scholarly journals Autonomous Growth of Isolated Single Listeria monocytogenes and Salmonella enterica Serovar Typhimurium Cells in the Absence of Growth Factors and Intercellular Contact

2010 ◽  
Vol 76 (8) ◽  
pp. 2600-2606 ◽  
Author(s):  
Barbara Roeder ◽  
Martin Wagner ◽  
Peter Rossmanith
Keyword(s):  
Growth Factors ◽  
Listeria Monocytogenes ◽  
Salmonella Enterica ◽  
Single Cells ◽  
Intercellular Contact ◽  
Bacterial Cells ◽  
Chi Square ◽  
Significant Difference ◽  
Serovar Typhimurium ◽  
Autonomous Growth

ABSTRACT The aim of this study was to observe growth of isolated single bacterial cells in the absence of growth factors and intercellular contact. In order to exclude stochastic uncertainties induced by dilution series, a new micromanipulation method was developed to ensure explicit results under visual control. This was performed with particular care for production of single prokaryotic cells and subsequent investigation of their autonomous growth. Over 450 single isolated Listeria monocytogenes and Salmonella enterica subsp. enterica serovar Typhimurium cells in lag, log, and stationary growth phases were investigated by this method, which included thoroughly washing the cells. The proportion of living cells within the initial cultures was compared to the proportion of positive samples after enrichment of the separated single cells. This resulted in P values of ≥0.05 using the chi-square test for statistical analysis, indicating no significant difference, and clearly demonstrates reproduction of isolated single bacterial cells without the need for growth factors or intercellular contact. Ease of handling of the apparatus and good performance of the cleaning procedures were achieved, as was validation of the method, demonstrating its suitability for routine laboratory use.

TolA mediates the differential detergent resistance pattern between the Salmonella enterica subsp. enterica serovars Typhi and Typhimurium

Microbiology ◽  
2011 ◽  
Vol 157 (5) ◽  
pp. 1402-1415 ◽  
Author(s):  
Amit Lahiri ◽  
T. K. Ananthalakshmi ◽  
Arvindhan G. Nagarajan ◽  
Seemun Ray ◽  
Dipshikha Chakravortty
Keyword(s):  
Outer Membrane ◽  
Salmonella Enterica ◽  
Single Cells ◽  
Membrane Integrity ◽  
Luria Broth ◽  
Resistance Pattern ◽  
Significant Difference ◽  
Serovar Typhimurium ◽  
Bile Resistance

The tol–pal genes are essential for maintaining the outer membrane integrity and detergent resistance in various Gram-negative bacteria, including Salmonella. The role of TolA has been well established for the bile resistance of Salmonella enterica subsp. enterica serovar Typhimurium. We compared the bile resistance pattern between the S. enterica serovars Typhi and Typhimurium and observed that Typhi is more resistant to bile-mediated damage. A closer look revealed a significant difference in the TolA sequence between the two serovars which contributes to the differential detergent resistance. The tolA knockout of both the serovars behaves completely differently in terms of membrane organization and morphology. The role of the Pal proteins and difference in LPS organization between the two serovars were verified and were found to have no direct connection with the altered bile resistance. In normal Luria broth (LB), S. Typhi ΔtolA is filamentous while S. Typhimurium ΔtolA grows as single cells, similar to the wild-type. In low osmolarity LB, however, S. Typhimurium ΔtolA started chaining and S. Typhi ΔtolA showed no growth. Further investigation revealed that the chaining phenomenon observed was the result of failure of the outer membrane to separate in the dividing cells. Taken together, the results substantiate the evolution of a shorter TolA in S. Typhi to counteract high bile concentrations, at the cost of lower osmotic tolerance.

scholarly journals Clarithromycin Exerts an Antibiofilm Effect against Salmonella enterica Serovar Typhimurium rdar Biofilm Formation and Transforms the Physiology towards an Apparent Oxygen-Depleted Energy and Carbon Metabolism

