Interactions between Pseudomonas aeruginosa and iodophor germicides in milking parlor udder wash water systems

In a field study of 29 dairy farms, Pseudomonas aeruginosa was isolated more frequently (P = 0.05) from milking parlor udder wash water systems containing iodophor germicides than from those with no germicide. Most available iodine (AI2) concentrations were below the recommended level of 25 ppm (25 microgram/ml). Rubber and polyvinyl chloride hoses caused rapid decreases in the AI2 concentrations of 25 ppm iodophor solutions. AI2 dropped from 25 ppm to 6 ppm or less in 240 min for solutions contained in either polyvinyl chloride or rubber, compared with solutions in glass, which were unchanged in 240 min. Addition of inactivated iodophor solution to aqueous cultures resulted in significantly higher (P less than 0.05) numbers of P. aeruginosa at 10 and 24 h postinoculation. P. aeruginosa was grown in polyvinyl chloride tubing and exposed twice daily to 0, 10, or 25 ppm of AI2. None of the exposure concentrations eliminated the bacteria from the hoses, and bacterial numbers were not significantly different in hoses exposed to 0 and 10 ppm by the eighth treatment day. Bacteria taken from the water in these hoses were exposed to different concentrations of iodophor solution. Iodophor concentrations which will kill 50% of P. aeruginosa cultures previously exposed to 0, 10, and 25 ppm of AI2 were predicted to be 3.0, 11.8, and 20.8 ppm, respectively.     

Interactions between Pseudomonas aeruginosa and iodophor germicides in milking parlor udder wash water systems.

In a field study of 29 dairy farms, Pseudomonas aeruginosa was isolated more frequently (P = 0.05) from milking parlor udder wash water systems containing iodophor germicides than from those with no germicide. Most available iodine (AI2) concentrations were below the recommended level of 25 ppm (25 microgram/ml). Rubber and polyvinyl chloride hoses caused rapid decreases in the AI2 concentrations of 25 ppm iodophor solutions. AI2 dropped from 25 ppm to 6 ppm or less in 240 min for solutions contained in either polyvinyl chloride or rubber, compared with solutions in glass, which were unchanged in 240 min. Addition of inactivated iodophor solution to aqueous cultures resulted in significantly higher (P less than 0.05) numbers of P. aeruginosa at 10 and 24 h postinoculation. P. aeruginosa was grown in polyvinyl chloride tubing and exposed twice daily to 0, 10, or 25 ppm of AI2. None of the exposure concentrations eliminated the bacteria from the hoses, and bacterial numbers were not significantly different in hoses exposed to 0 and 10 ppm by the eighth treatment day. Bacteria taken from the water in these hoses were exposed to different concentrations of iodophor solution. Iodophor concentrations which will kill 50% of P. aeruginosa cultures previously exposed to 0, 10, and 25 ppm of AI2 were predicted to be 3.0, 11.8, and 20.8 ppm, respectively.     

Evaluation of preservative effectiveness in pharmaceutical products : the use of a wild strain of Pseudomonas cepacia

A sodium benzoate-sorbic acid preservative system of a pharmaceutical product was proved effective against a wild strain of Pseudomonas cepacia , following the official method of the Italian and British Pharmacopoeias. However, this preservative system was ineffective against a challenge of Ps. cepacia wild strain cells grown in the unpreserved pharmaceutical product and on culture media different from those described by the Pharmacopoeias. The adaptive resistance of the wild strain of Ps. cepacia was not demonstrated with a laboratory strain (ATCC 25609). In contrast, p- hydroxybenzoate-based preservative systems proved to be efficient in protecting the pharmaceutical product against a challenge of wild and laboratory strains of Ps. cepacia grown in the different conditions described above. The results obtained suggest the usefulness, in the official methods for testing pharmaceutical preservatives, of using wild microbial strains isolated from the pharmaceutical environment. Metabolic adaptive responses, capable of affecting the antimicrobial sensitivity of wild micro-organisms used to challenge the preserved product, can be detected by using cells grown in the unpreserved pharmaceutical product.     

