Oct 5, 1990 - State University, University Park, Pennsylvania 16802 ... one potential P. aeruginosa isolate from each farm was ..... man, and R. W. Haley.
Vol. 57, No. 2
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1991, p. 568-572
0099-2240/91/020568-05$02.00/0 Copyright © 1991, American Society for Microbiology
Interactions between Pseudomonas aeruginosa and lodophor Germicides in Milking Parlor Udder Wash Water Systems ROBERT J. EBERHART,' RONALD J. ERSKINE,lt STEPHEN B. SPENCER2 Departments of Veterinary Science' and Dairy and Animal Science,2 The Pennsylvania State University, University Park, Pennsylvania 16802
CATHLEEN R.
HICKS,'*
AND
Received
5
October 1990/Accepted 6 December 1990
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 (Al2) concentrations were below the recommended level of 25 ppm (25 ,ug/ml). Rubber and polyvinyl chloride hoses caused rapid decreases in the Al2 concentrations of 25 ppm iodophor solutions. Al2 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 < 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 Al2. 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. lodophor concentrations which will kill 50% of P. aeruginosa cultures previously exposed to 0, 10, and 25 ppm of Al2 were predicted to be 3.0, 11.8, and 20.8 ppm, respectively.
Each farm was visited at least 6 h after the morning milking; samples of both standing and running water were taken from wash hoses in the parlor. Individual 10-ml samples were collected from four hoses in each parlor, and a 500-ml composite sample was made of approximately equal volumes from each hose in the parlor. Samples were transported on ice in a styrofoam cooler. A preliminary study indicated that this maintained original Al2 concentrations for over 12 h. AT2 concentrations in standing and running water samples were measured by duplicate sodium thiosulfate titrations (3) immediately upon return to the laboratory (within 8 h of collection). Eighteen to 24 h after collection, water samples from all farms were tested for the presence of P. aeruginosa. Several isolation techniques were used, including direct culture of 0.1 ml of each 10-ml sample and filtration of 300 ml of water through a 0.2-p.m-pore-size filter which was then cultured. In this report, a system yielding P. aeruginosa by any method is considered positive. All samples were cultured on Pseudomonas isolation agar (Difco, Detroit, Mich.). After incubation of the cultures at 37°C for 48 h, at least one potential P. aeruginosa isolate from each farm was identified to species level with the API 20E chemical testing system (Analytab Products Inc., Plainview, N.Y.). Differences in frequency of isolation of P. aeruginosa between farms using and not using iodophor were tested by using the Fisher's exact test of the Statistical Analysis System (SAS, Cory, N.C.). Laboratory studies. Upon completion of the field study, interactions among hose materials, iodophor germicide, and P. aeruginosa were examined. Bacterial strains and equipment. P. aeruginosa isolated from the udder wash system of Penn State's Dairy Research and Production Center was maintained in a 0.1% glucose aqueous solution and provided P. aeruginosa cultures throughout the laboratory experiments. Since the organism
Pseudomonas aeruginosa is not generally resistant to iodine, but contamination of iodophor solutions by Pseudomonas species has been reported (1, 4-7, 12). These reports all involve hospital-use solutions of approximately 10% available iodine (AT2). Survival of P. aeruginosa in iodophor solutions in milking parlor udder wash water systems has recently been reported as well (13). Such systems are composed of the pipes and hoses used to wash the teats and udders of dairy cows before milking. Colonization of udder wash systems by the organism has been reported on several occasions (2, 8, 15, 17); however, Erskine et al. (13) were the first to mention iodophor usage in such systems colonized by P. aeruginosa. Use of a germicide in the wash water, in accordance with Food and Drug Administration recommendations (19), should prevent bacterial contamination, but Erskine et al. (13) reported four herd epizootics of P. aeruginosa mastitis in which an iodophor germicide was used in the udder wash system. Examination of the wash hoses revealed heavy colonization of the hose interiors and high numbers of bacteria present in the standing wash water (that water present in pipes and hoses between milkings). This suggests a possible relationship between iodophor usage and colonization of the udder wash system. The present study attempts to verify this relationship and offers possible explanations for it. MATERIALS AND METHODS Field study. Samples were collected from 29 dairy farms to examine a possible relationship between the use of iodophor germicide in the udder wash water and the presence of P. aeruginosa. Eleven farms used iodophor germicide in the udder wash water and 18 used no germicide in the water. Corresponding author. t Present address: Department of Large Animal Surgery and Medicine, Auburn University, Auburn, AL 36849. *
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VOL. 57, 1991
P. AERUGINOSA AND IODOPHORS IN UDDER WASH SYSTEMS
