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PubMed2005

Alternative luciferase for monitoring bacterial cells under adverse conditions.

The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod Gaussia princeps (Gluc) has recently been cloned. We expressed the Gluc gene in Mycobacterium smegmatis using a shuttle vector and compared its performance with that of an existing luxAB reporter. In contrast to luxAB, the Gluc luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.

Animals↗

PubMed2005

Diversity of bacteria growing in natural mineral water after bottling.

Bacterial growth occurs in noncarbonated natural mineral waters a few days after filling and storage at room temperature, a phenomenon known for more than 40 years. Using the full-cycle rRNA approach, we monitored the development of the planktonic bacterial community in a noncarbonated natural mineral water after bottling. Seven 16S rRNA gene libraries, comprising 108 clones in total, were constructed from water samples taken at various days after bottling and from two different bottle sizes. Sequence analyses identified 11 operational taxonomic units (OTUs), all but one affiliated with the betaproteobacterial order Burkholderiales (6 OTUs) or the class Alphaproteobacteria (4 OTUs). Fluorescence in situ hybridization (FISH) was applied in combination with DAPI (4',6'-diamidino-2-phenylindole) staining, viability staining, and microscopic counting to quantitatively monitor changes in bacterial community composition. A growth curve similar to that of a bacterium grown in a batch culture was recorded. In contrast to the current perception that Gammaproteobacteria are the most important bacterial components of natural mineral water in bottles, Betaproteobacteria dominated the growing bacterial community and accounted for 80 to 98% of all bacteria detected by FISH in the late-exponential and stationary-growth phases. Using previously published and newly designed genus-specific probes, members of the betaproteobacterial genera Hydrogenophaga, Aquabacterium, and Polaromonas were found to constitute a significant proportion of the bacterial flora (21 to 86% of all bacteria detected by FISH). For the first time, key genera responsible for bacterial growth in a natural mineral water were identified by applying molecular cultivation-independent techniques.

Bacteria↗

PubMed1987

[Toxic action of the polyene antibiotic levorin on the superproducer Streptomyces levoris].

Along with an increase in the activity of a superproductive strain of Str. levoris there was observed in its population an increase in the number of low active cells sensitive to high concentrations of the own antibiotic. Levorin had an especially high toxic action on viability and antibiotic activity of the low productive strain 78/19 as compared to the highly productive strain 78/72. Resistance of the levorin-producing organism to its own antibiotic was associated with composition and properties of the cytoplasmic membrane. Acridine orange had no effect on survival and morphological variation of the strains studied. It had either no effect on the antibiotic production by the low active strain 78/19. However, under the effect of the dye there was induced marked variation in the highly productive strain 78/72 with respect to the antibiotic production property: decreasing of the population activity due to elimination of highly productive variants and accumulation of low productive variants. The data suggested the presence of plasmid genes in the levorin-producing organism determining biosynthesis of the antibiotic and resistance to it.

Acridine Orange↗

PubMed1999

Enhancement of anti-Aeromonas salmonicida activity in Atlantic salmon (Salmo salar) macrophages by a mannose-binding lectin.

We investigated the effects of a calcium-dependent mannose-binding lectin isolated from the serum of Atlantic salmon on Aeromonas salmonicida viability and the anti-A. salmonicida activity of Atlantic salmon macrophages. In the absence of other factors, binding of this lectin at concentrations of 0.8, 4.0 and 20.0 ng ml(-1) to virulent A. salmonicida failed to significantly reduce (P> 0.05) cell viability. However, binding of the lectin to A. salmonicida did result in significant (P < or = 0.05) dose-dependent increases in phagocytosis, and bactericidal activity. Significant increases (P < or = 0.05) were also observed in phagocyte respiratory burst activity within the lectin concentration range of 4.0-20.0 ng ml(-1) but the stimulation was not dose dependent at these lectin concentrations. At the lowest lectin concentration tested (0.32 ng ml(-1)), a significant decrease (P < or = 0.05) in respiratory burst was observed. The structure and activity of this lectin are similar to that of mammalian mannose-binding lectins, which are known to play a pivotal role in innate immunity. The presence of this lectin may be an important defense mechanism against Gram-negative bacteria such as A. salmonicida.

