Resuscitation effects of catalase on airborne bacteria.

Catalase incorporation into enumeration media caused a significant increase (greater than 63%) in the colony-forming abilities of airborne bacteria. Incubation for 30 to 60 min of airborne bacteria in collection fluid containing catalase caused a greater than 95% increase in colony-forming ability. However, catalase did not have any effects on enumeration at high relative humidities (80 to 90%).     

Effects of betaine on enumeration of airborne bacteria

The osmoprotectant betaine was incorporated into collection fluid and enumeration medium to determine its effects on the colony-forming abilities of airborne bacteria, which were collected from three separate locations: a wastewater treatment plant, the roof of a laboratory building, and an unobstructed farmland. At all locations, addition of 2 to 5 mM betaine caused a significant increase (from 21.6 to 61.3%) in colonial outgrowth, compared with the growth rate of controls without betaine. The presence of betaine in both the collection fluid and the enumeration medium had an additive effect on the colony-forming ability of airborne bacteria compared with the presence of betaine in either one alone. The effect of various betaine concentrations on the enumeration of aerosolized Pseudomonas syringae was determined. Betaine showed a threshold for maximum effect at a concentration of 2 to 5 mM. At higher concentrations (10 to 20 mM), the effects of betaine were negligible or possibly inhibitory. The significance of these results with respect to the development of protocols for monitoring airborne microorganisms, including genetically engineered microorganisms, is discussed.     

Effects of betaine on enumeration of airborne bacteria.

The osmoprotectant betaine was incorporated into collection fluid and enumeration medium to determine its effects on the colony-forming abilities of airborne bacteria, which were collected from three separate locations: a wastewater treatment plant, the roof of a laboratory building, and an unobstructed farmland. At all locations, addition of 2 to 5 mM betaine caused a significant increase (from 21.6 to 61.3%) in colonial outgrowth, compared with the growth rate of controls without betaine. The presence of betaine in both the collection fluid and the enumeration medium had an additive effect on the colony-forming ability of airborne bacteria compared with the presence of betaine in either one alone. The effect of various betaine concentrations on the enumeration of aerosolized Pseudomonas syringae was determined. Betaine showed a threshold for maximum effect at a concentration of 2 to 5 mM. At higher concentrations (10 to 20 mM), the effects of betaine were negligible or possibly inhibitory. The significance of these results with respect to the development of protocols for monitoring airborne microorganisms, including genetically engineered microorganisms, is discussed.     

Plants as Sources of Airborne Bacteria, Including Ice Nucleation-Active Bacteria

Vertical wind shear and concentration gradients of viable, airborne bacteria were used to calculate the upward flux of viable cells above bare soil and canopies of several crops. Concentrations at soil or canopy height varied from 46 colony-forming units per m³ over young corn and wet soil to 663 colony-forming units per m³ over dry soil and 6,500 colony-forming units per m³ over a closed wheat canopy. In simultaneous samples, concentrations of viable bacteria in the air 10 m inside an alfalfa field were fourfold higher than those over a field with dry, bare soil immediately upwind. The upward flux of viable bacteria over alfalfa was three- to fourfold greater than over dry soil. Concentrations of ice nucleation-active bacteria were higher over plants than over soil. Thus, plant canopies may constitute a major source of bacteria, including ice nucleation-active bacteria, in the air.     

Investigation and application of methods for enumerating heterotrophs and Escherichia coli in the air within piggery sheds

