scholarly journals Optical Tweezers Cause Physiological Damage to Escherichia coli and Listeria Bacteria

2008 ◽  
Vol 74 (8) ◽  
pp. 2441-2446 ◽  
Author(s):  
M. B. Rasmussen ◽  
L. B. Oddershede ◽  
H. Siegumfeldt
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Optical Tweezers ◽  
Laser Power ◽  
Fluorescent Protein ◽  
Growth Conditions ◽  
Ph Gradient ◽  
Bacterial Cells ◽  
E Coli

ABSTRACT We investigated the degree of physiological damage to bacterial cells caused by optical trapping using a 1,064-nm laser. The physiological condition of the cells was determined by their ability to maintain a pH gradient across the cell wall; healthy cells are able to maintain a pH gradient over the cell wall, whereas compromised cells are less efficient, thus giving rise to a diminished pH gradient. The pH gradient was measured by fluorescence ratio imaging microscopy by incorporating a pH-sensitive fluorescent probe, green fluorescent protein or 5(6)-carboxyfluorescein diacetate succinimidyl ester, inside the bacterial cells. We used the gram-negative species Escherichia coli and three gram-positive species, Listeria monocytogenes, Listeria innocua, and Bacillus subtilis. All cells exhibited some degree of physiological damage, but optically trapped E. coli and L. innocua cells and a subpopulation of L. monocytogenes cells, all grown with shaking, showed only a small decrease in pH gradient across the cell wall when trapped by 6 mW of laser power for 60 min. However, another subpopulation of Listeria monocytogenes cells exhibited signs of physiological damage even while trapped at 6 mW, as did B. subtilis cells. Increasing the laser power to 18 mW caused the pH gradient of both Listeria and E. coli cells to decrease within minutes. Moreover, both species of Listeria exhibited more-pronounced physiological damage when grown without shaking than was seen in cells grown with shaking, and the degree of damage is therefore also dependent on the growth conditions.

Relationship of growth conditions to desiccation tolerance of Salmonella enterica, Escherichia coli, and Listeria monocytogenes

2021 ◽  
Author(s):  
Rachel K Streufert ◽  
Susanne E Keller ◽  
Joelle K Salazar
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Desiccation Tolerance ◽  
Salmonella Enterica ◽  
Growth Conditions ◽  
Initial Population ◽  
Desiccation Resistance ◽  
E Coli ◽  
Solid Media ◽  
Agar Media

Growth on solid media as sessile cells is believed to increase the desiccation tolerance of Salmonella enterica . However, the reasons behind increased resistance have not been well explored. In addition, the same effect has not been examined for other foodborne pathogens such as pathogenic Escherichia coli or Listeria monocytogenes . The purpose of this research was two-fold: first, to determine the role of oxygenation during growth on the desiccation resistance of S. enterica , E. coli , and L. monocytogenes , and second, to determine the effect of sessile versus planktonic growth on the desiccation resistance of these pathogens. Three different serotypes each of Salmonella , E. coli , and L. monocytogenes were cultured in trypticase soy broth with 0.6% yeast extract (TSBYE), with (aerobic) shaking or on TSBYE with agar (TSAYE) under either aerobic or anaerobic conditions and harvested in stationary phase. After adding cell suspensions to cellulose filter disks, pathogen survival was determined by enumeration at 0 and after drying for 24 h. Results showed statistical differences in harvested initial populations prior to drying (0 h). For Salmonella , a correlation was found between high initial population and greater survival on desiccation (p = 0.05). In addition, statistical differences (p ≤ 0.05) between survival based on growth type were identified. However, differences found were not the same for the three pathogens, or between their serotypes. In general, Salmonella and E. coli desiccation resistance followed the pattern of aerobic agar media ≥ liquid media ≥ anaerobic agar media. For L. monocytogenes serotypes, resistance to desiccation was not statistically different based on mode of growth. These results indicate growth on solid media under aerobic conditions is not always necessary for optimal desiccation survival but may be beneficial when the desiccation resistance of the test serotype is unknown.