2020 ◽  
Vol 88 (11) ◽  
Author(s):  
Munirah Zafar ◽  
Humera Jahan ◽  
Sulman Shafeeq ◽  
Manfred Nimtz ◽  
Lothar Jänsch ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Salmonella Enterica ◽  
Biofilm Formation ◽  
Antibiofilm Activity ◽  
Bacterial Cells ◽  
Content Type ◽  
Regulatory Pathways ◽  
Heat Shock Stress ◽  
Extracellular Matrix Components ◽  
Serovar Typhimurium

ABSTRACT Upon biofilm formation, production of extracellular matrix components and alteration in physiology and metabolism allows bacteria to build up multicellular communities which can facilitate nutrient acquisition during unfavorable conditions and provide protection toward various forms of environmental stresses to individual cells. Thus, bacterial cells within biofilms become tolerant against antimicrobials and the immune system. In the present study, we evaluated the antibiofilm activity of the macrolides clarithromycin and azithromycin. Clarithromycin showed antibiofilm activity against rdar (red, dry, and rough) biofilm formation of the gastrointestinal pathogen Salmonella enterica serovar Typhimurium ATCC 14028 (Nalr) at a 1.56 μM subinhibitory concentration in standing culture and dissolved cell aggregates at 15 μM in a microaerophilic environment, suggesting that the oxygen level affects the activity of the drug. Treatment with clarithromycin significantly decreased transcription and production of the rdar biofilm activator CsgD, with biofilm genes such as csgB and adrA to be concomitantly downregulated. Although fliA and other flagellar regulon genes were upregulated, apparent motility was downregulated. RNA sequencing showed a holistic cell response upon clarithromycin exposure, whereby not only genes involved in the biofilm-related regulatory pathways but also genes that likely contribute to intrinsic antimicrobial resistance, and the heat shock stress response were differentially regulated. Most significantly, clarithromycin exposure shifted the cells toward an apparent oxygen- and energy-depleted status, whereby the metabolism that channels into oxidative phosphorylation was downregulated, and energy gain by degradation of propane 1,2-diol, ethanolamine and l-arginine catabolism, potentially also to prevent cytosolic acidification, was upregulated. This analysis will allow the subsequent identification of novel intrinsic antimicrobial resistance determinants.

scholarly journals Gene-Specific Effects of Antisense Phosphorodiamidate Morpholino Oligomer-Peptide Conjugates on Escherichia coli and Salmonella enterica Serovar Typhimurium in Pure Culture and in Tissue Culture

2006 ◽  
Vol 50 (8) ◽  
pp. 2789-2796 ◽  
Author(s):  
Lucas D. Tilley ◽  
Orion S. Hine ◽  
Jill A. Kellogg ◽  
Jed N. Hassinger ◽  
Dwight D. Weller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Luciferase Reporter ◽  
Bacterial Cells ◽  
Luciferase Reporter Gene ◽  
E Coli ◽  
Specific Effects ◽  
Covalently Bonded ◽  
Serovar Typhimurium ◽  
Free Translation

ABSTRACT The objective was to improve efficacy of antisense phosphorodiamidate morpholino oligomers (PMOs) by improving their uptake into bacterial cells. Four different bacterium-permeating peptides, RFFRFFRFFXB, RTRTRFLRRTXB, RXXRXXRXXB, and KFFKFFKFFKXB (X is 6-aminohexanoic acid and B isβ -alanine), were separately coupled to two different PMOs that are complementary to regions near the start codons of a luciferase reporter gene (luc) and a gene required for viability (acpP). Luc peptide-PMOs targeted to luc inhibited luciferase activity 23 to 80% in growing cultures of Escherichia coli. In cell-free translation reactions, Luc RTRTRFLRRTXB-PMO inhibited luciferase synthesis significantly more than the other Luc peptide-PMOs or the Luc PMO not coupled to peptide. AcpP peptide-PMOs targeted to acpP inhibited growth of E. coli or Salmonella enterica serovar Typhimurium to various extents, depending on the strain. The concentrations of AcpP RFFRFFRFFXB-PMO, AcpP RTRTRFLRRTXB-PMO, AcpP KFFKFFKFFKXB-PMO, and ampicillin that reduced CFU/ml by 50% after 8 h of growth (50% inhibitory concentration [IC50]) were 3.6, 10.8, 9.5, and 7.5μ M, respectively, in E. coli W3110. Sequence-specific effects of AcpP peptide-PMOs were shown by rescuing growth of a merodiploid strain that expressed acpP with silent mutations in the region targeted by AcpP peptide-PMO. In Caco-2 cultures infected with enteropathogenic E. coli (EPEC), 10 μM AcpP RTRTRFLRRTXB-PMO or AcpP RFFRFFRFFXB-PMO essentially cleared the infection. The IC50 of either AcpP RTRTRFLRRTXB-PMO or AcpP RFFRFFRFFXB-PMO in EPEC-infected Caco-2 culture was 3 μM. In summary, RFFRFFRFFXB, RTRTRFLRRTXB, or KFFKFFKFFXB, when covalently bonded to PMO, significantly increased inhibition of expression of targeted genes compared to PMOs without attached peptide.

