Mixed culture fermentation studies on the effects of synbiotics on the human intestinal pathogens Campylobacter jejuni and Escherichia coli

Batch and continuous culture anaerobic fermentation systems, inoculated with human faeces, were utilised to investigate the antimicrobial actions of two probiotics, Lactobacillus plantartan 0407, combined with oligofructose and Bifidobacterium bifidum Bb12, combined with a mixture of oligofructose and xylo-oligosaccharides (50:50 w/w) against E coli and Campylobacter jejuni. In batch fermenters, both E coli and C jejuni were inhibited by the synbiotics, even when the culture pH was maintained at around neutral. In continuous culture C jejuni was inhibited but the synbiotic failed to inhibit E coli. Although no definitive answer in addressing the mechanisms underlying antimicrobial activity was derived, results suggested that acetate and lactate directly were conferring antagonistic action, rather than as a result of lowering culture pH. In the course of the study culturing and fluorescent in situ hybridisation (FISH) methodologies for the enumeration of bacterial populations were compared. Bifidobacterial populations were underestimated using plating techniques, suggesting the non-culturability of certain bifidobacterial species. (C) 2003 Elsevier Ltd. All rights reserved.

The influence of ribosome modulation factor on the survival of stationary-phase Escherichia coli during acid stress

Ribosome modulation factor (RMF) was shown to have an influence on the survival of Escherichia coli under acid stress during stationary phase, since the viability of cultures of a mutant strain lacking functional RMF decreased more rapidly than that of the parent strain at pH 3. Loss of ribosomes was observed in both strains when exposed to low pH, although this occurred at a higher rate in the RMF-deficient mutant strain, which also suffered from higher levels of rRNA degradation. It was concluded that the action of RMF in limiting the damage to rRNA contributed to the protection of E coli under acid stress. Expression of the rmf gene was lower during stationary phase after growth in acidified media compared to media containing no added acid, and the increased rmf expression associated with transition from exponential phase to stationary phase was much reduced in acidified media. It was demonstrated that RMF was not involved in the stationary-phase acid-tolerance response in E coli by which growth under acidic conditions confers protection against subsequent acid shock. This response was sufficient to overcome the increased vulnerability of the RMF-deficient mutant strain to acid stress at pH values between 6.5 and 5.5.

The activity of ribosome modulation factor during growth of Escherichia coli under acidic conditions

Expression of the gene encoding ribosome modulation factor (RMF), as measured using an rmf-lacZ gene fusion, increased with decreasing pH in exponential phase cultures of Escherichia coli. Expression was inversely proportional to the growth rate and independent of the acidifying agent used and it was concluded that expression of rmf was growth rate controlled in exponential phase under acid conditions. Increased rmf expression during exponential phase was not accompanied by the formation of ribosome dimers as occurs during stationary phase. Nor did it appear to have a significant effect on cell survival under acid stress since the vulnerability of an RMF-deficient mutant strain was similar to that of the parent strain. Ribosome degradation was increased in the mutant strain compared to the parent strain at pH 3.75. Also, the peptide elongation rate was reduced in the mutant strain but not the parent during growth under acid conditions. It is speculated that the function of RMF during stress-induced reduction in growth rate is two-fold: firstly to prevent reduced elongation efficiency by inactivating surplus ribosomes and thus limiting competition for available protein synthesis factors, and secondly to protect inactivated ribosomes from degradation.

rRNA probes used to quantify the effects of glycomacropeptide and alpha-lactalbumin supplementation on the predominant groups of intestinal bacteria of infant rhesus monkeys challenged with enteropathogenic Escherichia coli

Objectives: Certain milk factors may help to promote the growth of a host-friendly colonic microflora (e.g. bifidobacteria, lactobacilli) and explain why breast-fed infants experience fewer and milder intestinal infections than those who are formula-fed. The effects of supplementation of formula with two such milk factors was investigated in this study. Materials and Methods: Infant rhesus macaques were breastfed, fed control formula, or formula supplemented with glycomacropeptide (GMP) or alpha-lactalburnin (alpha-LA) from birth to 5 months of age. Blood was drawn monthly and rectal swabs were collected weekly. At 4.5 months of age, 10(8) colonyforming units of enteropathogenic E.coli O127, strain 2349/68 (EPEC) was given orally and the response to infection assessed. The bacteriology of rectal swabs pre- and post-infection was determined by culture independent fluorescence in situ hybridization. Results: Post-challenge, breast-fed infants and infants fed alpha-LA-supplemented formula had no diarrhea, whilst those infants fed GMP-supplemented formula had intermittent diarrhea. In infants fed control formula the diarrhea was acute. Conclusions: Supplementation of infant formula with appropriate milk proteins may be useful for improving the infant's ability to resist acute infection caused by E.coli.

