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PubMed2003

Rod shape determination by the Bacillus subtilis class B penicillin-binding proteins encoded by pbpA and pbpH.

The peptidoglycan cell wall determines the shape and structural integrity of a bacterial cell. Class B penicillin-binding proteins (PBPs) carry a transpeptidase activity that cross-links peptidoglycan strands via their peptide side chains, and some of these proteins are directly involved in cell shape determination. No Bacillus subtilis PBP with a clear role in rod shape maintenance has been identified. However, previous studies showed that during outgrowth of pbpA mutant spores, the cells grew in an ovoid shape for several hours before they recovered and took on a normal rod shape. It was postulated that another PBP, expressed later during outgrowth, was able to compensate for the lack of the pbpA product, PBP2a, and to guide the formation of a rod shape. The B. subtilis pbpH (ykuA) gene product is predicted to be a class B PBP with greatest sequence similarity to PBP2a. We found that a pbpH-lacZ fusion was expressed at very low levels in early log phase and increased in late log phase. A pbpH null mutant was indistinguishable from the wild-type, but a pbpA pbpH double mutant was nonviable. When pbpH was placed under the control of an inducible promoter in a pbpA mutant, viability was dependent on pbpH expression. Growth of this strain in the absence of inducer resulted in conversion of the cells from rods to ovoid/round shapes and lysis. We conclude that PBP2a and PbpH play redundant roles in formation of a rod-shaped peptidoglycan cell wall.

Amino Acid Sequence↗

PubMed2003

Surfactant proteins A and D inhibit the growth of Gram-negative bacteria by increasing membrane permeability.

The pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D), have been reported to bind lipopolysaccharide (LPS), opsonize microorganisms, and enhance the clearance of lung pathogens. In this study, we examined the effect of SP-A and SP-D on the growth and viability of Gram-negative bacteria. The pulmonary clearance of Escherichia coli K12 was reduced in SP-A-null mice and was increased in SP-D-overexpressing mice, compared with strain-matched wild-type controls. Purified SP-A and SP-D inhibited bacterial synthetic functions of several, but not all, strains of E. coli, Klebsiella pneumoniae, and Enterobacter aerogenes. In general, rough E. coli strains were more susceptible than smooth strains, and collectin-mediated growth inhibition was partially blocked by coincubation with rough LPS vesicles. Although both SP-A and SP-D agglutinated E. coli K12 in a calcium-dependent manner, microbial growth inhibition was independent of bacterial aggregation. At least part of the antimicrobial activity of SP-A and SP-D was localized to their C-terminal domains using truncated recombinant proteins. Incubation of E. coli K12 with SP-A or SP-D increased bacterial permeability. Deletion of the E. coli OmpA gene from a collectin-resistant smooth E. coli strain enhanced SP-A and SP-D-mediated growth inhibition. These data indicate that SP-A and SP-D are antimicrobial proteins that directly inhibit the proliferation of Gram-negative bacteria in a macrophage- and aggregation-independent manner by increasing the permeability of the microbial cell membrane.

Animals↗

PubMed1978

[Bacteriocinogenic activity of strains of the genus Erwinia].

Antibacterial activity of 272 Erwinia strains was studied. It was found that 182 or 66.9 per cent of the strains were capable of producing spontaneously antibacterial substances belonging to the class of bacteriocins; 125 bacteriocynogenic strains were divided into 25 groups on the basis of their antibacterial spectrum similarity; 57 bacteriocynogenic strains were not included into any of these groups because of their significant heterogenicity with respect to the feature studied. It was shown that most of the strains inhibited viability of the bacteria of both its own and other species. Investigation of the antagonistic activity of the Erwinia strains with broad antibacterial spectra with respect to E. coli indicative for colicins gave negative results. The study of the Erwinia strains sensitivity to the antibacterial effect of the bacteriocynogenic cultures showed that 210 out of 272 cultures were sensitive to separate bacteriocins.

Antibiosis↗

PubMed1995

Antibacterial properties of lansoprazole alone and in combination with antimicrobial agents against Helicobacter pylori.

