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PubMed1998

Bacteriophages show promise as antimicrobial agents.

The emergence of antibiotic-resistant bacteria has prompted interest in alternatives to conventional drugs. One possible option is to use bacteriophages (phage) as antimicrobial agents. We have conducted a literature review of all Medline citations from 1966-1996 that dealt with the therapeutic use of phage. There were 27 papers from Poland, the Soviet Union, Britain and the U.S.A. The Polish and Soviets administered phage orally, topically or systemically to treat a wide variety of antibiotic-resistant pathogens in both adults and children. Infections included suppurative wound infections, gastroenteritis, sepsis, osteomyelitis, dermatitis, empyemas and pneumonia; pathogens included Staphylococcus, Streptococcus, Klebsiella, Escherichia, Proteus, Pseudomonas, Shigella and Salmonella spp. Overall, the Polish and Soviets reported success rates of 80-95% for phage therapy, with rare, reversible gastrointestinal or allergic side effects. However, efficacy of phage was determined almost exclusively by qualitative clinical assessment of patients, and details of dosages and clinical criteria were very sketchy. There were also six British reports describing controlled trials of phage in animal models (mice, guinea pigs and livestock), measuring survival rates and other objective criteria. All of the British studies raised phage against specific pathogens then used to create experimental infections. Demonstrable efficacy against Escherichia, Acinetobacter, Pseudomonas and Staphylococcus spp. was noted in these model systems. Two U.S. papers dealt with improving the bioavailability of phage. Phage is sequestered in the spleen and removed from circulation. This can be overcome by serial passage of phage through mice to isolate mutants that resist sequestration. In conclusion, bacteriophages may show promise for treating antibiotic resistant pathogens. To facilitate further progress, directions for future research are discussed and a directory of authors from the reviewed papers is provided.

Animals↗
PubMed2003

Study on interaction between T4 phage and Escherichia coli B by microcalorimetric method.

The process that T4 phages multiply in host cells of Escherichia coli B was determined using LKB-2277 Bioactivity Monitor by means of stopped-flow method, and the growth was measured turbidometrically at the same time at 37 degrees C. By analyzing thermo-curves, quantitative parameters could be obtained to characterize the interactions of host cells and phages. The parameters such as k(a), P(max), G etc. change regularly with the decrease of multiplicity of infection (MOI) value. Infection-lysis equations were fitted and the lytic rate constant k(L) was obtained. The results show that the metabolic activity of infected cells is more intensive than that of normal cells. The phenomenon of lysis inhibition (LIN) was first detected with the microcalorimetric method, and the mechanism is discussed.

Bacteriolysis↗
PubMed2000

Chromogenic plate assay distinguishing bacteriolytic from bacteriostatic activity of an antibiotic agent.

A solid agar plate assay was devised to discriminate bacteriolytic from bacteriostatic activity for a given antibacterial agent. The assay uses a bacterial culture harboring beta-galactosidase enzyme as reporter of cellular lysis. When a drop of bacteriolytic compound is placed on the agar, beta-galactosidase is released from the bacteria to the external solid medium where it hydrolyzes X-Gal substrate analogue, developing a blue halo at the edge of the inhibition growth zone. The assay was successfully evaluated against several antibiotics with well-known mechanism of action. It was found that bacteriostatic compounds consistently did not display blue halo at the inhibition zone.

Anti-Bacterial Agents↗
PubMed2003

Application of sonication to release DNA from Bacillus cereus for quantitative detection by real-time PCR.

