Membrane damage and microbial inactivation by chlorine in the absence and presence of a chlorine-demanding substrate

The relationship between cell inactivation and membrane damage was studied in two gram-positive organisms, Listeria monocytogenes and Bacillus subtilis, and two gram-negative organisms, Yersinia enterocolitica and Escherichia coli, exposed to chlorine in the absence and presence of 150 ppm of organic matter (Trypticase soy broth). L. monocytogenes and B. subtilis were more resistant to chlorine in distilled water. The addition of small amounts of organic matter to the chlorination medium drastically increased the resistance of both types of microorganisms, but this effect was more marked in Y. enterocolitica and E. coli. In addition, the survival curves for these microorganisms in the presence of organic matter had a prolonged shoulder. Sublethal injury was not detected under most experimental conditions, and only gram-positive cells treated in distilled water showed a relevant degree of injury. The exposure of bacterial cells to chlorine in distilled water caused extensive permeabilization of the cytoplasmic membrane, but the concentrations required were much higher than those needed to inactivate cells. Therefore, there was no relationship between the occurrence of membrane permeabilization and cell death. The addition of organic matter to the treatment medium stabilized the cytoplasmic membrane against permeabilization in both the gram-positive and gram-negative bacteria investigated. Exposure of E. coli cells to the outer membrane-permeabilizing agent EDTA increased their sensitivity to chlorine and caused the shoulders in the survival curves to disappear. Based on these observations, we propose that bacterial envelopes could play a role in cell inactivation by modulating the access of chlorine to the key targets within the cell.     

Resistance of early stationary phase E. coli to membrane permeabilization by the antimicrobial peptide Cecropin A

Antimicrobial peptides (AMPs) cause bacterial membrane permeabilization and ultimately cell death at low μM concentrations. The membrane permeabilization action of a moth derived AMP Cecropin A on E. coli cells in exponential growth (mid-log phase) is well studied. At 1× MIC concentration, Cecropin A penetrates the lipopolysaccharide (LPS) barrier and causes outer membrane (OM) and cytoplasmic membrane (CM) permeabilization. For non-septating cells, permeabilization of both membranes begins at one pole. For septating cells, OM permeabilization begins at the septal region and CM permeabilization begins at one pole. However, in nature bacteria are frequently found in nutrient-starved conditions. Here we extend our single-cell microscopy assays to the attack of Cecropin A on E. coli cells in early stationary phase. Stationary phase E. coli is much more resistant to membrane permeabilization by Cecropin A than mid-log phase E. coli. A tenfold higher concentration of Cecropin A is required to observe CM permeabilization in the majority of stationary phase cells, and even then permeabilization proceeds more slowly. In addition, the spatial pattern of initial CM permeabilization changes from localized at one pole to global. Studies of lipid mutant strains suggest that a sufficient localized concentration of the anionic phospholipid phosphatidylglycerol (PG) guides the position of initial attack of the cationic AMP Cecropin A on the CM.     

Lipoteichoic acid is a major component of the Bacillus subtilis periplasm

Cryo-electron microscopy (cryo-EM) of frozen-hydrated specimens allows high-resolution observation of structures in optimally preserved samples. In gram-positive bacteria, this method reveals the presence of a periplasmic space between the plasma membrane and an often differentiated cell wall matrix. Since virtually nothing is known about the composition of its constituent matter (i.e., the periplasm), it is still unclear what structures (or mechanism) sustain a gram-positive periplasmic space. Here we have used cryo-EM of frozen-hydrated sections in combination with various labels to probe the model gram-positive organism Bacillus subtilis for major periplasmic components. Incubation of cells with positively charged gold nanoparticles showed almost similar levels of gold binding to the periplasm and the cell wall. On cells whose cell walls were enzymatically hydrolyzed (i.e., on protoplasts), a surface diffuse layer extending ~30 nm from the membrane was revealed. The thickness and density of this layer were not significantly altered after treatment with a nonspecific protease, whereas it was labeled with anti-lipoteichoic acid (LTA) antibodies conjugated to nanogold. Further, the LTA layer spans most of the thickness of the periplasmic space, which strongly suggests that LTA is a major component of the B. subtilis periplasm.     

