Crucial role for central carbon metabolism in the bacterial L-form switch and killing by ß-lactam antibiotics.

The peptidoglycan cell wall is an essential structure for the growth of most bacteria. However, many are capable of switching into a wall-deficient L-form state in which they are resistant to antibiotics that target cell wall synthesis under osmoprotective conditions, including host environments. L-form cells may have an important role in chronic or recurrent infections. The cellular pathways involved in switching to and from the L-form state remain poorly understood. This work shows that the lack of a cell wall, or blocking its synthesis with ß-lactam antibiotics, results in an increased flux through glycolysis. This leads to the production of reactive oxygen species from the respiratory chain, which prevents L-form growth. Compensating for the metabolic imbalance by slowing down glycolysis, activating gluconeogenesis or depleting oxygen enables L-form growth in Bacillus subtilis, Listeria monocytogenes and Staphylococcus aureus. These effects do not occur in Enterococcus faecium, which lacks the respiratory chain pathway. Our results collectively show that when cell wall synthesis is blocked under aerobic and glycolytic conditions, perturbation of cellular metabolism causes cell death. We provide a mechanistic framework for many anecdotal descriptions of the optimal conditions for L-form growth and non-lytic killing by ß-lactam antibiotics.

Lysozyme Counteracts ß-Lactam Antibiotics by Promoting the Emergence of L-Form Bacteria.

ß-lactam antibiotics inhibit bacterial cell wall assembly and, under classical microbiological culture conditions that are generally hypotonic, induce explosive cell death. Here, we show that under more physiological, osmoprotective conditions, for various Gram-positive bacteria, lysis is delayed or abolished, apparently because inhibition of class A penicillin-binding protein leads to a block in autolytic activity. Although these cells still then die by other mechanisms, exogenous lytic enzymes, such as lysozyme, can rescue viability by enabling the escape of cell wall-deficient "L-form" bacteria. This protective L-form conversion was also observed in macrophages and in an animal model, presumably due to the production of host lytic activities, including lysozyme. Our results demonstrate the potential for L-form switching in the host environment and highlight the unexpected effects of innate immune effectors, such as lysozyme, on antibiotic activity. Unlike previously described dormant persisters, L-forms can continue to proliferate in the presence of antibiotic.

L-form transformation phenomenon in Mycobacterium tuberculosis associated with drug tolerance to ethambutol.

OBJECTIVE/BACKGROUND: Cell wall-deficient bacterial forms (L-forms) may occur along with resistance to factors that trigger their appearance. It is of interest to study the relationship between the L-form transformation of Mycobacterium tuberculosis and the exhibition of drug tolerance to ethambutol (EMB), an inhibitor of cell wall synthesis. METHODS: L-form variant was produced from a sensitive EMB strain of M. tuberculosis through a cryogenic stress treatment protocol and was subsequently cultivated in Middlebrook 7H9 semisolid medium, containing EMB in a minimal inhibitory concentration of 2mg/L. Susceptibility to EMB of the parental strain and its L-form variant was evaluated phenotypically and using polymerase chain reaction-restriction fragment length polymorphism assay targeting a mutation in the embB306 gene fragment. RESULTS: In contrast to the sensitivity to EMB of the parental strain, its L-form variant showed phenotypic resistance to high concentrations of EMB (16mg/L), but the mutation in embB306 was not found. Electron microscopy observation of the L-form variant showed a heterogenic population of bacteria, with different degrees of cell wall deficiency, as well as cells of protoplastic type without cell walls. Of special interest were the observed capsule-like structures around the L-form cells and the biofilm-like matrix produced by the L-form population. CONCLUSION: We suggest that the expression of phenotypic resistance to EMB in M. tuberculosis can be associated with alterations or loss of cell walls in L-form bacteria, respectively, which results in a lack of a specific target for EMB action. In addition, production of capsule-like structures and biofilm matrix by L-forms could contribute to their resistance and survival in the presence of antibacterial agents.

[Induction of L-forms of Mycobacterium abscess with isoniazid].

