protoplasts

The possibility that the plant cell wall influences the severity of freezing injury was examined by comparing the freeze stress response of intact cells and protoplasts from four different suspension cultures. In no case did the intact cells suffer more injury than the respective wall-less protoplasts, showing that mechanical strain imposed by the cell wall during freeze-thaw stress is not a major determinant of injury. For three of the four species studied, cells from which the wall was removed showed significantly greater freezing injury, indicating that the plant cell wall may have a protective role. Other researchers have suggested that cell wall rigidity may minimize freezing injury by slowing freeze-induced loss of cell water. We found that decreased enzyme digestibility (perhaps indicating greater rigidity) of cell walls accompanied cold acclimation in various tissues. These results provide impetus to research which will characterize low-temperature-induced cell wall modification in cold acclimating tissues.     

N-acetylmuramyl-L-alanine amidase of Bacillus licheniformis and its L-form

A cell wall lytic enzyme has been demonstrated to be a component of the membrane of Bacillus licheniformis NCTC 6346 and an l-form derived from it. The lytic enzyme, characterized as an N-acetylmuramyl-l-alanine amidase, is solubilized from membranes by nonionic detergents. Ionic detergents inactivate the enzyme. In the bacterium the specific activities of amidase and d-alanine carboxypeptidase in mesosomes are approximately 65% of those in membranes. Selective transfer of lytic enzyme from nongrowing L-forms, L-form membranes, and protoplasts to added walls occurred after mixing, and 31 to 77% of the enzyme lost from L-form membranes was recovered on the walls. Membranes isolated from L-forms growing in the presence of added walls contained as little as 13% of the amidase found in membranes of a control culture. These results have been interpreted as showing that in vivo the amidase is "bound" to the surface of the bacterial cell membrane in such a location that it can be readily accessible to the cell wall.     

Bioconversions of a macrolide glycoside by growing L-form cells of Streptomyces hygroscopicus

Growing L-form cells of Streptomyces hygroscopicus were shown to carry out 3-0-acylation and 14-C-hydroxylation of a macrolide glycoside suggesting that both types of bioconversion do neither require the intact cell wall nor the periplasmic space.     

Development of protoplasts of Streptomycetes on media favoring the regeneration and formation of L-forms

The protoplasts of three Streptomyces species and their regenerative ability were studied using light microscopy. When Streptomyces lividans and S. erythraeus protoplasts are cultivated on regeneration media, their regeneration is not synchronous during the first day; some protoplasts revert to yield the mycelial form and also L-forms of these cultures are produced. If the protoplasts are transferred to a medium inducing L-forms, they grow and multiply for a long time with the production of L-form colonies. This process is maintained if S. lividans L-form cells are passaged on the medium inducing L-forms, but the protoplasts revert to yield the mycelial form on the regeneration medium.     

Effect of antibacterial factors and cell metabolism biostimulants on bacterial luminescence

Bioluminescence was used as an index of effects of a series of factors (differing in origin and structure) on cell metabolism. Luminescence of intact cells of Escherichia coli lum+ (a genetically modified strain) was measured on exposure to antibiotics, probiotics, phages, and biostimulators. The sensitivity of E. coli lum+ to antibiotics correlated with the antibiotic-induced luminescence quenching. Bioluminescence-based assessment of the antagonistic activity of the main probiotics (bificoll, bifidobacterin, acylact, colibacterin, and a composite probiotic preparation), each taken at a concentration of 1 dose/ml demonstrated that bacterial luminescence was inhibited by 75-99.9% (exposure time, 30 min). Bioluminescence changes reflected cell damage associated with phage infection. It was shown that bioluminescence stimulation could be used as an index of the effect of immunomodulators (olexin and vermin) on cell metabolism in bacterial cultures.     

Experimental inhibition of cell wall formation and of reversion inNadsonia elongata andSchizosaccharomyces pombe protoplasts

Summary The growth, cell wall regeneration, and the reversion of the protoplasts ofNadsonia elongata andSchizosaccbaromyces pombe cultivated in nutrient media containing snail enzyme was studied by light and electron microscopy. The protoplasts grew in the presence of snail enzyme and an incomplete cell wall composed of fibrils was formed on their surface. Thus, the presence of snail enzyme inhibited the completion of cell wall structure and, consequently, the reversion of the protoplasts to normal cells. The transfer of these protoplasts to medium free from snail enzyme led first to the completion of the cell wall and then to the reversion of the protoplasts to normal cells. The reported experiments confirmed that the regeneration of the complete cell wall preceded the protoplast reversion.     

