Inhibition of Injured Escherichia coli by Several Selective Agents

A population of Escherichia coli ML30 cells was exposed to a quaternary ammonium compound, and injury to the cells was measured by a comparison of counts on Trypticase Soy Agar and Violet Red Bile Agar. Substantial injury could not be detected with a minimal medium. The ingredients of Violet Red Bile Agar were tested against damaged cells. The bile salts mixture alone in the medium prevented as many injured cells from growing as did any combination of the selective agents and inhibited as many injured bacteria as were inhibited by Violet Red Bile Agar itself. These dyes and salts were similarly assayed in minimal agar, and comparable results were obtained. Individual bile salts and other potential selective agents were added to the minimal medium, and the media were tested for inhibition of injured E. coli. Sodium deoxycholate was the bile salt most inhibitory to damaged E. coli cells.     

The Recovery of Indicator Bacteria on Selective Media

S ummary . The recovery of Pseudomonas aeruginosa, Escherichia coli , and Streptococcus faecalis from aqueous suspending media has been studied with a rich plating medium (trypticase-soy agar) and selective media. Tap water was highly toxic to all strains investigated. Recovery of Ps. aeruginosa was most successful when phosphate buffer was the diluent. Phosphate buffer did not improve the recovery of E. coli. Streptococcus faecalis remained viable when suspended in double distilled water, deionized distilled water or phosphate buffer. Following a lag period all strains grew in 0.1% peptone water or stream water. Injury preventing recovery of viable cells on selective media occurred during suspension in all aqueous media tested, including those which supported growth. These observations suggest difficulties inherent in the interpretation of bacteriological results obtained during surveys of water sources and a need to reduce the selectivity of recovery media against injured cells.     

Injury and recovery of Escherichia coli after sublethal acidification.

Over 99% of the viable cells of Escherichia coli K-12 were injured after a 60-min exposure to 0.3 M sodium acetate buffer at pH 4.2. Injured cells were those able to grow on Trypticase soy agar but unable to grow on violet red bile agar. The extent of both death and injury of acid-treated cells increased with decreasing pH or increasing concentration of acid. Injured cells were able to recover their colony-forming ability on violet red bile agar by incubation in Trypticase soy broth or potassium phosphate buffer before plating on the agar media. Direct neutralization of the injury medium did not allow recovery and, in fact, was lethal to the population. The addition of metabolic inhibitors to the Trypticase soy recovery broth was used to study the repair process. It was not affected by the presence of inhibitors of protein, cell wall, deoxyribonucleic acid, or ribonucleic acid syntheses. The addition of 2,4-dinitrophenol to the recovery medium also did not inhibit recovery. Actinomycin D and N,N'-dicyclohexylcarbodiimide were lethal to a proportion of the acidified cells but allowed another fraction of the population to recover. There were no detectable amounts of 260- or 280-nm-absorbing materials leaked during the course of acid injury.     

Injury and recovery of Escherichia coli after sublethal acidification.

Over 99% of the viable cells of Escherichia coli K-12 were injured after a 60-min exposure to 0.3 M sodium acetate buffer at pH 4.2. Injured cells were those able to grow on Trypticase soy agar but unable to grow on violet red bile agar. The extent of both death and injury of acid-treated cells increased with decreasing pH or increasing concentration of acid. Injured cells were able to recover their colony-forming ability on violet red bile agar by incubation in Trypticase soy broth or potassium phosphate buffer before plating on the agar media. Direct neutralization of the injury medium did not allow recovery and, in fact, was lethal to the population. The addition of metabolic inhibitors to the Trypticase soy recovery broth was used to study the repair process. It was not affected by the presence of inhibitors of protein, cell wall, deoxyribonucleic acid, or ribonucleic acid syntheses. The addition of 2,4-dinitrophenol to the recovery medium also did not inhibit recovery. Actinomycin D and N,N'-dicyclohexylcarbodiimide were lethal to a proportion of the acidified cells but allowed another fraction of the population to recover. There were no detectable amounts of 260- or 280-nm-absorbing materials leaked during the course of acid injury.     

