Susceptibilities of Bacillus subtilis, Bacillus cereus, and avirulent Bacillus anthracis spores to liquid biocides.

The susceptibility of spores of Bacillus subtilis, Bacillus cereus, and avirulent Bacillus anthracis to treatment with hydrogen peroxide, peroxyacetic acid, a peroxy-fatty acid mixture, sodium hypochlorite, and acidified sodium chlorite was investigated. Results indicated that B. cereus spores may be reasonable predictors of B. anthracis spore inactivation by peroxyacetic acid-based biocides. However, B. cereus was not a reliable predictor of B. anthracis inactivation by the other biocides. In studies comparing B. cereus and B. subtilis, B. cereus spores were more resistant (by 1.5 to 2.5 log CFU) than B. subtilis spores to peroxyacetic acid, the peroxy-fatty acid mixture, and acidified sodium chlorite. Conversely, B. subtilis spores were more resistant than B. cereus spores to hydrogen peroxide. These findings indicated the relevance of side-by-side testing of target organisms and potential surrogates against categories of biocides to determine whether both have similar properties and to validate the use of the surrogate microorganisms.

Inactivation of Yersinia pseudotuberculosis, as a surrogate for Yersinia pestis, by liquid biocides in the presence of food residue.

The efficacy of liquid biocides is influenced by surface cleanliness, treatment time, and temperature. Experiments were completed to measure the impact of these variables on the ability of commercial biocides to inactivate Yersinia pseudotuberculosis ATCC 29910, as a surrogate for Yersinia pestis, in the presence of food residues. The test organism was mixed with water, milk, flour, or egg yolk and then dried onto stainless steel coupons. Coupons were then exposed to sodium hypochlorite, acidified sodium chlorite, a quaternary ammonium compound, an iodophor, hydrogen peroxide, peroxyacetic acid, or a peroxy-fatty acid mixture, for 10 or 30 min at 10, 20, or 30 degrees C. For all biocides except the iodophor, manufacturer-recommended disinfection levels applied for 10 min at 20 degrees C resulted in 5-log reductions of the test organism dried alone or with flour. However, in the presence of whole milk or egg yolk residue, markedly higher sodium hypochlorite, peroxyacetic acid, peroxy-fatty acid mixture, quaternary ammonium compound, and iodophor concentrations were needed to achieve the 5-log reductions. Further, the quaternary ammonium compound was incapable of achieving 5-log reductions in 10 min in the presence of milk and egg yolk residues. Hydrogen peroxide and acidified sodium chlorite disinfection levels (7.5% and 2500 ppm, respectively) achieved 5-log reductions under all test conditions. These results suggest that commercial disinfectants can adequately decontaminate clean surfaces contaminated with Y. pseudotuberculosis and Y. pestis. These results also provide guidance on the feasibility of overcoming the negative influence of food residues on disinfection by adjusting biocide exposure time, temperature, and concentration.

Inactivation of Bacillus anthracis spores by liquid biocides in the presence of food residue.

Biocide inactivation of Bacillus anthracis spores in the presence of food residues after a 10-min treatment time was investigated. Spores of nonvirulent Bacillus anthracis strains 7702, ANR-1, and 9131 were mixed with water, flour paste, whole milk, or egg yolk emulsion and dried onto stainless-steel carriers. The carriers were exposed to various concentrations of peroxyacetic acid, sodium hypochlorite (NaOCl), or hydrogen peroxide (H(2)O(2)) for 10 min at 10, 20, or 30 degrees C, after which time the survivors were quantified. The relationship between peroxyacetic acid concentration, H(2)O(2) concentration, and spore inactivation followed a sigmoid curve that was accurately described using a four-parameter logistic model. At 20 degrees C, the minimum concentrations of peroxyacetic acid, H(2)O(2), and NaOCl (as total available chlorine) predicted to inactivate 6 log(10) CFU of B. anthracis spores with no food residue present were 1.05, 23.0, and 0.78%, respectively. At 10 degrees C, sodium hypochlorite at 5% total available chlorine did not inactivate more than 4 log(10) CFU. The presence of the food residues had only a minimal effect on peroxyacetic acid and H(2)O(2) sporicidal efficacy, but the efficacy of sodium hypochlorite was markedly inhibited by whole-milk and egg yolk residues. Sodium hypochlorite at 5% total available chlorine provided no greater than a 2-log(10) CFU reduction when spores were in the presence of egg yolk residue. This research provides new information regarding the usefulness of peroxygen biocides for B. anthracis spore inactivation when food residue is present. This work also provides guidance for adjusting decontamination procedures for food-soiled and cold surfaces.

