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PubMed2026

Meta-analysis of growth and inactivation kinetics of Legionella.

Quantitative risk assessments intended to inform evidence-based water management plans and public health targets for Legionella in engineered water systems are constrained by fragmented and heterogeneous growth and inactivation kinetics. We conducted a meta-analysis of 25 growth and 39 thermal- and chemical-inactivation studies, fitting microbial persistence models to harmonize parameters. Nonlinear models outperformed first-order formulations, indicating that lag phases and resistant or protected subpopulations are central to Legionella persistence. Random forest analysis identified environmental and methodological drivers of variability based on 226 growth rates and reduction times for thermal (209) and chemical (135) inactivation. Growth was primarily governed by temperature, nutrient availability, and compatible Legionella-host pairings; thermal inactivation by quantification method, temperature, and turbidity; and chemical inactivation by inoculum size, disinfectant type, concentration, and host-associations. Accordingly, temperature-dependent growth parameters and exposure metrics for heat, free-chlorine, and monochloramine, expressed as TT (Temperature×time) and CT (Concentration×time), were derived as condition-specific inputs for predictive models. Growth optima around 37-40 °C, together with lag-time estimates, indicate that hot-water temperature setbacks and energy-saving practices may favor Legionella proliferation under repeated or prolonged lukewarm exposure. Culture- and viability-based TT differences highlight the need to consider viable‑but-non-culturable persistence in monitoring programs. CT comparisons suggest monochloramine may be advantageous because of its lower apparent sensitivity to host-associated protection. Although limited by restricted experimental conditions, the findings show that predictive models should account for microbial ecology, water matrix effects, and quantification endpoints. Future kinetic studies should prioritize realistic multi-host systems, strain pre-adaptation, complementary viability measurements, and standardized protocols and reporting to ensure reproducibility and enable robust system-level predictive modeling.

Legionella↗
PubMed1975

Soil sterilization effects on in situ indigenous microbial cells in soil.

Soil was sterilized by various procedures, and then the resident microorganisms were physically separated and concentrated from the soil for viewing by transmission electron microscopy as thin sections and frozen-etched preparation. Remaining cell viability in the soil was tested by conventional plating before and after enrichment culture. The soil proved to be sterile after treatment with 60Co radiation, prolonged autoclaving, prolonged dry heat application at 200C, or glutaraldehyde (if followed by subsequent milk heating), and could be considered sterile after OsO4 treatment. Treatment with glutaraldehyde alone, or 160C dry heat for 3 h, did not sterilize the soil. Cellular fine structure was latered or destroyed by the heat treatments, but was not affected to any extent by any of the other treatments including glutaraldehyde followed by milk heating. These findings are considered in relation to the residual biological information observable by electron microscopy in soil samples which have been sterilized to eliminate possible pathogens before handling of the soil. These findings are also considered with the objective of obliterating the fine structure of the indigenous microorganisms during soil sterilization so that electron microscopy studies can be made of microorganisms inoculated into and grown in the presterilized soil. These findings are considered in relation to the residual biological information observable by electron microscopy in soil samples which have been sterilized to eliminate possible pathogens before handling of the soil. These findings are also considered with the objective of obliterating the fine structure of the indigenous microorganisms during soil sterilization so that electron microscopy studies can be made of microorganisms inoculated into and grown in the presterilized soil.

Bacteria↗
PubMed2005

Expanding the bactericidal action of the food color additive phloxine B to gram-negative bacteria.

