Comparative mycobactericidal efficacy of chemical disinfectants in suspension and carrier tests

The efficacy of nine disinfectants on Mycobacterium smegmatis was tested in the presence of sputum, using quantitative suspension and carrier tests. Glutaraldehyde, povidone iodine, and chlorhexidine gluconate produced at least a 6-log10 reduction in CFU in all tests. Four disinfectants (sodium dichloroisocyanurate, phenol, ethanol, and sodium hypochlorite) were not as effective in the carrier tests as in the suspension tests; this difference ranged from a 1- to a 5-log10 reduction in CFU. The efficacy of ethanol and sodium hypochlorite was further reduced (3- and 1-log10 reductions in CFU, respectively) in the presence of sputum. The quaternary ammonium compound and iodophor were ineffective in all tests. The findings of this study demonstrate the need for a quantitative carrier test such as the one presented here.     

Studies on prolyl endopeptidase from shakashimeji (Lyophyllum cinerascens): purification and enzymatic properties

High prolyl endopeptidase (post-proline cleaving enzyme) [EC 3.4.21.26] activity was detected in fruit bodies of shakashimeji (Lyophyllum cinerascens), tsukuritake (mushroom: Agaricus bisporus), hirohachichitake (Lactarius hygrophoroides), and yaburebenitake (Russula lepida) which belong to the genus Basidiomycetes. Cell-free extract of shakashimeji showed high activities of proline iminopeptidase and arylamidase as well as prolyl endopeptidase. The prolyl endopeptidase was purified from the extract of shakashimeji by sequential chromatographies on DEAE-Toyopearl, DEAE-Sephadex and hydroxyapatite, and high-performance liquid chromatography with a DEAE-5PW column. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 6.8 as checked with Z-Gly-Pro-beta-naphthylamide as a substrate and was stable in the range of pH 5.8-7.4. The isoelectric point of the enzyme was 5.2 and the molecular weight was estimated to be 76,000 by gel filtration on Sephadex G-150 and by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme was a monomer. The enzyme was completely inhibited by diisopropyl fluorophosphate (DFP), Z-Gly-Pro-CH2Cl, and Z-Pro-prolinal, while it was not inhibited by p-chloromercuribenzoate (PCMB), phenylmethylsulfonyl fluoride (PMSF), or metal chelators. It was estimated that at least five subsites were concerned with the enzyme-substrate binding. Among them, the S1, S2, and S1' sites showed high stereospecificity, as in mammalian, microbial, and plant enzymes. The enzyme hydrolyzed TRH at the carboxyl side of the proline residue. The mushroom enzyme, that was sensitive to DFP, Z-Pro-prolinal, and Z-Gly-Pro-CH2Cl, but not to PCMB, were quite similar in characteristics to the Flavobacterium enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term survival of human rotavirus in raw and treated river water

This study was aimed at assessing the role of water as a vehicle for rotavirus spread by determining how well these viruses survive in the water environment. A cell culture adapted strain of human rotavirus subgroup 2, grown in MA-104 cells, was used as a model. Virus survival was tested in the following types of water samples, derived from the Ottawa River, at two different times of the year: (i) raw water (RW), (ii) muncipally treated tap water (TW), and (iii) raw water that had been filtered (FW) through a membrane (0.22 micron). The water samples, with approximately 5.0 X 10(4) plaque-forming units (PFU) of the virus, were held at either 4 or 20 degrees C and tested for infectious virus over a period of 64 days. The TW samples had a total and free chlorine content of 0.05 and less than 0.05 mg/L, respectively. The chlorine in these samples was not neutralized before virus contamination. Irrespective of the holding temperature, the virus titre in FW remained essentially unaltered throughout the test period. In TW held at 4 degrees C, there was no significant drop in the virus titre even after 64 days, whereas at 20 degrees C the titre in TW was reduced by about 2 log10 over the same period. Even though the loss of virus infectivity was most rapid in RW held at 20 degrees C, it took about 10 days for a 99.0% reduction in the plaque titre of the virus. These findings, therefore, indicate that rotaviruses can survive for several days in raw and treated river water thus making recreational and potable waters potential vehicles for the transmission of rotavirus infections.     