Membrane Permeabilization in Relation to Inactivation Kinetics of Lactobacillus Species due to Pulsed Electric Fields

Membrane permeabilization due to pulsed electric field (PEF) treatment of gram-positive Lactobacillus cells was investigated by using propidium iodide uptake and single-cell analysis with flow cytometry. Electric field strength, energy input, treatment time, and growth phase affected membrane permeabilization of Lactobacillus plantarum during PEF treatment. A correlation between PEF inactivation and membrane permeabilization of L. plantarum cells was demonstrated, whereas no relationship was observed between membrane permeabilization and heat inactivation. The same results were obtained with a Lactobacillus fermentum strain, but the latter organism was more PEF resistant and exhibited less membrane permeabilization, indicating that various bacteria have different responses to PEF treatment. While membrane permeabilization was the main factor involved in the mechanism of inactivation, the growth phase and the acidity of the environment also influenced inactivation. By using flow cytometry it was possible to sort cells in the L. plantarum population based on different cell sizes and shapes, and the results were confirmed by image analysis. An apparent effect of morphology on membrane permeabilization was observed, and larger cells were more easily permeabilized than smaller cells. In conclusion, our results indicate that the ability of PEF treatment to cause membrane permeabilization is an important factor in determining inactivation. This finding should have an effect on the final choice of the processing parameters used so that all microorganisms can be inactivated and, consequently, on the use of PEF treatment as an alternative method for preserving food products.     

Antimicrobial activity of endogenous peptides of the moss <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Plant and animal cells contain pools of endogenous peptides, which are the degradation products of functionally active proteins. It is known that these peptides can possess biological activity; however, the functions of most of them are unknown. The goal of the present study was to estimate the antimicrobial potential of endogenous peptides resulting from the degradation of functional proteins in cells of the moss Physcomitrella patens. Earlier, 117 peptides possessing an antimicrobial potential predicted in silico have been identified in the peptidomes of three types of P. patens cells by mass spectrometry. In the present work, the antimicrobial activity of six of these peptides toward the gram-positive bacteria Bacillus subtilis SHgw and Clavibacter michiganensis pv. michiganensis and gram-negative bacteria Escherichia coli K12 and Xanthomonas arboricola 3004 has been revealed. The results have shown that three of six peptides inhibit the growth of the phytopathogenic bacteria X. arboricola and C. m. pv. michiganensis; four peptides inhibit the growth of the gram-negative bacterium E. coli K12, and one peptide inhibits the growth of the gram-positive bacterium B. subtilis. It has been found that the peptides inhibiting the bacterial growth are predominantly the fragments of ribosomal proteins. The work confirms the potential of the biological activity of peptides that are the degradation products of functional proteins.     

Study of ectoparasitism of ultramicrobacteria of the genus <Emphasis Type="Italic">Kaistia</Emphasis>, strains NF1 and NF3 by electron and fluorescence microscopy

Transmission electron and fluorescence microscopy was used to study the character of the interaction of free-living ultramicrobacterial (UMB) strains NF1 and NF3, affiliated with the genus Kaistia, and seven species of gram-positive and gram-negative heterotrophic bacteria. Strains NF1 and NF3 were found to exhibit parasitic activity against gram-positive Bacillus subtilis and gram-negative Acidovorax delafildii. UMB cells are tightly attached to the envelopes of the victim cells and induce their lysis, thus demonstrating the features of typical ectoparasitism. The selectivity of parasitism of the studied UMB to the victim bacteria has been shown: only two soil microorganisms of the seven test objects, B. subtilis ATCC 6633 and an aerobic gramnegative bacterium A. delafildii 39, were found to be sensitive to UMB attack. Other bacteria (Micrococcus luteus VKM Ac-2230, Staphylococcus aureus 209-P, Pseudomonas putida BS394, Escherichia coli C 600, and Pantoea agglomerans ATCC 27155) were not attacked by UMB. It was established for the first time that free-living UMB may be facultative parasites not only of phototrophic bacteria, as we have previously demonstrated [1], but of heterotrophic bacteria as well. The UMB under study seem to play an important role in the regulation of the quantity of microorganisms and in the functioning of microbial communities in some natural ecotopes.     