Enumeration of stressed cells of Escherichia coli.

Cells of Escherichia coli K-12 were stressed by heating at 48 degrees C or by acid treatment at pH 4.2 for periods up to 1h. The addition of catalase to the selective medium increased the count of heat-stressed cells by 2.3-fold and acid-stressed cells by 4.8-fold. However, these values represented only a small percentage (3% for heat-stressed and 6% for acid-stressed cells respectively) of the population of injured but still viable cells. The addition of mannitol to the selective medium used to count acid-stressed cells did not increase the count. Whilst the presence of H2O2 in media may cause significant errors in the estimation of E. coli in certain situations these errors are unlikely to be significant in physiological studies of populations of cells injured by stress.     

Survival and virulence of copper- and chlorine-stressed Yersinia enterocolitica in experimentally infected mice.

The effect of gastric pH on the viability and virulence of Yersinia enterocolitica O:8 after exposure to sublethal concentrations of copper and chlorine was determined in mice. Viability and injury were assessed with a nonselective TLY agar (tryptic soy broth containing lactose, yeast extract, and agar) and two selective media, TLYD agar (TLY agar plus sodium deoxycholate) and CIN agar (cefsulodin-Irgasan-novobiocin agar). Both copper and chlorine caused injury which was manifested by the inability of the cells to grow on selective media. CIN agar was more restrictive to the growth of injured cells than TLYD agar. Injury of the exposed cells was further enhanced in the gastric environment of mice. Besides injury, the low gastric pH caused extensive loss of viability in copper-exposed cells. Lethality in the chlorine-exposed cells was less extensive, and a portion of the inoculum (5.2 X 10(5) of 1 X 10(7) inoculated cells) reached the small intestine 5 min postinoculation. No adverse effect on the injured cells was apparent in the small intestine, and a substantial revival (approximately 70%) of the injury occurred in 3 to 4 h after intraluminal inoculation. The virulence of chlorine-stressed Y. enterocolitica in orally inoculated mice was similar to that of the control culture, but copper-stressed cells showed reduced virulence. Virulence was partly restored by oral administration of sodium bicarbonate before the inoculation of copper-exposed cells. Neutralization of gastric acidity had no effect on the virulence of the control or chlorine-stressed cells. The results of this study indicate that the extensive injury caused by the low gastric pH does not affect the virulence potential of chlorine-exposed cells. However, extensive cell death in the mouse stomach is responsible for the reduced virulence of the copper-stressed bacteria.     

Loss of viability in hybridoma cell culture—A kinetic study

In the cultivation of hybridoma cells HB8178 in suspension, exponential growth was followed by cell death after reaching a maximum cell concentration. We examined the cause of such transition from growth to death. Exogenously added lactate and ammonium did not cause cell death at the concentrations observed at the end of exponential growth. The rate of cell death decreases by diluting the conditioned medium with phosphate buffer saline, suggesting the presence of inhibitory factor(s) in the conditioned medium. This inhibitory factor(s) is dialyzable. Furthermore, the conditioned medium obtained from HB8178 culture also causes transition to death phase for another hybridoma cell line AFP.     

Survival and virulence of copper- and chlorine-stressed Yersinia enterocolitica in experimentally infected mice