The effects of alpha-lactalbumin and glycomacropeptide on the association of CaCo-2 cells by enteropathogenic Escherichia coli, Salmonella typhimurium and Shigella flexneri

Two milk components, alpha-lactalbumin (alpha-La) and glycomacropeptide (GMP) may inhibit intestinal infection/intoxification. (3)[H] thymidine-labeled enteropathogenic Escherichia coli (EPEC), Salmonella typhimurium (ATCC 6994) or Shigella flexneri (ATCC 9199) were introduced to CaCo-2 cultures and their association with CaCo-2 cells was assessed. Undigested, pepsin-digested and pepsin- and pancreatin-digested alpha-lactalbumin and glycomacropeptide inhibited association. Thus, milk supplemented with alpha-lactalbumin and glycomacropeptide might be effective in inhibiting associations of the pathogens EPEC, Salmonella typhimurium, and Shigella flexneri to intestinal cells.

A two-stage continuous culture system to study the effect of supplemental alpha-lactalbumin and glycomacropeptide on mixed cultures of human gut bacteria challenged with enteropathogenic Escherichia coli and Salmonella serotype Typhimurium

Aims: Certain milk factors may promote the growth of a gastrointestinal microflora predominated by bifidobacteria and may aid in overcoming enteric infections. This may explain why breast-fed infants experience fewer intestinal infections than their formula-fed counterparts. The effect of formula supplementation with two such factors was investigated in this study. Methods and Results: Infant faecal specimens were used to ferment formulae supplemented with glycomacropeptide (GMP) and alpha-lactalbumin (alpha-la) in a two-stage compound continuous culture model. At steady state, all fermenter vessels were inoculated with 5 ml of 0.1 M phosphate-buffered saline (pH 7.2) containing 10(8) CFU ml(-1) of either enteropathogenic Escherichia coli 2348/69 (O127:H6) or Salmonella serotype Typhimurium (DSMZ 5569). Bacteriology was determined by independent fluorescence in situ hybridization. Vessels that contained breast milk (BM), as well as alpha-la and GMP supplemented formula had stable total counts of bifidobacteria while lactobacilli increased significantly only in vessels with breast milk. Bacteroides, clostridia and E. coli decreased significantly in all three groups prior to pathogen addition. Escherichia coli counts decreased in vessels containing BM and alpha-la while Salmonella decreased significantly in all vessels containing BM, alpha-la and GMP. Acetate was the predominant acid. Significance and Impact of the Study: Supplementation of infant formulae with appropriate milk proteins may be useful in mimicking the beneficial bacteriological effects of breast milk.

The relationship between membrane damage, release of protein and loss of viability in Escherichia coli exposed to high hydrostatic pressure

The aim of this work was to examine a possible association between resistance of two Escherichia coli strains to high hydrostatic pressure and the susceptibility of their cell membranes to pressure-induced damage. Cells were exposed to pressures between 100 and 700 MPa at room temperature (~20C) in phosphate-buffered-saline. In the more pressure-sensitive strain E. coli 8164, loss of viability occurred at pressures between 100 MPa and 300 MPa and coincided with irreversible loss of membrane integrity as indicated by uptake of propidium iodide (PI) and leakage of protein of molecular mass between 9 and 78 kDa from the cells. Protein release increased to a maximum at 400 MPa then decreased, possibly due to intracellular aggregation at the higher pressures. In the pressure-resistant strain E. coli J1, PI was taken up during pressure treatment but not after decompression indicating that cells were able to reseal their membranes. Loss of viability in strain J1 coincided with the transient loss of membrane integrity between approximately 200 MPa and 600 MPa. In E. coli J1 leakage of protein occurred before loss of viability and the released protein was of low molecular mass, between 8 and 11 kDa and may have been of periplasmic origin. In these two strains differences in pressure resistance appeared to be related to differences in the ability of their membranes to withstand disruption by pressure. However it appears that transient loss of membrane integrity during pressure can lead to cell death irrespective of whether cells can reseal their membranes afterwards.

Inactivation of Escherichia coli by citral

Aims: The aim was to evaluate (i) the resistance of Escherichia coli BJ4 to citral in a buffer system as a function of citral concentration, treatment medium pH, storage time and initial inoculum size, (ii) the role of the sigma factor RpoS on citral resistance of E. coli, (iii) the role of the cell envelope damage in the mechanism of microbial inactivation by citral, and (iii) possible synergistic effects of mild heat treatment and pulsed-electric fields (PEF) treatment combined with citral. Methods and Results: The initial inoculum size greatly affected the efficacy of citral against E. coli cells. Exposure to 200 µl l-1of citral at pH 4.0 for 24 h at 20 ºC caused the inactivation of more than 5 log10 cycles of cells starting at an inoculum size of 106 or 107 CFU ml-1, whereas increasing the cell concentration to 109 CFU ml-1 caused less than 1 log10 cycle of inactivation. E. coli showed higher resistance to citral at pH 4.0 than pH 7.0. The rpoS null mutant strain E. coli BJ4L1 was less resistant to citral than the wild-type strain. Occurrence of sublethal injury to both, the cytoplasmic and outer membranes was demonstrated by adding sodium chloride or bile salts to the recovery media. The majority of sublethally-injured cells by citral required energy and lipid synthesis for repair. A strongly synergistic lethal effect was shown by mild heat treatment combined with citral but the presence of citral during the application of a PEF treatment did not show any advantage. Conclusions: This work confirms that cell envelope damage is an important event in citral inactivation of bacteria, and it describes the key factors on the inactivation of E. coli cells by citral. Significance and Impact of Study: Knowledge about the mechanism of microbial inactivation by citral helps establish successful combined preservation treatments.