Assay-dependent effect of silver nanoparticles to <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

We assess the microbial assay-dependent effect of AgNP on gram-negative Escherichia coli and gram-positive Bacillus subtilis. The experiment was conducted via three different assays: a growth inhibition assay, a colony forming unit assay, and a liquid-to-plate assay. AgNP were exposed either as liquid suspensions or in an agar state. Bacterial sensitivity to AgNP was found to be dependent on the microbial assay employed. E. coli was more sensitive than B. subtilis in the growth inhibition and CFU assays, but B. subtilis was more vulnerable than E. coli in the liquid-to-plate assay, ostensibly owing to the food stress mechanisms of B. subtilis in exposure medium. The dissolution of silver from AgNP could not explain the observed toxicity of AgNP. We detected clear evidence of AgNP uptake by cells. The results of this study showed that the microbial toxicity of AgNP and the effects of dissolved silver ions were influenced profoundly by the microbial test method employed.     

Photometric Application of the Gram Stain Method To Characterize Natural Bacterial Populations in Aquatic Environments

The Gram stain method was applied to the photometric characterization of aquatic bacterial populations with a charge-coupled device camera and an image analyzer. Escherichia coli and Bacillus subtilis were used as standards of typical gram-negative and gram-positive bacteria, respectively. A mounting agent to obtain clear images of Gram-stained bacteria on Nuclepore membrane filters was developed. The bacterial stainability by the Gram stain was indicated by the Gram stain index (GSI), which was applicable not only to the dichotomous classification of bacteria but also to the characterization of cell wall structure. The GSI spectra of natural bacterial populations in water with various levels of eutrophication showed a distinct profile, suggesting possible staining specificity that indicates the presence of a particular bacterial population in the aquatic environment.Gram’s method is the most important and fundamental orthodox method for bacterial identification. It classifies bacteria into two groups, gram-negative and gram-positive. The mechanism of Gram staining is based on the fundamental structural and chemical attributes of bacterial cell walls. The cell walls of gram-positive bacteria have a high percentage of peptidoglycan, while those of gram-negative bacteria have only a thin peptidoglycan layer (1–3, 6). In Gram’s method, an insoluble dye-iodine complex is formed inside bacterial cells and is extracted by alcohol from gram-negative but not gram-positive bacteria (6, 12, 16). There are taxonomically gram-variable species, but some cells of gram-negative or gram-positive species may show gram-variable characteristics due to environmental stress, such as unsuitable nutrients, temperature, pH, or electrolytes (3).Functional differences between gram-positive and gram-negative cell walls have been studied with special emphasis on nutrient uptake from the ambient environment. Gram-negative bacteria have a periplasmic space between the lipopolysaccharide layer and the plasma membrane. In this space, binding proteins initially attach to nutrients and take them to a membrane carrier. Gram-positive bacteria lack the periplasmic space and are believed to have no binding proteins (9). Therefore, nutrient uptake from the environment is easier for gram-negative bacteria than for gram-positive bacteria. Because of this difference, the population density of gram-negative bacteria in more oligotrophic environments could be higher than that of gram-positive bacteria (20).Gram staining is commonly used only to reflect cell wall structure. If Gram staining characterizes not only simple taxonomical dichotomy but also multiple biological functions, it may also be used to correlate bacterial cell wall structure with related physiological responses to the environment. In particular, Gram staining could supply ecological information on natural bacterial populations that are difficult to culture by the present technology.Membrane filter methods are widely used for microscopy in aquatic microbiology because of the low population densities of bacteria in many aquatic environments (4, 11, 16). However, these methods sometimes have problems associated with microscopic observations, causing unclear images of bacterial cells on Nuclepore filters when used with the conventional mounting medium (immersion oil; refractive index [nd] = 1.514). Hence, a suitable mounting agent must be applied to obtain precise image analyses of Gram-stained bacteria on Nuclepore filters.In this study, we have established a distinct method to characterize photometric Gram stain images; it involves the Gram stain index (GSI) for specifying natural bacterial populations in various aquatic environments. For this purpose, we have standardized the GSI of typical gram-negative and gram-positive bacteria by using Escherichia coli and Bacillus subtilis, respectively, and compared these GSI values to those of natural bacterial populations of several freshwater environments. The natural waters we investigated were Hyoutaro-ike pond, Matsumi-ike bog, and Lake Kasumigaura, which are oligotrophic, mesotrophic, and eutrophic water bodies, respectively, as previously determined (8, 10, 13, 18, 22, 23).     

