The antimicrobial effects of ozonated water in a recirculating concurrent reactor were evaluated against four gram-positive and four gram-negative bacteria, two yeasts, and spores of Aspergillus niger. More than 5 log units each of Salmonella typhimurium and Escherichia coli cells were killed instantaneously in ozonated water with or without addition of 20 ppm of soluble starch (SS). In ozonated water, death rates among the gram-negative bacteria-S. typhimurium, E. coli, Pseudomonas aeruginosa, and Yersinia enterocolitica-were not significantly different (P > 0.05). Among gram-positive bacteria, Listeria monocytogenes was significantly P < 0.05) more sensitive than either Staphylococcus aureus or Enterococcus faecalis. In the presence of organic material, death rates of S. aureus compared with L. monocytogenes and E. coli compared with S. typhimurium in ozonated water were not significantly (P > 0.05) affected by SS addition but were significantly reduced (P < 0.05) by addition of 20 ppm of bovine serum albumin (BSA). More than 4.5 log units each of Candida albicans and Zygosaccharomyces bailii cells were killed instantaneously in ozonated water, whereas less than 1 log unit of Aspergillus niger spores was killed after a 5-min exposure. The average ozone output levels in the deionized water (0.188 mg/ml) or water with SS (0.198 mg/ml) did not differ significantly (P < 0.05) but were significantly lower in water containing BSA (0.149 mg/ml). ''The treatment of domestic water to provide a microbiologically safe, aesthetic, potable end product has been normal practice since before the turn of the twentieth century'' (5). Chlorine at low concentrations has been the usual agent of choice in drinking-water purification. At the low concentrations of chlorine used, however, its limitations as a bactericide (3, 4) as well as limitations in its effectiveness against certain eukaryotic pathogens and viruses (18, 20, 21, 25, 27) have been recognized. Furthermore, chlorine at low concentrations may alter certain organic compounds in water producing off tastes and odors as well as forming chloro-organic compounds with carcinogenic potential (22). As an alternative to chlorination in drinking-water disinfection, ozonation of water supplies, which was done first a century ago (29), has become an established means of disinfection and has been reviewed by Rice et al. (25). Bacteria, including Escherichia coli, Staphylococcus aureus, Bacillus cereus, Bacillus megaterium, Salmonella typhimurium, Shigella flexneri, and Vibrio cholerae are sensitive to ozonated water under various conditions (5, 6, 13, 17). Limited information on ozone's effectiveness against bacterial endospores (5, 17) and viruses (6, 20) as well as against eukaryotic pathogens including Cryptosporidium parvum (11, 21, 24) and Giardia lamblia and Giardia muris (12, 31, 32) also exists. The use of ozone in the food industry has been investigated for food preservation, shelf life extension, equipment sterilization, and improvement of food plant effluents (9, 14, 19, 2...
A chromogenic agar, R&F Enterobacter sakazakii chromogenic plating medium (ESPM), was developed for isolating presumptive colonies of E. sakazakii from foods and environmental sources. ESPM contains two chromogenic substrates (5-bromo-4-chloro-3-indoxyl-alpha-D-glucopyranoside and 5-bromo-4-chloro-3-indoxyl-beta-D-cellobioside), three sugars (sorbitol, D-arabitol, and adonitol), a pH indicator, and inhibitors (bile salts, vancomycin, and cefsulodin), which all contribute to its selectivity and differential properties. On ESPM, 79 pure culture strains of E. sakazakii (10 clinical isolates and others from food and environmental sources) yielded blue-black (three strains were blue-gray) raised colonies, 1 to 2 mm in diameter with and without halos after 24 h at 35 degrees C. Other enteric organisms plus Pseudomonas aeruginosa yielded white, yellow, green, or clear colonies with and without clear halos. Of these genera, only Shigella sonnei and one Pantoea strain produced blue-black to blue-gray colonies. ESPM was used to isolate E. sakazakii from a variety of foods: corn, wheat, and rice flours; powdered infant formula; dairy products (dried milk, whey, and caseinates); cereals; and environmental sources. Most false-positive results on ESPM were eliminated by observing acid production on either sucrose or melibiose after 6 h at 35 degrees C on a R&F E. sakazakii screening medium (ESSM) biplate. In an analysis of 240 samples, the number of samples positive for E. sakazakii by the ESPM-ESSM method and the U.S. Food and Drug Administration protocols (violet red bile glucose agar and tryptic soy agar) were 27 and 16, respectively, with sensitivity and specificity values of 100.0 and 96.9% versus 59.3 and 43.7%, respectively. These data support the fact that E. sakazakii confirmation should be based on more than one confirmation system. Both the API 20E and Biolog Microlog3 4.20 systems should be used for confirmation of E. sakazakii isolates.
The BCM Listeria monocytogenes detection system (LMDS) consists of a selective preenrichment broth (LMPEB), selective enrichment broth (LMSEB), selective/differential plating medium (LMPM), and identification on a confirmatory plating medium (LMCM). The efficacy of the BCM LMDS was determined using pure cultures and naturally and artificially contaminated environmental sponges. The BCM LMPEB allowed the growth of Listeria and resuscitation of heat-injured L. monocytogenes. The BCM LMSEB, which contains the fluorogenic substrate 4-methylumbelliferyl-myo-inositol-1-phosphate and detects phosphatidylinositol phospholipase C (PI-PLC) activity, provided a presumptive positive test for the presence of pathogenic Listeria (L. monocytogenes and L. ivanovii) after 24 h at 35 degrees C. An initial inoculum of 10 to 100 CFU/ml of L. monocytogenes in BCM LMSEB yielded a fluorogenic response after 24 h. On BCM LMPM, L. monocytogenes and L. ivanovii were the two Listeria species forming turquoise convex colonies (1.0 to 2.5 mm in diameter) from PI-PLC activity on the chromogenic substrate, 5-bromo-4-chloro-3-indoxyl-myo-inositol-1-phosphate. L. monocytogenes was distinguished from L. ivanovii by either its fluorescence on BCM LMCM or acid production from rhamnose. False-positive organisms (Bacillus cereus, Staphylococcus aureus, Bacillus thuringiensis, and yeasts) were eliminated by at least one of the media in the BCM LMDS. Using a pure culture system, the BCM LMDS detected one to two L. monocytogenes cells from a sponge rehydrated in 10 ml of DE neutralizing broth. In an analysis of 162 environmental sponges from facilities inspected by the U.S. Department of Agriculture (USDA), the values for identification of L. monocytogenes by BCM LMDS and the USDA method were 30 and 14 sites, respectively, with sensitivity and specificity values of 85.7 and 100.0% versus 40.0 and 66.1%, respectively. No false-positive organisms were isolated by BCM LMDS, whereas 26.5% of the sponges tested by the USDA method produced false-positive results.
A 24-h direct plating method for Escherichia coli using Peptone-Tergitol agar was used to compare the effectiveness of the chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GLUC) with the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide (MUG) for β-glucuronidase activity. Values obtained for enumeration of two strains of E. coli recovered from artificially inoculated raw minced chicken (i.e., plating efficiencies on the inoculum, cells per g, and recovery percentages elated to those on Plate Count Agar) indicate that X-GLUC at 50 μg/ml was as effective as MUG in an agar medium. Unlike MUG, X-GLUC does not require ultraviolet light illumination, and the color reaction produced remains localized in the positive colonies.
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