Prolonged survival of Pseudomonas cepacia in commercially manufactured povidone-iodine

Pseudomonas cepacia organisms were recently recovered from a povidone-iodine antiseptic solution. During the subsequent investigation, laboratory studies were initiated to determine the survival time of these organisms in the iodophor solution, which contains 1% titratable iodine. The solution was sampled weekly upon receipt in our laboratory, and P. cepacia was subsequently recovered through 29 weeks of sampling. Current laboratory data and lot production date information from the manufacturer indicate that P. cepacia survived for up to 68 weeks from the time of manufacture. Scanning electron microscopic examination of contaminated solution demonstrated bacterial cells embedded in extracellular material.     

Prolonged survival of Pseudomonas cepacia in commercially manufactured povidone-iodine.

Pseudomonas cepacia organisms were recently recovered from a povidone-iodine antiseptic solution. During the subsequent investigation, laboratory studies were initiated to determine the survival time of these organisms in the iodophor solution, which contains 1% titratable iodine. The solution was sampled weekly upon receipt in our laboratory, and P. cepacia was subsequently recovered through 29 weeks of sampling. Current laboratory data and lot production date information from the manufacturer indicate that P. cepacia survived for up to 68 weeks from the time of manufacture. Scanning electron microscopic examination of contaminated solution demonstrated bacterial cells embedded in extracellular material.     

Identification of Pseudomonas tolaasi: the White Line in Agar and Mushroom Tissue Block Rapid Pitting Tests

A sharply defined white line of precipitate forms in Pseudomonas Agar F (Difco) between the opaque white colonies of Pseudomonas tolaasi and translucent colonies of certain unidentified pseudomonads. This visible interaction has been utilized in a specific and reliable method for the identification of Ps. tolaasi. The white line test was positive when 113 isolates of Ps. tolaasi from five different countries were examined, whereas 154 isolates of pseudomonads other than Ps. tolaasi , including Ps. corrugata, Ps. delphinii, Ps. fluorescens, Ps. lachrymans, Ps. marginalis, Ps. pastinaceae, Ps. phaseolicola, Ps. aeruginosa, Ps. putida, Ps. syringae, Ps. mors-prunorum, Ps. cichorii, Ps. antirrhini, Ps. viridiflava, Ps. caryophylli, Ps. cepacia, Ps. mendocina, Ps. stutzeri, Ps. acidivorus and Ps. lemoignei did not give the white line reaction with a reacting translucent colony pseudomonad. Browning of mushrooms in host tests does not help in the identification of Ps. tolaasi , but a conspicuous pitting produced in less than 10 min at the cut surface of mushroom tissue is as specific as the white line test in detecting Ps. tolaasi in suspension in distilled water.     

Molecular characterization of natural biofilms from household taps with different materials: PVC, stainless steel, and cast iron in drinking water distribution system

Microorganism in drinking water distribution system may colonize in biofilms. Bacterial 16S rRNA gene diversities were analyzed in both water and biofilms grown on taps with three different materials (polyvinyl chloride (PVC), stainless steel, and cast iron) from a local drinking water distribution system. In total, five clone libraries (440 sequences) were obtained. The taxonomic composition of the microbial communities was found to be dominated by members of Proteobacteria (65.9–98.9 %), broadly distributed among the classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. Other bacterial groups included Firmicutes, Acidobacteria, Bacteroidetes, Cyanobacteria, and Deinococcus-Thermus. Moreover, a small proportion of unclassified bacteria (3.5–10.6 %) were also found. This investigation revealed that the bacterial communities in biofilms appeared much more diversified than expected and more care should be taken to the taps with high bacterial diversity. Also, regular monitor of outflow water would be useful as potentially pathogenic bacteria were detected. In addition, microbial richness and diversity in taps ranked in the order as: PVC?<?stainless steel?<?cast iron. All the results interpreted that PVC would be a potentially suitable material for use as tap component in drinking water distribution system.     