Chemical disinfection of hepatitis A virus on environmental surfaces

Hepatitis A virus disinfection was assessed on contaminated stainless-steel disks. Ten microliters of fecally suspended hepatitis A virus was deposited on the center of each disk, dried for 20 min, and then covered with 20 microliters of the test product for 1 min. Of the 20 formulations tested, only 2% glutaraldehyde, a quaternary ammonium formulation containing 23% HCl (toilet bowl cleaner), and sodium hypochlorite (greater than 5,000 ppm [greater than 5,000 micrograms/ml] of free chlorine) reduced the virus titer by greater than 99.9%; phenolics, iodine-based products, alcohols, and solutions of acetic, peracetic, citric, and phosphoric acids were unable to do so.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Chemical disinfection to interrupt transfer of rhinovirus type 14 from environmental surfaces to hands.

Rhinoviruses can survive on environmental surfaces for several hours under ambient conditions. Hands can readily become contaminated after contact with such surfaces, and self-inoculation may lead to infection. Whereas hand washing is crucial in preventing the spread of rhinovirus colds, proper disinfection of environmental surfaces may further reduce rhinovirus transmission. In this study, the capacities of Lysol Disinfectant Spray (0.1% o-phenylphenol and 79% ethanol), a domestic bleach (6% sodium hypochlorite diluted to give 800 ppm of free chlorine), a quaternary ammonium-based product (7.05% quaternary ammonium diluted 1:128 in tap water), and a phenol-based product (14.7% phenol diluted 1:256 in tap water) were compared in interrupting the transfer of rhinovirus type 14 from stainless steel disks to fingerpads of human volunteers upon a 10-s contact at a pressure of 1 kg/cm2. Ten microliters of the virus, suspended in bovine mucin (5 mg/ml), was placed on each disk, and the inoculum was dried under ambient conditions; the input number on each disk ranged from 0.5 x 10(5) to 2.1 x 10(6) PFU. The dried virus was exposed to 20 microliters of the test disinfectant. The Lysol spray was able to reduce virus infectivity by > 99.99% after a contact of either 1 or 10 min, and no detectable virus was transferred to fingerpads from Lysol-treated disks. The bleach (800 ppm of free chlorine) reduced the virus titer by 99.7% after a contact time of 10 min, and again no virus was transferred from the disks treated with it.(ABSTRACT TRUNCATED AT 250 WORDS)     

Basolateral plasma membrane localization of ouabain-sensitive sodium transport sites in the secretory epithelium of the avian salt gland

The distribution of Na+ pump sites (Na+-K+-ATPase) in the secretory epithelium of the avian salt gland was demonstrated by freeze-dry autoradiographic analysis of [(3)H] ouabain binding sites. Kinetic studies indicated that near saturation of tissue binding sites occurred when slices of salt glands from salt-stressed ducks were exposed to 2.2 μM ouabain (containing 5 μCi/ml [(3)H]ouabain) for 90 min. Washing with label-free Ringer's solution for 90 min extracted only 10% of the inhibitor, an amount which corresponded to ouabain present in the tissue spaces labeled by [(14)C]insulin. Increasing the KCl concentration of the incubation medium reduced the rate of ouabain binding but not the maximal amount bound. In contrast to the low level of ouabain binding to salt glands of ducks maintained on a freshwater regimen, exposure to a salt water diet led to a more than threefold increase in binding within 9-11 days. This increase paralleled the similar increment in Na+-K+-ATPase activity described previously. [(3)H]ouabain binding sites were localized autoradiographically to the folded basolateral plasma membrane of the principal secretory cells. The luminal surfaces of these cells were unlabeled. Mitotically active peripheral cells were also unlabeled. The cell-specific pattern of [(3)H]ouabain binding to principal secretory cells and the membrane-specific localization of binding sites to the nonluminal surfaces of these cells were identical to the distribution of Na+-K+-ATPase as reflected by the cytochemical localization of ouabain-sensitive and K+-dependent nitrophenyl phosphatase activity. The relationship between the nonluminal localization of Na+-K+-ATPase and the possible role of the enzyme n NaCl secretion is considered in the light of physiological data on electrolyte transport in salt glands and other secretory epithelia.     

Rotavirus concentration from cell culture harvests: trypsin treatment followed by hydroextraction.

An 80- to 150-ml amount of calf or simian rotavirus-containing cell culture harvests of MA-104 cells were treated with 50 microgram of trypsin per ml and hydroextracted overnight (4 degrees C) with polyethylene glycol 6,000. The concentrate was resuspended in 8 to 10 ml of tryptose phosphate broth and plaque assayed. Between 85 and 97% of the input virus could be recovered with a concentration of up to 15-fold.