Phloxine B (D&C red no. 28) is a color additive for food, drugs, and cosmetics. It has been previously shown to have anti-Staphylococcus aureus activities. In this work, the effect of Phloxine B on various gram-negative bacteria and other gram-positive bacteria including Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus subtilis, Bacillus aureus, Salmonella, Escherichia coli and Shigella was studied, along with the mechanism of anti-microbial activity. In the presence of fluorescent light, the viable count for gram-positive bacteria, (Bacillus spp. and S. aureus) decreased in a dose and time dependent manner when incubated with Phloxine B. The viability of gram-positive bacteria was reduced by 99.99% in 40 min, while there was no effect on gram-negative bacteria (Salmonella choleraesuis, E. coli and Shigella flexneri). However, the use of ethylenediaminetetraacetic acid (EDTA) expands the spectrum of activity for Phloxine B to include gram-negative bacteria. EDTA increased membrane-permeability by releasing lipopolysaccharide. Overall, in an Agar diffusion test the light-dependent bactericidal activity of 1 microg of Phloxine B had a potency of 0.64 units of chloramphenicol and 0.5 units of tetracycline when tested on B. cereus, and had a potency of 0.7 units of chloramphenicol and 0.2 units of tetracycline when tested on S. aureus. The data suggest that the dye may have some potential anti-microbial applications.

Anti-Bacterial Agents↗
PubMed2001

Microbial ecotoxicity and mutagenicity of 1-hydroxypyrene and its photoproducts.

1-Hydroxypyrene (1-HP) is a carcinogenic and slightly water-soluble polycyclic aromatic hydrocarbon. Ecotoxicity and mutagenicity of 1-HP and its photoproducts, and the effect of Mn2+ and Cu2+ on their mutagenicity were measured with microbial assay in this study. The assay includes spread plate counting, direct counting, microbial mineralization of 14C-UL-D-glucose and Mutatox Test. At the concentration examined (0.8 microM), the photoproducts (after 1.5 h solar irradiation) of 1-HP inhibited microbial glucose mineralization activity (by 64%) after microbial assemblages of a local reservoir site were exposed for 1 day. However, heterotrophic bacteria were able to utilize 1-HP photoproducts as the growth substrates and increase viability counts by up to 4.75-folds. 1-HP exhibited positive response to Mutatox Test in both direct medium and S-9 medium, with the lowest observable effective concentration of 0.625 microM in the test with direct medium. After photolysis, 1-HP decreased its mutagenicity. Mn2+ (312.5 microM-5 mM) and Cu2+ (6.25-100 microM) themselves are not mutagenic. However, addition of the metal ions before or after photolysis modifies the light readings of 1-HP during the test. Therefore, presence of metal ions could affect the genotoxicity of 1-HP in aquatic environments, depending on timing of the addition.

Biological Assay↗
PubMed1999

Toxicity of selected plant volatiles in microbial and mammalian short-term assays.

In this study, several short-term microbial and mammalian in vitro assays were used to evaluate cytotoxicity and genotoxicity of four plant volatiles showing antifungal activity: cinnamaldehyde, carvacrol, thymol and S(+)-carvone. All inhibited viability and proliferation of Hep-2 cells in a dose-dependent manner. IC50 ranged from 0.3 mM (cinnamaldehyde) to 0.7 mM (thymol) in viability tests and from 0.2 mM (carvacrol) to 0.9 mM (carvone) in the proliferation test. The morphological analysis suggested an involvement of apoptosis in the cases of carvone, carvacrol and cinnamaldehyde. At nontoxic doses, carvacrol and thymol increased the number of revertants in the Ames test by 1.5-1.7 times, regardless of metabolic activation. In the SOS-chromotest, none of the four plant volatiles caused DNA damage at non-toxic doses. In the DNA repair test, a marked dose-dependent differential toxicity was observed with carvone and, to a lesser extent, with cinnamaldehyde, while with thymol and carvacrol, this effect was less pronounced. In conclusion, the considered in vitro cytotoxicity assays have shown to be sensitive enough to highlight a variety of toxic effects at the cellular level, which can be rather different between chemically closely related compounds, such as isomers.

Acrolein↗
PubMed1985

Killed Escherichia coli stimulates macrophage-mediated alterations in hepatocellular function during in vitro coculture: a mechanism of altered liver function in sepsis.