A rapid sonication method for lysis of Gram-positive bacteria was evaluated for use in combination with quantitative real-time polymerase chain reaction (PCR) analyses for detection. Other criteria used for evaluation of lysis were microscopic cell count, colony forming units (cfu), optical density at 600 nm and total yield of DNA measured by PicoGreen fluorescence. The aim of this study was complete disruption of cellular structures and release of DNA without the need for lysing reagents and time-consuming sample preparation. The Gram-positive bacterium Bacillus cereus was used as a model organism for Gram-positive bacteria. It was demonstrated by real-time PCR that maximum yield of DNA was obtained after 3 to 5 min of sonication. The yield of DNA was affected by culture age and the cells from a 4-h-old culture in the exponential phase of growth gave a higher yield of DNA after 5 min of sonication than a 24-h-old culture in the stationary phase of growth. The 4-h-old culture was also more sensitive for lysis caused by heating. The maximum yield of DNA, evaluated by real-time PCR, from a culture of the Gram-negative bacterium Escherichia coli, was obtained after 20 s of sonication. However, the yield of target DNA from E. coli rapidly decreased after 50 s of sonication due to degradation of DNA. Plate counting (cfu), microscopic counting and absorbance at 600 nm showed that the number of viable and structurally intact B. cereus cells decreased rapidly with sonication time, whereas the yield of DNA increased as shown by PicoGreen fluorescence and real-time PCR. The present results indicate that 3-5 min of sonication is sufficient for lysis and release of DNA from samples of Gram-positive bacteria.

Bacillus cereus↗
PubMed2000

Characterization of Streptococcus thermophilus strains that undergo lysis under unfavourable environmental conditions.

The autolysis of starter lactic acid bacteria appears as a promising way to enhance the flavour of fermented dairy products. The present work was aimed at investigating the autolysis phenomenon in Streptococcus thermophilus, a thermophilic lactic acid bacteria involved in the starters used for the production of yoghurts, Italian and Swiss-type cheeses. Out of 146 strains screened for their aptitude to spontaneously lyse at the end of growth in M17 medium containing lactose in limited concentration, six strains, among which is the type strain CNRZ 1358, were found to be highly autolytic. These autolytic strains are characterized by a typical bell-shaped growth curve. Lysis of the type strain, which was studied as the model, was triggered under unfavourable environmental conditions, such as lactose depletion and NaCl or organic solvents addition. The lysogenic character of this strain was evidenced. Taken together, our results indicate that the autolytic phenotype in S. thermophilus is linked to the lysogenic character but does not result from the massive prophage induction under stressing conditions.

Bacteriolysis↗
PubMed2001

Structural and ultrastructural changes in yeast cells during autolysis in a model wine system and in sparkling wines.

This study shows the changes that occur during the autolysis of yeast in a model wine medium and in a sparkling wine after 12 months of aging, using Nomarsky Light Microscopy and Low Temperature Scanning Electron Microscopy (LTSEM). The size of the yeasts after 24 h of autolysis in a model medium is much smaller than when they are in the growth stage. With LTSEM. a large number of folds can be observed on the surface of the yeast and practically empty cells. Greater morphological changes, both structural and ultrastructural, can be observed in the yeast after 12 months of aging in wine than in the yeast after 24 h of induced autolysis. However, less of the cytoplasmic content of the yeast that has undergone autolysis in the wine was solubilized than that of the yeast after 24 h of autolysis in the model wine system. These findings indicate that autolysis of yeast in wine is a long-lasting process, which continues for at least 12 months.

Bacteriolysis↗
PubMed2003

Early lysis of Lactobacillus helveticus CNRZ 303 in Swiss cheese is not prophage-related.

Lactobacillus helveticus is mainly used as starter in Swiss-type cheeses. Often, lysogenic strains are eliminated because of the risk of early lysis and acidification failure due to phage expression. On the other hand, L. helveticus lysis was shown to positively influence cheese proteolysis during ripening. In order to better assess the relationship between lysis and lysogeny, a prophage-cured derivative of L. helveticus CNRZ 303 was isolated (LH 303-G11) and relysogenised (LH 303-G11R), as demonstrated by hybridisation using the whole phage DNA as probe. The growth, lysis in buffered solutions and lytic activities in zymogram using either Micrococcus luteus or L. helveticus as substrate were identical between the mother strain and its cured derivatives. Only morphological differences were observed by scanning electron microscopy: the cells of the cured derivative were shorter in length. The mother strain and its cured and relysogenised derivatives were assayed in triplicate in experimental Swiss cheeses (scale 1:100). No differences were noted during the cheese making: the three strains exhibited identical kinetics of acidification, leading to similar cheeses at day 1 in terms of gross composition and pH. Phages were detected only in the cheeses made with the mother strain and the relysogenised derivative. The lysis of L. helveticus, estimated by viability decrease and release of the intracellular marker D-lactate deshydrogenase, started early before brining and continued during the cold room ripening. No obvious differences of lysis extent were observed. These results demonstrated for the first time that, in the case of LH 303, the extensive lysis observed in cheese is mainly due to autolysin activity and not to prophage induction.

