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Viability analysis of alginate encapsulated micro-organisms using fluorescent stains.

The encapsulation of micro-organisms such as bacteria and fungi in biopolymers is currently being evaluated as delivery systems in many fields. Information about the viability and morphology of the organisms in the microparticle is often required to ascertain the longevity of the systems. A rapid method using fluorescent stains for microbial viability has been validated for organisms within alginate microparticles. Usually viability is assessed by dissolving the microparticles and cell culturing. This new method is advantageous for slow growing or filamentous organisms because these are not quickly or accurately enumerated by plate counts. In addition, the technique also allows the morphology of the organism to be monitored over time.

Alginates↗

Inhibition of Escherichia coli viability by external guide sequences complementary to two essential genes.

Narrow spectrum antimicrobial activity has been designed to reduce the expression of two essential genes, one coding for the protein subunit of RNase P (C5 protein) and one for gyrase (gyrase A). In both cases, external guide sequences (EGS) have been designed to complex with either mRNA. Using the EGS technology, the level of microbial viability is reduced to less than 10% of the wild-type strain. The EGSs are additive when used together and depend on the number of nucleotides paired when attacking gyrase A mRNA. In the case of gyrase A, three nucleotides unpaired out of a 15-mer EGS still favor complete inhibition by the EGS but five unpaired nucleotides do not.

Bacterial Proteins↗

The ability of direct fluorescence-based, two-colour assays to detect different physiological states of oral streptococci.

AIMS: To investigate the ability of six fluorescent-based, two-colour viability assays to detect different physiological growth stages of two oral streptococci species. METHODS AND RESULTS: The growth of Streptococcus sanguinis and Strep. mutans from 0 to 73 h culture periods was monitored by cell labelling with six mixtures of fluorescent stains, in addition to the growth parameters optical density (O.D.), log values of the total cell counts (log BC ml(-1)) and of the colony-forming units (log cfu ml(-1)). CONCLUSION: In comparison with the corresponding cfu values as control, the vitality proportions determined by the Syto 9/PI test best reflected the dynamic growth pattern of both test strains. The direct fluorescent-based, two-colour assay Syto 9/PI provides valuable information about microbial viability stages. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection of viable but non-culturable bacteria requires more precise direct methods such as the fluorescent staining technique presented here, in addition to the classical plate count method.

Colony Count, Microbial↗

Comparison of antiadhesive and antibacterial effects of antiseptics on Streptococcus sanguinis.

Three antiseptic (chlorhexidine, Olaflur, Octenisept) and one putative antiadhesive (chitosan) agent were investigated for their effect on viable planktonic and attached Streptococcus sanguinis cells. The bacterial pretreatment with each chemotherapeutic was performed in two steps: (i) After the exposure of planktonic streptococci to the antiseptics, the cells were suspended in human sterile saliva and allowed to attach to human enamel for 60 min; (ii) After 60 min in the flow chamber system, initially attached streptococci were treated with these agents. The microbial viability was monitored by the percentage of vital streptococci determined by fluorescence microscopy and cell reproduction. In comparison with the negative control NaCl, the non-bactericidal chitosan derivative showed distinctive antiadhesive properties. For both treatment procedures, the efficacy of the antiseptics in reducing the viability of planktonic and attached streptococci was Octenisept > Olaflur > chlorhexidine > saline > chitosan. Further studies appear warranted to develop new antiplaque/antibiofilm strategies involving highly efficient bactericidals with antiadhesive formulations.

Anti-Infective Agents, Local↗

Antibiotic susceptibility testing by flow cytometry.

The first significant development in antibiotic susceptibility testing in recent years may be the flow cytofluorometric susceptibility test (FCST). The advanced analytical capability of the flow cytometer has provided the means to measure microbial diversity in culture. Membrane integrity and other indicators of microbial viability can be evaluated on a cell-by-cell basis. The FCST measures subtle dosage-response effects as well as the conventional minimum inhibitory and minimum bactericidal concentrations, simultaneously, in rapid tests which have the potential to supersede conventional techniques in terms of sensitivity and reproducibility.