Analysis of the Crushing Effect about Ultrasound on E. coli and B. subtilis

2012 ◽  
Vol 260-261 ◽  
pp. 1017-1021
Author(s):  
Xin Ying Wang ◽  
Yong Tao Liu ◽  
Min Hui ◽  
Ji Fei Xu
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Bacillus Subtilis ◽  
Bacterial Cell ◽  
Logarithmic Phase ◽  
Growth Stages ◽  
Bacterial Cells ◽  
E Coli ◽  
Different Growth Stages ◽  
Main Factor

Escherichia coli and Bacillus subtilis as objects of the study, ultrasonic fragmentation acted on the bacterial cells in different growth stages, results showed that, it’s similar to the crushing effect of ultrasound on E. coli and B. subtilis cells of different growth stages, the highest crushing rate in the logarithmic phase, reached to 95.8% and 94.3% respectively, the crushing rate of adjustment phase is lowest, maintained at around 60%, the crushing rate stability cell was centered, which can be achieved 90%. The structure of the bacterial cell wall didn’t the main factor to decide the ultrasonic fragmentation effect, but different growth periods of bacterial cells did the determinant.

scholarly journals The active repertoire of Escherichia coli peptidoglycan amidases varies with physiochemical environment

2020 ◽  
Author(s):  
Elizabeth A. Mueller ◽  
Abbygail G. Iken ◽  
Mehmet Ali Öztürk ◽  
Mirko Schmitz ◽  
Barbara Di Ventura ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Separation ◽  
Antimicrobial Agents ◽  
Model Organism ◽  
Growth Conditions ◽  
Low Ph ◽  
E Coli ◽  
Ph Dependent ◽  
Stimulation Of

ABSTRACTNearly all bacteria are encased in a peptidoglycan cell wall, an essential crosslinked matrix of polysaccharide strands and short peptide stems. In the Gram-negative model organism Escherichia coli, more than forty cell wall synthases and autolysins coordinate the growth and division of the peptidoglycan sacculus in the periplasm. The precise contribution of many of these enzymes to cell wall metabolism remains unclear due to significant apparent redundancy, particularly among the cell wall autolysins. E. coli produces three major LytC-type-N-acetylmuramoyl-L-alanine amidases, which share a role in separating the newly formed daughter cells during cytokinesis. Here, we reveal two of the three amidases exhibit growth medium-dependent changes in activity. Specifically, we report acidic growth conditions stimulate AmiB—and to a lesser extent, AmiC—activity. Combining computational and genetic analysis, we demonstrate that low pH-dependent stimulation of AmiB requires three periplasmic amidase activators: EnvC, NlpD, and YgeR. Altogether, our findings support overlapping, but not redundant, roles for the E. coli amidases in cell separation and illuminate the physiochemical environment as an important mediator of cell wall enzyme activity.IMPORTANCEPenicillin and related β-lactam antibiotics targeting the bacterial cell wall synthesis are among the most commonly prescribed antimicrobials worldwide. However, rising rates of antibiotic resistance and tolerance jeopardize their continued clinical use. Development of new cell wall active therapeutics, including those targeting cell wall autolysins, has been stymied in part due to high levels of apparent enzymatic redundancy. In this study, we report a subset of E. coli amidases involved in cell separation during cell division are not redundant and instead are preferentially active during growth in distinct pH environments. Specifically, we discover E. coli amidases AmiB and AmiC are activated by acidic pH. Three semi-redundant periplasmic regulators—NlpD, EnvC, and YgeR—collectively mediate low pH-dependent stimulation of amidase activity. This discovery contributes to our understanding of how the cell wall remains robust across diverse environmental conditions and reveals opportunities for the development of condition-specific antimicrobial agents.

Validation of Apple Cider Pasteurization Treatments against Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes

2001 ◽  
Vol 64 (11) ◽  
pp. 1679-1689 ◽  
Author(s):  
PEGGY P. MAK ◽  
BARBARA H. INGHAM ◽  
STEVEN C. INGHAM
Keyword(s):  
Escherichia Coli ◽  
New York ◽  
Listeria Monocytogenes ◽  
Fluorescent Protein ◽  
Escherichia Coli O157 ◽  
Salmonella Spp ◽  
Apple Cider ◽  
E Coli ◽  
Log Reduction ◽  
Green Fluorescent