Preliminary X-ray diffraction analysis of YqjH from Escherichia coli: a putative cytoplasmic ferri-siderophore reductase

YqjH is a cytoplasmic FAD-containing protein from Escherichia coli; based on homology to ViuB of Vibrio cholerae, it potentially acts as a ferri-siderophore reductase. This work describes its overexpression, purification, crystallization and structure solution at 3.0 A resolution. YqjH shares high sequence similarity with a number of known siderophore-interacting proteins and its structure was solved by molecular replacement using the siderophore-interacting protein from Shewanella putrefaciens as the search model. The YqjH structure resembles those of other members of the NAD(P)H:flavin oxidoreductase superfamily.

Role of rpoS in the Development of Cell Envelope Resilience and Pressure Resistance in Stationary-Phase Escherichia coli

This work investigated the role of rpoS in the development of increased cell envelope resilience and enhanced pressure resistance in stationary phase cells of Escherichia coli. Loss of both colony-forming ability and membrane integrity, measured as uptake of propidium iodide (PI), occurred at lower pressures in E. coli BW3709 (rpoS) than in the parental strain (BW2952). The rpoS mutant also released much higher concentrations of protein under pressure than the parent. We propose that RpoS-regulated functions are responsible for the increase in membrane resilience as cells enter stationary phase and that this plays a major role in the development of pressure resistance. Strains from the Keio collection with mutations in two RpoS-regulated genes, cfa (cyclopropane fatty acyl phospholipid synthase) and osmB (outer membrane lipoprotein), were significantly more pressure-sensitive and took up more PI than the parent strains with cfa having the greatest effect. Mutations in the bolA morphogene and other RpoS-regulated lipoprotein genes (osmC, osmE, osmY and ybaY) had no effect on pressure resistance. The cytoplasmic membranes of the rpoS mutant failed to reseal after pressure treatment and strains with mutations in osmB and nlpI (new lipoprotein) were also somewhat impaired in the ability to reseal their membranes. The cfa mutant, though pressure-sensitive, was unaffected in membrane resealing implying that the initial transient permeabilization event is critical for loss of viability rather than the failure to reseal. The enhanced pressure sensitivity of polA, recA and xthA mutants suggested that DNA may be a target of oxidative stress in pressure-treated cells.

Induction of the ferritin gene (ftnA) of Escherichia coli by Fe2+–Fur is mediated by reversal of H-NS silencing and is RyhB independent

FtnA is the major iron-storage protein of Escherichia coli accounting for < or = 50% of total cellular iron. The FtnA gene (ftnA) is induced by iron in an Fe(2+)-Fur-dependent fashion. This effect is reportedly mediated by RyhB, the Fe(2+)-Fur-repressed, small, regulatory RNA. However, results presented here show that ftnA iron induction is independent of RyhB and instead involves direct interaction of Fe(2+)-Fur with an 'extended' Fur binding site (containing five tandem Fur boxes) located upstream (-83) of the ftnA promoter. In addition, H-NS acts as a direct repressor of ftnA transcription by binding at multiple sites (I-VI) within, and upstream of, the ftnA promoter. Fur directly competes with H-NS binding at upstream sites (II-IV) and consequently displaces H-NS from the ftnA promoter (sites V-VI) which in turn leads to derepression of ftnA transcription. It is proposed that H-NS binding within the ftnA promoter is facilitated by H-NS occupation of the upstream sites through H-NS oligomerization-induced DNA looping. Consequently, Fur displacement of H-NS from the upstream sites prevents cooperative H-NS binding at the downstream sites within the promoter, thus allowing access to RNA polymerase. This direct activation of ftnA transcription by Fe(2+)-Fur through H-NS antisilencing represents a new mechanism for iron-induced gene expression.

Mechanism of the Escherichia coli DNA T:G-mismatch endonuclease (Vsr protein) probed with thiophosphate-containing oligodeoxynucleotides

The mechanism of the Escherichia coli DNA T:G mismatch endonuclease (Vsr) has been investigated using oligodeoxynucleotides substituted, at the scissile phosphate, with isomeric phosphorothioates and a 3'-phosphorothiolate. Binding and kinetic data with the phosphorothioates/phosphorothiolate indicate that the two magnesium ions, which constitute essential co-factors, are required to stabilise the extra negative charge developed on the phosphate as the transition state is formed. Additionally one of the magnesium ions serves to activate the leaving group (the non-bridging 3'-oxygen atom of the scissile phosphate) during the hydrolysis reaction. Stereochemical analysis, using the R-p phosphorothioate isomer, indicates that Vsr carries out a hydrolytic reaction with inversion of stereochemistry at phosphorus, compatible with an in-line attack of water and a pentacovalent transition state with trigonal bipyramidal geometry. In conjunction with structures of Vsr bound to its products, these data allow the reconstruction of the enzyme-substrate complex and a comprehensive description of the hydrolysis mechanism. (c) 2005 Elsevier Ltd. All rights reserved.