Aeromonas↗

PubMed1997

Antibiotic MICs and short time-killing against Helicobacter pylori: therapeutic potential of kanamycin.

We compared the susceptibility of Helicobacter pylori to several antibiotics, expressed as MICs and as bactericidal effectiveness in short (3 h) time-killing studies. Of the antimicrobial agent tests, clarithromycin and amoxycillin had the lowest MIC50, 0.063 and 0.125 mg/L respectively, for 24 strains of H. pylori. Minocycline, levofloxacin and lansoprazole followed, with MIC50s of 0.5, 1, and 2 mg/L, respectively. Three-hour time-killing studies using a standard strain demonstrated a different pattern. At 4 x MIC, kanamycin, metronidazole and clarithromycin produced 4.4, 2.6 and 2.1 log decreases in viability, whereas the remaining seven antibiotics (including amoxycillin) were less bactericidal. Amoxycillin's lack of bactericidal activity during brief incubations was confirmed by examining several different clinically isolated H. pylori strains. Clarithromycin's effect, on the other hand, was strain- and concentration-dependent. Kanamycin was the most potent antibiotic in short time-killing studies, with concentrations of 1 x MIC and 4 x MIC producing a reduction of more than 2 and 4 log respectively in all ten strains. Our data suggest that the MIC of antimicrobial agents against H. pylori does not necessarily predict their activity in short time-killing studies. Furthermore, our short time-kill data suggest kanamycin as a potential therapeutic choice for H. pylori infection, even though this agent's MIC would suggest limited activity.

Amoxicillin↗

PubMed1998

The mode of action of metronidazole in Helicobacter pylori: futile cycling or reduction?

The effects of metronidazole on catalase-positive and spontaneous catalase-negative mutants of Helicobacter pylori were studied to investigate whether the action of metronidazole on this microaerophilic organism occurs by reactive oxygen species generated by futile cycling or by the reduction of metronidazole to its active form. Increased sensitivity would be expected to occur in catalase-negative mutants if the mode of action of metronidazole was mediated through reactive oxygen species that may result from futile cycling of metronidazole. Two strains, RU1 and N6, were found to mutate spontaneously to a catalase-negative phenotype. The catalase-positive strain RU1(KatA+) and its catalase-negative counterpart RU1(KatA-) were sensitive to metronidazole, with MICs of 0.5 mg/L. The metronidazole-sensitive strain RU1(KatA-) lost viability at a rate similar to the parent RU1(KatA+) strain in the presence of 10 mg/L of metronidazole. Stable resistance to metronidazole was induced in RU1(KatA+) and RU1(KatA-) by passaging these strains in the presence of metronidazole. The catalase-positive and catalase-negative strains, N6(KatA+) and N6(KatA-), were resistant to metronidazole, with MICs of 96 mg/L. These observations indicated that the presence or absence of catalase activity did not affect the susceptibility of strains to metronidazole. The metabolism of metronidazole by H. pylori was investigated by 14N-NMR spectroscopy. Metronidazole was reduced in sensitive, catalase-positive and catalase-negative strains. Metronidazole-resistant cells reduced the 5-nitroimidazole more slowly, suggesting that resistance is achieved through the prevention or inhibition of metronidazole reduction.

Anti-Bacterial Agents↗

PubMed1999

Acid adaptation of Listeria monocytogenes strains does not offer cross-protection against an activated lactoperoxidase system.