OBJECTIVE: To explore the induction of L-forms of Mycobacterium abscess using isoniazid. METHODS: Mycobacterium abscess were cultured in aqueous culture media in the presence or absence of 128 µg/ml isoniazid. The culture media containing isoniazid were filtered with 0.45 µm membrane, and the filtrate was subcultured in nutrient agar media for reversion. The isoniazid-free and isoniazid-containing media and the reversion bacteria were observed for cell wall integrity by cell wall staining and transmission electron microscopy, and the microstructures of the cell surfaces were observed by scanning electron microscopy. The isoniazid-containing culture was subcultured in L-form agar media, and the isoniazid-free culture and the reversed bacteria in nutrient agar media to observe the colony morphology. The reversed and non-induced bacteria were identified for 16S rDNA. RESULTS: The bacteria induced with 128 µg/ml isoniazid showed cell wall defect, presenting with a spherical cell morphology and typical fried egg-like colonies in L-form agar media, while in isoniazid-free cultures, the cells showed intact cell walls with rod-like shapes and round colony morphologies on nutrient agar. The reversed bacteria, showing also intact cell walls with rod-like shapes and round colonies on nutrient agar, had identical 16S rDNA with the non-induced bacteria. CONCLUSION: Isoniazid can induce the L-forms of mycobacterium abscess for use in studies of multidrug resistance and treatment of the bacteria.

Cell envelope stress response in cell wall-deficient L-forms of Bacillus subtilis.

L-forms are cell wall-deficient bacteria that can grow and proliferate in osmotically stabilizing media. Recently, a strain of the Gram-positive model bacterium Bacillus subtilis was constructed that allowed controlled switching between rod-shaped wild-type cells and corresponding L-forms. Both states can be stably maintained under suitable culture conditions. Because of the absence of a cell wall, L-forms are known to be insensitive to ß-lactam antibiotics, but reports on the susceptibility of L-forms to other antibiotics that interfere with membrane-anchored steps of cell wall biosynthesis are sparse, conflicting, and strongly influenced by strain background and method of L-form generation. Here we investigated the response of B. subtilis to the presence of cell envelope antibiotics, with regard to both antibiotic resistance and the induction of the known LiaRS- and BceRS-dependent cell envelope stress biosensors. Our results show that B. subtilis L-forms are resistant to antibiotics that interfere with the bactoprenol cycle, such as bacitracin, vancomycin, and mersacidin, but are hypersensitive to nisin and daptomycin, which both affect membrane integrity. Moreover, we established a lacZ-based reporter gene assay for L-forms and provide evidence that LiaRS senses its inducers indirectly (damage sensing), while the Bce module detects its inducers directly (drug sensing).

Membrane aggregation and perturbation induced by antimicrobial peptide of S-thanatin.

Thanatin, a 21-residue peptide, is an inducible insect peptide. In our previous study, we have identified a novel thanatin analog of S-thanatin, which exhibited a broad antimicrobial activity against bacteria and fungi with low hemolytic activity. This study was aimed to delineate the antimicrobial mechanism of S-thanatin and identify its interaction with bacterial membranes. In this study, membrane phospholipid was found to be the target for S-thanatin. In the presence of vesicles, S-thanatin interestingly led to the aggregation of anionic vesicles and sonicated bacteria. Adding S-thanatin to Escherichia coli suspension would result in the collapse of membrane and kill bacteria. The sensitivity assay of protoplast elucidated the importance of outer membrane (OM) for S-thanatin's antimicrobial activity. Compared with other antimicrobial peptide, S-thanatin produced chaotic membrane morphology and cell debris in electron microscopic appearance. These results supported our hypothesis that S-thanatin bound to negatively charged LPS and anionic lipid, impeded membrane respiration, exhausted the intracellular potential, and released periplasmic material, which led to cell death.

New insights into bacterial persistence in reactive arthritis.

Persistence of arthritis-triggering bacteria can cause chronization of reactive arthritis (ReA). In the evaluation of bacterial persistence in ReA, the persistence of both the triggering bacteria and also of the other bacteria residing in the foci of chronic infection, are important. Two forms of bacterial persistence, cell wall-deficient bacteria (L-forms) and bacterial biofilms, are characterized, and the possible links between these forms and ReA are revealed. Data showing the possibility of bacterial ReA triggers to enter the cell wall-deficient state and to persist in bacterial biofilms, and evidence, suggesting that cell wall-deficient bacteria and bacterial biofilms are involved in the foci of chronic infection, are discussed. The understanding of the properties of microbes when they exist in cell wall-deficient state and bacterial biofilms may expand our knowledge on the clinical value of persisting microorganisms in ReA. In conclusion, both modes of persistence, cell wall-deficient state of bacteria and bacterial biofilms, deserve rheumatologists' attention, as their investigation, applying modern standardized methods, may contribute to the elaboration of new beneficial schemes of antibacterial ReA therapy.

Unstable Escherichia coli L forms revisited: growth requires peptidoglycan synthesis.