AIMS: To investigate methods for the recovery of airborne bacteria within pig sheds and to then use the appropriate methods to determine the levels of heterotrophs and Escherichia coli in the air within sheds. METHODS AND RESULTS: AGI-30 impingers and a six-stage Andersen multi-stage sampler (AMS) were used for the collection of aerosols. Betaine and catalase were added to impinger collection fluid and the agar plates used in the AMS. Suitable media for enumerating E. coli with the Andersen sampler were also evaluated. The addition of betaine and catalase gave no marked increase in the recovery of heterotrophs or E. coli. No marked differences were found in the media used for enumeration of E. coli. The levels of heterotrophs and E. coli in three piggeries, during normal pig activities, were 2.2 x 10(5) and 21 CFU m(-3) respectively. CONCLUSIONS: The failure of the additives to improve the recovery of either heterotrophs or E. coli suggests that these organisms are not stressed in the piggery environment. The levels of heterotrophs in the air inside the three Queensland piggeries investigated are consistent with those previously reported in other studies. Flushing with ponded effluent had no marked or consistent effect on the heterotroph or E. coli levels. SIGNIFICANCE AND IMPACT OF THE STUDY: Our work suggests that levels of airborne heterotrophs and E. coli inside pig sheds have no strong link with effluent flushing. It would seem unlikely that any single management activity within a pig shed has a dominant influence on levels of airborne heterotrophs and E. coli.     

Bioaerosols and Particle Release During Composting of Contaminated Sawmill Soil

Compost windrows for bioremediation of soil were built at a wood-preserving site contaminated with chlorophenols, polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs). Sampling of airborne particles during the mixing of the compost windrows found concentrations of PCDDs and PCDFs in different particle sizes. The congener distribution of PCDDs and PCDFs in the collected air particle fractions was similar to that in the compost windrows, and the level of PCDDs and PCDFs was 1000-fold higher than the atmospheric background values reported previously. Viable particle-sizing samplers and several selective growth media were used to enumerate bacteria and fungi in the airborne particles. From the collected air samples, 40 bacteria were isolated and identified. Among the isolated bacteria, 80% were Gram-positive and spore-forming. Two of the identified airborne bacteria, Pseudomonas aeruginosa and Bacillus cereus, may cause human disease and are classified in biological agent hazard group 2. The amounts of airborne fungi, molds, and yeasts were 1000 to 2000 colony-forming units (CFUs) per m³. The number of actinomycetes was up to 6-fold, and the number of bacteria was 2- to 20-fold compared to background values. The overall level of airborne bacteria (200 to 3500 CFUs per m³) was low compared to the level of bacteria (10⁵ to 10⁸ CFUs per m³) found when composting municipal waste.     

Medical Applications of Dust-free Rooms. III. Use in an Animal Care Laboratory

Bacterial air sampling in an animal care laboratory showed that dense aerosols are generated during cage changing and cage cleaning. Reyniers and Andersen sampling showed that the airborne bacteria numbered 50 to 200 colony-forming units (CFU)/ft³ of air. Of the viable particles collected by Andersen samplers, 78.5% were larger than 5.5 μm. A low velocity laminar air flow system composed of high-efficiency particulate air (HEPA) filters and a ceiling distribution system maintained the number of airborne viable particles at low levels, generally less than 2 CFU/ft³. Vertical air flow of 15 ft/min significantly reduced the rate of airborne infection by a strain of Proteus mirabilis. Other factors shown to influence airborne infection included type of cage utilized, the use of bedding, the distance between cages, and the number of animals per cage.     

Effects of growth temperature and strictly anaerobic recovery on the survival of Listeria monocytogenes during pasteurization

Listeria monocytogenes F5069 was suspended in either Trypticase soy broth-0.6% yeast extract (TSBYE) or sterile, whole milk and heated at 62.8 degrees C in sealed thermal death time tubes. Severely heat-injured cells were recovered in TSBYE within sealed thermal death time tubes because of the formation of reduced conditions in the depths of the TSBYE. Also, the use of strictly anaerobic Hungate techniques significantly increased recovery in TSBYE containing 1.5% agar compared with aerobically incubated controls. The exogenous addition of catalase, but not superoxide dismutase, slightly increased the recovery of heat-injured cells in TSBYE containing 1.5% agar incubated aerobically. Growth of cells at 43 degrees C caused a greater increase in heat resistance as compared with cells heat shocked at 43 degrees C or cells grown at lower temperatures. Growth of L. monocytogenes at 43 degrees C and enumeration by the use of strictly anaerobic Hungate techniques resulted in D62.8 degrees C values that were at least sixfold greater than those previously obtained by using cells grown at 37 degrees C and aerobic plating. Results indicate that, under the conditions of the present study, high levels of L. monocytogenes would survive the minimum low-temperature, long-time treatment required by the U.S. Food and Drug Administration for pasteurizing milk. The possible survival of low levels of L. monocytogenes during high-temperature, short-time pasteurization and enumeration of injured cells by recovery on selective media under strictly anaerobic conditions are discussed.     