Listeria monocytogenes has been implicated in foodborne illness outbreaks involving several types of cheeses made from acidified milk. Acid shock response (ASR) and acid tolerance response (ATR) could be possible reasons for its survival. The ASR and ATR of three strains of L. monocytogenes (V7, V37, and CA) in skim milk acidified to pH 4.0 and 3.5 with lactic acid and held at 32 degrees C were studied. Studies were also done to determine if acid adaptation of the organism enhanced survival in the presence of an activated lactoperoxidase system. The cells were directly shocked at pH 4.0 and 3.5 in skim milk to study the ASR. To study the ATR, cells were initially adapted in skim milk at a mild pH of 5.5 for the equivalent of one generation before being shocked at pH 4.0 and 3.5 in skim milk. Cells adapted at pH 5.5 in tryptic soy broth without dextrose and nonadapted cells were challenged at pH 4.5 in skim milk with or without an activated lactoperoxidase system. In all cases, viability and pH were measured 24 or 48 h after challenge. In pH 4.0 skim milk, for all three strains, the adapted cell population survived better (0.5 to 1.0 log higher) than that of nonadapted cells for 24 h. In pH 3.5 skim milk, the acid-adapted populations of all three strains were 3 to 4 logs greater than those of nonadapted cells at 6 h. The acid adapted cells of all three strains had survival rates similar to those of the nonadapted cells at pH 4.5 both in the presence and absence of an activated lactoperoxidase system. It was also evident that these strains do not exhibit an adaptive ATR at pH 4.5, although they do at lower pH levels (pH 4.0 and 3.5). Survival due to the ATR was better seen at pH 3.5 than at pH 4.0.

Adaptation, Physiological↗

PubMed1986

Perfusion technique of suckling rat liver, and comparison of cytologic and biochemical properties between hepatocytes isolated from suckling and adult rats.

Collagenase-liver-perfusion technique, currently used with adult rat liver, was applied to isolation of hepatocytes from suckling rat liver. The total hepatocyte numbers isolated from suckling rats by collagenase-liver-perfusion technique were 9-fold higher than those by non-perfusion technique. The yield and viability of isolated hepatocytes from suckling rats were 18.1 X 10(7) cells per gram liver and 95%, respectively. The cell yield per gram liver and viability from suckling rats were 185% and 112% of those from adult ones, respectively. In comparison with adult rat hepatocytes, suckling rat hepatocytes were smaller and more homogeneous. The percentage (3.1%) of binucleate cells in the hepatocytes isolated from suckling rats was about one tenth of that (30.7%) in the hepatocytes isolated from adult rats. The isolated suckling rat hepatocytes had higher tyrosine aminotransferase (TAT) and glucose-6-phosphate (G6Pase) activities than adult ones. Suckling and adult rat hepatocytes were transferred to primary culture to compare their cell number kinetics and functional longevities. The functional longevities of those hepatocytes were assessed by their capacity to secrete albumin and alpha-fetoprotein (AFP) into the culture medium and to express TAT and G6Pase activities up to day 6 of primary culture.

Albumins↗

PubMed2001

Performance of mycological media in enumerating desiccated food spoilage yeasts: an interlaboratory study.

Dichloran 18% glycerol agar (DG18) was originally formulated to enumerate nonfastidious xerophilic moulds in foods containing rapidly growing Eurotium species. Some laboratories are now using DG18 as a general purpose medium for enumerating yeasts and moulds, although its performance in recovering yeasts from dry foods has not been evaluated. An interlaboratory study compared DG18 with dichloran rose bengal chloramphenicol agar (DRBC), plate count agar supplemented with chloramphenicol (PCAC), tryptone glucose yeast extract chloramphenicol agar (TGYC), acidified potato dextrose agar (APDA), and orange serum agar (OSA) for their suitability to enumerate 14 species of lyophilized yeasts. The coefficient of variation for among-laboratories repeatability within yeast was 1.39% and reproducibility of counts among laboratories was 7.1%. The order of performance of media for recovering yeasts was TGYC > PCAC = OSA > APDA > DRBC > DG 18. A second study was done to determine the combined effects of storage time and temperature on viability of yeasts and suitability of media for recovery. Higher viability was retained at -18 degrees C than at 5 degrees C or 25 degrees C for up to 42 weeks, although the difference in mean counts of yeasts stored at -18 degrees C and 25 degrees C was only 0.78 log10 cfu/ml of rehydrated suspension. TGYC was equal to PCAC and superior to the other four media in recovering yeasts stored at -18 degrees C, 5 degrees C, or 25 degrees C for up to 42 weeks. Results from both the interlaboratory study and the storage study support the use of TGYC for enumerating desiccated yeasts. DG18 is not recommended as a general purpose medium for recovering yeasts from a desiccated condition.