Ruminal infusion of ammonium chloride in lactating cows to determine effect of pH on ammonia trapping.

Milking rations containing 16 (control), 13.2, and 10.4% protein were fed to four midlactation, rumen-fistulated Holstein cows. Ammonium chloride was infused ruminally for 5 consecutive days after morning feeding when cows were fed milking rations containing 13.2 and 10.4% protein. Amount infused was equivalent to the ammonia in 1 or 2% dietary urea. Rumen and blood samples were taken prior to and following morning feedings. Intake of milking ration was the same across treatments. Initial rumen pH was higher for ammonium chloride treatments. It then declined, as did the control, to the same, lowest pH at 1.5 to 3 h postfeeding. Rumen ammonia increased rapidly for cows receiving both ammonium chloride treatments to .5 h postfeeding and then declined rapidly. Blood urea nitrogen was highest for cows fed the control ration, peaked .5 h postfeeding for cows infused with the low ammonium chloride, then dropped and peaked again 6 h postfeeding. Blood ammonia was highest among treatments for control cows and differed by sampling time only for control cows with a peak .5 h postfeeding. Because lower rumen pH traps ammonia preventing rapid absorption into blood, interpretation of high rumen ammonia must consider effect of rumen pH.

Factors influencing intake of high urea-containing rations by lactating dairy cows.

In a series of experiments we investigated effects of several factors on intake of urea by lactating dairy cows. Cows given an unfamiliar ingredient or altered moisture in the ration reduced their intake, and this effect was attributed to a newness factor. Addition of urea to a ration may have a similar effect. An experimental design providing both no-choice and two-choice data was devised to minimize the effect of newness. When urea was isolated physically by pellets coated with ground corn, cows selected against urea-containing pellets on a two-choice basis and reduced intake on a no-choice basis. When the choice was between two urea-containing rations, cows preferred the pellets in which urea flavor and odor should have been most evident. Urea odor in the feedbox or urea in the drinking water did not reduce feed intake. Eating rate of a 2.5% urea-containing ration over two daily 30-min feeding periods was about one-half that of a non-urea ration. This effect was evident during the first 5-min eating interval. Administration of urea solution into the rumen prior to feeding a nonurea ration markedly reduced subsequent intake. Reticulum pH and ammonia indicated possible sublethal ammonia toxicity. Intake and eating rate were depressed and reticulum pH was elevated when cows were fed a ration with 2.5% urea versus 1% urea or nonurea rations. Elevated reticulum ammonia occurred on both 1 and 2.5% urea-containing rations. Cows not previously exposed to urea-containing rations reduced intake and eating rate when fed rations with 2.0 and 2.5% urea. Intake reduction was not comparable to that by cows preconditioned to urea rations. Upon third exposure to 2.5% urea in the ration, cows reduced and ceased intake but readily consumed a nonurea ration. Cows require preconditioning to develop a negative aversion to rations containing high urea, perhaps through a mechanism of sublethal ammonia toxicity.

Characterization of lactose-fermenting revertants from lactose-negative Streptococcus lactis C2 mutants.