The activities of various types of antiulcer agents against Helicobacter pylori strains were determined by an agar dilution method. Among the compounds tested, benzimidazole proton pump inhibitors were found to have significant activity against this organism. The activity of lansoprazole was fourfold more potent than that of omeprazole and bismuth subsalicylate, with MICs ranging from 1.56 to 25 micrograms/ml. Exposure of Helicobacter pylori to lansoprazole led to an extensive loss of viability as well as suppression of virulence factors such as motility, adhesiveness to epithelial cells and urease activity. The combination of lansoprazole with antimicrobial agents such as penicillins, cephalosporins, macrolides, tetracyclines, aminoglycosides, quinolones, metronidazole and bismuth subsalicylate generally had an additive effect on inhibition of Helicobacter pylori growth.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1999

Oxygen starvation induces cell death in Candida shehatae fermentations of D-xylose, but not D-glucose.

Candida shehatae cells, cultivated on D-glucose and D-xylose, were subjected to a shift from fully aerobic to anaerobic fermentative conditions. After anaerobic conditions were imposed, growth was limited to approximately one doubling or less as C. shehatae rapidly entered a stationary phase of growth. Following the shift to anoxia, cell viability rapidly declined and the total cell volume declined in the D-xylose fermentations. Moreover, the cell volume distribution shifted to smaller volumes. Cell viability, measured by plate counts, declined nine times faster for D-xylose fermentations than for D-glucose fermentations. Anaerobic growth did not occur on either D-glucose or D-xylose. Selected vitamins and amino acids did not stimulate anaerobic growth in C. shehatae, but did enhance anaerobic growth on D-glucose in S. cerevisiae. The decline in cell viability and lack of anaerobic growth by C. shehatae were attributed to oxygen deficiency and not to ethanol inhibition. The results shed light on why C. shehatae anaerobic fermentations are not currently practical and suggest that research directed towards a biochemical understanding of why C. shehatae can not grow anaerobically will yield significant improvements in ethanol fermentations from D-xylose.

Aerobiosis↗

PubMed2005

Detection of Kluyveromyces marxianus and other spoilage yeasts in yoghurt using a PCR-culture technique.

A combined PCR-culture technique was developed for the detection of viable yeasts in yoghurt samples. Yoghurt samples were inoculated with either viable or heat-inactivated Kluyveromyces marxianus cells, and analyzed before and after incubation for 24 h at 25 degrees C under agitation. DNA was extracted from the samples and amplified using yeast-specific primers targeted at the gene coding for the 18S rRNA. A 251-bp fragment was amplified by the Polymerase Chain Reaction from the yoghurt samples containing initial yeasts counts of 10 cfu g(-1) or higher, whereas no PCR product was generated from control uninoculated yoghurt samples. Comparison of PCR results obtained before and after the incubation step was used to assess yeast viability. Viability was also confirmed by plating on Sabouraud-Dextrose-Chloramphenicol Agar. Moreover, comparison of the results obtained using PCR-culture and plate count methods for the analysis of commercial yoghurt samples, demonstrated that the PCR-culture technique developed in this work can be very useful for the rapid detection of viable spoilage yeasts in dairy industries.

Colony Count, Microbial↗

PubMed1998

Viability and activity in readily culturable bacteria: a review and discussion of the practical issues.

In microbiology the terms 'viability' and 'culturability' are often equated. However, in recent years the apparently self-contradictory expression 'viable-but-nonculturable' ('VBNC') has been applied to cells with various and often poorly defined physiological attributes but which, nonetheless, could not be cultured by methods normally appropriate to the organism concerned. These attributes include apparent cell integrity, the possession of some form of measurable cellular activity and the apparent capacity to regain culturability. We review the evidence relating to putative VBNC cells and stress our view that most of the reports claiming a return to culturability have failed to exclude the regrowth of a limited number of cells which had never lost culturability. We argue that failure to differentiate clearly between use of the terms 'viability' and 'culturability' in an operational versus a conceptual sense is fuelling the current debate, and conclude with a number of proposals that are designed to help clarify the major issues involved. In particular, we suggest an alternative operational terminology that replaces 'VBNC' with expressions that are internally consistent.

Animals↗

PubMed1996

Survival of Staphylococcus aureus and enterotoxin A in glassy and rubbery states of gelatin.