Agar↗

PubMed2003

Viability of Saccharomyces cerevisiae cells exposed to low-amperage electrolysis as assessed by staining procedure and ATP content.

Assessment of yeast viability by plate counts, ATP determination and FUN-1 viability staining was performed to study sublethal injury of Saccharomyces cerevisiae cells submitted to low-amperage electrolysis. Lethal effects of electrolysis were confirmed by all methods, demonstrated by the decrease in viable counts observed during electrolysis. FUN-1 viability staining and ATP determination appeared to demonstrate higher survivors than plate counts. To study possible recovery of certain yeast cells damaged by electrolysis thus rendering them nonculturable, yeast suspensions were stored in phosphate buffer at 4 and 20 degrees C. Increase in viable counts and ATP content of treated yeast cells was observed during storage at 20 degrees C, whereas viable counts of treated and control yeast cells were shown to decrease during storage at 4 degrees C. The increase in the number of viable cells appeared to be the result of repair of damaged cells rather than regrowth of few cells remaining culturable. The lethal efficacy of electrolysis might be overestimated by plate counts. Further experiments must be done to evaluate the lethal efficacy of electrolysis on microorganisms in real conditions encountered in food products.

Adenosine Triphosphate↗

PubMed1986

Primary cultures of human livers and their albumin-producing capacity.

Primary cultures of surgically obtained noncancerous portions of human liver tissues were made. Liver tissues were poorly dissociated with collagenase, but well dissociated with dispase. The yield and viability of cells were improved somewhat when dissociated with collagenase followed by dispase. The mean cell yield was 1.1 X 10(6) cells/g liver. The epithelial-like morphology of the dissociated liver cells was maintained for about one week, but thereafter degenerative alteration of cells was observed. In liver explant culture, an active outgrowth of cells was observed for more than one month. Albumin production in culture fluids from dissociated livers was detectable for about 2 weeks, but later became undetectable, while that from explant culture was detectable for at least one month. These data demonstrate that adult human hepatocytes can be isolated from noncancerous portions of livers with relatively high yield, and that albumin production of the dissociated cells is detectable for several days.

Adult↗

PubMed2001

Chronological lifespan of stationary phase yeast cells; a model for investigating the factors that might influence the ageing of postmitotic tissues in higher organisms.

Budding yeast can be considered to have two distinct lifespans: (a) a replicative (budding, non-chronological) lifespan, measured as the number of daughters produced by each actively dividing mother cell; and (ii) a chronological lifespan, measured as the ability of stationary cultures to maintain viability over time. In non-dividing cells, essential components that become damaged cannot be diluted out through cell division but must, of necessity, be turned over and renewed. By elevating stress resistances, many of the activities needed for such renewal should be elevated with commensurate reduction in the steady-state levels of damaged cell components. Therefore, chronological lifespan in particular might be expected to relate to stress resistance. For yeast to attain a full chronological lifespan requires the expression of the general stress response. It is more important, though, that the cells should be efficiently adapted to respiratory maintenance, since it is cultures grown to stationary phase on respiratory media that usually display the longest chronological lifespans. For this reason, respiration-adapted cells potentially provide a better model of chronological ageing than cultures pre-grown on glucose.

Animals↗

PubMed1995

Evaluation of the polarographic technique for assay of the viability of freeze-dried BCG vaccine: II. Viability of the vaccine assessed by polarography, Warburg respirometry and colony counting.

A comparative study of the viability of 34 lots of freeze-dried BCG vaccine has been carried out using an oxygen electrode polarographic technique, Warburg respirometry and colony counting. There were no statistical differences between the results obtained with the polarographic and Warburg techniques. Both methods gave reliable and similar results and showed a positive correlation (r = 0.8615). Comparison between the polarographic and colony-counting methods showed a positive correlation (r = 0.6530); for comparison between the Warburg and colony-counting methods, the correlation value was r = 0.6868. All the tests were significant at the level of alpha = 0.05. The advantages of the polarographic technique are that it is much less time- and material-consuming than other methods. It is a reliable, inexpensive and convenient method for BCG vaccine quality control.