Time and temperature pasteurization conditions common in the Wisconsin cider industry were validated using a six-strain cocktail of Escherichia coli O157:H7 and acid-adapted E. coli O157:H7 in pH- and ∘Brix-adjusted apple cider. Strains employed were linked to outbreaks (ATCC 43894 and 43895, C7927, and USDA-FSIS-380–94) or strains engineered to contain the gene for green fluorescent protein (pGFP ATCC 43894 and pGFP ATCC 43889) for differential enumeration. Survival of Salmonella spp. (CDC 0778, CDC F2833, and CDC HO662) and Listeria monocytogenes (H0222, F8027, and F8369) was also evaluated. Inoculated cider of pH 3.3 or 4.1 and 11 or 14°Brix was heated under conditions ranging from 60°C for 14 s to 71.1°C for 14 s. A 5-log reduction of nonadapted and acid-adapted E. coli O157:H7 was obtained at 68.1°C for 14 s. Lower temperatures, or less time at 68.1°C, did not ensure a 5-log reduction in E. coli O157:H7. A 5-log reduction was obtained at 65.6°C for 14 s for Salmonella spp. L. monocytogenes survived 68.1°C for 14 s, but survivors died in cider within 24 h at 4°C. Laboratory results were validated with a surrogate E. coli using a bench-top plate heat-exchange pasteurizer. Results were further validated using fresh unpasteurized commercial ciders. Consumer acceptance of cider pasteurized at 68.1°C for 14 s (Wisconsin recommendations) and at 71.1°C for 6 s (New York recommendations) was not significantly different. Hence, we conclude that 68.1°C for 14 s is a validated treatment for ensuring adequate destruction of E. coli O157:H7, Salmonella spp., and L. monocytogenes in apple cider.

scholarly journals A MurF Inhibitor That Disrupts Cell Wall Biosynthesis in Escherichia coli

2007 ◽  
Vol 51 (12) ◽  
pp. 4420-4426 ◽  
Author(s):  
Ellen Z. Baum ◽  
Steven M. Crespo-Carbone ◽  
Alexandra Klinger ◽  
Barbara D. Foleno ◽  
Ignatius Turchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Antibacterial Activity ◽  
Binding Assay ◽  
Pharmacophore Model ◽  
Fold Increase ◽  
Cell Wall Biosynthesis ◽  
Bacterial Cells ◽  
E Coli ◽  
Essential Enzyme

ABSTRACT MurF is an essential enzyme of bacterial cell wall biosynthesis. Few MurF inhibitors have been reported, and none have displayed measurable antibacterial activity. Through the use of a MurF binding assay, a series of 8-hydroxyquinolines that bound to the Escherichia coli enzyme and inhibited its activity was identified. To derive additional chemotypes lacking 8-hydroxyquinoline, a known chelating moiety, a pharmacophore model was constructed from the series and used to select compounds for testing in the MurF binding and enzymatic inhibition assays. Whereas the original diverse library yielded 0.01% positive compounds in the binding assay, of which 6% inhibited MurF enzymatic activity, the pharmacophore-selected set yielded 14% positive compounds, of which 37% inhibited the enzyme, suggesting that the model enriched for compounds with affinity to MurF. A 4-phenylpiperidine (4-PP) derivative identified by this process displayed antibacterial activity (MIC of 8 μg/ml against permeable E. coli) including cell lysis and a 5-log10-unit decrease in CFU. Importantly, treatment of E. coli with 4-PP resulted in a 15-fold increase in the amount of the MurF UDP-MurNAc-tripeptide substrate, and a 50% reduction in the amount of the MurF UDP-MurNAc-pentapeptide product, consistent with inhibition of the MurF enzyme within bacterial cells. Thus, 4-PP is the first reported inhibitor of the MurF enzyme that may contribute to antibacterial activity by interfering with cell wall biosynthesis.

scholarly journals The Mechanism of Chlorine Damage Using Enhanced Green Fluorescent Protein-Expressing Escherichia coli

Water ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 2156
Author(s):  
Mizozoe ◽  
Otaki ◽  
Aikawa
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Green Fluorescent Protein ◽  
Cell Membrane ◽  
Fluorescence Intensity ◽  
Enhanced Green Fluorescent Protein ◽  
Hypochlorous Acid ◽  
Fluorescent Protein ◽  
E Coli ◽  
Green Fluorescent