Effects of growth temperature and strictly anaerobic recovery on the survival of Listeria monocytogenes during pasteurization.

Listeria monocytogenes F5069 was suspended in either Trypticase soy broth-0.6% yeast extract (TSBYE) or sterile, whole milk and heated at 62.8 degrees C in sealed thermal death time tubes. Severely heat-injured cells were recovered in TSBYE within sealed thermal death time tubes because of the formation of reduced conditions in the depths of the TSBYE. Also, the use of strictly anaerobic Hungate techniques significantly increased recovery in TSBYE containing 1.5% agar compared with aerobically incubated controls. The exogenous addition of catalase, but not superoxide dismutase, slightly increased the recovery of heat-injured cells in TSBYE containing 1.5% agar incubated aerobically. Growth of cells at 43 degrees C caused a greater increase in heat resistance as compared with cells heat shocked at 43 degrees C or cells grown at lower temperatures. Growth of L. monocytogenes at 43 degrees C and enumeration by the use of strictly anaerobic Hungate techniques resulted in D62.8 degrees C values that were at least sixfold greater than those previously obtained by using cells grown at 37 degrees C and aerobic plating. Results indicate that, under the conditions of the present study, high levels of L. monocytogenes would survive the minimum low-temperature, long-time treatment required by the U.S. Food and Drug Administration for pasteurizing milk. The possible survival of low levels of L. monocytogenes during high-temperature, short-time pasteurization and enumeration of injured cells by recovery on selective media under strictly anaerobic conditions are discussed.     

Catalase and enumeration of stressed Staphylococcus aureus cells.

The effects of catalase on the enumeration of stressed (heated, reduced water activity, or freeze-dried) Staphylococcus aureus cells on several selective media were examined. The addition of catalase greatly increased the enumeration of stressed cells. The beneficial effects of catalase were most pronounced on those media least efficient in enumeration of stressed staphylococci, showing increases in enumeration of up to 1,100-fold. The effects of catalase appear to be due to the reduced ability of stressed cells to repair and form colonies in the absence of an exogenous decomposer of H2O2. Thermally stressed cells were more sensitive to H2O2 than unstressed cells. During recovery, stressed cells overcame the requirement for catalase. These findings implicate H2O2 as a factor in the failure of certain selective media to adequately enumerate stressed cells and demonstrate that the addition of catalase to these media markedly increases their productivity.     

New water disinfection system using UVA light-emitting diodes

AIM: To evaluate the ability of high-energy ultraviolet A (UVA) light-emitting diode (LED) to inactivate bacteria in water and investigate the inactivating mechanism of UVA irradiation. METHODS AND RESULTS: We developed a new disinfection device equipped with high-energy UVA-LED. Inactivation of bacteria was determined by colony-forming assay. Vibrio parahaemolyticus, enteropathogenic Escherichia coli, Staphylococcus aureus and Escherichia coli DH5alpha were reduced by greater than 5-log(10) stages within 75 min at 315 J cm(-2) of UVA. Salmonella enteritidis was reduced greater than 4-log(10) stages within 160 min at 672 J cm(-2) of UVA. The formation of 8-hydroxy-2'-deoxyguanosine in UVA-LED irradiated bacteria was 2.6-fold higher than that of UVC-irradiated bacteria at the same inactivation level. Addition of mannitol, a scavenger of hydroxyl radicals (OH(*)), or catalase, an enzyme scavenging hydrogen peroxide (H(2)O(2)) to bacterial suspensions significantly suppressed disinfection effect of UVA-LED. CONCLUSION: This disinfection system has enough ability to inactivate bacteria and OH(*) and H(2)O(2) participates in the disinfection mechanism of UVA irradiation. SIGNIFICANCE AND IMPACT OF THE STUDY: We newly developed UVA irradiation system and found that UVA alone was able to disinfect the water efficiently. This will become a useful disinfection system.     

