Investigations into the survival of Pseudomonas aeruginosa in poloxamer-iodine

Laboratory investigations were conducted to study potential mechanisms for prolonged survival of Pseudomonas aeruginosa in poloxamer-iodine (PxI). P. aeruginosa organisms isolated from PxI and adapted for growth in distilled water or found as part of a mixed microbial population from water in a manufacturing plant did not survive more than 15 s after challenge in stock PxI solution. Batches of PxI were compounded in the laboratory to determine the survival and growth of P. aeruginosa during the various stages of preparation. No P. aeruginosa organisms were recovered from the finished product at 1 min after the addition of iodine-iodide. However, we found P. aeruginosa in PxI 48 h after adding sterile PxI to the inside of a naturally contaminated polyvinyl chloride water distribution pipe. These organisms (10(4) CFU/ml) survived for as long as 98 days in contaminated stock PxI after it was removed from the polyvinyl chloride pipe. Both decreasing the free iodine level through addition of potassium iodide and increasing the free iodine level through dilution of the product resulted in an increased length of survival of P. aeruginosa in contaminated PxI solution. Comparative survival studies with pipes of different composition revealed that other materials may exert an effect similar to polyvinyl chloride. We concluded that polyvinyl chloride and perhaps other materials may play an important role in the survival of P. aeruginosa in iodophors and may be one source of resistant microbial populations when used in manufacturing plants which produce these antimicrobial solutions.     

The colonization of solid PVC surfaces and the acquisition of resistance to germicides by water micro-organisms

Six common water bacteria were examined for their ability to colonize polyvinyl chloride (PVC) surfaces, survive various germicidal treatment, and re-establish themselves in sterile distilled water (SDW). For each test, two 30·4 cm PVC pipes attached to a 90° PVC elbow were filled with 600 ml of distilled water inoculated with either Pseudomonas aeruginosa, Ps. cepacia, Ps. mesophilica, Acinetobacter anitratus, Mycobacterium chelonae or M. chelonae var. abscessus. After 8 weeks contaminated water was removed and the pipes were exposed to 600 ml of 1 : 213 iodophor disinfectant (ID), 1 : 128 phenolic detergent (P), 1 : 256 quaternary ammonium compound (QA), stock iodophor antiseptic (IA), 2% formaldehyde (F), 10–15 ppm free chlorine (C), 2% glutaraldehyde (G) and 70% ethanol (E). These germicides were periodically sampled, neutralized and examined for surviving organisms. After exposure for 7 d the germicides were removed and each pipe was refilled with SDW. This was assayed at 7 d intervals to determine microbial re-establishment. Samples were removed during microbial conditioning and examined by scanning electron microscopy (SEM). Pseudomonads were isolated directly from ID, QA, C, P and F, and mycobacteria from QA, IA, ID, P, G, C and F. Pseudomonas aeruginosa and Ps. cepacia survived in PVC pipes after 7 d of exposure to P, ID and C; Ps. mesophilica, after C and ID; and both mycobacteria, after C. SEM examination of PVC remnants revealed bacterial attachment and formation of extracellular material with embedded cells. These studies show that common water bacteria can attach and colonize the interior surface of PVC pipes and develop significant resistance to the action of certain germicides. Specific disinfection strategies are needed to control microbial populations that form in water distribution systems.     

Intrinsic Bacterial Contamination of a Commercial Iodophor Solution: Investigation of the Implicated Manufacturing Plant

After an outbreak of peritoneal infections attributed to intrinsic contamination of a poloxamer-iodine solution with Pseudomonas aeruginosa, the manufacturer of the contaminated solution permitted investigation and sampling of materials within the plant. Pseudomonas spp. were recovered from two different unopened lots of solution and from numerous water samples obtained at the plant. The isolates from water identical to those of an isolate recovered from Prepodyne solution (West-Agro Chemical Co., Inc., Westwood, Kans., manufactured for AMSCO Medical Products Div., Erie, Pa.) manufactured 1 month earlier at the same plant. P. aeruginosa was not recovered from incoming city water. P. aeruginosa was recovered from sterile water and poloxamer-iodine after 48 h of incubation in a plant polyvinyl chloride pipe. Scanning electron micrographs of polyvinyl chloride pipe used in the plant showed massive concentrations of rod-shaped and coccobacillary cells apparently embedded in interior deposits of the pipe. Manufacturers of iodophors should be aware that pipes or other surfaces colonized with bacteria may be a source of contamination of their products.     

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.     