This study investigated how chlorine inactivates and damages Escherichia coli cells. E. coli that had transformed to express enhanced green fluorescent protein (EGFP) at the cytoplasm was treated with chlorine. Damage to the cell membrane and cell wall was analyzed by measuring the fluorescence intensity of the leaked EGFP, then accounting for the fluorescence deterioration. At pH 7, E. coli was lethally damaged after treatment with chlorine, but significant leakage of EGFP was not observed. In contrast, significant leakage of EGFP was observed at pH 9, even though E. coli was not as inactivated as it was at pH 7. Flow cytometry was used to confirm the fluorescence intensity of the remaining EGFP inside the cells. No significant fluorescence loss was observed in the cells at pH 7. However, at pH 9, the fluorescence intensity in the cells decreased, indicating leakage of EGFP. These results suggest that hypochlorous acid inactivates E. coli without damaging its cell membrane and cell wall, whereas the hypochlorite ion inactivates E. coli by damaging its cell membrane and cell wall. It was possible to confirm the chlorine damage mechanism on E. coli by measuring the fluorescence intensity of the leaked EGFP.

scholarly journals Assessment of Photodynamic Destruction of Escherichia coli O157:H7 and Listeria monocytogenes by Using ATP Bioluminescence

2003 ◽  
Vol 69 (11) ◽  
pp. 6393-6398 ◽  
Author(s):  
N. A. Romanova ◽  
L. Y. Brovko ◽  
L. Moore ◽  
E. Pometun ◽  
A. P. Savitsky ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Yeast Cells ◽  
Bacterial Cells ◽  
E Coli ◽  
Intracellular Atp ◽  
Microdilution Method ◽  
Wide Range ◽  
Atp Bioluminescence ◽  
Time Courses

ABSTRACT Antimicrobial photodynamic therapy was shown to be effective against a wide range of bacterial cells, as well as for fungi, yeasts, and viruses. It was shown previously that photodestruction of yeast cells treated with photosensitizers resulted in cell destruction and leakage of ATP. Three photosensitizers were used in this study: tetra(N-methyl-4-pyridyl)porphine tetratosylate salt (TMPyP), toluidine blue O (TBO), and methylene blue trihydrate (MB). A microdilution method was used to determine MICs of the photosensitizers against both Escherichia coli O157:H7 and Listeria monocytogenes. To evaluate the effects of photodestruction on E. coli and L. monocytogenes cells, a bioluminescence method for detection of ATP leakage and a colony-forming assay were used. All tested photosensitizers were effective for photodynamic destruction of both bacteria. The effectiveness of photosensitizers (in microgram-per-milliliter equivalents) decreased in the order TBO > MB > TMPyP for both organisms. The MICs were two- to fourfold higher for E. coli O157:H7 than for L. monocytogenes. The primary effects of all of the photosensitizers tested on live bacterial cells were a decrease in intracellular ATP and an increase in extracellular ATP, accompanied by elimination of viable cells from the sample. The time courses of photodestruction and intracellular ATP leakage were different for E. coli and L. monocytogenes. These results show that bioluminescent ATP-metry can be used for investigation of the first stages of bacterial photodestruction.

scholarly journals Dynamic Localization of MreB in Vibrio parahaemolyticus and in the Ectopic Host Bacterium Escherichia coli

2008 ◽  
Vol 74 (21) ◽  
pp. 6739-6745 ◽  
Author(s):  
Shen-Wen Chiu ◽  
Shau-Yan Chen ◽  
Hin-chung Wong
Keyword(s):  
Escherichia Coli ◽  
Vibrio Parahaemolyticus ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Normal Growth ◽  
Growth Conditions ◽  
Host Bacterium ◽  
E Coli ◽  
Division Cell