About 1% of Staphylococcus aureus cells survived the production of gelatin sheets containing nutrient broth. Those cells which survived showed no evidence of injury. Growth occurred in rubbery state gelatin with a(w) values of 0.98 and 0.93 ; viability decreased during storage at a(w) values of 0.89, 0.62 and 0.36 but there was little loss of viability in gelatin at an aw of 0.25 over 27 d storage at 26 degrees C. Assays for enterotoxin A detected no synthesis of new toxin but no loss in pre-formed toxin. The results suggest that high levels of Staph. aureus and its toxins should be excluded from glassy and rubbery state food products in order to prevent illness.

Colony Count, Microbial↗

PubMed1997

Antimicrobial action of rabbit leukocyte CAP18(106-137).

CAP18 is a cationic antimicrobial protein originally isolated from rabbit neutrophils, of which a 32-mer sequence from its C-terminal and (CAP18(106-137)) has been found to be the most active. The bactericidal action of this peptide has been characterized by conventional culture techniques and flow cytometry. Cultures of Escherichia coli NCTC10418 were exposed to the MBC (12 microM) of the peptide for up to 60 min and stained with a fluorochrome sensitive to changes in either membrane potential (bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)), or membrane integrity (propidium iodide [PI]) before flow cytometric analysis. Addition of CAP18(106-137) to E. coli in broth culture resulted in immediate collapse of membrane potential [as determined by uptake of DiBAC4(3)] and loss of membrane integrity (as indicated by uptake of PI), with a corresponding 6- to 8-log decrease in viable counts as determined by colony formation on solid media. In identical experiments, the presence of Mg2+ (1 to 10 mM), K+ (50 to 250 mM), or EDTA (5 mM) or incubation in nutrient-free buffer or at 4 degrees C had no effect on peptide-induced dye uptake. In contrast, addition of Ca2+ (1 to 10 mM) or the respiratory chain poison carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 microM) inhibited the uptake of both dyes. These findings, however, did not relate to bacterial recovery on solid media, where (unless in the presence of K+ 150 to 250 mM) CAP18(106-137) at 12 microM fulfilled the MBC criteria (99.9% killing). We conclude that CAP18(106-137) exerts a rapid and profound action on E. coli cytoplasmic membranes and viability as measured by colony formation. The results suggest, however, that CAP18(106-137) may exert its action at sites additional to the cell membrane and that its activity profile is unique among cationic antimicrobial proteins.

Animals↗

PubMed2001

Growth phase variation in cell and nucleoid morphology in a Bacillus subtilis recA mutant.

The major role of RecA is thought to be in helping repair and restart stalled replication forks. During exponential growth, Bacillus subtilis recA cells exhibited few microscopically observable nucleoid defects. However, the efficiency of plating was about 12% of that of the parent strain. A substantial and additive defect in viability was also seen for addB and recF mutants, suggesting a role for the corresponding recombination paths during normal growth. Upon entry into stationary phase, a subpopulation (approximately 15%) of abnormally long cells and nucleoids developed in B. subtilis recA mutants. In addition, recA mutants showed a delay in, and a diminished capacity for, effecting prespore nucleoid condensation.

Bacillus subtilis↗

PubMed2005

Effect of nitric oxide on the growth of Chlamydophila pneumoniae.

Chlamydophila pneumoniae is an important human intracellular pathogen; however, the pathogenesis of C. pneumoniae infection is poorly understood and the immune control mechanism versus host cells is not completely known. The role of the nitric oxide (NO) synthase pathway in inhibiting the ability of C. pneumoniae to infect macrophage J774 cells and the ability of NO to damage isolated C. pneumoniae were investigated. Exposure of infected cultures to recombinant murine gamma interferon (MurIFN-gamma) resulted in increased production of NO and reduced viability. Addition of 2-(N,N-diethylamino)-diazenolase-2-oxide before infection of J774 cells or during chlamydial cultivation released NO, both resulting in a reduction in the viability of C. pneumoniae in a dose-dependent way. These results indicate that immune control of chlamydial growth in murine macrophage cells may trigger a mechanism that includes NO release with effects on the multiplication of the microorganism, thus suggesting that NO may play a role in preventing the systemic spread of Chlamydia.

Animals↗

PubMed2005

[The distribution and viability changes of the Agrobacteria in the vacuum infiltrated plants of Chinese cabbage].