was known to thrive in water from the udder wash system at Penn State, autoclave-sterilized barn water served as growth medium for the experiments. One bottle of Agway udder wash (Agway Farm Supplies, Syracuse, N.Y.) provided iodophor for all experiments. Polyvinyl chloride (PVC) milk line tubing and a rubber garden hose were purchased from Agway Farm Supplies. These two materials are commonly used as udder wash hoses in milking parlors. Interactions among udder wash system components. (i) lodophor concentrations in wash hoses. Each hose was filled with an iodophor solution providing 25 ppm (25 ,ug/ml) of Al2, and 200 ml was drained from the hose for sodium thiosulfate titration at 15, 60, 120, 180, and 240 min, unless iodine concentrations had already fallen below 5 ppm. A control solution of iodophor in a clear glass jug was sampled for AT2 determination at the same intervals. The hoses were thoroughly rinsed with distilled water between each of four repetitions. Both hoses were filled with concentrated iodophor solutions (250 ppm of AT2) which were replaced at 4-day intervals for 20 days. On day 21, after thorough rinsing, four more repetitions of the experiment were conducted by using the same protocol. Multiple Student's t tests were conducted to detect differences among the Al2 concentrations found in the controls and in each hose, both before and after soaking with iodophor (n = 4). (ii) Inactivated iodophor effect on bacterial numbers. Eighteen 100-ml volumes of autoclave-sterilized barn water were prepared, and 8 ml of 0.005 N sodium thiosulfate (twice the amount needed to inactivate 25 ppm of AT2) was added to each. lodophor providing 25 ppm of Al2 was added to nine of the cultures; the others served as controls. Each culture was inoculated with approximately 1,000 CFU of P. aeruginosa per ml and incubated at room temperature. Bacterial numbers were determined by serial plate counts 0, 10, and 24 h after inoculation. Log2 transformed data were analyzed by paired Student's t tests (n = 9). (iii) P. aeruginosa and iodophors in PVC tubing. To provide a laboratory model of the hose portion of the milking parlor wash system, lengths of PVC tubing (20 cm) were partially filled with a suspension of P. aeruginosa and exposed to different iodophor concentrations. Each piece of PVC tubing was circled at 1-cm intervals with indelible marker, and rubber stoppers were placed in both ends. A cotton-plugged glass pipette was inserted through a hole near the middle of, each length of hose (this allowed for oxygen penetration to the hose interior), and the hoses were gas sterilized. For each of three replications, nine hoses were filled with 15 ml of P. aeruginosa suspension. Hoses were stored horizontally, with pipettes positioned vertically, and water levels in each hose were marked. To allow for bacterial colonization of the hose walls, the hoses remained undisturbed for two consecutive 9-day periods. .After the first 9 days, bacterial suspensions in the hoses were replaced with fresh suspensions. After this colonization period, water in each hose was replaced twice daily for 10 days with solution containing 0, 10, or 25 ppm of Al2; this simulated replacement of water in the hoses during milking in the parlor. At each water change, nonadherent bacteria were resuspended by gently tipping the tubing lengthwise five times. Water within the hose was then decanted into a sterile 50-ml vial. Each hose section was rinsed twice with approximately 15 ml of autoclave-sterilized barn water and then refilled with 15 ml of the appropriate iodophor solution.
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Numbers of nonadherent bacteria (those suspended in the decanted water) and bacteria adhered to the hose interior were determined at day zero (as the water in the hoses was changed after the 18-day colonization period) and at the a.m. water change on days 1 through 4, 6, 8, and 10. Nonadherent bacterial numbers were determined by duplicate plate counts of 10-fold dilutions of the decanted water. Adherent bacterial numbers were determined by swabbing a 1-cm section of the hose (below the water line) after it had been rinsed. Since absolute numbers of adherent bacteria could not be determined, a standard swabbing pattern was developed. The soluble calcium alginate swabs (Fisher Scientific, Pittsburgh, Pa.) were dissolved in a 2.5-ml volume of 1% sodium citrate with 4% 0.005 N sodium thiosulfate. After serial dilutions and duplicate plate counts, bacterial numbers were recorded as CFU per swab. Least-square means of bacterial numbers for the three treatments (n = 9) on each day were compared by using the Scheffe test procedure (SAS). (iv) Determination of bactericidal iodophor concentrations. On days 9 and 11 of the third replication of experiment iii, water decanted from the hoses in each treatment group was pooled. Suspensions from hoses exposed to 0 and 10 ppm of Al2 were diluted (on the basis of the previous morning's results) to approximately equal bacterial numbers in the 25 ppm Al2 pool. Exact bacterial concentrations of each pool were determined by serial dilution. Bacteria from each pooled sample (total n = 6) were exposed to 0, 5, 10, 15, and 25 ppm of A12. At 5, 10, and 15 min, 0.1 ml of the test solution was transferred to a 5-ml tryptic soy broth culture. Presence of turbidity in the broths was recorded after 48 h of incubation at 37°C. Turbid broths were subcultured on blood agar, and the growth was confirmed as P. aeruginosa by oxidase testing and examination of colony morphology. After determining which variables (previous iodophor exposure, challenge iodophor concentration, bacterial numbers in challenge solution, and time of sampling after challenge) significantly affected bacterial survival, a model predicting survival percentages for each treatment group was developed, and predicted 50% bactericidal concentrations were determined. RESULTS Field study.
AT2 concentrations in both standing and
were found to be lower than the recommended level of 25 ppm (19). Since the accuracy of sodium thiosulfate titration is questionable at very low levels, concentrations of less than 5 ppm of AI2 were recorded only as