Growing bacterial L forms are reputed to lack peptidoglycan, although cell division is normally inseparable from septal peptidoglycan synthesis. To explore which cell division functions L forms use, we established a protocol for quantitatively converting a culture of a wild-type Escherichia coli K-12 strain overnight to a growing L-form-like state by use of the beta-lactam cefsulodin, a specific inhibitor of penicillin-binding proteins (PBPs) 1A and 1B. In rich hypertonic medium containing cefsulodin, all cells are spherical and osmosensitive, like classical L forms. Surprisingly, however, mutant studies showed that colony formation requires d-glutamate, diaminopimelate, and MurA activity, all of which are specific to peptidoglycan synthesis. High-performance liquid chromatography analysis confirmed that these L-form-like cells contain peptidoglycan, with 7% of the normal amount. Moreover, the beta-lactam piperacillin, a specific inhibitor of the cell division protein PBP 3, rapidly blocks the cell division of these L-form-like cells. Similarly, penicillin-induced L-form-like cells, which grow only within the agar layers of rich hypertonic plates, also require d-glutamate, diaminopimelate, and MurA activity. These results strongly suggest that cefsulodin- and penicillin-induced L-form-like cells of E. coli-and possibly all L forms-have residual peptidoglycan synthesis which is essential for their growth, probably being required for cell division.

L-forms of Staphylococcus epidermidis induced by penicillin.

L-forms of S. epidermidis were induced at 35 degrees C with the use of an L-form medium with penicillin. The aim of this study was to evaluate the frequency of L-form induction and demonstrate whether the origin of the clinical strains affects the frequency of L-forms induction, as well as to study whether the time of action of the antibiotic has an influence on frequency of L-form induction.

[The composition and drug sensitivity of a mycobacterial population in patients with suspected tuberculosis].

By using the diagnostic material (175 sputum samples and 103 bronchoalveolar lavage fluid samples) taken from 39 patients with suspected tuberculous infection during a 2.5-month follow-up, the authors traced the time course of changes in the composition and drug sensitivity of a mycobacterial population to rifampicin. Along with the traditional microbiological studies, the latest molecular biological studies, a TB-BIOCHIP test system (enzyme immunoassay) in particular, were employed to detect the bacterial and L-transformed forms of the causative agent. A molecular biological assay was first developed to detect the drug sensitivity of L-forms of Mycobacterium tuberculosis.

[Observations of properties of the L-form of M. tuberculosis induced by the antituberculosis drugs].

OBJECTIVES: To investigate the mechanism of generation of L-form of M. tuberculosis and its significance on the development, diagnosis and treatment of tuberculosis. METHODS: M. tuberculosis was inoculated into the non-high osmotic medium with rifampin, isoniazid or ethambutol and then the L-form was observed by microscopy daily. The cultures were filtrated to get the pure cultures of stable L-form by subculture with the non-high osmotic medium and characteristics of morphology, growth, susceptibilities to the antibacterial drugs and the special gene of M. tuberculosis were observed when the pure subcultures of the L-form were isolated. RESULTS: L-form of M. tuberculosis was induced by the concentrations of routine inhibition test of rifampin, isoniazid or ethambutol. The L-form would not be susceptible to the above mentioned antituberculosis drugs but susceptible to streptomycin, erythromycin, ofloxacin, norfloxacin and others. The morphologies of L-form were irregular or spherical with single, paired or chain form, and growth under the bottom of the medium but not movement or adhere to the glass. The L-form was negative by acid-fast stain and negative or positive by Gram stain. The gene of L-form reacted with the PCR kit for the M. tuberculosis and showed the same band. CONCLUSIONS: M. tuberculosis could be killed by rifampin, isoniazid or ethambutol but also could be induced to become the L-form by these antituberculosis drugs, and it is one of the important factor that affecting the effect of treatment of the tuberculosis. The cell wall deficient variants of M. tuberculosis could be determined by the PCR of M. tuberculosis. It is recommended that the L-forms should be noticed during the treatment with the antituberculosis drugs and combination treatment with antituberculous drugs to which the L-forms were susceptible, is also very important.

Introduction of a mini-gene encoding a five-amino acid peptide confers erythromycin resistance on Bacillus subtilis and provides temporary erythromycin protection in Proteus mirabilis.

