Reduced redox potential during growth of some Gram-negative bacteria (original) (raw)
A re-appraisal of the biological activity of bacteroides LPS
Journal of Medical Microbiology, 1995
Lipopolysaccharides (LPS) were extracted from seven Bacteroides strains by three different techniques: the phenol-water (PW), phenol-chloroform-petroleum (PCP) and Triton-Mg2+ methods. The strains selected included two different B. fragilis strains, one of which was grown in two different media. Yields varied between the strains, growth media and extraction technique, but generally the highest yield by weight was from the PCP method and the lowest from the PW method. The PW method was selected for the greatest amounts of carbohydrate and KDO, and the PCP method for the least. Phosphorus levels were more uniform among all extraction methods. Protein contamination was found in all Bacteroides LPS extracts, with extremely low levels in PW-LPS and the highest levels in material extracted by the PCP and Triton-Mg2+ techniques. No protein contamination could be detected after proteinase K treatment. After silver staining LPS PAGE profiles showed ladder patterns characteristic of smooth LPS for B. uulgatus, B. thetaiotaomicron and the control Escherichia coli 018 : K-strains, whereas the other Bacteroides strains showed mainly rough and low M, material only. The PCP method did not select for high M, material in the B. fragilis strains; otherwise the LPS profiles for all extraction methods were identical. The biological activities of native and sodium salt form LPS were investigated on a weight for weight basis and compared to that of E. coli 0 1 8 : K-PW-LPS. Amongst the LPS from Bacteroides strains, those prepared by the PW method were found to have a significantly higher activity in a galactosamine mouse lethality model, in induction of TNF and the Limulus amoebocyte lysate (LAL) assay, than LPS extracted by the PCP or Triton-Mg2+ methods. LPS from Bacteroides strains extracted by the PCP method had consistently low activity in all assays. Comparing PW-LPS from Bacteroides strains with that from E. coli 018:K-in the galactosamine mouse model, the E. coli 018 : K-LPS was c. 5000-fold more active than the most active bacteroides LPS. However, in the LAL assay native PW-LPS from both the B. fragilis strains, and B. caccae had higher activities (up to 30-fold) than E. coli 0 18 : K-LPS, with the PW-LPS from the other Bacteroides spp. being up to 15-fold less active than the E. coli 0 1 8 : K-PW-LPS. In the TNF induction assay, E. coli 0 18 : K-PW-LPS was 4-50-fold more active than bacteroides PW-LPS. In the LAL assay and galactosamine mouse model, native LPS had more activity (c. twofold) than sodium salt form LPS. There was no clear difference in activity between native and sodium salt form LPS in the TNF induction assay. The results for the LAL and TNF induction assay were re-evaluated relative to KDO concentration. In the TNF induction assay, previously low activities seen on a weight for weight basis were due in part to less KDO being present. However, LAL activity for PCP-LPS was still low after re-evaluation relative to KDO concentration. The molecular basis for the differences in biological activity of bacteroides LPS in relation to extraction methods and chemical composition is not yet understood.
Food Microbiology, 2009
The utilization of sub-lethal decontamination treatments gains more and more interest due to the increased consumers' demand for fresh, minimally processed and convenient food products. These products rely on cold chain and hurdle (combination) technology to provide microbiological safety and quality during their shelf life. To investigate the ability of surviving cells to resuscitate and grow in a food simulating environment, sub-lethal decontamination treatments were coupled with subsequent storage under sub-optimal growth conditions. For this purpose chlorine dioxide (ClO 2) and neutralized electrolyzed oxidizing water (NEW)-treated cultures of Escherichia coli O157:H7 were inoculated in TSB-YE of pH 5.8 and a w 0.99, and stored at 10 C, 12.5 C and 15 C, under four different atmospheres (0%, 30% and 60% CO 2 balanced with N 2 , and air). Due to the severity of injury, lactic acid-treated cells were inoculated in TSB-YE pH 7.0. Data obtained reveal that the fraction of sub-lethally injured E. coli O157:H7 undergoes an additional inhibitory effect during the storage period under of sub-optimal conditions. Observed extension in the lag growth phase was a direct consequence prior sub-lethal injury. The effects of liquid ClO 2 and NEW were less pronounced in comparison to lactic acid. The current study signifies the potential utilization of appropriate combination of different extrinsic and intrinsic factors in the elimination or growth inhibition of food-borne pathogens.