Bacteria↗

Measurement of sputum Mycobacterium tuberculosis messenger RNA as a surrogate for response to chemotherapy.

Effective treatment regimens for pulmonary tuberculosis are difficult to assess because of the slow growth rate of Mycobacterium tuberculosis in culture and its protracted clearance from sputum. A rapid method that reflects effective antimicrobial activity would markedly advance evaluation of treatment and promote the assessment of new antituberculosis drugs. Conventional methods measure the progressive reduction of numbers of acid-fast bacilli in the sputum smear and the clearance of organisms in sputum culture. In this study, we measured levels of M. tuberculosis 85B (alpha antigen) messenger RNA (mRNA), 16S ribosomal RNA (rRNA), and IS6110 DNA in patients' sputa to ascertain whether they could serve as potential surrogate markers of response to chemotherapy. Sputum specimens were sequentially collected for up to a year from 19 smear-positive pulmonary tuberculosis patients receiving an optimal drug treatment regimen. Nucleic acids were isolated from these specimens, and two M. tuberculosis molecular targets (mRNA, DNA) were quantified, using the ABI Prism 7700 Sequence Detection System. The Mycobacterium genus-specific 16S rRNA was quantified with a limiting dilution RT-PCR assay. Results show that levels of 85B mRNA declined after initiation of therapy, as did viable M. tuberculosis colony counts, with 90% of patients becoming negative for both markers after 2 mo of treatment. The rapid disappearance of M. tuberculosis mRNA from sputum suggests that it is a good indicator of microbial viability and a useful marker for rapid assessment of response to chemotherapy.

Adult↗

Probiotic-derived extracellular vesicles as food-based nanocarriers: Mechanisms, functional applications, and future perspectives in food systems.

Probiotic-derived extracellular vesicles (PDEVs) are a promising type of postbiotic nanoparticle derived by fermentation of probiotics, and have gained growing interest as a potential application in food science and nutrition. These are lipid bilayer vesicles of nanoscale, which are naturally released by probiotic cells and contain a wide variety of bioactive molecules, such as proteins, nucleic acids, and metabolites. Moreover, PDEVs are highly stable, biocompatible, and can be easily engineered to have surfaces with high functionality, which makes them good candidates in functional engineering. In contrast to traditional live probiotics, PDEVs overcome the difficulties of preserving microbial viability during processing and storage, thus providing superior safety, stability, and predictable biological performance. This is a systematic review of the various functions of PDEVs in food systems. We conclude on the processes through which PDEVs control intestinal barrier integrity, alter gut microbiota composition, and alter host immune responses, and their potential to enhance gut health when added to functional foods. In addition to their health-promoting effects, PDEVs have shown significant potential as natural antimicrobial agents to preserve food and as effective nanocarriers of hydrophobic bioactive compounds, including fucoxanthin, to improve their stability, bioavailability, and targeted delivery. Moreover, PDEVs can be used as new regulators of microbial fermentation. However, it should be noted that a lot of the evidence that is available is still preliminary and the effectiveness of these applications in real food-processing and storage conditions has not been fully proven. Although they have potential, there are a number of challenges that still hinder the widespread use of PDEVs in the food industry. These involve the creation of scalable and cost-effective production processes, batch-to-batch consistency, vesicle stability in a variety of food matrices, and regulatory and safety considerations. Other emerging engineering approaches, such as surface functionalization and cargo loading, are also discussed in this review and could further increase the specificity, functionality, and application versatility of PDEVs in food systems. Moving forward, the incorporation of PDEVs into the next generation functional foods, novel food preservation methods, and customized nutrition plans should be prioritized in future studies. Further developments in these fields can make PDEVs useful platforms at the interface of food microbiology, nanotechnology, and human health.

Probiotics↗

Bacterial responses to ultraviolet irradiation.