慢性肾盂肾炎L型细菌感染及耐药分析

目的了解L型细菌在慢性肾盂肾炎的感染及耐药状况。方法对71例患者清洁中段尿做普通细菌培养(B型)、L型细菌培养(L型)及耐药分析。结果细菌阳性率为77.5%,其中单独L型阳性率为49.3%、B型与L型混合感染为15.5%,而B型阳性率仅为12.7%。主要是大肠埃希菌,其次是葡萄球菌;青霉素及头孢噻肟均有较高的耐药率(88.9%及73.6%)。结论L型细菌在慢性肾盂肾炎感染中占主导,β-内酰胺类药物有较高的耐药性,临床治疗应据药敏结果合理选择及时调整抗生素。     

Effect of polyene antibiotics on bacterial protoplasts

The carbonyl-conjugated pentaenes flavofungin, nigrofungin and flavopentin exhibit considerable lytic activity toward Micrococcus lysodeikticus and Bacillus megaterium protoplasts. The antibiotics at concentrations of 5 to 14 microgram/ml cause lysis of 50% of the protoplasts within 15 min of their incubation. The antibiotics inhibit the activity of NADH oxidase and malate oxidase by 50% in the lysates of Micrococcus lysodeikticus and Bacillus megaterium protoplasts at concentrations of 30 to 50 microgram/ml; preincubation of the lysates with the antibiotics intensify the inhibiting action of the polyenes. Growth of the bacteria is inhibited when the minimal concentration of the polyenes is 75 to 100 microgram/ml. Interaction of the polyenes with bacterial membranes lacking sterols indicates that resistance of at least some bacteria to polyenes is caused by impermeability of the cell wall for these substances rather than by the absence of sterols in the membranes.     

Lysis of Saccharomyces cerevisiae with 2-deoxy-2-fluoro-D-glucose, an inhibitor of the cell wall glucan synthesis

The effect of a synthetic glucose analogue, 2-deoxy-2-fluoro-d-glucose (FG) on growth and glucose metabolism of Saccharomyces cerevisiae was studied. The addition of FG (0.005-0.05%) to a 2% glucose medium resulted in reduction of the initial growth rate and, after several hours, in a complete cessation of the culture growth. These two events were due to extensive lysis of the population which continued long after the period when no more growth was recorded. Electron microscope examination of lysed cells showed that the lysis was a consequence of a dissolution of the cell walls. FG inhibited to a similar extent the initial growth rate and the incorporation of radioactivity from labeled glucose into growing population. The inhibition of radioactivity incorporation from glucose by growing protoplasts was much less. The yeast was found to be extremely FG sensitive whenever the synthesis of new cell wall material was involved. All observations imply that FG interferes mainly with the cell wall formation of S. cerevisiae. A comparison of the FG effects on metabolic activity of protoplasts, simultaneous secretion of mannan-proteins into the growth medium, and the formation of glucan fibrils on the surface of protoplasts demonstrated that the cell wall glucan synthesis is the most FG-sensitive process and evidently the growth-limiting factor in intact cells. FG-resistant cells were selected during growth experiments. They exhibited an altered mode of cell division when grown in the presence of FG.     

Effects of non-ionogenic surface active compounds on yeast protoplasts]

The lytic effect of non-ionogenic surface active compounds (SAC), based on polyoxyethylated fatty acids and alkylphenols on the yeast protoplast cytoplasmic membranes and the extracting ability of the SAC with respect to intracellular proteins of intact yeast cells, were studied. It was shown that the lytic activity of the SAC under study depends on the overall effect of the size of their hydrophobic and hydrophylic fragments rather than on the level of the hydrophylic-lypolytic equilibrium of SAC. The absence of correlation between the lytic activity and the extracting ability of SAC is accounted for by the differences in the mechanisms of membrane degradation under the action of SAC on the protoplasts and intact cells. The data obtained support the previously made assumption that the correlation between the size of SAC mycelles and that of the cell wall pores is the limiting factor of the SAC induced protein extraction from the intact cells.