Bacteriolysis↗
PubMed1997

Autolysis of Lactococcus lactis ssp. lactis and Lactobacillus casei ssp. casei. Cell lysis induced by a crude bacteriocin.

Autolytic properties of Lactococcus lactis subsp. lactis IFPL359, its Lac Prt derivative Lc. lactis Tl and Lactobacillus casei subsp. casei IFPL731, used as starter and adjunct starter in goat's milk cheese making, have been studied. The lytic effect of a bacteriocin produced by a lactic acid bacterium isolated from raw goat's milk has also been analyzed. Lactococcal cells resuspended in phosphate buffer showed a peak of autolysis when they were harvested in the early growth phase. A more stable autolytic pattern through the exponential growth was obtained for Lb. casei IFPL731. Optimal autolysis was found in 0.1 M sodium phosphate buffer during incubation at 40 degrees C for Lb. casei IFPL731 and at 35 degrees C for the lactococci. Thermoinduction of cell lysis was not obtained in any of the cases under the conditions studied. Lytic effect of the crude bacteriocin assayed was strongest against Lc. lactis Tl. Lysis response to the bacteriocin seemed to be strain-dependent and related to growth conditions.

Bacteriocins↗
PubMed1998

Enhancement of bacteriocin production by Carnobacterium divergens AS7 in the presence of a bacteriocin-sensitive strain Carnobacterium piscicola.

The effect of Carnobacterium piscicola in the growth medium of Carnobacterium divergens on divercin production was studied. C. piscicola cultures were added in the form of living cultures, thermally inactivated cultures and pretreated autolyzed cultures. Each form was applied as whole culture comprising growth medium with cells, culture supernatants and cell pellets. It was found that the divercin-sensitive bacterium enhanced significantly the divercin production by C. divergens. The highest stimulating effect was shown by C. piscicola culture autoclaved at 121 degrees C. It enhanced the divercin activity about 64-times compared to the control. The nonautolyzed cultures stimulated divercin biosynthesis to a greater extent than autolyzed cultures, independent of the culture pretreatment. The form of addition was the main external factor affecting divercin production. The possible biochemical mechanisms involved in this enhancement of production are examined.

Bacteriocins↗
PubMed2000

Gentle lysis of mucous producing cold-adapted bacteria by surfactant treatment combined with mechanical disruption.

A procedure for the enhanced lysis of mucous producing psychrotrophic gram positive bacteria for subsequent enzyme studies is described. An initial washing of bacterial cells with Tween 80 was found to improve the degree of cell disruption in subsequent sonication or grinding with glass beads, resulting in about 20-200% increase in total soluble protein content. However, in terms of lactate dehydrogenase (LDH) activity present in the lysate, pretreatment with Tween 80 was more effective in combination with grinding, especially in the highly mucous producing strain GY11. The type of surfactant used in the pretreatment procedure before grinding strongly influenced the percentage lysis of tested strains, both in terms of released soluble protein and enzyme activity. Zymograms of LDH and glutamate dehydrogenase (GDH) activity present in the lysates also very well supported the results obtained by total protein and enzyme activity measurements.

Adaptation, Physiological↗
PubMed1998

Aqueous release and purification of poly(beta-hydroxybutyrate) from Escherichia coli.