To understand further the mechanism of agrobacteria transformation by vacuum infiltration, the distribution and viability changes of the agrobacteria in infiltrated Chinese cabbage( B. rapa ssp. chinesis) plants were investigated by histochemical staining and bacteria plating techniques. Results revealed that there were different quantities of Agrobacteria in different organs, among which the most quantity occurred in flowers, especially in ovules. The Agrobacteria lied in the space between cells and concentrated in the vascular bundle no matter the type of the organs examined. The CFUs (colony forming unit) of the Agrobacteria derived from the flowers were always the most in every experiment. The viabilities of the agrobacteria in the plants declined dramatically along with the elongation of the time after plant infiltration in all organs, but there were still 10(3) CFUs per gram fresh tissue in the flowers after 15 days of infiltration. These results may explain the fact that the ovules were the targets for the Agrobacteria mediated vacuum infiltration transformation.

Brassica↗

PubMed2005

Campylobacter jejuni loss of culturability in aqueous microcosms and ability to resuscitate in a mouse model.

Water is known as one of the main transmission routes of Campylobacter and contributes to increase the number of sporadic infections and outbreaks. Campylobacter jejuni persists in the environment, especially in water, in a viable but non-culturable (VBNC) form that is thought to be a possible cause of water-borne outbreaks. In this study, we evaluated the loss of culturability and viability of 9 C. jejuni strains of clinical origin and one ATCC reference strain when kept at 4 degrees C in artificial sea water (ASW). Culturability was measured as colony-forming units while viability was evaluated by CTC-DAPI double staining and the combined CTC-specific fluorescent antibody technique (CTC-FA). When cultured on Columbia Agar plates, strains exhibited different growth profiles which allowed to classify them into three different groups. Both techniques used to monitor the viability of the bacterial cells showed that C. jejuni strains survived in the VBNC form in the microcosms through a period lasting from 138 to 152 days. The recovery of C. jejuni VBNC forms to culturability, as evidenced by cell division, was obtained by passage in the mouse intestine. Our results indicate that C. jejuni VBNC cells were able to remain in this state for a few months and regain their culturability after in vivo passage depending on their lasting in the VBNC state, which affects the number of respiring bacteria. In fact, the resuscitation was achieved when the number of respiring bacteria became higher than 10(4) cell/ml. Therefore, a relatively high microbial titer of respiring bacteria in the VBNC state seems to be important for the resuscitation and subsequent intestinal colonisation.

Animals↗

PubMed2003

Identification and quantitation of allelochemicals from the lichen Lethariella canariensis: phytotoxicity and antioxidative activity.

Phytotoxicity-based extraction and fractionation were employed to separate allelochemicals contained in an extract of Lethariella canariensis. Twelve phenolic substances were isolated from the phytotoxic fraction "Letharal" of the thalli. These were identified by spectroscopic methods, physicochemical constants, and HPLC chemical correlation, and determined to be atranol (2), chloroatranol (3), hematommic acid (4), chlorohematommic acid (5), methyl hematommate (6), methyl chlorohematommate (7) (new compound), ethyl hematommate (8), ethyl chlorohematommate (9), methyl beta-orsellinate (10), atranorin (11), chloroatranorin (12), and (+)-usnic acid (13). Further identification and quantification of these allelochemicals in the environment were conducted by HPLC. Several phenolic compounds showed moderate antimicrobial activity. The cytostatic activity of the polyphenols was investigated on U937 and HL-60 cells. All compounds were assayed, with the exception of 10. The "Letharal" mixture decreased cell viability in both cell lines. Protection against lipid peroxidation was investigated using brain homogenates. Compounds 2, 3, 6, 8, 11, and Letharal decreased H2O2/Fe+2 induced lipid peroxidation in a concentration-dependent manner, while 10 and 13 were unable to protect tissue against oxidative stress.

Animals↗

PubMed1969

Control of fatty acid composition in phospholipids of Escherichia coli: response to fatty acid supplements in a fatty acid auxotroph.

The effect of exogenous unsaturated fatty acids on the fatty acid composition of phospholipids of a mutant of E. coli has been examined. These exogenous acids serve as growth factors for an auxotroph requiring unsaturated fatty acids. When each member of a structurally homologous series of cis-unsaturated fatty acids serves as a growth factor, the percentage of unsaturated fatty acid present in phospholipids increases with increasing chain length or decreasing number of double bonds in the apolar chain of the supplement. At 37 degrees , trans-octadecenoic acids support growth and are incorporated into phospholipids at levels higher than the corresponding cis-acids. However, a temperature shift to 27 degrees with trans-acids results in loss of viability and lysis. Utilization of cis-acids as supplements at decreasing temperatures between 42 degrees and 27 degrees results in increasing amounts of unsaturated fatty acids in phospholipids with decreasing temperature. These observations suggest the operation of a regulatory mechanism which controls the composition of saturated versus unsaturated acids in order to maintain the physical properties of phospholipids within narrow limits.