Nonperturbative Imaging of Nucleoid Morphology in Live Bacterial Cells during an Antimicrobial Peptide Attack

Studies of time-dependent drug and environmental effects on single, live bacterial cells would benefit significantly from a permeable, nonperturbative, long-lived fluorescent stain specific to the nucleoids (chromosomal DNA). The ideal stain would not affect cell growth rate or nucleoid morphology and dynamics, even during laser illumination for hundreds of camera frames. In this study, time-dependent, single-cell fluorescence imaging with laser excitation and a sensitive electron-multiplying charge-coupled-device (EMCCD) camera critically tested the utility of “dead-cell stains” (SYTOX orange and SYTOX green) and “live-cell stains” (DRAQ5 and SYTO 61) and also 4′,6-diamidino-2-phenylindole (DAPI). Surprisingly, the dead-cell stains were nearly ideal for imaging live Escherichia coli, while the live-cell stains and DAPI caused nucleoid expansion and, in some cases, cell permeabilization and the halting of growth. SYTOX orange performed well for both the Gram-negative E. coli and the Gram-positive Bacillus subtilis. In an initial application, we used two-color fluorescence imaging to show that the antimicrobial peptide cecropin A destroyed nucleoid-ribosome segregation over 20 min after permeabilization of the E. coli cytoplasmic membrane, reminiscent of the long-term effects of the drug rifampin. In contrast, the human cathelicidin LL-37, while similar to cecropin A in structure, length, charge, and the ability to permeabilize bacterial membranes, had no observable effect on nucleoid-ribosome segregation. Possible underlying causes are suggested.     

Effects of chemically and electrochemically dosed chlorine on Escherichia coli and Legionella beliardensis assessed by flow cytometry

The present study reports the disinfection effects of chemically and electrochemically dosed chlorine on two models for typical water-borne bacteria (Escherichia coli and Legionella beliardensis) by plating and flow cytometry (FCM) in combination with different fluorescence dyes. The residual effect on various cell functions, including cultivability, esterase activity, membrane polarization, and integrity, was tested at different free chlorine concentrations. In comparison, chemical disinfection yielded on average 60% more E. coli cells entering the viable but nonculturable (VBNC) state than electrochemical disinfection. Here, VBNC is defined as those cells with intact cell membrane but which cannot be cultured on solid nutrient agar plates. L. beliardensis was about five times more resistant to chlorine disinfection than E. coli. The results also suggested the two methods result in different disinfection mechanisms on L. beliardensis, i.e., chemically dosed chlorine targeted cell membrane integrity before enzyme activity, while electrochemically dosed chlorine acted the other way round. In addition, both bacteria lost the integrity of their cell membranes at three times lower chlorine concentration over a longer contact time (i.e., 40 vs. 10 min) by the chemical method. Our results showed that FCM is an appropriate tool to evaluate the effects of water disinfection and the percentage of cells in VBNC in a matter of hours. Electrochemical disinfection is suggested to be a favorable alternative for chemical disinfection.     

In Situ Characterization of Differences in the Viscoelastic Response of Individual Gram-Negative and Gram-Positive Bacterial Cells