The poly(beta-hydroxybutyrate) (PHB) biosynthetic genes of Ralstonia eutropha that are organized in a single operon (phaCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type pha operon from plasmid pTZ18U-PHB leads to the formation of 50-80% PHB/celldry mass when the cells are grown in Luria-Bertani medium supplemented with 1% glucose (w/v). In combination with the phaCAB genes, expression of cloned lysis gene E of bacteriophage PhiX174 from plasmid pSH2 has been used to release PHB granules produced in E. coli. It was shown that small PHB granules in a semiliquid stage are squeezed out of the cells through the E-lysis tunnel structure which is characterized by a small opening in the envelope with borders of fused inner and outer membranes. All envelope components remain intact after E-lysis and can be removed from the mixture of released PHB granules by density gradient centrifugation. In addition, a modified E-lysis procedure is described which enables the release of PHB from cell pellets in pure water or low ionic strength buffer. PHB granules in aqueous solution can be aggregated by divalent cations. Addition of glassmilk speeds up the agglomeration of PHB granules and binding to glass beads can either be used for collection or further purification of PHB in aqueous solutions.

Bacteriolysis↗
PubMed1999

Effect of phenylacetic acid feeding on the process of cellular autolysis in submerged batch cultures of Penicillium chrysogenum.

The effects of feeding the 'toxic' penicillin precursor, phenylacetic acid (PAA) at varying rates, upon the process of cellular autolysis, was assessed in batch bioreactor cultures of an industrial strain of Penicillium chrysogenum. Five processes were fed at rates which resulted in extracellular concentrations of PAA ranging from zero (the control) to approximately ten times levels said to be optimal for penicillin biosynthesis. The culture response was assessed chemically and morphologically, using computerised image analysis. High concentrations of PAA reduced biomass and penicillin production, and were associated with increased cellular autolysis. However, the values of classical morphological indices (branch length, main hyphal length and hyphal growth unit) varied little in cultures which showed extensive autolysis and biomass loss. Lower precursor concentrations (0.01 to 1.0 g l-1) had little effect on biomass, penicillin, or upon the levels of autolysis compared with the control process. Therefore, precursor concentration controlled within the optimal range for penicillin production, has little impact upon differentiation or degradation within an industrial culture of P. chrysogenum. By contrast, exploitation of the toxicity of PAA is proposed as a means to bring forward or enhance autolysis, providing a reliable method of 'induction' with which to study the phenomenon in P. chrysogenum.

Bacteriolysis↗
PubMed1984

Analysis of cephalosporin-induced changes in the volume distribution of Escherichia coli cultures by an electronic counter-channelanalyser.

The demonstration of morphological alterations of the bacterial cell together with bactericidal kinetics are of value for the description of the antibacterial activity of beta-lactam antibiotics. One of the indicators of antibiotic effect on bacterial morphology is the change in bacterial cell volume. This can be demonstrated graphically and numerically by means of electronic cell counting and volume distribution analysis using the Coulter Counter-Channelanalyser system connected to a computer. After registration of the distribution of the relative volume of the Escherichia coli population, the mean cell volume was calculated. The latter parameter increased more than 6 fold with increasing cefotaxim-exposure times. The onset of the delayed (2 h after cefotaxim administration) bacteriolytic effect was reorganized by the appearance of small cell breakdown particles and quantified by counting. In order to demonstrate the therapeutic process graphically the distribution curves were transformed to a common scale. Since a complete storage of the data per sample is performed normally within one minute, the Coulter Counter-Channelanalyser technique can be carried out simultaneously with the bactericidal kinetic experiment. The data demonstrate the speed and intensity of the morphological cell alterations induced by the beta-lactam antibiotic.

Bacteriological Techniques↗
PubMed1984

Different mechanisms of TEM-1 and Oxa-1 mediated resistance to piperacillin in E. coli.

Clinical isolates of Oxa-1 and TEM-1 producing strains of E. coli were studied. Susceptibility to piperacillin was determined by the agar and broth dilution procedure, and beta-lactam hydrolysis rates measured by the iodometric method. The beta-lactamases were identified by isoelectric focusing. Our data on TEM-1 producing strains showed a statistically significant correlation between the MIC, if determined by the agar dilution test, and the specific beta-lactamase activity. The majority of Oxa-1 producing E. coli was resistant to piperacillin although the inactivation rate of piperacillin was usually low. Cell wall permeability of TEM-1 producing strains of E. coli to piperacillin was below the lower limit of detectability, but preincubation of the E. coli strains in piperacillin containing broth led to increased cell wall permeability. Bactericidal kinetics of an Oxa-1 and TEM-1 E. coli were studied. It revealed that regrowth of the Oxa-1 strain in piperacillin containing broth was associated with a 25% decrease of piperacillin concentrations without the formation of degradation products, suggesting binding of piperacillin to bacterial cells. The TEM-1 plasmid bearing strain inactivated piperacillin, and the degradation products (penicilloate) could be detected.