Microbial contamination of water-soaked cotton gauze and its cause

Seven in-use cotton gauze samples and three cotton balls soaked in sterile distilled water in canisters were investigated 7 days after they were prepared in hospital. All samples were contaminated with bacteria including 10(6) to 10(7) colony forming units/ml of Pseudomonas aeruginosa. In vitro viability tests using cotton gauze and cotton balls soaked in sterile distilled water revealed rapid proliferation of P. aeruginosa, Serratia marcescens and Candida albicans. Since the cotton gauze and the cotton balls were soaked in water containing nutrients, such as protein and glucose, these materials may be readily contaminated with bacteria including P. aeruginosa. Thus, when using cotton gauze and cotton balls containing water, microbial contamination should be expected.     

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.     

Factors associated with Pseudomonas pickettii intrinsic contamination of commercial respiratory therapy solutions marketed as sterile.

Laboratory investigations were conducted to study the growth dynamics of Pseudomonas pickettii in commercial 0.9% sodium chloride solution under various environmental conditions and to determine the retention of these organisms after challenge through a 0.2-micron cartridge filter system. Low numbers of P. pickettii (1 to 10 CFU/ml of test solution) inoculated into commercial vials containing 5 ml of 0.9% sodium chloride solution and 500-ml volumes of 0.9% sodium chloride solution were shown to proliferate over a 168-h incubation period. These organisms demonstrated growth over a wide range of temperatures (15 to 42 degrees C) in this salt solution, and survival studies at 50, 55, and 60 degrees C indicated that this strain was not unusually resistant to heat (with the times required at a given temperature to reduce the surviving microbial population 10-fold [D-values] being 26.0, 1.9, and 0.7 min, respectively). A challenge test demonstrated that P. pickettii organisms were not completely retained by a 0.2-micron cartridge filter. The number of organisms detected increased from 1 CFU/liter of effluent at 1 to 2 min to a maximum of 176 CFU/liter at 4 to 5 min. Our results indicate that P. pickettii can penetrate a 0.2-micron filtration system and that the passage of organisms and subsequent microbial growth in the filter effluent probably are the mechanisms by which these organisms were recovered from "sterile" commercial 0.9% sodium chloride solution.     

Comparison of chemical dehydration and critical point drying for the stabilization and visualization of aging biofilm present on interior surfaces of PVC distribution pipe

In this study, fixation of attached glycocalyx on the interior surfaces of polyvinyl chloride distribution pipe remnants was compared with and without ruthenium red/osmium tetroxide and, in the final preparatory phase, with chemical dehydration and critical point drying. SEM examination of interior surface of the polyvinyl chloride pipe showed varying concentrations of adherent bacteria, depending on the preparatory technique used. It was concluded that using a combination of ruthenium red/osmium tetroxide and critical point drying is the optimum method for visually demonstrating aging biofilm on the interior surface of contaminated polyvinyl chloride pipe.     

In situ colonization of polyvinyl chloride, brass, and copper by Legionella pneumophila.

A sampling device (Robbins device) was used to expose brass, copper, and polyvinyl chloride plugs to potable water contaminated by Legionella pneumophila serogroup 1. Plugs were removed at approximately 1-week intervals and cultured. The colonization rates were polyvinyl chloride, 70; copper, 31; and brass, 25%. Quantitative cultures revealed that polyvinyl chloride was most heavily colonized, whereas brass was least colonized. We conclude that materials used in plumbing systems are readily colonized by Legionella and that the Robbins device provides a means for testing such materials in an in situ setting.     

Techniques for the Assessment of Growth of Micro-organisms on Plumbing Materials Used in Contact with Potable Water Supplies

Water samples from plumbing installations have often been of poorer microbiological quality than samples collected from the distribution main. This deterioration is often associated with a musty taste or visible turbidity in the water and slimes have sometimes been observed on water fittings. A technique to assess the ability of plumbing materials to support microbial growth is described and the most common categories of materials capable of supporting growth are identified. The most significant micro-organisms frequently found to grow on unsuitable materials were coliform organisms, especially members of the genus Citrobacter. Aeromonas hydrophila, Pseudomonas aeruginosa and fungi.     