Selective media for the specific isolation and enumeration of Botrytis cinerea conidia

AIMS: To develop selective media for the enumeration of Botrytis cinerea. METHODS AND RESULTS: Two new media, Botrytis Selective Medium (BSM) and Botrytis Spore Trap Medium (BSTM), were developed and compared with currently available media for the enumeration of B. cinerea conidia from the environment. The new Botrytis media proved advantageous over previous media because they were easier to prepare, had greater selectivity and allowed enumeration when a greater number of colony-forming units were present on individual plates. CONCLUSION: BSM and BSTM were shown to be suitable selective media for the enumeration of B. cinerea conidia from the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The media developed can be used to monitor the population of the pathogen B. cinerea and will allow detailed studies within the crop environments.     

Collection of airborne micro-organisms on Nuclepore filters, estimation and analysis--CAMNEA method

The total number of airborne micro-organisms collected on Nuclepore filters was determined by acridine orange staining and epifluorescence microscopy. The viable fraction of the total numbers varied significantly when actinomycete and fungal spores from different environments were stored on the filter surface for 1 week, although the microflora composition was not altered. A high correlation between viable and total counts was noted in environments where the airborne flora was dominated by fungal spores, while a low correlation was found for airborne bacteria. Peak values of the total counts registered in some work environments varied between 10(7) and 10(11) micro-organisms/m3. Size analysis showed a dominating fraction of respirable micro-organisms (aerodynamical diameter less than 5 micron). The investigation shows that it is of the utmost importance to combine viable counts with total count enumeration in the study of exposure to micro-organisms in work-related situations.     

Beneficial effects of catalase or pyruvate in a most-probable-number technique for the detection of Staphylococcus aureus.

The effects of the addition of catalase (EC 1.11.1.6) or pyruvate on the enumeration of Staphylococcus aureus in Trypticase soy broth with 10% NaCl were examined using a most-probable-number technique. Addition of catalase or pyruvate to the broth increased enumeration of all heat-stressed S. aureus strains tested. Increases were also observed with nonstressed cells. Catalase and pyruvate were similarly effective when added to Trypticase soy broth-10% NaCl in enumerating staphylococci naturally present in low-temperature-rendered ground-beef samples.     

Beneficial effects of catalase or pyruvate in a most-probable-number technique for the detection of Staphylococcus aureus.

The effects of the addition of catalase (EC 1.11.1.6) or pyruvate on the enumeration of Staphylococcus aureus in Trypticase soy broth with 10% NaCl were examined using a most-probable-number technique. Addition of catalase or pyruvate to the broth increased enumeration of all heat-stressed S. aureus strains tested. Increases were also observed with nonstressed cells. Catalase and pyruvate were similarly effective when added to Trypticase soy broth-10% NaCl in enumerating staphylococci naturally present in low-temperature-rendered ground-beef samples.     

The induction and repair of DNA damage and its influence on cell death in primary human fibroblasts exposed to UV-A or UV-C irradiation

Irradiation with UV-A of normal human fibroblasts in phosphate-buffered saline induced cell death, measured as lack of colony-forming ability. A specially filtered sunlamp, emitting wavelengths greater than 330 nm, was used as UV-A source. After UV-A irradiation, single-strand breaks (alkali-labile bonds) could be detected in DNA; these lesions were rapidly repaired. The induction of these single-strand breaks was almost eliminated when irradiation was performed in the presence of catalase. However, catalase, when present during UV-A irradiation, did not reduce cell death of the fibroblasts. Excision repair, monitored as unscheduled DNA synthesis, was induced strongly by irradiation with UV-C (predominantly 254 nm), but could not be detected after UV-A irradiation. Moreover, very little accumulation of incision breaks during post-irradiation incubation with hydroxyurea and 1-beta-D-arabinofuranosylcytosine (araC) was detected after UV-A. This is consistent with the low amount of pyrimidine dimers (measured as UV-endonuclease susceptible sites) induced by UV-A. Xeroderma pigmentosum fibroblasts of complementation group A, which are extremely sensitive to UV-C irradiation, showed the same sensitivity to UV-A as normal fibroblasts. The results indicate that lethality by UV-A wavelengths greater than 330 nm is caused by lesions other than single-strand breaks (alkali-labile bonds) and pyrimidine dimers.     