ABSTRACT MreB, a homolog of eukaryotic actin, participates in morphogenesis, cell division, cell polarity, and chromosome segregation in prokaryotes. In this study, a yellow fluorescent protein conjugate (YFP-MreBVp) was generated to investigate the behavior of MreB in merodiploid strain SC9 of the enteropathogen Vibrio parahaemolyticus. Under normal growth conditions, YFP-MreBVp formed helical filaments with a pitch of 0.64 � 0.09 μm in about 85% of exponential-phase cells, and different clusters, relaxed coils, and ring configurations were observed in a small proportion of the cells. Overexpression of YFP-MreBVp substantially altered the structure of the MreB cytoskeleton and resulted in swollen and pleomorphic cells. Disturbing the activities of penicillin-binding proteins or adding magnesium suppressed the morphological distortions. These results indicate that mislocalization of cell wall-synthesizing machinery was responsible for morphological abnormality. By expressing YFP-MreBVp in the ectopic host bacterium Escherichia coli, shrinkage, fragmentation, and annealing of MreBVp filaments were directly observed. This work revealed the dynamic pattern of the localization of YFP-MreBVp in V. parahaemolyticus and its relationship to cell morphogenesis, and the YFP-MreBVp-E. coli system may be used to investigate the dynamic spatial structures of the MreB cytoskeleton in vivo.

Sites of Adsorption of the Bacteriophages T3 and T5 on the Wall of Escherichia Coli B.

1967 ◽  
Vol 25 ◽  
pp. 96-97
Author(s):  
Manfred E. Bayer
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Electron Microscope ◽  
Uranyl Acetate ◽  
E Coli ◽  
Receptor Sites ◽  
Rigid Cell Wall ◽  
Host Cell Surface ◽  
Bacterial Viruses ◽  
Escherichia Coli B

Bacterial viruses adsorb specifically to receptors on the host cell surface. Although the chemical composition of some of the cell wall receptors for bacteriophages of the T-series has been described and the number of receptor sites has been estimated to be 150 to 300 per E. coli cell, the localization of the sites on the bacterial wall has been unknown.When logarithmically growing cells of E. coli are transferred into a medium containing 20% sucrose, the cells plasmolize: the protoplast shrinks and becomes separated from the somewhat rigid cell wall. When these cells are fixed in 8% Formaldehyde, post-fixed in OsO4/uranyl acetate, embedded in Vestopal W, then cut in an ultramicrotome and observed with the electron microscope, the separation of protoplast and wall becomes clearly visible, (Fig. 1, 2). At a number of locations however, the protoplasmic membrane adheres to the wall even under the considerable pull of the shrinking protoplast. Thus numerous connecting bridges are maintained between protoplast and cell wall. Estimations of the total number of such wall/membrane associations yield a number of about 300 per cell.

scholarly journals A Metzincin and TIMP-Like Protein Pair of a Phage Origin Sensitize Listeria monocytogenes to Phage Lysins and Other Cell Wall Targeting Agents

2021 ◽  
Vol 9 (6) ◽  
pp. 1323
Author(s):  
Etai Boichis ◽  
Nadejda Sigal ◽  
Ilya Borovok ◽  
Anat A. Herskovits
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Mammalian Cells ◽  
Protein Pair ◽  
Growth Conditions ◽  
Wall Structure ◽  
Antibiotic Resistant ◽  
Extracellular Milieu ◽  
Virion Release ◽  
Genes Encoding

Infection of mammalian cells by Listeria monocytogenes (Lm) was shown to be facilitated by its phage elements. In a search for additional phage remnants that play a role in Lm’s lifecycle, we identified a conserved locus containing two XRE regulators and a pair of genes encoding a secreted metzincin protease and a lipoprotein structurally similar to a TIMP-family metzincin inhibitor. We found that the XRE regulators act as a classic CI/Cro regulatory switch that regulates the expression of the metzincin and TIMP-like genes under intracellular growth conditions. We established that when these genes are expressed, their products alter Lm morphology and increase its sensitivity to phage mediated lysis, thereby enhancing virion release. Expression of these proteins also sensitized the bacteria to cell wall targeting compounds, implying that they modulate the cell wall structure. Our data indicate that these effects are mediated by the cleavage of the TIMP-like protein by the metzincin, and its subsequent release to the extracellular milieu. While the importance of this locus to Lm pathogenicity remains unclear, the observation that this phage-associated protein pair act upon the bacterial cell wall may hold promise in the field of antibiotic potentiation to combat antibiotic resistant bacterial pathogens.

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