Effects of diverse water pipe materials on bacterial communities and water quality in the annular reactor

To investigate the effects of pipe materials on biofilm accumulation and water quality, an annular reactor with the sample coupons of four pipe materials (steel, copper, stainless steel, and polyvinyl chloride) was operated under hydraulic conditions similar to a real plumbing system for 15 months. The bacterial concentrations were substantially increased in the steel and copper reactors with progression of corrosion, whereas those in stainless steel (STS) and polyvinyl chloride (PVC) reactors were affected mainly by water temperature. The heterotrophic plate count (HPC) of biofilms was about 100 times higher on steel pipe than other pipes throughout the experiment, with the STS pipe showing the lowest bacterial number at the end of the operation. Analysis of the 16S rDNA sequences of 176 cultivated isolates revealed that 66.5% was Proteobacteria and the others included unclassified bacteria, Actinobacteria, and Bacilli. Regardless of the pipe materials, Sphingomonas was the predominant species in all biofilms. PCR-DGGE analysis showed that steel pipe exhibited the highest bacterial diversity among the metallic pipes, and the DGGE profile of biofilm on PVC showed three additional bands not detected from the profiles of the metallic materials. Environmental scanning electron microscopy showed that corrosion level and biofilm accumulation were the least in the STS coupon. These results suggest that the STS pipe is the best material for plumbing systems in terms of the microbiological aspects of water quality.     

A pilot study of bacteriological population changes through potable water treatment and distribution

This pilot study compares the compositions of bacterial biofilms in pipe networks supplied with water containing either high levels of biodegradable organic matter (BOM) or low levels of BOM (conventionally or biologically treated, respectively). The Microbial Identification System for fatty acid analysis was utilized in this study to identify a large number of organisms (>1,400) to determine population changes in both conventionally and biologically treated water and biofilms. Data generated during this study indicated that suspended bacteria have little impact on biofilms, and despite treatment (conventional or biological), suspended microbial populations were similar following disinfection. Prechlorination with free chlorine resulted not only in reduced plate count values but also in a dramatic shift in the composition of the bacterial population to predominately gram-positive bacteria. Chlorination of biologically treated water produced the same shifts toward gram-positive bacteria. Removal of assimilable organic carbon by the biologically active filters slowed the rate of biofilm accumulation, but biofilm levels were similar to those found in conventionally treated water within several weeks. Iron pipes stimulated the rate of biofilm development, and bacterial levels on disinfected iron pipes exceeded those for chlorinated polyvinyl chloride pipes. The study showed that the iron pipe surface dramatically influenced the composition, activity, and disinfection resistance of biofilm bacteria.     

A Pilot Study of Bacteriological Population Changes through Potable Water Treatment and Distribution

This pilot study compares the compositions of bacterial biofilms in pipe networks supplied with water containing either high levels of biodegradable organic matter (BOM) or low levels of BOM (conventionally or biologically treated, respectively). The Microbial Identification System for fatty acid analysis was utilized in this study to identify a large number of organisms (>1,400) to determine population changes in both conventionally and biologically treated water and biofilms. Data generated during this study indicated that suspended bacteria have little impact on biofilms, and despite treatment (conventional or biological), suspended microbial populations were similar following disinfection. Prechlorination with free chlorine resulted not only in reduced plate count values but also in a dramatic shift in the composition of the bacterial population to predominately gram-positive bacteria. Chlorination of biologically treated water produced the same shifts toward gram-positive bacteria. Removal of assimilable organic carbon by the biologically active filters slowed the rate of biofilm accumulation, but biofilm levels were similar to those found in conventionally treated water within several weeks. Iron pipes stimulated the rate of biofilm development, and bacterial levels on disinfected iron pipes exceeded those for chlorinated polyvinyl chloride pipes. The study showed that the iron pipe surface dramatically influenced the composition, activity, and disinfection resistance of biofilm bacteria.     

Iodine sensitivity of bacteria isolated from iodinated water systems

Fourteen bacterial isolates, predominantly Pseudomonas sp., from two water systems disinfected by iodinated anion-exchange resins were studied and compared with an isolate of Pseudomonas aeruginosa from a povidone-iodine solution and four other isolates. Pseudomonas cepacia and P. aeruginosa grown in brain heart infusion were 3 to 5 logs less sensitive to 1 mg/L I2 (pH 7.2, 1 min) when compared with cultures grown in phosphate buffer. Another P. cepacia isolate was the least sensitive culture when grown in brain heart infusion (1 log decrease) but was more sensitive after cultivation in phosphate buffer (5 logs). Isolates from an iodinated potable water system, including P. cepacia, Staphyloccus warneri, and a Bacillus sp., were all less sensitive to iodine than a "resistant" P. aeruginosa and three other isolates when grown in brain heart infusion. A clinical isolate of P. aeruginosa exhibited intermediate sensitivity. The sensitivity of bacteria to iodine is thus highly variable, depending on the organism as well as the growth conditions.     

