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PubMed2003

Inactivation of bacteria and spores by pulse electric field and high pressure CO2 at low temperature.

The common methods for inactivation of bacteria involve heating or exposure to toxic chemicals. These methods are not suitable for heat-sensitive materials, food, and pharmaceutical products. Recently, a complete inactivation of many microorganisms was achieved with high-pressure carbon dioxide at ambient temperature and in the absence of organic solvent and irradiation. The inactivation of spores with CO(2) required long residence time and high temperatures, such as 60 degrees C. In this study the synergistic effect of pulsed electric field (PEF) in combination with high-pressure CO(2) for inactivation was investigated. The bacteria Escherichia coli, Staphylococcus aureus, and Bacillus cereus were suspended in glycerol solution and treated in the first step with PEF (up to 25 KV/cm) and then with high-pressure CO(2) not higher than 40 degrees C and 200 bar. The inactivation efficiency was determined by counting the colony formation units of control and sample. Samples of the cells subjected to PEF treatment alone and in combination with CO(2) treatment were examined by scanning electron microscopy to determine the effect of the processes on the cell wall. Experimental results indicate that the viability decreased with increasing electrical field strength and number of pulses. A further batch treatment with supercritical CO(2) lead to complete inactivation of bacterial species and decreased the count of the spores by at least three orders of magnitude, the inactivation being enhanced by an increase of contact time between CO(2) and the sample. A synergistic effect between the pulsed electric field and the high-pressure CO(2) was evident in all the species treated. The new low temperature process is an alternative for pasteurization of thermally labile compounds such as protein and plasma and minimizes denaturation of important nutrient compounds in the liquid media.

Bacillus cereus↗

PubMed1986

Analysis of resistance to pyrimethamine/sulphadoxine of isolates of Plasmodium falciparum from Eastern Thailand.

The in vitro susceptibility of five field isolates of Plasmodium falciparum from the region of the Thai-Kampuchean border to pyrimethamine, sulphadoxine, and their combination, was determined using a microtitre test system and media deficient in p-aminobenzoic acid and folic acid. Two-fold serial dilutions of pyrimethamine ranging from concentrations of 8.0 to 0.125 microM and sulphadoxine ranging from 800 to 50 microM were evaluated for antimalarial activity. Viability was based on the maturation of the ring stages to normally-appearing schizonts. Tested singly the parasites had an average ID90 of 3.82 microM for pyrimethamine and greater than 800 microM for sulphadoxine. Analysis of the drugs interaction showed maximum potentiation at approximately 0.8 microM of pyrimethamine and 80 microM of sulphadoxine. These results suggest that resistance to Fansidar is due to the resistance to both components. Although there was a potentiating effect it was probably not sufficient enough for the drugs to be effective in vivo. This may, in part, explain the reduction in clinical cures with the sulphadoxine-pyrimethamine combination in eastern Thailand.

Drug Resistance, Microbial↗

PubMed2004

Influence of the growth phase and culture medium on the survival of Mannheimia haemolytica during storage at different temperatures.

AIMS: To quantify the influence of the growth phase, storage temperature and nutritional quality of the plate count medium on the apparent viability of Mannheimia haemolytica during storage at different temperatures. METHODS AND RESULTS: Mannheimia haemolytica was grown in shake flasks and in aerobic continuous culture to investigate factors affecting cell viability during storage, which was determined using plate counts on different media and epifluorescence microscopy. The high specific death rates of cells harvested after cessation of exponential growth and stored at 22, 4, -18 and -75 degrees C could be related to the rapid onset of exponential death in batch cultures. Yeast extract supplementation of the culture medium increased the viability of cells at most of the above-mentioned storage temperatures. Of the total cell count in continuous culture, only 48% could be recovered on brain-heart infusion agar, whereas supplementation of the agar medium with foetal calf serum increased the plate count to 71% of the total count. CONCLUSIONS: Mannheimia haemolytica cells harvested from the exponential growth phase had the highest survival rate during storage at low temperatures. Plate count values also depended on the nutritional quality of the agar medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Results presented here impact on the procedures for culture preservation and plate count enumeration of this fastidious animal pathogen.

Bacteriological Techniques↗

PubMed2002

Susceptibility of Escherichia coli O157 and non-O157 isolates to lactate.