Numerical systematics of bacteria of the genus Pseudomonas

In 290 strains of bacteria belonging to the genus Pseudomonas, 120 morphological and physiologo-biochemical characters were studied and the results obtained thereby were analyzed by the methods of numerical taxonomy using computers. The majority of strains were subdivided into 11 clusters: Ps. aeruginosa (1), Ps. putida (2), Ps. rathonis (5), Ps. syringae (8), Ps. pseudoalcaligenes (9), Ps. maltophilia (10), Ps. acidovorans (11), Ps. testosteroni (12), Ps. mendocina (13), Ps. cepacia (14), Ps. fluorescens (3). The latter cluster included also the strains identified earlier as Ps. aurantiaca, Ps. lemonnieri, Ps. fluoro-violaceus, and Ps. aureofaciens. Three clusters contained strains which could not be identified and probably should be regarded as distinct species. The characteristics have been selected useful for diagnostics of the above Pseudomonas bacteria and the subgroups of Ps. fluorescens.     

Occurrence of mycobacteria in water treatment lines and in water distribution systems

The frequency of recovery of atypical mycobacteria was estimated in two treatment plants providing drinking water to Paris, France, at some intermediate stages of treatment. The two plants use two different filtration processes, rapid and slow sand filtration. Our results suggest that slow sand filtration is more efficient for removing mycobacteria than rapid sand filtration. In addition, our results show that mycobacteria can colonize and grow on granular activated carbon and are able to enter distribution systems. We also investigated the frequency of recovery of mycobacteria in the water distribution system of Paris (outside buildings). The mycobacterial species isolated from the Paris drinking water distribution system are different from those isolated from the water leaving the treatment plants. Saprophytic mycobacteria (present in 41.3% of positive samples), potentially pathogenic mycobacteria (16.3%), and unidentifiable mycobacteria (54.8%) were isolated from 12 sites within the Paris water distribution system. Mycobacterium gordonae was preferentially recovered from treated surface water, whereas Mycobacterium nonchromogenicum was preferentially recovered from groundwater. No significant correlations were found among the presence of mycobacteria, the origin of water, and water temperature.     

Occurrence of Mycobacteria in Water Treatment Lines and in Water Distribution Systems

The frequency of recovery of atypical mycobacteria was estimated in two treatment plants providing drinking water to Paris, France, at some intermediate stages of treatment. The two plants use two different filtration processes, rapid and slow sand filtration. Our results suggest that slow sand filtration is more efficient for removing mycobacteria than rapid sand filtration. In addition, our results show that mycobacteria can colonize and grow on granular activated carbon and are able to enter distribution systems. We also investigated the frequency of recovery of mycobacteria in the water distribution system of Paris (outside buildings). The mycobacterial species isolated from the Paris drinking water distribution system are different from those isolated from the water leaving the treatment plants. Saprophytic mycobacteria (present in 41.3% of positive samples), potentially pathogenic mycobacteria (16.3%), and unidentifiable mycobacteria (54.8%) were isolated from 12 sites within the Paris water distribution system. Mycobacterium gordonae was preferentially recovered from treated surface water, whereas Mycobacterium nonchromogenicum was preferentially recovered from groundwater. No significant correlations were found among the presence of mycobacteria, the origin of water, and water temperature.     

Production of extracellular nucleic acids by genetically altered bacteria in aquatic-environment microcosms.

The factors which affect the production of extracellular DNA by genetically altered strains of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas cepacia, and Bradyrhizobium japonicum in aquatic environments were investigated. Cellular nucleic acids were labeled in vivo by incubation with [3H]thymidine or [3H]adenine, and production of extracellular DNA in marine waters, artificial seawater, or minimal salts media was determined by detecting radiolabeled macromolecules in incubation filtrates. The presence or absence of the ambient microbial community had little effect on the production of extracellular DNA. Three of four organisms produced the greatest amounts of extracellular nucleic acids when incubated in low-salinity media (2% artificial seawater) rather than high-salinity media (10 to 50% artificial seawater). The greatest production of extracellular nucleic acids by P. cepacia occurred at pH 7 and 37 degrees C, suggesting that extracellular-DNA production may be a normal physiologic function of the cell. Incubation of labeled P. cepacia cells in water from Bimini Harbor, Bahamas, resulted in labeling of macromolecules of the ambient microbial population. Collectively these results indicate that (i) extracellular-DNA production by genetically altered bacteria released into aquatic environments is more strongly influenced by physiochemical factors than biotic factors, (ii) extracellular-DNA production rates are usually greater for organisms released in freshwater than marine environments, and (iii) ambient microbial populations can readily utilize materials released by these organisms.     