Survival of potentially pathogenic human-associated bacteria in the rhizosphere of hydroponically grown wheat

Plants may serve as reservoirs for human-associated bacteria (H-AB) in long-term space missions containing bioregenerative life support systems. The current study examined the abilities of five human-associated potential pathogens, Pseudomonas aeruginosa, Pseudomonas cepacia, Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli, to colonize and grow in the rhizosphere of hydroponically grown wheat, a candidate crop for life support. All of these bacteria have been recovered from past NASA missions and present potential problems for future missions. The abilities of these organisms to adhere to the roots of axenic five-day-old wheat (Triticum aestivum L. cv. Yecora rojo) were evaluated by enumeration of the attached organisms after a one hour incubation of roots in a suspension (approximately 10(8) cfu ml-1) of the H-AB. Results showed that a greater percentage of P. aeruginosa cells adhered to the wheat roots than the other four H-AB. Similarly incubated seedlings were also grown under attempted axenic conditions for seven days to examine the potential of each organism to proliferate in the rhizosphere (root colonization capacity). P. cepacia and P. aerogiunosa showed considerable growth, E. coli and S. aureus showed no significant growth, and S. pyogenes died off in the wheat rhizosphere. Studies examining the effects of competition on the survival of these microorganisms indicated that P. aeruginosa was the only organism that survived in the rhizosphere of hydroponically grown wheat in the presence of different levels of microbial competition.     

Factors Influencing the Occurrence of High Numbers of Iodine-Resistant Bacteria in Iodinated Swimming Pools

It has been shown that, although iodinated swimming-pool waters are usually free from coliform bacteria and enterococci, the total counts frequently become relatively high. Pseudomonas alcaligenes and Alcaligenes faecalis have been shown to account for most of these high counts. It was of interest, therefore, to compare the microbial flora of four alternately chlorinated and iodinated swimming pools. By means of the membrane filter method and suitable selective media, examinations were made for total viable counts, coliform bacteria, enterococci, staphylococci, Streptococcus salivarius, and P. aeruginosa. Colonies also were picked from membrane filters incubated on standard plate count agar and identified. The results showed that, although viable counts were significantly higher during the iodinated periods, the specific types of bacteria determined were either fewer than or the same as in chlorinated periods. During chlorination, the predominant microbial flora consisted of staphylococci and members of the genus Bacillus. During iodination, however, the P. alcaligenes-A. faecalis group accounted for 92 to 99% of the microbial flora. The accumulation of high numbers of these bacteria was shown to be due to their iodine resistance and their ability to grow rapidly in pool water in the absence of free iodine.     

Transport and survival of alginate-encapsulated and free lux-lac marked Pseudomonas aeruginosa UG2Lr cells in soil

Transport and survival of alginate-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr through soil microcosms was examined. Bacterial cells encapsulated in alginate beads or mixed with soil were introduced into soil microcosms. Microbial cell survival and cell transport were monitored by destructive sampling and selective plating of the microcosms over a 9-week period. Survival rates were greatest when using encapsulated P. aeruginosa UG2Lr cells. Water flow increased microbial cell dispersal from the site of inoculation. After 3 weeks, encapsulated and free cells showed similar distribution patterns. However, after 9 weeks microbial cell distribution was more extensive throughout the soil in the encapsulated treatments under all conditions. Therefore, alginate encapsulation is a suitable method to enhance survival and distribution of microbial inocula in the soil environment.     

Factors affecting the lytic susceptibility of some marine and terrestrial bacteria

Eighteen gram-negative marine bacteria and two terrestrial species, Escherichia coli and Pseudomonas aeruginosa, were examined for their sensitivity to lysis in distilled water after exposure to a salt solution containing a sea water concentration of Mg2+ (0.05 M) or to 0.5 M NaCl. A spectrum of lytic susceptibility was observed among the marine bacteria ranging from those organisms which lysed in distilled water after exposure to the Mg2+-containing solution, through organisms which could be sensitized to lysis by washing with the NaCl solution, to organisms which failed to lyse in distilled water even after having been washed with a solution of 0.5 M NaCl. Pseudomonas aeruginosa and E. coli fell within this spectrum, the former being capable of being induced to lyse in distilled water by washing with 0.5 M NaCl, while the latter failed to lyse in distilled water after this treatment. It was thus concluded that no overall distinction could be made between marine and terrestrial bacteria on the basis of the sensitivity of the two groups of organisms to lysis in freshwater. Quite large decreases in optical density and increases in the release of ultraviolet-absorbing material took place when cells preexposed to the Mg2+-containing solution or to 0.5 M NaCl were subsequently suspended in distilled water even though in some cases no loss of cell numbers could be detected. In most cases two to three times as much K+ as Na+ and 1/10 to 1/100 as much Mg2+ was required to prevent these changes. For three of the marine bacteria and P. aeruginosa grown in a terrestrial type medium little difference in the requirements for Na+ and K+ to prevent the optical density changes was noted. For P. aeruginosa grown in a marine type medium, cells required more K+ than Na+ to prevent these changes.