AIMS: To examine the effect of temperature on the antimicrobial efficacy of lactate and propionate against O157 and non-O157 Escherichia coli isolates. METHODS AND RESULTS: Lactate and, to a lesser extent, propionate effectively reduced viability at 37 degrees C. Ethanol enhanced this effect. Reducing the temperature to 20 or 5 degrees C caused an increase in survival in the presence of these organic acids with or without ethanol. At 20 degrees C the deltapH, membrane potential and intracellular lactate anion concentration were less than at 37 degrees C. CONCLUSIONS: The efficacy of lactate and propionate against E. coli O157 and non-O157 isolates is reduced at lower temperatures, perhaps due to the reduction in the deltapH, membrane potential and intracellular lactate anion concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that the usefulness of organic acids as decontaminants for E. coli O157 is temperature dependent.

Animals↗

PubMed2006

In vitro biofilm formation on the surface of resin-based dentine adhesives.

Prevention of root caries on exposed root surfaces in the aging population is a significant challenge. Bonding resins can be applied to exposed root surfaces as sealants; however, minimal data exists regarding biofilm formation on the surface of these resins. We hypothesized that an antibacterial dentine-bonding resin containing methacryloxydodecyl-pyridiniumbromide (MDPB) may reduce biofilm formation. Biofilms were produced in pooled stimulated natural whole saliva, supplemented with 1% sucrose, on the surface of 5 dentine-bonding resins (Clearfil SE, OptiBond Solo, Protect Bond, Protect Bond Primer, and Xeno III) using untreated root surfaces as controls. Biofilms were stained using the Live:Dead Baclight bacterial viability stain, viewed with confocal microscopy, and analyzed using ImageJ image-analysis software. Resin surfaces encouraged attachment of live bacteria compared with root surfaces. All resins showed similar bacterial colonization in sections adjacent to the resin surface, but in the central and outer portions of biofilms, Xeno III and Protect Bond Primer showed a viable bacterial load similar to that of the root surface. Fluoride-releasing resins (OptiBond Solo/Protect Bond) did not show reduced biofilm formation. Thus, antibacterial agents within the resins have a minimal effect on biofilm formation, particularly when directly adjacent to the root surface.

Anti-Bacterial Agents↗

PubMed1993

Activities of several benzimidazoles and tubulin inhibitors against Giardia spp. in vitro.

Previous studies in our laboratory have shown that albendazole is effective against Giardia spp. in vitro and in vivo, prompting an investigation of the effects of several related benzimidazoles (BZs) on the viability of this protozoan parasite. A range of BZs was tested, and their effects were compared with those of a number of microtubule inhibitors. The effects produced by the two types of drugs were markedly similar, namely, trophozoite detachment and distortion of morphology and general structure, indicating a potential antimicrotubule mode of action for BZs. Mebendazole, albendazole, and fenbendazole proved to be among the most effective BZs tested, exhibiting apparent irreversibility. Nocodazole, oxfendazole, and albendazole sulfoxide, among others, produced transient inhibitions only. Further studies are required to evaluate all available BZs and other antigiardial agents to ensure the development of the most effective and safest antigiardial agent possible.

Albendazole↗

PubMed1999

Uptake and intracellular activity of moxifloxacin in human neutrophils and tissue-cultured epithelial cells.

The penetration by moxifloxacin of human neutrophils (polymorphonuclear leukocytes [PMN]) and tissue-cultured epithelial cells (McCoy cells) was evaluated by a fluorometric assay. At extracellular concentrations of 5 mg/liter, the cellular-to-extracellular concentration ratios (C/E) of moxifloxacin in PMN and McCoy cells were 10.9 +/- 1.0 and 8.7 +/- 1.0, respectively (20 min; 37 degrees C). The uptake of moxifloxacin by PMN was rapid, reversible, nonsaturable (at extracellular concentrations ranging from 1 to 50 microg/ml), and not affected by cell viability. The uptake of moxifloxacin was affected by external pH and the environmental temperature. The incubation of PMN in the presence of sodium fluoride, sodium cyanide, and carbonyl cyanide m-chlorophenylhydrazone significantly decreased the C/E of this agent. Neither PMN stimulation nor phagocytosis of opsonized Staphylococcus aureus significantly affected the uptake of moxifloxacin by human PMN. This agent, at concentrations of 0.5, 1, and 5 mg/liter, induced a significant reduction in the survival of intracellular S. aureus in human PMN. In summary, moxifloxacin reaches much higher intracellular concentrations within phagocytic and nonphagocytic cells than extracellular ones, remaining active inside the neutrophils.