Production of extracellular nucleic acids by genetically altered bacteria in aquatic-environment microcosms

The factors which affect the production of extracellular DNA by genetically altered strains of Escherichia coli, Pseudomonas aeruginosa, Pseudomonas cepacia, and Bradyrhizobium japonicum in aquatic environments were investigated. Cellular nucleic acids were labeled in vivo by incubation with [3H]thymidine or [3H]adenine, and production of extracellular DNA in marine waters, artificial seawater, or minimal salts media was determined by detecting radiolabeled macromolecules in incubation filtrates. The presence or absence of the ambient microbial community had little effect on the production of extracellular DNA. Three of four organisms produced the greatest amounts of extracellular nucleic acids when incubated in low-salinity media (2% artificial seawater) rather than high-salinity media (10 to 50% artificial seawater). The greatest production of extracellular nucleic acids by P. cepacia occurred at pH 7 and 37 degrees C, suggesting that extracellular-DNA production may be a normal physiologic function of the cell. Incubation of labeled P. cepacia cells in water from Bimini Harbor, Bahamas, resulted in labeling of macromolecules of the ambient microbial population. Collectively these results indicate that (i) extracellular-DNA production by genetically altered bacteria released into aquatic environments is more strongly influenced by physiochemical factors than biotic factors, (ii) extracellular-DNA production rates are usually greater for organisms released in freshwater than marine environments, and (iii) ambient microbial populations can readily utilize materials released by these organisms.     

Chlorine disinfection of atypical mycobacteria isolated from a water distribution system

We studied the resistance of various mycobacteria isolated from a water distribution system to chlorine. Chlorine disinfection efficiency is expressed as the coefficient of lethality (liters per minute per milligram) as follows: Mycobacterium fortuitum (0.02) > M. chelonae (0.03) > M. gordonae (0.09) > M. aurum (0.19). For a C.t value (product of the disinfectant concentration and contact time) of 60 mg.min.liter(-1), frequently used in water treatment lines, chlorine disinfection inactivates over 4 log units of M. gordonae and 1.5 log units of M. fortuitum or M. chelonae. C.t values determined under similar conditions show that even the most susceptible species, M. aurum and M. gordonae, are 100 and 330 times more resistant to chlorine than Escherichia coli. We also investigated the effects of different parameters (medium, pH, and temperature) on chlorine disinfection in a chlorine-resistant M. gordonae model. Our experimental results follow the Arrhenius equation, allowing the inactivation rate to be predicted at different temperatures. Our results show that M. gordonae is more resistant to chlorine in low-nutrient media, such as those encountered in water, and that an increase in temperature (from 4 degrees C to 25 degrees C) and a decrease in pH result in better inactivation.     

Rapid screening method for Mycobactericidal activity of chemical germicides that uses Mycobacterium terrae expressing a green fluorescent protein gene

The slow growth of mycobacteria in conventional culture methods impedes the testing of chemicals for mycobactericidal activity. An assay based on expression of the green fluorescent protein (GFP) by mycobacteria was developed as a rapid alternative. Plasmid pBEN, containing the gene encoding a red-shifted, high-intensity GFP mutant, was incorporated into Mycobacterium terrae (ATCC 15755), and GFP expression was observed by epifluorescence microscopy. Mycobactericidal activity was assessed by separately exposing a suspension of M. terrae(pBEN) to several dilutions of test germicides based on 7.5% hydrogen peroxide, 2.4% alkaline glutaraldehyde, 10% acid glutaraldehyde, and 15.5% of a phenolic agent for contact times ranging from 10 to 20 min (22 degrees C), followed by culture of the exposed cells in broth (Middlebrook 7H9) and measurement of fluorescence every 24 h. When the fluorescence was to be compared with CFU, the samples were plated on Middlebrook 7H11 agar and incubated for 4 weeks. No increase in fluorescence or CFU occurred in cultures in which the cells had been inactivated by the germicide concentrations tested. Where the test bacterium was exposed to ineffective levels of the germicides, fluorescence increased after a lag period of 1 to 7 days, corresponding to the level of bacterial inactivation. In untreated controls, fluorescence increased rapidly to reach a peak in 2 to 4 days. A good Pearson correlation coefficient (r > or =0.85) was observed between the intensity of fluorescence and the number of CFU. The GFP-based fluorescence assay reduced the turnaround time in the screening of chemical germicides for mycobactericidal activity to < or =7 days.     