Chemiluminescence Induced by Phagocytosis of Escherichia coli by Polymorphonuclear Leucocytes
Microbiology, 1984
Chemiluminescence emitted by phagocytosing human polymorphonuclear leucocytes stimulated by Escherichia coli was measured using a liquid scintillation counter equipped with a multichannel analyser. In the presence of the amplifying agent luminol, light emission can be divided into two channels, one of which ('high energy') appears to correlate directly with phagocytic activity of the PMNL, and the other ('low energy') with the background luminol dioxygenation by the cells. Measuring in the 'high energy' window also eliminates the normal 'out of coincidence' background. The method is applicable to measuring opsonizing capacity of different sera, and responds to PMNL number, age, composition of assay medium and the integrity of the stimulating bacteria. Other bacterial strains produce a similar response, as does the artificial stimulator zymosan. Low temperature and anaerobiosis, which inhibit phagocytic killing, also suppress light emission.
Journal of Clinical Investigation, 1982
bacterial survival in either condition indicating that intracellular O2-independent bactericidal system(s) of rabbit polymorphonuclear leukocytes can at least match the leukocyte's ingestive capacity. Whole homogenates and crude acid extracts manifest similar bactericidal capacity toward S. typhimurium 395. This activity can be accounted for by the BPI content of these cell fractions and is virtually eliminated by immune (anti-BPI), but not by preimmune goat IgG- rich fractions. Opsonization of smooth MS, required for bacterial killing by intact leukocytes, does not alter bacterial sensitivity to BPI in crude or purified form. Leukocytes of a patient with chronic granulomatous disease killed ingested S. typhimurium 395 MS nearly as well as did normal leukocytes. The bactericidal activity toward E. coli (J5) of crude acid extracts of the CGD and normal human leuko- cytes was virtually the same and was nearly completely inhibited by anti-BPI IgG-rich fractions, but not by preimmune IgG-rich fractions. These findings suggest that the killing of gram-negative bacteria such as S. typhimurium by intact polymorphonuclear leukocytes may also be attributed to the action of BPI.
Variable Bacterial Responses to Oxidative Stress in Different Bacterial Species
Al-Azhar Medical Journal
Background: Living organisms are exposed to oxidative stress due to internal or external stimuli. It results from the imbalance between the production and elimination of reactive oxygen species. This leads to loss of homeostasis. Objective: To test the effect of oxidative stress on the level of the production of reduced glutathione (GSH) as an antioxidant, malondialdehyde (MDA) as a measure of lipid peroxidation, and of the siderophore enterobactin as an oxidative stress response, in different bacterial species. Materials and Methods: H2O2 minimum inhibitory concentration (MIC) was determined in Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603, using broth-macrodilution method. The levels of GSH and MDA were measured in E. coli ATCC 25922 and K. pneumoniae ATCC 700603 and in clinical isolates of E. coli, K. pneumoniae and Staphylococcus aureus after exposure to lethal H2O2 concentration, using Glutathione Reduced Kit and Lipid Peroxide-Malondialdehyde Kit, respectively. The level of expression of entC gene, involved in enterobactin biosynthesis, in presence of 0.25 and 0.5 MIC of H2O2 was determined using quantitative reverse transcription-polymerase chain reaction. Results: H2O2 MIC for both E. coli ATCC 25922 and K. pneumoniae ATCC 700603 was 1.5 mM. Exposure of E. coli to H2O2 resulted in a significant increase in GSH (p=0.0001) and MDA (p=0.0001) levels. However, in K. pneumoniae, a significant decrease in the GSH (p=0.0001) and MDA levels (p=0.0001) was recorded upon H2O2 exposure. No change in MDA and GSH levels was detected in S. aureus isolates exposed to H2O2. The expression of entC gene in both E. coli ATCC 25922 and K. pneumoniae ATCC 700603 was reduced in presence of 0.25 and 0.5 H2O2 MIC. Conclusion: Bacteria responded differently to oxidative stress, with S. aureus bacteria as the least affected by oxidative stress. Enterobactin role in oxidative stress needs reevaluation.