We used a novel atomic force microscopy (AFM)-based technique to compare the local viscoelastic properties of individual gram-negative (Escherichia coli) and gram-positive (Bacillus subtilis) bacterial cells. We found that the viscoelastic properties of the bacterial cells are well described by a three-component mechanical model that combines an instantaneous elastic response and a delayed elastic response. These experiments have allowed us to investigate the relationship between the viscoelastic properties and the structure and composition of the cell envelope. In addition, this is the first report in which the mechanical role of Lpp, the major peptidoglycan-associated lipoprotein and one of the most abundant outer membrane proteins in E. coli cells, has been quantified. We expect that our findings will be helpful in increasing the understanding of the structure-property relationships of bacterial cell envelopes.The surface layers that isolate the interior of a bacterial cell from its external environment play a crucial mechanical role in the survival of the cell. They must be strong enough to maintain the cellular shape and resist turgor pressure yet, at the same time, be flexible enough to allow cell growth and division. Their elastic response is evident from their ability to recover from transient deformations, such as those induced by the incorporation of additional surface components (e.g., proteins) in response to changes in environmental conditions and the passage of small molecules across the cell boundary. It is therefore clear that understanding many aspects of cell physiology requires knowledge of the mechanical properties of cells.The mechanical properties of the cell originate from the structural organization of the constituent lipids, sugar polymers, and proteins. Lipid molecules are brought together by their hydrophobic domains to form bilayers (membranes) that also incorporate different types of proteins. Polymeric strands of sugar molecules are typically cross-linked by flexible peptide molecules to form the peptidoglycan layer (27). Sometimes, an additional layer of proteins (S layer) is found on the outermost surface of the cell (7, 8, 40). Depending on the structural organization of the peptidoglycan and lipid bilayers, bacteria can generally be divided into gram-positive and gram-negative bacteria. In gram-positive cells, there is a relatively thick (20- to 35-nm) peptidoglycan layer that, together with the plasma membrane, sandwiches a viscous compartment called the periplasm (31, 32), whereas the envelope of gram-negative cells is made up of two lipid bilayers, the inner and outer membranes, separated by the periplasm, which contains a thin (3- to 8-nm) peptidoglycan layer (5, 33). In gram-negative bacteria, lipoproteins are associated with both the peptidoglycan layer and either the inner or outer membrane. Here, the “lipo” substituent is inserted into the hydrophobic domain of the membrane and the “protein” portion is linked to the peptidoglycan layer by either covalent or electrostatic bonds (18). Loss or altered expression of lipoproteins has been shown to affect cell shape generation and/or membrane integrity (10, 11, 13, 36, 43, 46), suggesting a possible mechanical role for these peptidoglycan-associated proteins.Although the structure and chemistry of the gram-negative and gram-positive bacterial cell envelopes are well known, information about their mechanical properties has been difficult to elucidate. The simple stretching model used by Isaac and Ware (21) to describe the flexibility of bacterial cells indicated differences in the deformability of bacterial cells. Further advances in the characterization of the mechanical properties of bacterial cells were achieved by using bacterial threads, which are so-called macrofibers obtained from cultures of a cell-separation-suppressed mutant that were investigated by standard fiber-testing techniques (34, 48, 49). The requirement to use filament-forming mutants for this mechanical measurement has restricted the studies to date to the gram-positive bacterium Bacillus subtilis. In these studies, bacterial threads of B. subtilis were shown to be viscoelastic by performing creep experiments, a transient rheological technique in which a known force is applied to the material and the resulting extension (or deformation) is measured over time. The properties measured in these experiments were extrapolated to those of the individual cells, often with tenuous lines of inference. Recently, remarkable advances have been made in applying atomic force microscopy (AFM) to quantify the mechanical properties of individual microbial cells (15, 55). Typically, AFM force-indentation curves, which represent the relationship between a loading force and the depth of the indentation as the tip at the end of the AFM cantilever pushes onto the sample surface, are measured. Quantitative information on the elasticity of the sample is then obtained from the force required to achieve a certain depth of penetration (3, 16, 37, 51). It is only recently that direct creep measurements have become possible at the individual cell level by using AFM (50). In AFM creep experiments, the loading force is maintained at a constant value by controlling the cantilever deflection, while the displacement of the cantilever base generated by the sample response to the applied load is measured as a function of time. The sample creep response can be then analyzed with theoretical mechanical models to provide quantitative information on sample viscoelasticity.In the present study, we used AFM creep experiments to probe and compare for the first time the local viscoelastic properties of individual gram-positive (B. subtilis) and gram-negative (Escherichia coli) bacterial cells. These experiments have allowed us to investigate the relationship between the viscoelastic properties and the structure and composition of the cell envelope. In addition, this is the first report in which the mechanical role of Lpp, the major peptidoglycan-associated lipoprotein and one of the most abundant outer membrane proteins in E. coli cells, has been quantified. We expect that our findings will be helpful in increasing the understanding of the structure-property relationships of bacterial cell envelopes.     

The exclusive use of flow cytometry to evaluate the antibiotic-susceptibility

Background

Live and injured bacteria cannot be successfully discriminated using flow cytometric methods (FCM) with commercial live/dead staining agents because injured cells have intact cell membranes and are counted as live cells. We previously reported that photoactivated ethidium monoazide (EMA) directly cleaves bacterial DNA both in vivo and in vitro (Microbiol. Immunol. 51:763-775, 2007).