Hepatic dysfunction is a poorly understood and highly lethal component of multiple-system organ failure. Both in vivo and in vitro studies of "liver" function have generally neglected hepatocyte-Kupffer cell interactions. In the following experiments, isolated hepatocytes were cocultivated with unstimulated peritoneal cells, predominately macrophages, which served as a readily available Kupffer cell analog. Coculture of hepatocytes with peritoneal cells resulted in little or no change in [3H]leucine incorporation into hepatocyte protein. When gentamicin-killed Escherichia coli cells (GKEC) were added to coculture, there was a marked decrease in hepatocyte [3H]leucine incorporation. In contrast, GKEC added to hepatocytes alone had no effect. Kinetic data revealed an 8-h delay before any significant decrease in leucine incorporation into hepatocyte protein after the addition of GKEC to the coculture. The maximal decrease in hepatocyte [3H]leucine incorporation occurred 24 h after GKEC were added. The decrease observed 24 h after GKEC were added disappeared almost completely after 48 h of coculture. Similar alterations in cocultured hepatocyte protein synthesis were observed after the addition of phorbol myristate acetate, lipopolysaccharide, or muramyl dipeptide, a component of bacterial peptidoglycan. Hepatocyte viability by trypan blue exclusion was unchanged, and gross morphology by light or electron microscopy was unaffected. We propose that during sepsis, macrophages (Kupffer cells) respond to circulating microbial products and mediate alterations in hepatocyte function. These experiments underscore the important role of Kupffer cell function in attempts to understand hepatic malfunction in multiple-system organ failure.

Animals↗
PubMed2004

Assessing cytotoxicity of photosensitized transformation products of 2,4,6-trinitrotoluene (TNT) and atrazine with freshwater microbial assemblages.

In this study of riboflavin-sensitized photolysis of 2,4,6-trinitrotoluene (TNT), the final photoproducts were found to include 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, and 3,5-dinitroaniline. After exposure to TNT (10 mg/L) for 90 min, in a river water sample there was inhibition of the viability count of heterotrophic bacterial assemblages by 28.3% and 24.3% and of bacterial heterotrophic mineralization of glucose by 99.5% and 93.6% relative to the light control and dark control groups, respectively. After exposure separately to 10 mg/L 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, or 3,5-dinitroaniline, the viability count of heterotrophic bacterial assemblages was enhanced by 30.0%, 98.2%, and 148.7%, respectively, relative to the TNT group in light and by 12.2%, 40.2%, and 36.5%, respectively, relative to TNT group in dark. After exposure to the same aforementioned test chemicals, heterotrophic activity of bacterial assemblages was inhibited by 75.7%, 72.4%, and 56.0%, respectively, relative to light control group, and there were no significant changes in the dark. A similar study was conducted with atrazine (10 mg/L). The main products of atrazine riboflavin-sensitized phototransformation include desethylatrazine (DEA) and desisopropylatrazine (DIA). It was found that DEA and DIA did not inhibit microbial heterotrophic activity. However, after 72 h the viable count was enhanced by 52.8% and 63.3% in the DEA and DIA exposure groups, respectively, in comparison to the control. These results suggested that photosensitized transformation decreased TNT and atrazine cytotoxicity to microbial assemblages in the natural water. Photoproducts of atrazine--DEA and -DIA, and of TNT could become growth substrates for bacterial assemblages in natural water.

Atrazine↗
PubMed2003

Evaluation of sputum decontamination methods for Mycobacterium tuberculosis using viable colony counts and flow cytometry.

Continuous monitoring systems for the detection of Mycobacterium tuberculosis are reported to have higher contamination rates than traditional radiometric technologies. Multiple decontamination methods have recently been reported in an attempt to optimize contamination rates for these systems. In this study, several decontamination methods for sputum were evaluated using viable colony count and flow cytometry. The decontamination protocols evaluated include N-Acetyl-L-Cysteine-Sodium Hydroxide (NALC-NaOH), modified Petroffs's method, and the Yamane procedure. Several parameters of the NALC-NaOH method were analyzed including final NaOH concentrations of 0.5-3%, NaOH exposure times of 0-30 min, and variations in resuspension media for the resultant pellet. All decontamination methods were performed on pooled and sterilized sputum seeded separately with either a mixture of common contaminating bacteria or M. tuberculosis H37Ra. Viability of organisms following decontamination was assessed by both colony counts and flow cytometric analysis. Flow cytometry viability assays utilized a combination of viability dyes and reference beads to determine viable organism concentrations. The results indicated that no decontamination method was clearly superior, however a concentration of 1-2% NaOH and an increase in the time of NaOH exposure to 30 min will effectively kill contaminating bacteria without significantly affecting the viability of M. tuberculosis H37Ra. While flow cytometry viability analysis did not directly correspond to viable colony counts, it was a useful tool for rapid viability analysis M. tuberculosis.