A 15-bp mini-gene was introduced into Bacillus subtilis and into stable protoplast-like L-forms of Proteus mirabilis. This mini-gene encoded the peptide MVLFV and modeled a fragment of Escherichia coli 23S rRNA responsible for E. coli erythromycin (Ery) resistance. Expression of the introduced mini-gene conferred permanent Ery resistance on B. subtilis. In L-forms of P. mirabilis, the Ery-protective effect was maintained in the course of several generations. Herewith, the mechanism of Ery resistance mediated by expression of specific short peptides was shown to exist in evolutionary distant bacteria. Three new plasmids were constructed containing the gene under study transcriptionally fused with the genes encoding glutamylendopeptidase of Bacillus licheniformis or delta-endotoxin of Bacillus thuringiensis. The Ery resistance pentapeptide (E-peptide) mini-gene served as an efficient direct transcriptional reporter and allowed to select bacillar glutamylendopeptidase with improved productivity. The mini-genes encoding E-peptides may be applied as selective markers to transform both Gram-positive and Gram-negative bacteria. The small size of the E-peptide mini-genes makes them attractive selective markers for vector construction.

[Study on pathogenicity of sputum from cavity of sputum negative patients with pulmonary tuberculosis after short course chemotherapy].

OBJECTIVE: To find out whether or not the sputa of pulmonary tuberculosis(PTB) patients with cavitation but sputum negative have pathogenicity after short course chemotherapy. METHODS: Guinea pigs were inoculated with sputa of PTB patients who were with cavitation but sputum negative after having finished short course chemotherapy. Then their body weight, enlargement of local lymph nodes and other ordinary symptom were observed. Six weeks later, pathological changes of TB in the internal organs were examined by dissecting these guinea pigs. Culture and drug resistance test of tubercle bacillus were also conducted. All of which were with negative and positive controls. RESULTS: Of the 63 cases included, 3(5%) patients' sputa resulted in tuberculous nodulation varying in amount in lung, liver and spleen of these guinea pigs, and the culture for tubercle bacillus of these sputa was positive too. CONCLUSIONS: 5% of sputum collected from PTB patients with cavitation but sputum negative still show pathogenicity after short course chemotherapy. For the cases with drug-resistant PTB and slow sputum negative conversion, the treatment should be prolonged and tubercle bacilli in their sputa should be monitored.

Sphingosines: antimicrobial barriers of the skin.

Among the factors that control the survival of microorganisms on human stratum corneum are skin lipids, including sphingosines. Because the antibacterial spectrum of sphinganine resembles that of cell wall antibiotics, electron microscopy of sphinganine-treated and untreated S. aureus was performed; the lipid induced multiple lesions of cell wall, membrane evaginations and loss of ribosomes. However, comparisons of minimal inhibitory concentration of sphinganine for coccal forms and L-forms of S. aureus, which lack cell walls, and of the respective dose-related reductions in colony-forming units demonstrated both the susceptibility of L-forms and their superior resistance. Therefore, cell wall lesions are sequelae of a probable membrane reaction. Candida albicans was susceptible to sphinganine, sphingosine, dimethylsphingosine, and to a lesser degree, stearylamine. Liquid assays of these lipids against Trichophyton mentagrophytes, T. tonsurans and Epidermatophyton floccosum established their high susceptibility to sphingosine and stearylamine. Sphinganine was the least effective, perhaps due to the presence of L isomers; T. tonsurans was the most sensitive. These four lipids were found to be fungistatic, preventing germination and retarding thalli. Antifungal efficacy was confirmed in vitro on stratum corneum.

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.

[Clinical significance of drug resistance of tuberculosis mycobacteria and L-forms]. / Klinicheskoe znachenie lekarstvenno-ustoichivykh mikobakterii tuberkuleza i L-form.

The clinical features of a tuberculous process were studied in 101 patients with active pulmonary tuberculosis among whom 76% of the patients lost their social adaptation. The patients were divided into 2 groups: group 1 comprising 61 (60.4%) subjects who excreted tuberculosis mycobacteria and L-forms and group 2 consisting of 40 (39.6%) who excreted only tuberculosis mycobacteria. The frequency and degree of drug resistance were more manifest in 63.8% of the patients of group 1 and in 40% of those of group 2 (p less than 0.01). Drug resistance to relatively new antituberculosis drugs (ethambutol and rifampicin) was found in 30% of the patients. Mixed bacillary population was found 2 times more frequently in patients with serious progressive forms of tuberculosis, which gives evidence to consider it as a prognostically unfavourable indicator.

[The ultrastructural organization of Brucella L forms and revertant cultures]. / Ul'trastrukturnaia organizatsiia L-form brutsell i kul'tur-revertantov.

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.