The effect of gastric pH on the viability and virulence of Yersinia enterocolitica O:8 after exposure to sublethal concentrations of copper and chlorine was determined in mice. Viability and injury were assessed with a nonselective TLY agar (tryptic soy broth containing lactose, yeast extract, and agar) and two selective media, TLYD agar (TLY agar plus sodium deoxycholate) and CIN agar (cefsulodin-Irgasan-novobiocin agar). Both copper and chlorine caused injury which was manifested by the inability of the cells to grow on selective media. CIN agar was more restrictive to the growth of injured cells than TLYD agar. Injury of the exposed cells was further enhanced in the gastric environment of mice. Besides injury, the low gastric pH caused extensive loss of viability in copper-exposed cells. Lethality in the chlorine-exposed cells was less extensive, and a portion of the inoculum (5.2 X 10(5) of 1 X 10(7) inoculated cells) reached the small intestine 5 min postinoculation. No adverse effect on the injured cells was apparent in the small intestine, and a substantial revival (approximately 70%) of the injury occurred in 3 to 4 h after intraluminal inoculation. The virulence of chlorine-stressed Y. enterocolitica in orally inoculated mice was similar to that of the control culture, but copper-stressed cells showed reduced virulence. Virulence was partly restored by oral administration of sodium bicarbonate before the inoculation of copper-exposed cells. Neutralization of gastric acidity had no effect on the virulence of the control or chlorine-stressed cells. The results of this study indicate that the extensive injury caused by the low gastric pH does not affect the virulence potential of chlorine-exposed cells. However, extensive cell death in the mouse stomach is responsible for the reduced virulence of the copper-stressed bacteria.     

Proceedings: Repair of injury in Salmonella anatum cells after freezing and thawing in milk

Fast freezing and slow thawing of Salmonella anatum cells in nonfat milk solids resulted in about 20% death and 50% injury of the cells surviving the treatment. Death was defined as the inability to form colonies on a nonselective plating medium [xylose-lysine-peptone agar (XLP)] after freezing and thawing. Injury was defined as the inability to form colonies on a selective plating medium (XLP with 0.2% sodium desoxycholate added). The injured cells repaired rapidly and within 2 hr at 25 °C, in the presence of 0.1% milk solids; all the injured cells regained the ability to form colonies on the selective medium. The treated cells showed a 1-hr extended lag phase of growth as compared to the unfrozen cells. Milk solids concentration in the freezing and repair menstrua influenced injury, repair of injury, and death. The repair process was affected by the pH and temperature of environment in which the injured cells were incubated. Maximum repair occurred at pH values between 6.0 and 7.4 and temperatures from 25 to 42 °C. The data suggested repair did not require the synthesis of protein, ribonucleic acid, or cell-wall mucopeptide but did require energy synthesis.     

The effect of free chlorine onEscherichia coli populations

The effect of free chlorine onEscherichia coli populations was studied by chlorination of a population of 10⁵ cells/ml. This cell density was low enough for the free-to-combined chlorine ratio to be 6.01 or greater. The predominance of free chlorine resulted in rapid and complete population death.Survivors obtained by dechlorination prior to complete population death were recovered equally well on nonselective and selective media. Although this suggests that survivors are not injured, evidence of survivor injury was observed.Colonies resulting from growth of these survivors had a smaller diameter than colonies from unchlornated controls. This suggests that the chlorinated cells have an increased lag and provides indirect evidence of survivor injury. Injury was indicated directly by an increase in the lag time of surviving cells in slide culture. Variability in the severity of free-chlorine-induced injury was indicated by a broadened range in the survivor lag times.     

Thermal injury of Yersinia enterocolitica

Procedures were developed to evaluate thermal injury to three strains of Yersinia enterocolitica (serotypes 0:3, 0:8, and 0:17). Serotype 0:17 (atypical strain) was more sensitive to bile salts no. 3 (BS) and to sublethal heat treatment than the typical strains, 0:3 and 0:8. When the 0:3, 0:8, and 0:17 serotypes were thermally stressed in 0.1 M PO4 buffer, pH 7.0, at 47 degrees C for 70, 60, and 12 min, respectively, greater than 99% of the total viable cell population was injured. Injury was determined by the ability of cells to form colonies on brain heart infusion (BHI) agar, but not on Trypticase soy agar (TSA) plus 0.6% BS for serotypes 0:3 and 0:8 and TSA plus 0.16% BS for 0:17. Heat injury of serotype 0:17 cells for 15 min in 0.1 M PO4 buffer caused an approximate 1,000-fold reduction in cell numbers on selective media as compared with cells heated in pork infusion (PI), BHI broth, and 10% nonfat dry milk (NFDM). The extended lag and resuscitation period in BHI broth was 2.5 times greater for 0:17 cells injured in 0.1 M PO4 than for cells injured in BHI or PI. The rate and extent of repair of Y. enterocolitica 0:17 cells in three recovery media were directly related to the heating menstruum used for injury. The use of metabolic inhibitors demonstrated that ribonucleic acid synthesis was required for repair, whereas deoxyribonucleic, cell wall, and protein synthesis were not necessary for recovery of 0:17 cells injured in 0.1 M PO4 buffer, BHI, or PI. Inhibition of respiration by 2,4-dinitrophenol slowed repair only for 0:17 cells injured in 0.1 M PO4 buffer, not for cells injured in PI or BHI.     