Expanding the mycobacterial diversity of metalworking fluids (MWFs): evidence showing MWF colonization by Mycobacterium abscessus

Nontuberculous mycobacteria (NTM) have been associated with hypersensitivity pneumonitis in machinists. Only two species of NTM, namely Mycobacterium immunogenum and Mycobacterium chelonae, have been reported thus far to have the ability to colonize contaminated metalworking fluids (MWFs). Here, we report, for the first time, the presence and characterization (phenotypic and genotypic) of a third species, Mycobacterium abscessus, colonizing these harsh alkaline machining fluids. Two Mycobacterium morphotypes, smooth (S) and rough (R), were isolated (two isolates each) from an in-use industrial MWFs. Biocide susceptibility analysis using triclosan as a model yielded the same minimal inhibitory concentration for the two morphotypes. PCR-restriction analysis-based speciation of the morphotypes confirmed their identity as M. abscessus. Genotyping based on partial DNA sequences corresponding to the variable regions of the hsp65 gene and 16S-23S rRNA operon internal transcribed spacer region and randomly amplified polymorphic DNA-PCR analysis showed that both morphotypes belong to a single genotype. In addition, we isolated and confirmed two novel mycobacterial genotypes, one each of M. immunogenum and M. chelonae from additional in-use MWF screening. Taken together, this study expands the known mycobacterial species- and strain-diversity colonizing MWF. Furthermore, the study emphasizes the need for including M. abscessus species in the existing mycobacterial screening of contaminated MWF.     

Microbial communities of printing paper machines

The microbial content of printing paper machines, running at a temperature of 45–50 °C and at pH 4·5–5, was studied. Bacteria were prevalent colonizers of the machine wet end and the raw materials. A total of 390 strains of aerobic bacteria were isolated and 86% of these were identified to genus and species by biochemical, chemotaxonomic and phylogenetic methods. The most common bacteria found at the machine wet end were Bacillus coagulans and other Bacillus species, Burkholderia cepacia , Ralstonia pickettii , and in pink slimes, accumulating in the wire area and press section, species of Deinococcus, Aureobacterium and Brevibacterium . Paper-making chemicals also contained species of Aureobacterium , B. cereus , B. licheniformis , B. sphaericus , Bordetella, Hydrogenophaga , Klebsiella pneumoniae , Pantoea agglomerans , Pseudomonas stutzeri , Staphylococcus and sometimes other enteric bacteria, but these did not colonize the process water. Yeasts and moulds were not present in significant numbers . A total of 131 strains were tested for their potential to degrade paper-making raw materials; 91 strains were found to have degradative activity, mainly species of Burkholderia and Ralstonia , Sphingomonas and Bacillus , and enterobacteria produced enzymes which degraded paper-making chemicals. Stainless steel adhering strains occurred in slimes and wire water and were identified as Burkholderia cepacia , B. coagulans and Deinococcus geothermalis . Coloured slimes were formed on the machine by species of Deinococcus , Acinetobacter and Methylobacterium (pink), Aureobacterium , Pantoea and Ralstonia (yellowish) and Microbulbifer -related strains (brown). The impact of the strains and species found in the printing paper machine community on the technical quality of paper, machine operation, and as a potential biohazard (Hazard Group 2 bacteria), is discussed.     