Anti-Infective Agents↗

PubMed1997

Effect of alum on free-living and copepod-associated Vibrio cholerae O1 and O139.

The effects of alum [KAl(SO4)2] on free-living and copepod-associated Vibrio cholerae O1 and O139 were investigated by using plate counts and immunofluorescence direct viable counting (DVC). Growth of alum-treated cells in 0.5/1000 Instant Ocean seawater was inhibited, i.e., no growth was obtained on Luria-Bertani (LB) agar or thiosulfate-citrate-bile salt-sucrose (TCBS) agar. However, a significant number of the inhibited cells maintained viability, as measured by DVC. In comparison, a significant number of V. cholerae organisms associated with zooplankton, most of which were crustacean copepods, were viable but nonculturable, with only a small number of cells retaining culturability on LB and TCBS agar. Both DVC and viable plate counts (CFU) were significantly greater for V. cholerae O1 and O139 associated with zooplankton than for V. cholerae in water alone, i.e., without copepods. It is concluded that alum is an effective coagulant but not an effective killing agent for V. cholerae and that association with copepods offers protection for V. cholerae O1 and O139 against alum and chlorine treatments.

Alum Compounds↗

PubMed2005

Influence of nicotine and cotinine on epithelial colonization by periodontopathogens.

BACKGROUND: Since smoking is an established risk factor for the development of periodontitis, the present study investigated whether nicotine and cotinine can make epithelial cells more prone to colonization by periodontopathogens. METHODS: Primary epithelial cell mono-layers were inoculated with nicotine and cotinine prior to adhesion experiments with Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis. The number of bacteria associated with cells inoculated or not with nicotine or cotinine were assessed by an indirect culture viability assay. The same experimental set-up was used for assessing HeLa cells exposed to cigarette smoke extract (CSE). RESULTS: Primary epithelial cells inoculated with concentrations of nicotine and cotinine, found in smokers and non smokers, did not show significant differences (P>0.05) in colonization susceptibility to A. actinomycetemcomitans. When these concentrations were increased to 1 mg/ml, a significant (P<0.05) and species-specific effect of the colonization susceptibility of epithelial cells was observed: It increased for A. actinomycetemcomitans, while it decreased for P. gingivalis. For both species the effects were more pronounced for nicotine, although this was not statistically significant. The change in colonization susceptibility did not result from alterations of the bacterial viability due to nicotine or cotinine. Treatment of HeLa cells with CSE also led to a species-specific variation in colonization tendency; i.e., increased for A. actinomycetemcomitans (P<0.05), but not for P. gingivalis. CONCLUSIONS: The susceptibility of epithelial cells to become colonized by either A. actinomycetemcomitans or P. gingivalis could be altered by nicotine, cotinine, or CSE in a time-dependent, species-specific manner. Whether these findings that support the hypothesis of an increased patient susceptibility for bacterial adhesion to epithelial cells in smokers are clinically relevant remains to be proven.

Aggregatibacter actinomycetemcomitans↗

PubMed2006

Waterborne microbial risk assessment: a population-based dose-response function for Giardia spp. (E.MI.R.A study).