The UV dose-response behavior of laboratory cultures of waterborne bacteria were examined for UV doses ranging from ca. 0-100 mW.s/cm2 using a collimated-beam reactor. Specific physiological responses measured in these tests included viability (ability to reproduce) and respiration (oxygen uptake rate). The results of these exposures indicated that resistance to UV-imposed loss of viability in E. coli cultures can be partially attributed to agglomeration during the irradiation process. From these results, it is conjectured that a bacterial population may be comprised of two sub-populations: one with low resistance (discrete or paired cells) and a second with high resistance (bacterial aggregates). A small fraction of the high-resistance portion of the population appears to be essentially unaffected by UV irradiation, thereby causing a discontinuity in the measured dose-response behavior. Moreover, the dose-response behavior of the highly resistant fraction is variable and difficult to describe quantitatively. The basis of these statements and most information in the literature is microbial viability as quantified by the membrane filtration assay. In contrast to these findings, the results of analyses for bacterial activity (respiration) suggest that comparatively little change in the population can be found to result from UV irradiation. This suggests that UV radiation accomplishes inactivation of the bacteria, but does not "kill" the bacterial cells per se, thereby highlighting the importance of considering bacterial repair processes in the design of UV disinfection systems.

Bacteria↗

Bactericidal properties of ozone and its potential application as a terminal disinfectant.

The efficacy of ozone as a terminal disinfectant was evaluated under laboratory conditions. Different microorganisms of importance to the food industry were inoculated onto stainless steel squares and incubated at various temperatures and relative humidities for up to 4 h. Survival of microorganisms from these controls was compared with identically incubated squares exposed to ozone. Exposure of the contaminated surfaces to ozone (2 ppm for 4 h) resulted in a reduction in microbial viability that ranged, depending on organism type, from 7.56 to 2.41 log values. For all the microorganisms tested, this loss in viability was significantly greater (P < 0.05) than that observed in the absence of ozone. Gram-negative bacteria were more sensitive to ozone than gram-positive organisms; bacteria were more sensitive than the yeast strain tested. Exposure to ozone (2 ppm for 4 h) in the presence of ultra-high temperature (UHT) milk resulted in a reduction in bacterial viability that ranged from 5.64 to 1.65 log values. In most cases, this reduction was significantly less (P < 0.05) than that achieved in the absence of organic material, although still significantly greater (P < 0.05) than that observed in the absence of ozone. The presence of a meat-based broth reduced the effectiveness of ozone to a greater extent, although the number of surviving gram-negative organisms was still significantly less (P < 0.05) than in the absence of ozone. Less than 1 log unit of yeast cells was destroyed when exposed to ozone in the presence of UHT milk or meat-based broth. Results of this investigation suggest that if applied after adequate cleaning ozone could be used as an effective disinfectant.

Animals↗

Bacterial adherence to surgical sutures: can antibacterial-coated sutures reduce the risk of microbial contamination?

BACKGROUND: Surgical site infections are associated with severe morbidity and mortality. The role of surgical sutures in the etiology of surgical site infection has been the objective of discussion for decades. This study used a standardized in vitro microbiologic model to assess bacterial adherence and the antibacterial activity of a triclosan-coated polyglactin 910 (braided) suture against selected Gram-positive and Gram-negative clinical isolates that may infect surgical wounds. STUDY DESIGN: Standardized cultures (2.0 log(10) colony forming units/mL and 5.0 log(10) colony forming units/mL of three clinical strains, Staphyllococcus aureus (methicillin-resistant S aureus [MRSA]), S epidermidis (biofilm-positive) and Escherichia coli (extended-spectrum beta-lactamase [ESBL]-producer) were inoculated to triclosan-coated and noncoated polyglactin 910 sutures to evaluate comparative adherence of bacterial isolates to the antibacterial coated and noncoated surgical sutures; to assess the impact of serum proteins (bovine serum albumin) on antibacterial activity of triclosan-coated suture; and to document the duration of antibacterial activity of the triclosan-coated material. Selected suture samples were prepared for scanning electron microscopy to demonstrate bacterial adherence. RESULTS: Substantial (p < 0.01) reductions in both Gram-positive and Gram-negative bacterial adherence were observed on triclosan-coated sutures compared with noncoated material. Pretreatment of surgical sutures with 20% BSA did not diminish antibacterial activity of the triclosan-coated braided device compared with noncoated suture (p < 0.01), and antibacterial activity was documented to persist for at least 96 hours compared with controls (p < 0.01). CONCLUSIONS: The in vitro model demonstrated a considerable reduction (p < 0.01) in Gram-positive and Gram-negative bacterial adherence to a triclosan-coated braided suture, which was associated with decreased microbial viability (p < 0.001). Because bacterial contamination of suture material within a surgical wound may increase the virulence of a surgical site infection, treating the suture with triclosan provides an effective strategy for reducing perioperative surgical morbidity.