Identification of Pseudomonas spp. Isolated from Milk Produced in South Eastern Queensland

S ummary . Bacteria were isolated from raw and pasteurized milks produced throughout S. E. Queensland. Milk samples were plated initially on penicillin agar or on milk agar and incubated at 30° and 7°, respectively. On the basis of primary characterization, 167 of the 330 isolates obtained were identified as Pseudomonas spp. The pseudomonads were further characterized in accordance with the taxonomic studies of the genus by Stanier, Palleroni & Doudoroff (1966). Species designations were ascribed to the Pseudomonas isolates on the basis of distinctive species characteristics in conjunction with similarity coefficients between each isolate and the ideal species phenotype, as follows: Ps. fluorescens (121), Ps. aeruginosa (16), Ps. putida (12), Ps. maltophilia (9), Ps. pseudoalcaligenes (5), Ps. cepacia (3) and Ps. alcaligenes (1). A dendrogram obtained by cluster analysis of the Pseudomonas isolates is included.     

Water as a substrate for the development of Paracoccidiodes brasiliensis mycelial form

Two isolates of P. brasiliensis in the mycelial form were studied for their capacity to survive and grow in sterile distilled water (SDW). Inoculum for the experiments consisted of a spectrophotometrically-standardized suspension of washed and homogenized mycelial fragments; these had been obtained from 2-week old cultures grown in a synthetic medium (SM). Series of tubes with SDW and SM were incubated with the above suspension and kept stationary for 6 months at either 4 °C or room temperature (RT). Growth was measured by dry weight (DW) and turbidity (OD) determinations; additionally, CFU and ultrastructural appearance by transmission electron microscope (TEM) were assesed for one of the isolates. In general, cultures in SM at RT, grew exponentially after 2 weeks, becoming stationary for 7 weeks and then, declining abruptly. In SDW, fungal development was slow for 5 months when an increase in mass was recorded. When incubated at 4° C, both SDW and SM cultures required longer time to develop but mass also increased. Morphologically, mycelial elements in SDW at RT exhibited increased lipid vacuoles and glycogen deposits but were otherwise normal up to 6 weeks when they presented the inter-hyphae-hyphae phenomenum. In SDW P. brasiliensis appears to utilize debris from its degenerated fungal partners to continue growing.     

Draft genome sequence of Caloramator australicus strain RC3T, a thermoanaerobe from the Great Artesian Basin of Australia

Caloramator australicus strain RC3(T) (JCM 15081(T) = KCTC 5601(T)) is the type strain of a newly identified thermophilic species, which was isolated from red microbial mats that thrive at 66°C in the runoff channel of a Great Artesian Basin bore (New Lorne bore, registered number 17263) in outback Queensland, Australia. The ability of the C. australicus strain to use metals as terminal electron acceptors has led to concerns that it could colonize and enhance corrosion of the metal casing of Great Artesian Basin bore well pipes and that this could subsequently lead to bore failure and loss of water availability for the community which is so reliant on it. The genome of the C. australicus strain has been sequenced, and annotation of the ~2.65-Mb sequence indicates that the attributes are consistent with physiological and phenotypic traits.     

The antimicrobial activity of Fleroxacin RO 23-6240, a third generation fluoroquinolone derivative, tested in vitro

The broad antimicrobial spectrum of Fleroxacin RO 23-6240 was tested on a panel of bacterial and several mycobacterial species. Staph. aureus strains, gramnegative bacteria E. coli, Kl. pneumoniae, Salmonella spp. were found to be rather susceptible to the tested preparation in vitro. The same was true of those species which are known to feature strong natural resistance, e.g. Ps. aeruginosa and Acinetobacter calcoaceticus. As regards mycobacteria, complete susceptibility was recorded for 10 M. tuberculosis strains, 17 M. kansasii strains and 4 strains of M. fortuitum. Only two of the eleven tested strains of the complex M. avium-intracellulare were found to be susceptible and one M. chelonae strain tested proved resistant. Due to their broad spectrum and strong bactericidal effects, chinolone preparations can be anticipated to assume an everincreasing significance in the future.