Alkenes↗

PubMed2006

Effects of a high-molecular-weight cranberry fraction on growth, biofilm formation and adherence of Porphyromonas gingivalis.

BACKGROUND: Porphyromonas gingivalis is a major aetiological agent of periodontitis, a destructive disease affecting the tooth-supporting tissues. Recent reports have indicated that high-molecular-weight molecules from cranberry juice concentrate can prevent the attachment of human pathogens to host tissues. OBJECTIVES: The aim of the present study was to investigate the effect of non-dialysable material (NDM) prepared from cranberry juice concentrate on growth, biofilm formation and adherence properties of P. gingivalis. METHODS: The effect of cranberry NDM on biofilm formation was studied using a polystyrene microplate assay and by scanning electron microscopy. The effect of cranberry NDM on the attachment properties of P. gingivalis was evaluated by a microplate assay in which mammalian proteins were immobilized into wells. RESULTS: Our results indicated that cranberry NDM is a potent inhibitor of biofilm formation by P. gingivalis. However, it has no effect on growth and viability of bacteria. Cranberry NDM also prevented significantly the attachment of P. gingivalis to surfaces coated with type I collagen, fibrinogen or human serum. CONCLUSIONS: Our data suggest that cranberry constituents may have a beneficial effect for the prevention and treatment of periodontitis by reducing the capacity of P. gingivalis to colonize periodontal sites.

Anti-Bacterial Agents↗

PubMed1997

In vitro amphotericin B effects on growth, viability and dimorphism of Paracoccidioides brasiliensis: reversal of the treatment.

The in vitro effects of amphotericin B deoxycholate suspension (fungizone) on Paracoccidioides brasiliensis growth, cell viability and transformation were investigated. We also analyzed the protein synthesis patterns of both cellular forms, yeast and mycelium in the presence of AmB. This drug, at 30 micrograms/ml, highly inhibited yeast growth, which could be recovered depending on treatment time, where the most effective reversion was observed after 6 hr of incubation. The yeast cell viability, that had been partially affected by the drug, could also be efficiently recovered after AmB was removed. The effect of AmB on the cellular dimorphism process showed a strong reduction in the mycelium to yeast transformation (80% inhibition compared to the control without the drug). On the other hand, the transformation from yeast to mycelium in the presence of AmB was 50% affected, relative to the control. In contrast to the growth and cell viability experiments, the reversion effects on dimorphism were partial when the drug was removed, even with only 6 hr treatment. The two-dimensional gels of 35S-labeled proteins revealed a strong reduction in the three species of 80, 71 and 56 kDa in yeast and mycelium when treated with AmB.

Amphotericin B↗

PubMed1998

Comparison of fungal viability assays using Candida albicans yeast cells undergoing prolonged incubation in the absence of nutrients.

Staining methods for determining fungal viability are usually assessed by comparisons with enumeration of colony-forming units (CFU) on solid media. The purpose of the present study was to compare viability as assessed by the acridine orange (AO) and MTT methods with the numbers of CFUs obtained for Candida albicans yeast cells undergoing prolonged incubation in distilled water. In initial assessments of the assays using various proportions of control and heat-killed C. albicans, the AO and MTT methods consistently indicated significantly higher values for viability than did CFU determinations. Experiments using organisms cultured overnight revealed that approximately 95% of the cells were capable of dividing at least once in a microscopic proliferation assay, whereas only 69% were capable of forming colonies. Parallel assays comparing AO uptake and MTT reduction gave excellent agreement with the microscopic proliferation assay, but not with CFU determinations. Using organisms undergoing prolonged incubations in distilled water, much lower viabilities were obtained with the CFU method at 7 and 10 days than with the microscopic proliferation assay or the two staining methods. These results indicate that the AO and MTT assays correlate well with the ability of C. albicans to divide at least once, but may not accurately indicate the percentage of organisms actually able to form colonies.

Acridine Orange↗