Anti-Infective Agents, Local↗

Differential inactivation of Escherichia coli membrane dehydrogenases by a myeloperoxidase-mediated antimicrobial system.

Neutrophil myeloperoxidase, hydrogen peroxide, and chloride constitute a potent antimicrobial system with multiple effects on microbial cytoplasmic membranes. Among these is inhibition of succinate-dependent respiration mediated, principally, through inactivation of succinate dehydrogenase. Succinate-dependent respiration is inhibited at rates that correlate with loss of microbial viability, suggesting that loss of respiration might contribute to the microbicidal event. Because respiration in Escherichia coli can be mediated by dehydrogenases other than succinate dehydrogenase, the effects of the myeloperoxidase system on other membrane dehydrogenases were evaluated by histochemical activity stains of electrophoretically separated membrane proteins. Two bands of succinate dehydrogenase activity proved the most susceptible to inactivation with complete loss of staining activity within 20 min, under the conditions employed. A group with intermediate susceptibility, consisting of lactate, malate, glycerol-3-phosphate, and dihydroorotate dehydrogenases as well as three bands of glucose-6-phosphate dehydrogenase, was almost completely inactivated within 30 min. The relatively resistant group, including the dehydrogenases for glutamate, NADH, and NADPH and the remaining bands of glucose-6-phosphate dehydrogenase, retained substantial amounts of diaphorase activity for up to 60 min of incubation with the myeloperoxidase system. The differential effects of myeloperoxidase on dehydrogenase inactivation could not be correlated with published enzyme contents of flavin or iron-sulfur centers, potential targets of myeloperoxidase-derived oxidants. Despite the relative resistance of NADH dehydrogenase/diaphorase activity to myeloperoxidase-mediated inactivation, electron transport particles prepared from E. coli incubated for 20 min with the myeloperoxidase system lost 55% of their NADH oxidase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗

The alternative sigma factor sigmaE controls antioxidant defences required for Salmonella virulence and stationary-phase survival.

Bacteria must contend with conditions of nutrient limitation in all natural environments. Complex programmes of gene expression, controlled in part by the alternative sigma factors sigmaS (sigma38, RpoS) and sigmaH (sigma32, RpoH), allow a number of bacterial species to survive conditions of partial or complete starvation. We show here that the alternative sigma factor sigmaE (sigma24, RpoE) also facilitates the survival of Salmonella typhimurium under conditions of nutrient deprivation. Expression of the sigmaE regulon is strongly induced upon entry of Salmonella into stationary phase. A Salmonella mutant lacking sigmaE has reduced survival during stationary phase as well as increased susceptibility to oxidative stress. A Salmonella strain lacking both sigmaE and sigmaS is non-viable after just 24 h in stationary phase, but survival of these mutants is completely preserved under anaerobic stationary-phase conditions, suggesting that oxidative injury is one of the major mechanisms of reduced microbial viability during periods of nutrient deprivation. Moreover, the attenuated virulence of sigmaE-deficient Salmonella for mice can be largely restored by genetic abrogation of the host phagocyte respiratory burst, suggesting that the sigmaE regulon plays an important antioxidant role during Salmonella infection of mammalian hosts.