Effect of free-radical scavengers on enumeration of thermally stressed cells of Staphylococcus aureus MF-31.

Exposure of crude cell lysates of Staphylococcus aureus MF-31 to 5 or 10 mM hydrogen peroxide resulted in a linear decrease in superoxide dismutase activity. Approximately 13% of the superoxide dismutase activity was lost after 16 min. Thermally stressed and nonstressed cells were exposed to a photochemically generated exogenous flux of superoxide radicals (O2.-). The death of thermally stressed cells was linear with time. Addition of superoxide dismutase or catalase to the O2.- generating system resulted in protection of thermally stressed and nonstressed cells, with the protective effect being greater for thermally stressed cells. Incorporation of O2-, hydroxyl radical, or singlet oxygen scavengers or antioxidants to tryptic soy agar containing 7.5% NaCl did not increase the enumeration of thermally stressed cells.     

Sustained Photoproduction of Ammonia from Nitrate by Anacystis nidulans

Exposure of crude cell lysates of Staphylococcus aureus MF-31 to 5 or 10 mM hydrogen peroxide resulted in a linear decrease in superoxide dismutase activity. Approximately 13% of the superoxide dismutase activity was lost after 16 min. Thermally stressed and nonstressed cells were exposed to a photochemically generated exogenous flux of superoxide radicals (O2.-). The death of thermally stressed cells was linear with time. Addition of superoxide dismutase or catalase to the O2.- generating system resulted in protection of thermally stressed and nonstressed cells, with the protective effect being greater for thermally stressed cells. Incorporation of O2-, hydroxyl radical, or singlet oxygen scavengers or antioxidants to tryptic soy agar containing 7.5% NaCl did not increase the enumeration of thermally stressed cells.     

A Modification of the Andersen Sampler for Use in Occupied Environments

In occupied environments the median equivalent diameter of particles carrying airborne bacteria is frequently greater than the 50% cut off diameter, c. 12 μ, of the first stage of the Andersen sampler. By adding a prestage with a 50% cut off at an equivalent particle diameter of c. 19μ the sampler can be adapted for estimating the size distribution of the airborne bacteria-carrying particles found in these situations.     

Evaluation of the Airborne Bacterial Population in the Periodically Confined Antarctic Base Concordia

The environmental airborne bacterial population in relation to human confinement was investigated over a period of 1 year in the Concordia Research Station, which is located on the Eastern Antarctic plateau. The unique location of the station makes it suitable for different research domains such as glaciology, atmospheric sciences, astronomy, etc. Furthermore, it is used as a test bed for long-duration spaceflights to study the physiologic and psychological adaptation to isolated environments. A total of 96 samples were collected at eight different locations in the station at regular intervals. The airborne bacterial contamination was for 90% of the samples lower than 10.0 × 10² colony-forming units per cubic meter of air (CFU/m³) and the total bacterial contamination increased over time during confinement but diminished after re-opening of the base. Viable airborne bacteria with different morphology were identified by biochemical analyses. The predominant microflora was identified as Staphylococcus sp. (24.9% of total) and Bacillus sp. (11.6% of total) and was associated with human activity, but also environmental species such as Sphingomonas paucimobilis (belonging to the α-Proteobacteria) could establish themselves in the airborne population. A few opportunistic pathogens (6%) were also identified. Rob Van Houdt and Patrick De Boever contributed equally.