Methods

We report that EMA cleaves the chromosomal DNA of antibiotic-injured, but not live, Listeria monocytogenes. The combination of FCM and EMA treatment was evaluated as a rapid method to discriminate between live and antibiotic-injured L. monocytogenes. Additionally, we evaluated our methodology using blood from pediatric patients infected with other gram-negative and gram-positive bacteria.

Results

For antibiotic-injured, but not live, L. monocytogenes in blood, photoactivated EMA suppressed SYTO9 staining, as the SYTO9 staining of the antibiotic-injured L. monocytogenes was weak compared with that of live cells. Similarly, the rapid and clear discrimination between live and injured bacteria (gram-negative and gram-positive) was performed using the blood of pediatric patients administered antibiotics.

Conclusions

The combination of FCM with EMA treatment is a rapid method for evaluating the susceptibility of live pathogens in infants with bacteremia without the need for bacterial culture.

General significance

This assay is more rapid than other currently available techniques due to the elimination of the time-consuming culture step and could be used in clinical settings to rapidly determine the success of antibiotic treatment in pediatric bacteremia through the discrimination of injured (i.e., susceptible to the administered antibiotics) and live pathogens.     

Examination of Recovery In Vitro and In Vivo of Nonculturable Escherichia coli O157:H7

Escherichia coli O157:H7 (strains ATCC 43895 and FO46) became nonculturable in sterile, distilled, deionized water or after exposure to chlorine. Recovery of nonculturable E. coli O157:H7 was examined by in vitro and in vivo methods. The decline in culturability of starved E. coli O157:H7 was measured by plate count on rich medium. Recovery in vitro of nonculturable cells was conducted with media amended with catalase or sodium pyruvate; however, there was no apparent increase over culturable cell counts on amended versus nonamended media. Although nonculturable E. coli O157:H7 did not recover under in vitro conditions, a mouse model was used to determine if in vivo conditions would provide sufficient conditions for recovery of nonculturable E. coli O157:H7. In separate studies, mice were orally challenged with starvation-induced nonculturable cells (FO46) or chlorine-induced nonculturable cells (43895 and FO46). Passage through the mouse gastrointestinal tract had no effect on recovery of nonculturable (starvation or chlorine induced) E. coli O157:H7 (43895 or FO46), based on analysis of fecal samples. Mouse kidneys were assayed for the presence of Shiga toxin using the Vero cell assay. Differences in cytotoxicity towards Vero cells from kidney samples of mice receiving nonculturable cells and control mice were not significant, suggesting a loss of virulence.     

Damage in Escherichia coli Cells Treated with a Combination of High Hydrostatic Pressure and Subzero Temperature

The relationship between membrane permeability, changes in ultrastructure, and inactivation in Escherichia coli strain K-12TG1 cells subjected to high hydrostatic pressure treatment at room and subzero temperatures was studied. Propidium iodide staining performed before and after pressure treatment made it possible to distinguish between reversible and irreversible pressure-mediated cell membrane permeabilization. Changes in cell ultrastructure were studied using transmission electron microscopy (TEM), which showed noticeable condensation of nucleoids and aggregation of cytosolic proteins in cells fixed after decompression. A novel technique used to mix fixation reagents with the cell suspension in situ under high hydrostatic pressure (HHP) and subzero-temperature conditions made it possible to show the partial reversibility of pressure-induced nucleoid condensation. However, based on visual examination of TEM micrographs, protein aggregation did not seem to be reversible. Reversible cell membrane permeabilization was noticeable, particularly for HHP treatments at subzero temperature. A correlation between membrane permeabilization and cell inactivation was established, suggesting different mechanisms at room and subzero temperatures. We propose that the inactivation of E. coli cells under combined HHP and subzero temperature occurs mainly during their transiently permeabilized state, whereas HHP inactivation at room temperature is related to a balance of transient and permanent permeabilization. The correlation between TEM results and cell inactivation was not absolute. Further work is required to elucidate the effects of pressure-induced damage on nucleoids and proteins during cell inactivation.     