Aerobiosis↗

Type VI secretion system activity at lethal antibiotic concentrations leads to overestimation of weapon potency.

Competition assays are a mainstay of modern microbiology, offering a simple and cost-effective means to quantify microbe-microbe interactions in vitro. Here, we demonstrate a key weakness of this method that arises when competing microbes interact via toxins, such as those secreted via the type VI secretion system (T6SS). Time-lapse microscopy reveals that T6SS-armed Acinetobacter baylyi bacteria can maintain lethal T6SS activity against E. coli target cells, even under selective conditions intended to eliminate A. baylyi. Further, this residual killing creates a density- and T6SS-dependent bias in the apparent recovery of E. coli, leading to a misreporting of competition outcomes where target survival is low. We also show that incubating A. baylyi/E. coli co-cultures in liquid antibiotic prior to selective plating can substantially correct this bias. Our findings demonstrate the need for caution when using selective plating as part of T6SS competition assays, or assays involving other toxin-producing bacteria.

Type VI Secretion Systems↗

Hypochlorous acid and myeloperoxidase-catalyzed oxidation of iron-sulfur clusters in bacterial respiratory dehydrogenases.

Hypochlorous acid and related oxidants derived from myeloperoxidase-catalyzed reactions contribute to the microbicidal activities of phagocytosing neutrophils and monocytes. Microbial iron-sulfur (Fe/S) clusters have been suggested as general targets of myeloperoxidase-derived oxidations, but no susceptible Fe/S site has yet been identified. In this study, the effects of HOCl and myeloperoxidase-catalyzed peroxidation of chloride ion upon EPR-detectable Fe/S clusters in Escherichia coli and Pseudomonas aeruginosa were examined. Increasing amounts of oxidant produced progressive loss of signal amplitudes from the S-1 and S-3 Fe/S clusters of succinate:ubiquinone oxidoreductase in respiring membrane fragments. These changes were compared to loss of microbial viability, succinate uptake rates, succinate dehydrogenase activity and succinate-dependent respiration. The amounts of oxidant required to destroy Fe/S clusters exceeded the amounts required to kill organisms or inhibit respiratory function by factors of four or five. Power saturation characteristics of the S-1 signal indicated that the S-2 signal was also resistant to modification, even in highly oxidized membranes. Loss of succinate-dependent respiration was closely associated with HOCl and myeloperoxidase-mediated microbicidal activity against P. aeruginosa and was also an early event in the oxidant-mediated metabolic dysfunctions of E. coli. However, these effects were not caused by the destruction of the Fe/S clusters within the succinate:ubiquinone oxidoreductase. Rather, the major respiration-inhibiting lesion(s) appeared to reside at points in the respiratory chain between the Fe/S clusters and the ubiquinone reductase site.

Electron Spin Resonance Spectroscopy↗

Microbial activity at gigapascal pressures.

We observed physiological and metabolic activity of Shewanella oneidensis strain MR1 and Escherichia coli strain MG1655 at pressures of 68 to 1680 megapascals (MPa) in diamond anvil cells. We measured biological formate oxidation at high pressures (68 to 1060 MPa). At pressures of 1200 to 1600 MPa, living bacteria resided in fluid inclusions in ice-VI crystals and continued to be viable upon subsequent release to ambient pressures (0.1 MPa). Evidence of microbial viability and activity at these extreme pressures expands by an order of magnitude the range of conditions representing the habitable zone in the solar system.

Adaptation, Physiological↗

Inactivation of Escherichia coli penicillin-binding proteins by human neutrophils.

