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.

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.

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.

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.

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.

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.

[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.

Urinary infections: which antibacterial drug and regimen?

Urinary tract infection (UTI) is a common problem in clinical practice. The proper criteria for the diagnosis and treatment of individual and recurrent episodes of UTI are described. The success of antibacterial therapy is dependent upon the urinary concentration of the drug and not its plasma or lymphatic concentration.

Osmotically stable L forms of Haemophilus influenzae and their significance in testing sensitivity to penicillins.

The sensitivity of Haemophilus influenzae to penicillins in vitro, determined either by serial antibiotic dilution in broth or by the disc method on agar, is apparently profoundly influenced by inoculum size if the results are read by macroscopic inspection. Microscopic inspection of the growth, however, reveals that the turbidity in heavily inoculated broth containing concentrations higher than the minimal inhibitory concentration is the product of L forms which have failed to succumb to osmotic lysis. Similarly, minute colonies appearing in the ;inhibition zone' of disc tests are composed of L forms. In both broth and agar tests reduction of the osmolality of the medium from 340 to 144 mOsm per kg failed to bring about lysis of organisms exposed either to ampicillin or amoxycillin. The significance of this remarkable osmotic stability of haemophilus L forms is discussed in relation both to testing of sensitivity of this organism to penicillins and to persistence of chronic haemophilus infections of the lower respiratory tract.

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.

Induction of reversion from the L-form to the sporogenous phase of Bacillus licheniformis var. endoparasiticus (Benedek).

The rate of reversion from the L-form to the complete bacillus phase of Bacillus licheniformis var. endoparasiticus (BLE)was increased by a factor of c. x20, by growth in the presence of 1% diaminopimelic acid in a well plate, and c. x25 with a 1% hog gastric mucin spread on the plate surface. Saturated riboflavin solution and growth products of staphylococci in wells had a lesser effect. The revertants were subsequently stable when isolated in the absence of additive. The rate of reversion from a spheroplast to a diphtheroid phase was not significantly altered by these additives. These findings are of practical value in studies to distinguish between the BLE sporing bacillus and postulated phases of the organism that include diphtheroid and spheroplast L-forms and debated mycoplasma-like forms.

Synergistic effects of a macrolide and a cell wall-affecting antibiotic on Pseudomonas aeruginosa in vitro and in vivo. 3. Incorporation of [14C]midecamycin acetate (MOM) into P. aeruginosa pretreated with cell wall-affecting antibiotics.

The occurrence in beta-lactam treated patients of unstable L-forms of Pseudomonas aeruginosa insensitive to various antibiotics and synergistic effect of combined action of cell wall-affecting antibiotics and macrolide on Pseudomonas infection led us to examine the effects of macrolide on P. aeruginosa pretreated with cell wall-affecting antibiotics. The effects of macrolide antibiotics such as midecamycin acetate (MOM) on P. aeruginosa was investigated, a rapid killing effect by MOM was noted after treatment with suboptimal doses of cell wall-affecting antibiotics such as polymyxin B, carbenicillin, dibekacin or fosfomycin. Incorporation of [14C]MOM into intact P. aeruginosa cells was not significant, but was apparent into L-form cells or cells pretreated with cell wall-affecting antibiotics. The incorporated radioactivity was found in the 70 S ribosome fraction, binding with the 50 S subunits of ribosome in both cases. These results indicate that under certain conditions a macrolide antibiotic can enter the P. aeruginosa cell.

Effects of polyene macrolide antibiotics on normal and protoplast type L-form cells of Escherichia coli W1655F+.

The action of the polyene macrolide antibiotics mycotrienin, pimaricin, lucensomycin, tetramycin, rimocidin, nystatin, filipin, lagosin, pentaene antibiotic 2814P, flavomycoin, flavofungin, hexaene antibiotic 5001P, and candicidin, including perhydro derivatives of them, on wall-less stable protoplast type L-form and normal rod form cells of E. coli W1655F+ was studied. No inhibition of the normal rod form cells was detected. In contrast to these results the growth of the L-form cells was inhibited by all of the substances tested, with the exception of pimaricin. Further experiments have shown that the differences in sensitivity of normal and L-form cells cannot be explained by differences in sterol content, the target site of polyene antibiotics in sensitive eukaryotic cells. According to our results it is obvious that the cell wall of the normal cells functions as a penetration barrier to polyene antibiotics.

Evolution and taxonomy in the genus Brucella: steroid hormone induction of filterable forms with altered characteristics after reversion.

Several strains of Brucella abortus, Brucella suis, and Brucella melitensis were exposed to physiologic concentrations of testosterone, progesterone, and diethylstilbestrol by incorporating them into the growth medium. The hormones induced Brucella to form cell wall-defective organisms, including filterable forms. The filterable forms from 1 strain of B melitensis had altered characteristics when it reverted to growth as an intact cell. This is the 1st reported instance of induction of filterable forms of Brucella by progesterone, testosterone, and diethylstilbestrol.

Isolation of L-form variants after antibiotic treatment in Staphylococcus aureus bovine mastitis.

An unstable L-form of Staphylococcus aureus was identified in milk samples from 3 quarters of 2 cows after treatment with cloxacillin. Milk samples incubated on standard 5% blood agar plates were culture-negative for 7 to 30 days after treatment, but S aureus was reisolated in 80% of 66 samples by additional culturing on enriched L-form media when incubated in 10% CO2 at 37 C. The organism was identified at various phases of reversion of L-form agar and was confirmed on transfer to blood agar plates.

[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.

[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.

[Ultrastructure of Salmonella typhimurium forms with unbalanced growth induced by lithium chloride]. / Ul'trastruktura form nesbalansirovannogo rosta Salmonella typhimurium, poluchennykh pod deistviem khloristogo litiia.

The action of 1.0 and 1.5 M LiCl on S. typhimurium induces the appearance of unbalanced growth forms capable of growing and multiplication, when subcultured in a medium with this preparation. In this culture the prevalence of cells differing in their structure from the initial Salmonella cells and from stable L-form cultures is observed. Cells characteristic of the initial culture and cells resembling the L-forms occur in lesser numbers. LiCl seems to affect peptidoglycan and the cytoplasmic membrane, which brings about disturbances in the permeability of the surface structures of the cell.

[Production of L forms of the plague microbe]. / Poluchenie L-form chumnogo mikroba.

Experiments were conducted in vitro. The process of formation of unstable L-form cultures of Pasteurella pestis under the effect of penicillin was studied. A comparatively high frequency of this phenomenon in various strains was demonstrated. A possible role of the L-transformation process in the persistence of the causative agent in the organism of wild animals in endemic foci of infection is put forward.