Thermal injury of Yersinia enterocolitica.

Procedures were developed to evaluate thermal injury to three strains of Yersinia enterocolitica (serotypes 0:3, 0:8, and 0:17). Serotype 0:17 (atypical strain) was more sensitive to bile salts no. 3 (BS) and to sublethal heat treatment than the typical strains, 0:3 and 0:8. When the 0:3, 0:8, and 0:17 serotypes were thermally stressed in 0.1 M PO4 buffer, pH 7.0, at 47 degrees C for 70, 60, and 12 min, respectively, greater than 99% of the total viable cell population was injured. Injury was determined by the ability of cells to form colonies on brain heart infusion (BHI) agar, but not on Trypticase soy agar (TSA) plus 0.6% BS for serotypes 0:3 and 0:8 and TSA plus 0.16% BS for 0:17. Heat injury of serotype 0:17 cells for 15 min in 0.1 M PO4 buffer caused an approximate 1,000-fold reduction in cell numbers on selective media as compared with cells heated in pork infusion (PI), BHI broth, and 10% nonfat dry milk (NFDM). The extended lag and resuscitation period in BHI broth was 2.5 times greater for 0:17 cells injured in 0.1 M PO4 than for cells injured in BHI or PI. The rate and extent of repair of Y. enterocolitica 0:17 cells in three recovery media were directly related to the heating menstruum used for injury. The use of metabolic inhibitors demonstrated that ribonucleic acid synthesis was required for repair, whereas deoxyribonucleic, cell wall, and protein synthesis were not necessary for recovery of 0:17 cells injured in 0.1 M PO4 buffer, BHI, or PI. Inhibition of respiration by 2,4-dinitrophenol slowed repair only for 0:17 cells injured in 0.1 M PO4 buffer, not for cells injured in PI or BHI.     

Role of sublethal injury in decline of bacterial populations in lake water

Following their addition to lake water, the populations of Escherichia coli and of antibiotic-resistant strains of Pseudomonas fluorescens, Agrobacterium tumefaciens, Micrococcus flavus, Rhizobium meliloti, and Klebsiella pneumoniae declined rapidly, as determined by counting on media containing antibacterial compounds. The estimates of population sizes were occasionally higher if procedures were used that permitted possible resuscitation of injured cells. No resuscitation procedure yielded consistently higher estimates of populations of surviving cells than the use of selective media alone. The patterns of survival of the test bacteria in lake water amended with eucaryotic inhibitors were essentially the same whether a resuscitation procedure was used or not, and the patterns of survival in sterile lake water or buffer were the same whether counts were made on selective media or on media without antibacterial agents. On the basis of the methods used to show sublethal injury caused by stress, we suggest that such injury to the test bacteria is not a significant factor involved in their decline in lake water.     

Role of sublethal injury in decline of bacterial populations in lake water.

Following their addition to lake water, the populations of Escherichia coli and of antibiotic-resistant strains of Pseudomonas fluorescens, Agrobacterium tumefaciens, Micrococcus flavus, Rhizobium meliloti, and Klebsiella pneumoniae declined rapidly, as determined by counting on media containing antibacterial compounds. The estimates of population sizes were occasionally higher if procedures were used that permitted possible resuscitation of injured cells. No resuscitation procedure yielded consistently higher estimates of populations of surviving cells than the use of selective media alone. The patterns of survival of the test bacteria in lake water amended with eucaryotic inhibitors were essentially the same whether a resuscitation procedure was used or not, and the patterns of survival in sterile lake water or buffer were the same whether counts were made on selective media or on media without antibacterial agents. On the basis of the methods used to show sublethal injury caused by stress, we suggest that such injury to the test bacteria is not a significant factor involved in their decline in lake water.     