Neutrophils use a variety of microbicidal mechanisms in their role as one of the primary arms of the human host defense system. We have previously observed that a cell-free system containing myeloperoxidase (MPO), one of the major components of the neutrophil's oxidative antimicrobial systems, inactivated microbial penicillin-binding proteins (PBPs), which mediate the formation of the peptidoglycan layer of eubacterial cell walls. This is a potentially important mechanism of MPO-mediated bacterial toxicity. Since numerous other microbicidal systems, both oxidative and nonoxidative, are used by whole neutrophils, we investigated the effect of intact neutrophils on Escherichia coli PBPs. Penicillin binding activity was progressively reduced by neutrophil exposure for all PBPs. Loss of penicillin binding activity correlated well with loss of microbial viability for almost all PBPs. Azide-treated neutrophils, MPO-deficient neutrophils, and chronic granulomatous disease neutrophils inactivated E. coli PBPs in a manner similar to that of normal neutrophils, suggesting that MPO-independent, and even oxygen-independent, microbicidal systems are also involved in inactivation of PBPs. PBP inactivation, an antimicrobial strategy used by beta-lactam-producing molds (and now by physicians), may be an important microbicidal mechanism used by human neutrophils.

Azides↗

Inhibitory effect of saliva on glutamic acid accumulation by Lactobacillus acidophilus and the role of the lactoperoxidase-thiocyanate system.

Clem, W. H. (University of Washington, Seattle), and S. J. Klebanoff. Inhibitory effect of saliva on glutamic acid accumulation by Lactobacillus acidophilus and the role of the lactoperoxidase-thiocyanate system. J. Bacteriol. 91:1848-1853. 1966.-Saliva contains an antimicrobial system which inhibits the growth of Lactobacillus acidophilus, as well as a number of other organisms, in complete growth medium. This antimicrobial system consists of the salivary peroxidase (lactoperoxidase) and thiocyanate ions, and requires the presence of H(2)O(2). Saliva inhibits the accumulation of glutamic acid and certain other amino acids by resting cells. This effect of saliva is decreased by dialysis, and thiocyanate ions restore the inhibitory effect of dialyzed saliva. The inhibitory effect of saliva is decreased by heat (100 C, 10 min), and lactoperoxidase restores the inhibitory effect of heated saliva. Thus, the inhibition of glutamic acid accumulation by saliva appears to be due in part to the lactoperoxidase-thiocyanate antimicrobial system. H(2)O(2) increases the inhibitory effect of both saliva and the lactoperoxidase-thiocyanate system on glutamic acid accumulation. The inhibition of glutamic acid accumulation is not preceded by a loss in microbial viability. The glutamic acid accumulated by L. acidophilus under the conditions employed remains largely (over 90%) as free glutamic acid. This suggests that saliva and the lactoperoxidase-thiocyanate-H(2)O(2) system inhibit the net transport of glutamic acid into the cell.

Carbon Isotopes↗

Galectin-3 induces death of Candida species expressing specific beta-1,2-linked mannans.

Lectins play a critical role in host protection against infection. The galectin family of lectins recognizes saccharide ligands on a variety of microbial pathogens, including viruses, bacteria, and parasites. Galectin-3, a galectin expressed by macrophages, dendritic cells, and epithelial cells, binds bacterial and parasitic pathogens including Leishmania major, Trypanosoma cruzi, and Neisseria gonorrhoeae. However, there have been no reports of galectins having direct effects on microbial viability. We found that galectin-3 bound only to Candida albicans species that bear beta-1,2-linked oligomannans on the cell surface, but did not bind Saccharomyces cerevisiae that lacks beta-1,2-linked oligomannans. Surprisingly, binding directly induced death of Candida species containing specific beta-1,2-linked oligomannosides. Thus, galectin-3 can act as a pattern recognition receptor that recognizes a unique pathogen-specific oligosaccharide sequence. This is the first description of antimicrobial activity for a member of the galectin family of mammalian lectins; unlike other lectins of the innate immune system that promote opsonization and phagocytosis, galectin-3 has direct fungicidal activity against opportunistic fungal pathogens.

Candida↗