scholarly journals Differences in Enzymatic Properties Allow SodCI but Not SodCII To Contribute to Virulence in Salmonella enterica Serovar Typhimurium Strain 14028

2004 ◽  
Vol 186 (16) ◽  
pp. 5230-5238 ◽  
Author(s):  
Radha Krishnakumar ◽  
Maureen Craig ◽  
James A. Imlay ◽  
James M. Slauch
Keyword(s):  
Salmonella Enterica ◽  
Osmotic Shock ◽  
Acid Resistance ◽  
Noncovalent Interaction ◽  
Open Reading Frames ◽  
Striking Difference ◽  
Significant Difference ◽  
Serovar Typhimurium

ABSTRACT Salmonella enterica serovar Typhimurium produces two Cu/Zn cofactored periplasmic superoxide dismutases, SodCI and SodCII. While mutations in sodCI attenuate virulence eightfold, loss of SodCII does not confer a virulence phenotype, nor does it enhance the defect observed in a sodCI background. Despite this in vivo phenotype, SodCI and SodCII are expressed at similar levels in vitro during the stationary phase of growth. By exchanging the open reading frames of sodCI and sodCII, we found that SodCI contributes to virulence when placed under the control of the sodCII promoter. In contrast, SodCII does not contribute to virulence even when expressed from the sodCI promoter. Thus, the disparity in virulence phenotypes is due primarily to some physical difference between the two enzymes. In an attempt to identify the unique property of SodCI, we have tested factors that might affect enzyme activity inside a phagosome. We found no significant difference between SodCI and SodCII in their resistance to acid, resistance to hydrogen peroxide, or ability to obtain copper in a copper-limiting environment. Both enzymes are synthesized as apoenzymes in the absence of copper and can be fully remetallated when copper is added. The one striking difference that we noted is that, whereas SodCII is released normally by an osmotic shock, SodCI is “tethered” within the periplasm by an apparently noncovalent interaction. We propose that this novel property of SodCI is crucial to its ability to contribute to virulence in serovar Typhimurium.

scholarly journals Modeling Stochastic Variability in the Numbers of Surviving Salmonella enterica, Enterohemorrhagic Escherichia coli, and Listeria monocytogenes Cells at the Single-Cell Level in a Desiccated Environment

2016 ◽  
Vol 83 (4) ◽  
Author(s):  
Kento Koyama ◽  
Hidekazu Hokunan ◽  
Mayumi Hasegawa ◽  
Shuso Kawamura ◽  
Shigenobu Koseki
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Probability Distribution ◽  
Poisson Process ◽  
Poisson Distribution ◽  
Salmonella Enterica ◽  
Bacterial Cells ◽  
Content Type ◽  
Cell Numbers ◽  
Inactivation Process