BACKGROUND: Dose-response parameters based on clinical challenges are frequently used to assess the health impact of protozoa in drinking water. We compare the risk estimates associated with Giardia in drinking water derived from the dose-response parameter published in the literature and the incidence of acute digestive conditions (ADC) measured in the framework of an epidemiological study in a general population. METHODS: The study combined a daily follow-up of digestive morbidity among a panel of 544 volunteers and a microbiological surveillance of tap water. The relationship between incidence of ADC and concentrations of Giardia cysts was modeled with Generalized Estimating Equations, adjusting on community, age, tap water intake, presence of bacterial indicators, and genetic markers of viruses. The quantitative estimate of Giardia dose was the product of the declared amount of drinking water intake (in L) by the logarithm of cysts concentrations. RESULTS: The Odds Ratio for one unit of dose [OR = 1.76 (95% CI: 1.21, 2.55)] showed a very good consistency with the risk assessment estimate computed after the literature dose-response, provided application of a 20 % abatement factor to the cysts counts that were measured in the epidemiological study. Doing so, a daily water intake of 2 L and a Giardia concentration of 10 cysts/100 L, would yield an estimated relative excess risk of 12 % according to the Rendtorff model, against 11 % when multiplying the baseline rate of ADC by the corresponding OR. This abatement parameter encompasses uncertainties associated with germ viability, infectivity and virulence in natural settings. CONCLUSION: The dose-response function for waterborne Giardia risk derived from clinical experiments is consistent with epidemiological data. However, much remains to be learned about key characteristics that may heavily influence quantitative risk assessment results.

Adolescent↗

PubMed1991

Biochemical and morphological studies on perivenous and periportal liver parenchymal cells from female rats isolated by digitonin-collagenase method.

Periportal (pp) or perivenous (pv) liver parenchymal cells from female adult Uje: WIST rats were isolated after retro- or antegrade digitonin infusion followed by collagenase perfusion in the opposite direction. The morphological results revealed a distinct acinar-related destruction of the pv- or pp-zone by digitonin. The remaining cells of the respective other zone showed a good structural maintenance. After subsequent conventional collagenase perfusion the yield, viability and structural integrity of the isolated hepatocytes were high. The zonal cell separation was indicated by significant differences in the pp marker glucose-6-phosphatase and the pv marker glutamine synthetase found in the isolated pp or pv cell populations. Under our experimental conditions including the use of female rats, the alanine aminotransferase and glutamate dehydrogenase as well as ethylmorphine N-demethylase and ethoxycoumarin O-deethylase activities were evenly distributed in both preparations. Under stimulating conditions the capacity for urea synthesis was similar in both pv and pp cells.

7-Alkoxycoumarin O-Dealkylase↗

PubMed2000

Helicobacter pylori: ureA, cagA and vacA expression during conversion to the coccoid form.

As viability of coccoid forms of Helicobacter pylori can only be verified by demonstrating the integrity of the DNA and active protein synthesis, we analysed the expression of ureA, cagA, vacA genes after prolonged incubation in a liquid medium. Exponentially growing and ageing phase cultures were used. Our results showed that, although the coccoid forms had decreased DNA and RNA levels after 31 days, they were not degraded and still expressed the urease, cytotoxic island and vacuolating toxin genes. Coccoid forms are therefore viable and may act as a transmissible agent that plays a crucial role in disease relapses after antibiotic therapy.

Antigens, Bacterial↗

PubMed2004

Enhancement of amphotericin B activity against Candida albicans by superoxide radical.

This study aimed to examine the involvement of oxidative damage in amphotericin B (AmB) activity against Candida albicans using the superoxide (O2-) generator paraquat (PQ). The effects of PQ on AmB activities against growth, viability, membrane permeability and respiration were examined in a wild-type parent strain (K) and a respiration-deficient mutant (KRD-19) since PQ-induced superoxide generation depends on respiration. In the parent strain, the minimal inhibitory concentration (MIC) of AmB, 0.25 microg/ml, tested with a liquid culture was lowered to 0.025 microg/ml by 1 mM PQ. Such a PQ-induced decrease in the MIC value of AmB was minimal in the mutant. Similar PQ-induced enhancement of AmB activity toward the parent strain was also observed with growth on an agar medium. In viability tests, when candidal cells were exposed to AmB (0.1 microg/ml) for I h, the lethality of AmB was enhanced by 1 mM PQ only in the parent strain. Exogenous superoxide dismutase and catalase failed to diminish the enhancing effect of PQ on the growth inhibitory activity of AmB in the parent strain, suggesting an interaction between superoxide and AmB in candidal cells. The enhancement of AmB activity by PQ, observed preferentially in the wild-type strain, can be explained by extensive superoxide generation depending on respiration. These results suggest that oxidative damage induced by superoxide is involved in AmB activity against C. albicans.

Amphotericin B↗

PubMed1992

Antimicrobial susceptibility testing of Bilophila wadsworthia by using triphenyltetrazolium chloride to facilitate endpoint determination.