BCG Vaccine↗

PubMed2006

The growth of Propionibacterium cyclohexanicum in fruit juices and its survival following elevated temperature treatments.

This study investigated the growth of Propionibacterium cyclohexanicum in orange juice over a temperature range from 4 to 40 degrees C and its ability to multiply in tomato, grapefruit, apple, pineapple and cranberry juices at 30 and 35 degrees C. Survival after 10 min exposure to 50, 60, 70, 80, 85, 90 and 95 degrees C in culture medium and in orange juice was also assessed. In orange juice the organism was able to multiply by 2 logs at temperatures from 4 to 35 degrees C and survived for up to 52 days. However, at 40 degrees C viable counts were reduced after 6 days and no viable cells isolated after 17 days. The optimum growth temperature in orange juice over 6 days was 25 degrees C but over 4 days it was 35 degrees C. The growth of P. cyclohexanicum was monitored in tomato, grapefruit, cranberry, pineapple and apple juices at 30 and 35 degrees C over 29 days. Cranberry, grapefruit and apple juice did not support the growth of P. cyclohexanicum. At 30 degrees C no viable cells were detected after 8 days in cranberry juice or after 22 days in grapefruit juice while at 35 degrees C no viable cells were detected after 5 and 15 days, respectively. However, in apple juice, although a 5 log reduction occurred, viable cells could be detected after 29 days. P. cyclohexanicum was able to multiply in both tomato and pineapple juices. In tomato juice, there was a 2 log increase in viable counts after 8 days at 30 degrees C but no increase at 35 degrees C, while in pineapple juice there was a 1 log increase in numbers over 29 days with no significant difference between numbers of viable cells present at 30 and 35 degrees C. The organism survived at 50 degrees C for 10 min in culture medium without a significant loss of viability while similar treatment at 60, 70 and 80 degrees C resulted in approximately a 3-4 log reduction, with no viable cells detected after treatment at 85 or 90 or 95 degrees C but, when pre-treated at intermediate temperatures before exposure to higher temperatures, some cells survived. However, in orange juice a proportion of cells survived at 95 degrees C for 10 min without pre-treatment and there was no significant difference between numbers surviving with and without pre-treatment. The results from this study demonstrate that P. cyclohexanicum is able to grow in a number of juices, other than orange juice, and able to survive a number of high temperature procedures. Therefore, if initially present in the raw materials P. cyclohexanicum might survive the pasteurization procedures used in the fruit juice industry, contaminate and consequently spoil the final product.

Beverages↗

PubMedDate not supplied

Aerosolization of Newcastle disease vaccine virus and Enterococcus faecalis.

In order to study the aerosol transmission of arthropathic and amyloidogenic Enterococcus faecalis strains, preliminary aerosol experiments were performed. The experiments were carried out in empty isolators to assess the yield and viability of E. faecalis and Newcastle disease vaccine virus (NDVV) aerosol particles with time. NDVV was aerosolized because this virus would be used in combination with E. faecalis in a subsequent study. Concentrations of about 10(5) colony-forming units (CFU) of E. faecalis/ml of air were still found 30 min after the aerosol application. At 45 min, however, E. faecalis concentrations dropped below the detection level. The average E. faecalis concentration during the aerosol experiment was estimated at 10(5) CFU/liter. The NDVV aerosol generated an average of 10(4)-10(5) 50% embryo infective dose per liter of air. In these experiments, E. faecalis and NDVV aerosols were successfully generated despite considerable initial particle loss. The bacteria and virus uptakes per chick are discussed in case day-old chicks would be exposed to these aerosols.

Aerosols↗

PubMed2006

Fluconazole susceptibility testing of Candida species by flow cytometry.