Cell envelope damage in Escherichia coli caused by short-term stress in water.

Physiological and morphological changes in Escherichia coli exposed to oligotrophic natural waters and reagent grade water were studied. Several lines of evidence indicated that short-term exposure in water causes cellular envelope damage. Increasing susceptibility to lysozyme, lag time before cell division, and injury as defined by differential counts on selective and nonselective media occurred with exposure time. Electron micrographs of injured cells showed morphological changes in cell envelope.     

Normal serum cytotoxicity for P32-labeled smooth Enterobacteriaceae. I. Loss of label, death, and ultrastructural damage

Spitznagel, John K. (University of North Carolina, Chapel Hill), and Lawrence A. Wilson. Normal serum cytotoxicity for P(32)-labeled smooth Enterobacteriaceae. I. Loss of label, death, and ultrastructural damage. J. Bacteriol. 91:393-400. 1966.-Metabolically labeled smooth Escherichia coli lost between 10 and 90% of P(32), compared with control suspensions, when suspended for 60 min in normal serum at 37 C. Similar results were obtained with several other genera of Enterobacteriaceae. The structural nature of the cell injury in E. coli was shown by electron microscopic examination of ultrathin sections. Complex injury which included all peripheral cell structures occurred and various degrees of cytoplasmic loss resulted. Injured bacteria retained their essential shape and did not collapse, evidently because sufficient rigid cell wall remained to prevent this. The cell damage was more like that reported with ethylenediaminetetraacetic acid lysozyme than that reported with growth in penicillin. Damage sufficient to cause rupture of peripheral structures was unique in that it involved only localized areas of cell wall and left large sections relatively intact. The loss of P(32) in the fresh normal serum was closely related to bacterial viability loss.     

The enumeration of chlorine-injured Escherichia coli and Enterococcus faecalis is enhanced under conditions where reactive oxygen species are neutralized

AIM: To investigate the effect of neutralization of reactive oxygen species (ROS-neutralized conditions) on the enumeration of chlorine-injured Escherichia coli and Enterococcus faecalis using selective and nonselective media. METHODS: Pure cultures of E. coli NCTC8912 and Ent. faecalis NCTC775 were injured using dilute sodium hypochlorite, at free chlorine levels of 0.6 and 0.9 microg ml(-1), respectively, and then enumerated at 37 degrees C by surface plate counts on nonselective nutrient (N) agar and on several selective media, either under (i) standard aerobic conditions; (ii) aerobic conditions using growth medium, supplemented with 0.05%-w/v sodium pyruvate, to neutralize peroxides; or (iii) conditions designed to neutralize ROS, using a combination of 0.05%-w/v sodium pyruvate in the growth medium, together with incubation in an anaerobic jar. RESULTS: The counts obtained on the nonselective medium were lowest under aerobic conditions in unsupplemented medium, higher in pyruvate-supplemented (peroxide-neutralized) medium and highest for ROS-neutralized conditions. Counts for the selective media were often lower than those for nonselective N (nutrient) agar, with enhancement under peroxide-neutralized conditions and a further increase in counts under ROS-neutralized conditions. Broadly similar observations were made for three other strains of each organism. CONCLUSIONS: Chlorine-injured E. coli and Ent. faecalis become sensitive to ROS, giving higher counts under ROS-neutralized enumeration conditions than under conventional aerobic conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The enhancement in counts observed under ROS-neutralized conditions indicate that the addition of pyruvate to the growth medium may not fully counteract the effects of sublethal injury under aerobic conditions, which is a novel observation. Thus, ROS-neutralized conditions may be required for optimal enumeration of faecal indicator bacteria. Furthermore, the lower counts, obtained using selective media indicate that the sensitivity of chlorine-injured bacteria to selective agents is not necessarily reversed under ROS-neutralized conditions.     