Initial susceptibility studies of Bilophila wadsworthia indicated significant resistance to several beta-lactam antibiotics, including imipenem and cefoxitin. NO beta-lactamase production was detected. However, some B. wadsworthia strains may grow as a heavy "haze" at up to the highest concentration of an antibiotic on standard antimicrobial agent-containing plates, and it is often difficult to determine the point at which conventional growth stops and haze growth begins. We investigated the nature of the haze growth of B. wadsworthia by using triphenyltetrazolium chloride (TTC) as an indicator of viability during antimicrobial susceptibility testing, by determining viability counts on antimicrobial agent-containing plates at various times, and by microscopically inspecting stained preparations of the growth on the control plate and the haze area. TTC MICs were determined by applying a TTC solution over the growth on plates inside the anaerobic chamber or within 5 min after exposure to air (aerobic TTC MICs). The haze growth reduced TTC in the chamber but not under aerobic conditions, whereas TTC was reduced by the conventional growth in both atmospheres. The aerobic TTC MICs correlated with the viability counts. Separated proteins resolved by polyacrylamide gel electrophoresis and isoelectric focusing showed TTC-reactive bands only when stained under anaerobic conditions, further demonstrating the sensitivity of TTC reduction to aerobic conditions. Microscopic examination of the haze growth indicated spheroplast formation. A new antibiogram for B. wadsworthia has been established by use of aerobic TTC endpoints; we believe that the lower MICs obtained with the TTC method are likely the ones that are clinically relevant and should be used in tests of B. wadsworthia. Also, we found that when the organisms were grown on pyruvate-containing medium, 87% of 56 strains tested were Beta-lactamase positive.

Anti-Bacterial Agents↗

PubMed1994

Antibacterial activity of hydrogen peroxide and the lactoperoxidase-hydrogen peroxide-thiocyanate system against oral streptococci.

In secreted fluids, the enzyme lactoperoxidase (LP) catalyzes the oxidation of thiocyanate ion (SCN-) by hydrogen peroxide (H2O2), producing the weak oxidizing agent hypothiocyanite (OSCN-), which has bacteriostatic activity. However, H2O2 has antibacterial activity in the absence of LP and thiocyanate (SCN-). Therefore, LP may increase antibacterial activity by using H2O2 to produce a more effective inhibitor of bacterial metabolism and growth, or LP may protect bacteria against the toxicity of H2O2 by converting H2O2 to a less-potent oxidizing agent. To clarify the role of LP, the antibacterial activities of H2O2 and the LP-H2O2-SCN- system were compared by measuring loss of viability and inhibition of bacterial metabolism and growth. The relative toxicity of H2O2 and the LP system to oral streptococci was found to depend on the length of time that the bacteria were exposed to the agents. During incubations of up to 4 h, the LP system was from 10 to 500 times more effective than H2O2 as an inhibitor of glucose metabolism, lactic acid production, and growth. However, if no more H2O2 was added, the concentration of the inhibitor OSCN- fell because of slow decomposition of OSCN-, and when OSCN- fell below 0.01 mM, the bacteria resumed metabolism and growth. In contrast, the activity of H2O2 increased with time. H2O2 persisted in the medium for long periods of time because H2O2 reacted slowly with the bacteria and streptococci lack the enzyme catalase, which converts H2O2 to oxygen and water. After 24 h of exposure, H2O2 was as effective as the LP system as an inhibitor of metabolism. H2O2 also caused a time-dependent loss of viability, whereas the LP system had little bactericidal activity. The concentration of H2O2 required to kill half the bacteria within 15 s was 1.8 M (6%) but fell to 0.3 M (1%) at 2 min, to 10 mM (0.03%) at 1 h, and to 0.2 mM (0.0007%) with a 24-h exposure. The results indicate that if high levels of H2O2 can be sustained for long periods of time, H2O2 is an effective bactericidal agent, and the presence of LP and SCN- protects streptococci against killing by H2O2. Nevertheless, the combination of LP, H2O2, and SCN- is much more effective than H2O2 alone as an inhibitor of bacterial metabolism and growth.

Colony Count, Microbial↗

PubMed1991

Effects of lysozyme-thiocyanate combinations on the viability and lactic acid production of Streptococcus mutans and Streptococcus rattus.