Analysis of Variance↗
PubMed2006

Treatment of ballast water; how to test a system with a modular concept?

A variety of methods were successfully applied to examine the efficacy of a modular ballast water system according to the standards as adopted by the International Maritime Organization. The ballast water treatment system had a capacity of 530 m3 h(-1) consisted of a pump system, a hydrocyclone, a 50 microm mesh-size self-cleaning filter and an installation for the addition of a chemical disinfectant (PERACLEAN Ocean). The land-based testing facility used natural sea water of high turbidity during the spring phytoplankton bloom. The mesozooplankton fraction was inspected with a standard binocular. Larger zooplankton were effectively removed with the filter; the smaller sized fraction containing larvae and nauplia were killed after chemical treatment. The phytoplankton component was monitored using flow cytometry. The huge colonies of the phytoplankton Phaeocystis globosa were disrupted in the hydrocyclone liberating the colony cells which passed as single cells through the filter. These cells remained viable but were finally killed in the secondary (chemical) step. Bacteria also passed all mechanical treatment steps unharmed but were killed in the final step. Viability tests with SYTOX Green, which were specifically designed for phytoplankton, showed that mechanical treatment did not affect the percentage of viable cells a short-term, but after several hours the viable cell counts dropped down to 70%. Phytoplankton cells recovered within a single day and formed a new dense bloom rapidly. The bacteriostatic component of the chemical disinfectant (H2O2) remained present for several days preventing regrowth of bacteria for up to 15 days after addition. In conclusion, the IMO standards were met using the modular ballast water treatment unit and the applied instruments and assays were effective and rapid tools to qualify and quantify the organisms present as well as their viability.

Animals↗
PubMed1993

Effect of amplicon size on PCR detection of bacteria exposed to chlorine.

The effect of amplicon size on the PCR detection of Legionella pneumophila after chlorine inactivation was investigated. Two amplicons specific to the L. pneumophila mip gene were used for the PCR analyses: a 650-bp amplicon and smaller 168-bp amplicon within the 650-bp amplicon; a 108-bp amplicon specific to species rRNA coding sequence also was used. After exposure to chlorine, viable agar grown cells were not detected by plate counts or direct counts with p-iodonitrotetrazolium (INT) after 1 min for treatment at 10 mg/l, after 2 min for treatment at 5 mg/l, and after 4 min for treatment at 2.5 mg/l; viable water grown cells were present at least 4 min after biocide addition even with a chlorine dose of 5 mg/l. At the 10-mg/l dosage, PCR products from the 168-bp amplicon were detected on agarose gels up to 16 min after chlorination; even after 24 hr of PCR the 168-bp products were detectable using a capture probe hybridization assay. However, the 650-bp target was not detected after 4 min chlorine contact time at the same biocide dosage using agarose gels, and PCR products could not be detected by hybridization after 32 min. At lower chlorine concentrations, a similar pattern was seen with the 168-bp amplicon detectable longer after biocide addition than the 650-bp mip amplification target. On the basis of these data, larger amplicons appear to correlate better with viability of L. pneumophila in water samples.

Bacterial Proteins↗
PubMed2000

Anhydrobiotic engineering of gram-negative bacteria.

Anhydrobiotic engineering aims to improve desiccation tolerance in living organisms by adopting the strategies of anhydrobiosis. This was achieved for Escherichia coli and Pseudomonas putida by osmotic induction of intracellular trehalose synthesis and by drying from trehalose solutions, resulting in long-term viability in the dried state.

Biotechnology↗
PubMed2003

Development of reverse transcription (RT)-PCR and real-time RT-PCR assays for rapid detection and quantification of viable yeasts and molds contaminating yogurts and pasteurized food products.