Antimicrobial efficacy of extracts of Saudi Arabian desert Terfezia claveryi truffles

BackgroundTerfezia claveryi truffles are known for their nutritional value and have been considered among traditional treatments for ophthalmic infections and ailments.ObjectivesWe sought to investigate the in vitro antimicrobial efficacy of several T. claveryi extracts from Saudi Arabia. Certain pathogenic fungi and gram-negative and gram-positive bacteria were included.MethodsDry extracts were prepared using methanol, ethyl acetate, and distilled water, while the latter was used for preparing fresh extracts. The extracts were microbiologically evaluated through the disc-diffusion agar method; the zones of inhibition of microbial growth were measured post-incubation. The minimum bactericidal concentration (MBC) and minimum inhibitory concentration (MIC) were determined in Müller-Hinton Broth through the microdilution susceptibility method. anti-biofilm activity was assessed for potent extracts.ResultsDry extracts showed potent activity (>16-mm inhibition zones) against gram-positive (Bacillus subtilis IFO3007 and Staphylococcus aureus IFO3060) and gram-negative (Pseudomonas aeruginosa IFO3448 and Escherichia coli IFO3301) bacteria. The activity against fungi was moderate (12–16-mm inhibition zones) for both Aspergillus oryzae IFO4177 and Candida albicans IFO0583; there was no activity against Aspergillus niger IFO4414 growth. Methanolic extract had the lowest MIC and MBC, exhibiting remarkable activity against B. subtilis growth. Fresh extract showed moderate activity against bacterial growth and inactivity against fungal growth. Methanolic extract showed potent anti-biofilm activity (IC₅₀, 2.0 ± 0.18 mg/mL) against S. aureus.ConclusionsT. claveryi extracts showed antibacterial effects potentially suitable for clinical application, which warrants further in-depth analysis of their individual isolated compounds.     

Bacillus subtilis Homologs of MviN (MurJ), the Putative Escherichia coli Lipid II Flippase,Are Not Essential for Growth

Although peptidoglycan synthesis is one of the best-studied metabolic pathways in bacteria, the mechanism underlying the membrane translocation of lipid II, the undecaprenyl-disaccharide pentapeptide peptidoglycan precursor, remains mysterious. Recently, it was proposed that the essential Escherichia coli mviN gene encodes the lipid II flippase. Bacillus subtilis contains four proteins that are putatively homologous to MviN, including SpoVB, previously reported to be necessary for spore cortex peptidoglycan synthesis during sporulation. MviN complemented the sporulation defect of a ΔspoVB mutation, and SpoVB and another of the B. subtilis homologs, YtgP, complemented the growth defect of an E. coli strain depleted for MviN. Thus, these B. subtilis proteins are likely to be MviN homologs. However, B. subtilis strains lacking these four proteins have no defects in growth, indicating that they likely do not serve as lipid II flippases in this organism.Peptidoglycan synthesis is vital for cell growth and maintenance of cell shape in both gram-positive and gram-negative bacteria. This polymer of glycan chains that are cross-linked by peptide bridges forms an extracellular shell which provides protection against osmotic stresses as well as a sturdy scaffolding for extracellular appendages. The enzymes responsible for peptidoglycan synthesis are highly conserved in all bacteria with a cell wall. In the cytoplasm, the enzymes MurA to MurE synthesize the soluble MurNAc-pentapeptide starting with UDP-GlcNAc. MraY links this molecule to an isoprenoid chain, forming the membrane-associated lipid I precursor. MurG then adds UDP-GlcNAc to make lipid II, which is subsequently flipped across the cytoplasmic membrane and attached by penicillin-binding proteins via transglycosylation and transpeptidation reactions to the mature peptidoglycan.While these cytoplasmic and extracellular steps are well characterized, comparatively little is known about the mechanism of membrane translocation. Fluorescently tagged lipid II does not spontaneously flip in protein-free liposomes (31), as would be expected given its large hydrophilic carbohydrate and protein groups. This observation suggests that that flipping is a protein-mediated process, and, consistent with this prediction, fluorescent lipid II molecules were translocated across vesicles made from Escherichia coli membranes. Genetic data have pointed to proteins belonging to the SEDS family as potential lipid II flippases (14). These proteins are highly conserved and contain multiple membrane-spanning domains (generally 10 to 12 transmembrane helices). Since they are in most cases essential for viability, it has been problematic to demonstrate their function. However, depletion or temperature-sensitive mutations result in phenotypes consistent with a block in peptidoglycan synthesis. A nonessential SEDS protein, Bacillus subtilis SpoVE, is necessary for the formation of peptidoglycan during a later step in spore development (13), and point mutations in SpoVE block peptidoglycan synthesis without disturbing protein production or localization (24).Recently, the integral membrane protein MviN, encoded by an essential E. coli gene, was proposed to be the lipid II flippase (26). Strains carrying a temperature-sensitive mutation in MviN underwent lysis following incubation at the nonpermissive temperature and showed a twofold increase in lipid II accumulation (16). While the operon that includes mviN is essential in the gram-negative bacteria Sinorhizobium meliloti and Burkholderia pseudomallei (20, 25), mviN mutations in Rhizobium tropici, Salmonella enterica serovar Typhimurium, and Bdellovibrio bacteriovorus have not been fully characterized, and therefore the essentiality of MviN in these species remains to be demonstrated (4, 19, 21). Due to the high degree of conservation of other proteins involved in peptidoglycan synthesis between gram-positive and gram-negative bacteria and the essential nature of peptidoglycan synthesis, the protein(s) necessary for flipping of lipid II should also be essential and conserved in a gram-positive organism. We therefore set out to identify and examine the MviN (MurJ) homologs of B. subtilis.     