Effects of human lysozyme (HLZ) combined with thiocyanate (SCN-) ions on mutans streptococci, both in physiologic salivary concentrations, were studied. The bacteria were incubated for 75 min either in HLZ-supplemented sterilized human whole saliva (pH 5 and 7) or in neutral buffer in the presence or absence of HLZ (30 mg/l)-SCN- (1-5 mM) combinations. HLZ had no inhibitory effect on the viability of Streptococcus mutans, serotype c, either in saliva or in buffer, not even at pH 5, in the presence of salivary bicarbonate or in higher (up to 240 mg/l) concentrations of HLZ. In contrast, HLZ significantly decreased the viability of S. rattus in both media. HLZ also effectively blocked the lactic acid production of S. rattus but not that of S. mutans. Thiocyanate ions, which have been proposed to enhance the antimicrobial activity of lysozyme, did not affect the antibacterial activity of HLZ or HLZ-HCO3- combinations. It is concluded that the in vivo levels of SCN- ions, which constitute an integral part of the peroxidase antimicrobial system in saliva, may not be high enough to trigger the lysis of S. mutans by lysozyme in human saliva. The very low prevalence of S. rattus compared with S. mutans in human populations may be associated with their different susceptibility to lysozyme-mediated inhibition in saliva.

Buffers↗

PubMed2003

Impact of preheating on the behavior of Listeria monocytogenes in a broth that mimics Camembert cheese composition.

The effect of preheating on the survival of L. monocytogenes in Richard's broth, which mimics the composition of Camembert cheese composition, was examined. Experiments were carried out to reproduce contamination of cheese with environmental heat-stressed cells of L. monocytogenes surviving hot-cleaning procedures. Cells in mid-log phase were heated for 30 min at 56 degrees C before being inoculated into Richard's broth. The pHs and temperatures of Richard's broth were chosen to recreate the conditions of curd dripping (pH 5, 25 degrees C), of the beginning of cheese ripening (pH 5, 12 degrees C), and of the beginning (pH 5, 4 degrees C) and the end (pH 7, 4 degrees C) of cheese storage. Immediately after heat treatment, the viability loss was especially high for strain 306715, which exhibited only 0.6% +/- 0.2% survival, compared with 22% +/- 8.7% for strain EGD. The percentages of the surviving heated cells that were injured were 93% +/- 8% for strain 306715 and 98% +/- 3% for strain EGD. The destruction of the surviving L. monocytogenes cells was accelerated when they encountered the pH and temperature conditions of Camembert cheese during manufacturing, ripening, and cold storage (pH 5 at 25, 12, and 4 degrees C, respectively). The multiplication of the surviving heated cells was retarded under favorable growth conditions similar to those of storage by the distributor and the consumer (pH 7 at 4 and 12 degrees C, respectively).

Cheese↗

PubMed2000

Stable expression of varied levels of inducible nitric oxide synthase in primary cultures of endothelial cells.

Nitric oxide (NO*), generated by nitric oxide synthase (NOS II) from immunostimulated cells during infection, plays an important role in host immune defense against microbial invasion. The impact of different rates of NO* production on host cell function has not been defined. Herein, we describe the development of a method to express varied levels of murine NOS II in bovine pulmonary artery endothelial cells. A retroviral vector (pMFGSNOS) encoding NOS II was used to transduce primary cultures of endothelial cells. Bovine endothelial cells were susceptible to this transduction and up to 18% of the cells expressed immunodetectable murine NOS II. The NOS II-transduced endothelial cells were cultured on the three-dimensional matrix, Gelfoam, for 8-10 days. Stable expression of NOS II was assessed by measuring nitrite accumulation in media every 2 days. By day 10, endothelial cells on Gelfoam were found to secrete NO* at a rate exceeding 1.0 microM/h/10(6) cells, concomitant with an enhanced level of NOS II activity. Argininosuccinate synthetase, a key enzyme in the metabolism of l-citrulline to l-arginine, increased as well, perhaps in response to dimunition of the intracellular arginine pool corresponding to the observed high output of NO*. In spite of the continuous flux of NO*, endothelial cell viability was not effected. This system provides the opportunity to assess the impact of different levels of sustained NO* production on endothelial cell physiology.

Animals↗