OBJECTIVE: Currently, antifungal drug susceptibility testing is labor-intensive, limited by delays in obtaining results and high costs. The purpose of this study was to determine the usefulness of flow cytometry (FCM) antifungal drug susceptibility testing as a routine laboratory procedure. METHODS: A total of 24 clinical isolates of Candida spp. and reference strains were tested for susceptibility to fluconazole by FCM using propidium iodide (PI) as an indicator of viability. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of fluconazole that resulted in an increase of 30% in mean channel fluorescence (MCF), compared to the growth control. FCM results were compared with MIC results as determined by the Clinical and Laboratory Standards Institute (CLSI) method. RESULTS: An 8h incubation was sufficient for determination of the MICs. The results by FCM at 8h and the NCCLS methods at 24h showed 87.5% agreement to within two drug dilutions. However, the FCM method is labor-intensive in proportion to the larger number of samples. For Candida lusitaniae, MICs by the FCM method showed poor correlation with the CLSI method. CONCLUSIONS: Further evaluation is necessary to assess the usefulness of FCM as a technique for routine antifungal MIC testing in the clinical laboratory.

Antifungal Agents↗

PubMed2003

In vitro evaluation of a new treatment for urinary tract infections caused by nitrate-reducing bacteria.

Dietary and endogenous nitrates are excreted in urine, and during infection with nitrate-reducing bacteria they are reduced to nitrite. At a low pH nitrite is converted to a variety of nitrogen oxides that are toxic to bacteria. We hypothesized that acidification of nitrite-rich infected urine would result in the killing of the nitrate-reducing bacteria. An Escherichia coli control strain and a mutant lacking nitrate reductase activity were preincubated in urine supplemented with sodium nitrate (0 to 10 mM) at pH 7.0. Then, the nitrite-containing bacterial culture was transferred (and diluted 1/10) to slightly acidic urine (pH 5 and 5.5) containing ascorbic acid (10 mM) and growth was monitored. The control strain produced nitrite in amounts related to the amount of nitrate added. This strain was killed when the culture was transferred to acidic urine. In contrast, the mutant that did not produce nitrite retained full viability. When control bacteria were grown in acidic urine with nitrate and ascorbic acid present from the start of the experiment, no inhibition of growth was noted. The MICs and minimal bactericidal concentrations of sodium nitrite-ascorbic acid in acidic urine were comparable to those of conventional antibiotics. Preincubation of nitrate-reducing E. coli in nitrate-rich urine leads to the accumulation of nitrite. Subsequent acidification of the urine results in generation of nitrogen oxides that are bactericidal. Killing, however, requires a sequential procedure in which the bacteria are first allowed to grow in a nitrate-rich neutral environment, later followed by acidification. We speculate that ingestion of nitrate followed some hours later by acidification of urine could be a new therapeutic strategy for the treatment of urinary tract infections.

Adult↗

PubMed1975

Role of pyrimidine dimer excision in loss of potential streptomycin resistance mutations of ultraviolet-irradiated Escherichia coli on phosphate-buffered agar.

The frequency of ultraviolet (UV)-induced mutations to streptomycin resistance dropped rapidly when starved Escherichia coli strains WP-2 B/r and B/r T- were incubated on phosphate-buffered agar (PBA), but was reduced only slightly in a WP-2 hcr- mutant. During postirradiation, incubation viability remained approximately constant. Cells given an optimal recovery treatment with photo-reactivating light showed no further recovery if subsequently incubated on PBA. At least 70% of the mutations induced to streptomycin resistance by UV could be repaired. The loss of potential streptomycin-resistant mutants was markedly reduced in strain B/r T- when 5 mug of acriflavin or 700 mug of caffeine per ml was added to PBA. The excision of UV-induced thymine-containing dimers from E. coli tb/r T- was investigated. Dimer excision progressed more slowly when the cells were incubated on PBA containing acriflavin or caffeine. There was no congruity between the kinetics of dimer excision and the kinetics of mutant loss. Our results indicate that removal of potential streptomycin-resistant mutants is considerably faster than the excision of pyrimidine dimers.

Acriflavine↗