ABSTRACT Despite effective inactivation procedures, small numbers of bacterial cells may still remain in food samples. The risk that bacteria will survive these procedures has not been estimated precisely because deterministic models cannot be used to describe the uncertain behavior of bacterial populations. We used the Poisson distribution as a representative probability distribution to estimate the variability in bacterial numbers during the inactivation process. Strains of four serotypes of Salmonella enterica, three serotypes of enterohemorrhagic Escherichia coli, and one serotype of Listeria monocytogenes were evaluated for survival. We prepared bacterial cell numbers following a Poisson distribution (indicated by the parameter λ, which was equal to 2) and plated the cells in 96-well microplates, which were stored in a desiccated environment at 10% to 20% relative humidity and at 5, 15, and 25°C. The survival or death of the bacterial cells in each well was confirmed by adding tryptic soy broth as an enrichment culture. Changes in the Poisson distribution parameter during the inactivation process, which represent the variability in the numbers of surviving bacteria, were described by nonlinear regression with an exponential function based on a Weibull distribution. We also examined random changes in the number of surviving bacteria using a random number generator and computer simulations to determine whether the number of surviving bacteria followed a Poisson distribution during the bacterial death process by use of the Poisson process. For small initial cell numbers, more than 80% of the simulated distributions (λ = 2 or 10) followed a Poisson distribution. The results demonstrate that variability in the number of surviving bacteria can be described as a Poisson distribution by use of the model developed by use of the Poisson process. IMPORTANCE We developed a model to enable the quantitative assessment of bacterial survivors of inactivation procedures because the presence of even one bacterium can cause foodborne disease. The results demonstrate that the variability in the numbers of surviving bacteria was described as a Poisson distribution by use of the model developed by use of the Poisson process. Description of the number of surviving bacteria as a probability distribution rather than as the point estimates used in a deterministic approach can provide a more realistic estimation of risk. The probability model should be useful for estimating the quantitative risk of bacterial survival during inactivation.

scholarly journals Effect of Electropermeabilization by Ohmic Heating for Inactivation of Escherichia coli O157:H7, Salmonella enterica Serovar Typhimurium, and Listeria monocytogenes in Buffered Peptone Water and Apple Juice

2013 ◽  
Vol 79 (23) ◽  
pp. 7122-7129 ◽  
Author(s):  
Il-Kyu Park ◽  
Dong-Hyun Kang
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Electric Field ◽  
Salmonella Enterica ◽  
Apple Juice ◽  
Ohmic Heating ◽  
Conventional Heating ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Peptone Water

ABSTRACTThe effect of electric field-induced ohmic heating for inactivation ofEscherichia coliO157:H7,Salmonella entericaserovar Typhimurium, andListeria monocytogenesin buffered peptone water (BPW) (pH 7.2) and apple juice (pH 3.5; 11.8 °Brix) was investigated in this study. BPW and apple juice were treated at different temperatures (55°C, 58°C, and 60°C) and for different times (0, 10, 20, 25, and 30 s) by ohmic heating compared with conventional heating. The electric field strength was fixed at 30 V/cm and 60 V/cm for BPW and apple juice, respectively. Bacterial reduction resulting from ohmic heating was significantly different (P< 0.05) from that resulting from conventional heating at 58°C and 60°C in BPW and at 55°C, 58°C, and 60°C in apple juice for intervals of 0, 10, 20, 25, and 30 s. These results show that electric field-induced ohmic heating led to additional bacterial inactivation at sublethal temperatures. Transmission electron microscopy (TEM) observations and the propidium iodide (PI) uptake test were conducted after treatment at 60°C for 0, 10, 20, 25 and 30 s in BPW to observe the effects on cell permeability due to electroporation-caused cell damage. PI values when ohmic and conventional heating were compared were significantly different (P< 0.05), and these differences increased with increasing levels of inactivation of three food-borne pathogens. These results demonstrate that ohmic heating can more effectively reduce bacterial populations at reduced temperatures and shorter time intervals, especially in acidic fruit juices such as apple juice. Therefore, loss of quality can be minimized in a pasteurization process incorporating ohmic heating.

scholarly journals The Multi-Copper-Ion Oxidase CueO of Salmonella enterica Serovar Typhimurium Is Required for Systemic Virulence