This study intends to assess the cultivability of photo-treated Escherichia coli K-12 on media with different selectivity (PCA, LBA, T-7, T-7 + TTC, VRBA and MAC) and to establish optimal conditions for bacterial recuperation. For these purposes, immediate and long-term bacterial recovery after SODIS and photo-Fenton are evaluated. Moreover, the use of catalase and sodium pyruvate supplements in the medium is studied. The non-selective medium PCA showed the highest counts for the untreated and treated cells due to its content in nutrients (e.g. glucose) and lack of inhibitors. On the contrary, the selective media showed lower recovery, being the culture media effectiveness: PCA > LBA > T-7 > T-7 + TTC > VRBA > MAC. The presence of inhibitors, such as heptadecyl sulfate in T-7 or bile salts in VRBA and MAC, reduced the cultivability of the treated cells. These compounds can probably diffuse into the cells more easily after SODIS and photo-Fenton as a consequence of the loss of the membrane integrity. In addition, the lack of yeast extract in MAC had a detrimental effect on E. coli recovery. Sodium pyruvate was tested as supplement to PCA, leading to slightly enhanced bacterial immediate recovery after SODIS, SODIS + H2O2 and photo-Fenton. The addition of catalase and sodium pyruvate to the bulk was studied as well, considerably increasing bacterial survival in the long-term due to their ability to neutralize residual H2O2.
Applied and Environmental Microbiology, 1994
High pH has been shown to rapidly destroy gram-negative food-borne pathogens; however, the mechanism of destruction has not yet been elucidated. Escherichia coli O157:H7, Salmonella enteritidis ATCC 13706, and Listeria monocytogenes F5069 were suspended in NaHCO3-NaOH buffer solutions at pH 9, 10, 11, or 12 to give a final cell concentration of approximately 5.2 x 10(8) CFU/ml and then held at 37 or 45 degrees C. At 0, 5, 10, and 15 min the suspensions were sterilely filtered and each filtrate was analyzed for material with A260. Viability of the cell suspensions was evaluated by enumeration on nonselective and selective agars. Cell morphology was evaluated by scanning electron microscopy and transmission electron microscopy. A260 increased dramatically with pH and temperature for both E. coli and S. enteritidis; however, with L. monocytogenes material with A260 was not detected at any of the pHs tested. At pH 12, numbers of E. coli and S. enteritidis decreased at least 8 logs withi...
Electron microscopic study of phagocytosis of Escherichia coli by human polymorphonuclear leukocytes
Infection and immunity, 1985
The fate of Escherichia coli strains within the polymorphonuclear leukocytes was studied by determining the killing of bacteria, measuring the release of degradation products, and examining the phagocytic bacteria by electron microscopy. When sufficiently opsonized, both unencapsulated and encapsulated E. coli strains were rapidly phagocytized by polymorphonuclear leukocytes. Once phagocytized, the two unencapsulated E. coli strains (K-12 and O111) were rapidly killed (99% of the bacteria were killed during the first 5 min of phagocytosis) and extensively degraded (about 40% of the radiolabeled material was released from bacteria after 15 min of phagocytosis). Electron micrographs taken after 15 min of phagocytosis revealed extensive structural changes in most of the internalized bacteria. In contrast to the rapid killing and extensive breakdown of these strains, encapsulated E. coli O78:K80 was more resistant to killing and withstood degradation by polymorphonuclear leukocytes (onl...