Partial lactose-fermenting revertants from lactose-negative (lac(-)) mutants of Streptococcus lactis C2 appeared on a lawn of lac(-) cells after 3 to 5 days of incubation at 25 C. The revertants grew slowly on lactose with a growth response similar to that for cryptic cells. In contrast to lac(+)S. lactis C2, the revertants were defective in the accumulation of [(14)C]thiomethyl-beta-d-galactoside, indicating that they were devoid of a transport system. Hydrolysis of o-nitrophenyl-beta-d-galactoside-6-phosphate by toluene-treated cells confirmed the presence of phospho-beta-d-galactosidase (P-beta-gal) in the revertant. However, this enzyme was induced only when the cells were grown in the presence of lactose; galactose was not an inducer. In lac(+)S. lactis C2, enzyme induction occurred in lactose- or galactose-grown cells. The revertants were defective in EII-lactose and FIII-lactose of the phosphoenolpyruvate-dependent phosphotransferase system. Galactokinase activity was detected in cell extracts of lac(+)S. lactis C2, but the activity was 9 to 13 times higher in extracts from the revertant and lac(-), respectively. This suggested that the lac(-) and the revertants use the Leloir pathway for galactose metabolism and that galactose-1-phosphate rather than galactose-6-phosphate was being formed. This may explain why lactose, but not galactose, induced P-beta-gal in the revertants. Because the revertant was unable to form galactose-6-phosphate, induction could not occur. This compound would be formed on hydrolysis of lactose phosphate. The data also indicate that galactose-6-phosphate may serve not only as an inducer of the lactose genes in S. lactis C2, but also as a repressor of the Leloir pathway for galactose metabolism.

Extrachromosomal elements in group N streptococci.

The deoxyribonucleic acid (DNA) of Streptococcus lactis C2, S. cremoris B(1), and S. diacetilactis 18-16 was labeled by growing cells in Trypticase soy broth containing (3)H-labeled thymine. The cells were gently lysed with lysozyme, ethylenediaminetetraacetic acid, and sodium lauryl sulfate. The chromosomal DNA was separated from plasmid DNA by precipitation with 1.0 M sodium chloride. The existence of covalently closed circular DNA in the three organisms was shown by cesium chloride-ethidium bromide equilibrium density gradient centrifugation of the cleared lysate material. In an attempt to correlate the loss of lactose metabolism with the loss of plasmid DNA, lactose-negative mutants of these organisms were examined for the presence of extrachromosomal particles. Covalently closed circular DNA was detected in the lactose-negative mutants of S. lactis C2 and S. diacetilactis 18-16. In S. cremoris B(1), however, no covalently closed circular DNA was observed by using cesium chloride-ethidium bromide gradients. Electron micrographs of the satellite band material from S. lactis C2 and its lactose-negative mutant confirmed the presence of plasmid DNA. Three distinct plasmids having approximate molecular weights of 1.3 x 10(6), 2.1 x 10(6), and 5.1 x 10(6) were observed in both organisms.

Transduction of lactose metabolism in Streptococcus lactis C2.

Ultraviolet (UV)-induced phage lysates, from lactose-positive (lac(+)) Streptococcus lactis C2, transduced lactose fermenting ability to lac(-) recipient cells of this organism. Although the phage titer could not be determined due to the absence of an appropriate indicator strain, the number of transductants was proportional to the amount of phage lysate added. Treatment of the lysate with deoxyribonuclease had no effect on this conversion, indicating the observed genetic change was not mediated by free deoxyribonucleic acid. When the lac(+) transductants were isolated and exposed to UV irradiation, lysates with higher transducing ability were obtained. The transducing ability of this lysate was about 100-fold higher than that observed in the original lysates. The lac(+) transductants were unstable since lac(-) segregants occurred at high frequency. The phage lysate from S. lactis C2 also transduced maltose and mannose metabolism to the respective negative recipient cells. The results demonstrate the transduction of carbohydrate markers by a streptococcal phage and establish a genetic transfer system in group N streptococci.