Timed-release capsule method for coliform enumeration.

Wax-coated capsules containing selective ingredients (brilliant green and oxgall) were added at the time of inoculation of most-probable-number media (modified lactose broths). The inhibitory ingredients gradually diffused from the capsules into the nonselective media, imparting selectivity to the media. Concentrations of brilliant green did not reach inhibitory levels until 2 or more h had elapsed, which permitted repair of some injured cells. Resuscitation of heat-injured Escherichia coli B cells occurred in the capsule-containing media, but not in conventional brilliant green bile 2% broth or violet red bile agar. No statistically significant differences were noted between coliform counts obtained on two groups of water samples by using the capsule, most-probable-number, membrane filtration, and pour plate methods. The capsule method could be used, however, as a combined presumptive and confirmed test for the examination of water. Improvements are needed to adapt the capsule method to the analysis of some categories of food.     

Brain-derived neurotrophic factor can act as a pronecrotic factor through transcriptional and translational activation of NADPH oxidase

Several lines of evidence suggest that neurotrophins (NTs) potentiate or cause neuronal injury under various pathological conditions. Since NTs enhance survival and differentiation of cultured neurons in serum or defined media containing antioxidants, we set out experiments to delineate the patterns and underlying mechanisms of brain-derived neurotrophic factor (BDNF)-induced neuronal injury in mixed cortical cell cultures containing glia and neurons in serum-free media without antioxidants, where the three major routes of neuronal cell death, oxidative stress, excitotoxicity, and apoptosis, have been extensively studied. Rat cortical cell cultures, after prolonged exposure to NTs, underwent widespread neuronal necrosis. BDNF-induced neuronal necrosis was accompanied by reactive oxygen species (ROS) production and was dependent on the macromolecular synthesis. cDNA microarray analysis revealed that BDNF increased the expression of cytochrome b558, the plasma membrane-spanning subunit of NADPH oxidase. The expression and activation of NADPH oxidase were increased after exposure to BDNF. The selective inhibitors of NADPH oxidase prevented BDNF-induced ROS production and neuronal death without blocking antiapoptosis action of BDNF. The present study suggests that BDNF-induced expression and activation of NADPH oxidase cause oxidative neuronal necrosis and that the neurotrophic effects of NTs can be maximized under blockade of the pronecrotic action.     

Defined protein and animal component-free NS0 fed-batch culture

A chemically defined protein and animal component-free fed-batch process for an NS0 cell line producing a human IgG(1) antibody has been developed. The fed-batch feed profile was optimised in a step-wise manner. Depletion of measurable compounds was determined by direct analysis. The cellular need for non-measurable compounds was tested by continued culturing of cell suspension, removed from the bioreactor, in shake-flasks supplemented with critical substances. In the final fed-batch culture, 8.4 x 10(6) viable cells mL(-1) and 625 mg antibody L(-1) was obtained as compared to 2.3 x 10(6) cells mL(-1) and 70 mg antibody L(-1) in batch. The increase in cell density, in combination with a prolonged declining phase where antibody formation continued, resulted in a 6.2-fold increase in total cell yield, a 10.5-fold increase in viable cell hours and an 11.4-fold increase in product yield. These improvements were obtained by using a feed with glucose, glutamine, amino acids, lipids, sodium selenite, ethanolamine and vitamins. Specifically, supplementation with lipids (cholesterol) had a drastic effect on the maximum viable cell density. Calcium, magnesium and potassium were not depleted and a feed also containing iron, lithium, manganese, phosphorous and zinc did not significantly enhance the cell yield. The growth and death profiles in the final fed-batch indicated that nutrient deprivation was not the main cause of cell death. The ammonium concentration and the osmolality increased to potentially inhibitory levels, but an imbalance in the supply of growth/survival factors may also contribute to termination of the culture.