Reverse transcriptase PCR (RT-PCR) and real-time RT-PCR assays have been used to detect and quantify actin mRNA from yeasts and molds. Universal primers were designed based on the available fungal actin sequences, and by RT-PCR they amplified a specific 353-bp fragment from fungal species involved in food spoilage. From experiments on heat-treated cells, actin mRNA was a good indicator of cell viability: viable cells and cells in a nonculturable state were detected, while no signal was observed from dead cells. The optimized RT-PCR assay was able to detect 10 CFU of fungi ml(-1) in pure culture and 10(3) and 10(2) CFU ml(-1) in artificially contaminated yogurts and pasteurized fruit-derived products, respectively. Real-time RT-PCR, performed on a range of spoiled commercial food products, validated the suitability of actin mRNA detection for the quantification of naturally contaminating fungi. The specificity and sensitivity of the procedure, combined with its speed, its reliability, and the potential automation of the technique, offer several advantages to routine analysis programs that assess the presence and viability of fungi in food commodities.

Actins↗
PubMed2001

Nucleic acid sequence-based amplification of Aspergillus RNA in blood samples.

Nucleic acid sequence-based amplification (NASBA), an isothermal amplification technique, was established and evaluated for the detection of Aspergillus RNA and compared with a previously published, well-defined real-time PCR assay amplifying a region of the Aspergillus 18S rRNA gene. NASBA showed a lower detection limit of 1 CFU and detected RNA from five different clinically relevant Aspergillus species, including Aspergillus fumigatus. All 77 blood samples tested by PCR and NASBA showed identical results in both assays. Results with the NASBA technique were obtained within 6 h. Thus, the NASBA technique provided a valuable tool for sensitive, specific, fast, and reliable detection of Aspergillus RNA with potential for routine diagnosis, including the possibility to test the viability of cells.

Aspergillosis↗
PubMedDate not supplied

Post-antibiotic effects of cefdinir on Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Streptococcus pyrogenes.

The post-antibiotic effects (PAEs) of a new cephalosporin, cefdinir, were determined against a range of organisms using a viable counting technique. Cefdinir exerted considerable PAEs against Staphylococcus aureus and Streptococcus pyogenes, but no overall post-antibiotic inhibition of growth was detected against Escherichia coli or Klebsiella pneumoniae. Exposure to cefdinir made the gram-negative organisms susceptible to the washing procedure used for drug removal, but this was followed by rapid recovery of viability in drug-free broth.

Cefdinir↗
PubMed1996

Effects of freezing on the viability of nine pathogens from quarters with subclinical mastitis.

Milk samples from 45 quarters containing mastitis pathogens were collected from lactating cows to determine the viability of those pathogens after freezing. An initial bacteria count was conducted, and samples were divided into 2-ml portions and frozen. Weekly bacteria counts were conducted for 6 wk. Viability after freezing was determined on five isolates of nine bacterial species: Staphylococcus aureus, Staphylococcus hyicus, Staphylococcus chromogenes, Staphylococcus xylosus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Corynebacterium bovis, and Escherichia coli. Bacteria counts were converted to logarithm base 10, and analysis of variance was conducted to determine alterations in viability over the 6-wk period. Freezing of quarter milk samples for 6 wk did not affect viability of any of these pathogens.

Animals↗
PubMed2006

Effect of acacia (Robinia pseudo-acacia L.) honey on the characteristic microflora of yogurt during refrigerated storage.

The primary purpose of this research was to monitor the influence of acacia honey addition to yogurt milk on survival of the microbial flora of yogurt during refrigerated storage for 6 wk. Results showed that the presence of honey at 1.0% to 5.0% (w/v) did not significantly influence (P>0.05) the viability of characteristic microorganisms (i.e., Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) in yogurt during storage at 4 degrees C. Similarly, honey had no effect on pH and lactic acid levels of the final products. Despite these findings, enrichment of yogurt with honey is recommended because honey is a natural sweetener that possesses a wide range of beneficial nutritional properties. In addition, at a rate of approximately 3.0% (w/v), it highly improves the sensory quality of the finished product without having a detrimental effect on characteristic lactic acid bacteria.

Food Handling↗