2010 ◽  
Vol 78 (5) ◽  
pp. 2312-2319 ◽  
Author(s):  
Maud E. S. Achard ◽  
Jai J. Tree ◽  
James A. Holden ◽  
Kim R. Simpfendorfer ◽  
Odilia L. C. Wijburg ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Copper Ions ◽  
Copper Ion ◽  
Mesenteric Lymph Nodes ◽  
E Coli ◽  
Significant Difference ◽  
Serovar Typhimurium ◽  
Branched Chain ◽  
Periplasmic Enzyme

ABSTRACT Salmonella enterica serovar Typhimurium possesses a multi-copper-ion oxidase (multicopper oxidase), CueO (also known as CuiD), a periplasmic enzyme known to be required for resistance to copper ions. CueO from S. Typhimurium was expressed as a recombinant protein in Escherichia coli, and the purified protein exhibited a high cuprous oxidase activity. We have characterized an S. Typhimurium cueO mutant and confirmed that it is more sensitive to copper ions. Using a murine model of infection, it was observed that the cueO mutant was significantly attenuated, as indicated by reduced recovery of bacteria from liver and spleen, although there was no significant difference in recovery from Peyer's patches and mesenteric lymph nodes. However, the intracellular survival of the cueO mutant in unprimed or gamma-interferon-primed murine macrophages was not statistically different from that of wild-type Salmonella, suggesting that additional host factors are involved in clearance of the cueO mutant. Unlike a cueO mutant from E. coli, the S. Typhimurium cueO mutant did not show greater sensitivity to hydrogen peroxide and its sensitivity to copper ions was not affected by siderophores. Similarly, the S. Typhimurium cueO mutant was not rescued from copper ion toxicity by addition of the branched-chain amino acids and leucine.

Influence of the Treatment of Listeria monocytogenes and Salmonella enterica Serovar Typhimurium with Citral on the Efficacy of Various Antibiotics

2014 ◽  
Vol 11 (4) ◽  
pp. 265-271 ◽  
Author(s):  
Surama F. Zanini ◽  
Angela B. Silva-Angulo ◽  
Amauri Rosenthal ◽  
Dolores Rodrigo Aliaga ◽  
Antonio Martínez
Keyword(s):  
Listeria Monocytogenes ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Serovar Typhimurium

scholarly journals Effect of Frequency and Waveform on Inactivation of Escherichia coli O157:H7 and Salmonella enterica Serovar Typhimurium in Salsa by Ohmic Heating

2012 ◽  
Vol 79 (1) ◽  
pp. 10-17 ◽  
Author(s):  
Su-Yeon Lee ◽  
Sangryeol Ryu ◽  
Dong-Hyun Kang
Keyword(s):  
Escherichia Coli ◽  
Electrical Conductivity ◽  
Heating Rate ◽  
Salmonella Enterica ◽  
Treatment Time ◽  
Ohmic Heating ◽  
Content Type ◽  
Significant Difference ◽  
Serovar Typhimurium ◽  
The Impact

ABSTRACT The effect of frequency of alternating current during ohmic heating on electrode corrosion, heating rate, inactivation of food-borne pathogens, and quality of salsa was investigated. The impact of waveform on heating rate was also investigated. Salsa was treated with various frequencies (60 Hz to 20 kHz) and waveforms (sine, square, and sawtooth) at a constant electric field strength of 12.5 V/cm. Electrode corrosion did not occur when the frequency exceeded 1 kHz. The heating rate of the sample was dependent on frequency up to 500 Hz, but there was no significant difference ( P > 0.05) in the heating rate when the frequency was increased above 1 kHz. The electrical conductivity of the sample increased with a rise in the frequency. At a frequency of 60 Hz, the square wave produced a lower heating rate than that of sine and sawtooth waves. The heating rate between waveforms was not significantly ( P > 0.05) different when the frequency was >500 Hz. As the frequency increased, the treatment time required to reduce Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium to below the detection limit (1 log CFU/g) decreased without affecting product quality. These results suggest that ohmic heating can be effectively used to pasteurize salsa and that the effect of inactivation is dependent on frequency and electrical conductivity rather than waveform.

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