Growth of Listeria monocytogenes and Yersinia enterocolitica on Cooked Modified-Atmosphere-Packaged Poultry in the Presence and Absence of a Naturally Occurring Microbiota

Cooked poultry cuts were inoculated with five-strain composite mixtures of either Listeria monocytogenes or Yersinia enterocolitica (1,000 CFU/150-g piece), packaged in 44:56 CO₂-N₂, and stored at 3.5, 6.5, or 10°C for up to 5 weeks. Both L. monocytogenes and Y. enterocolitica grew under all test conditions. The presence of a naturally occurring microbiota did not influence the growth of either pathogen. Addition of lactate with the shelf life extender ALTA 2341 lengthened the lag phases of L. monocytogenes and Y. enterocolitica but did not prevent their growth.     

Comparison of Responses to Double-Strand Breaks between Escherichia coli and Bacillus subtilis Reveals Different Requirements for SOS Induction

DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in both Escherichia coli and Bacillus subtilis. In E. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell. E. coli strains incapable of SOS induction were sensitive to ionizing radiation. In striking contrast, we found that in B. subtilis both ionizing radiation and a site-specific double-strand break causes induction of prophage PBSX and SOS gene expression in only a small subpopulation of cells. These results show that double-strand breaks provoke global SOS induction in E. coli but not in B. subtilis. Remarkably, RecA-GFP focus formation was nearly identical following ionizing radiation challenge in both E. coli and B. subtilis, demonstrating that formation of RecA-GFP foci occurs in response to double-strand breaks but does not require or result in SOS induction in B. subtilis. Furthermore, we found that B. subtilis cells incapable of inducing SOS had near wild-type levels of survival in response to ionizing radiation. Moreover, B. subtilis RecN contributes to maintaining low levels of SOS induction during double-strand break repair. Thus, we found that the contribution of SOS induction to double-strand break repair differs substantially between E. coli and B. subtilis.     

Antimicrobial Peptide Trichokonin VI-Induced Alterations in the Morphological and Nanomechanical Properties of Bacillus subtilis

Antimicrobial peptides are promising alternative antimicrobial agents compared to conventional antibiotics. Understanding the mode of action is important for their further application. We examined the interaction between trichokonin VI, a peptaibol isolated from Trichoderma pseudokoningii, and Bacillus subtilis, a representative Gram-positive bacterium. Trichokonin VI was effective against B. subtilis with a minimal inhibitory concentration of 25 µM. Trichokonin VI exhibited a concentration- and time-dependent effect against B. subtilis, which was studied using atomic force microscopy. The cell wall of B. subtilis collapsed and the roughness increased upon treatment with trichokonin VI. Nanoindentation experiments revealed a progressive decrease in the stiffness of the cells. Furthermore, the membrane permeabilization effect of trichokonin VI on B. subtilis was monitored, and the results suggest that the leakage of intracellular materials is a possible mechanism of action for trichokonin VI, which led to alterations in the morphological and nanomechanical properties of B. subtilis.