Bacteriolysis↗
PubMed1985

The influence of penicillin on growth and morphology of Streptococcus pyogenes in vivo.

The influence of penicillin (pc) on the growth, phagocytosis and killing of Streptococcus pyogenes was studied for an M protein positive (M+) and an M protein negative (M-) strain in vivo as well as in vitro. In vivo studies were based on a tissue cage model and the analyses were performed by CFU determinations and electron microscopic investigations. The M- strain was easily phagocytized with and without pc, but killing only occurred after pc treatment and thus the number of viable bacteria rapidly decreased under the influence of pc. M+ streptococci were not reduced in numbers by pc-treatment in vivo, but morphological changes and at high pc concentrations, phagocytosis could be seen. When this strain (M+) was cultivated in the absence of pc, the phagocytic cells were totally destroyed - a reaction that was prevented by penicillin. Variations in surface morphology of the two strains seem to influence the differences in sensitivity to penicillin, phagocytosis and killing.

Animals↗
PubMed1986

Action of colistin (polymyxin E) on the lytic cycle of the mycobacteriophage D29 in Mycobacterium tuberculosis.

The antibiotic colistin (polymyxin E) inhibited the lytic cycle of the mycobacteriophage D29 in the tubercle bacilli, but not the D29 adsorption. The protein and nucleic acid synthesis in D29-infected bacteria were not affected significantly. The inhibitory activity was reversed by washing off the antibiotic, and by addition of Ca++, but not in media made iso-osmotic by addition of NaCl or sucrose. Transmission electron microscopy revealed an asymmetric to symmetric transition in the staining profile of the cytoplasmic membrane. Though no mature phage particles were ever observed in colistin-treated, D29-infected tubercle bacilli, loosely arranged aggregates resembling phage proheads were occasionally found. Judging from the above data, it was concluded that colistin inhibited D29 lytic cycle by causing molecular displacements in the inner leaflet of the cytoplasmic membrane, and consequently, the binding sites for D29 structural proteins were not available.

Adsorption↗
PubMed1987

Isolation and characterization of a staphylococcal enzyme bacteriolytic on streptococci.

Staphylococcus hyicus, strain CCM 2368, produced a bacteriolytic enzyme, highly effective on streptococci, some staphylococci and micrococci. Production of the enzyme increased significantly following addition of NaCl (at 1.5%) to the growth medium. The bacteriolytic enzyme could be precipitated from the cell-free staphylococcal culture supernatant with ammonium sulfate at 60% saturation and partially purified by DEAE chromatography. Subsequent isoelectric focusing resulted in complete removal of proteolytic activity and revealed 2 bacteriolytic fractions. One of these (LE IIIa) had an isoelectric point near pH 9.8, the other (LE IIIb) near pH 10.3. LE IIIa lyzed more effectively streptococcal and LE IIIb micrococcal test cells. HPLC gel permeation chromatography also yielded 2 bacteriolytic fractions. The isolated staphylococcal enzyme appeared to be suitable for lysis of streptococci and solubilization of their cell wall structures.

Bacteriolysis↗
PubMed1988

Mutanolysin-induced lysis of actinomyces pyogenes determined by aggregometry.

The lytic activity of mutanolysin from Streptomyces globisporus on 42 cultures of Actinomyces pyogenes could be effectively analyzed in an aggregometer. It was expressed as increase of transmittance at 546 nm after 20 min and 2 h at 37 degrees C. The A. pyogenes cultures revealed no uniform lysis pattern. Most of the cultures were lyzed within 20 to 40 min at 37 degrees C, others were lyzed only moderately or weakly within 2 h of incubation. The lytic activity was optimal at low (0.01 mol/l) molarity of the lysis buffer between pH 5.7 and 7 and could be inhibited by HgCl2.A. pyogenes was not lyzed by lysostaphin or lysozyme.

Actinomyces↗