Clifford H. Johnson

Isolation of Arcobacter butzleri from ground water.

Arcobacter butzleri was isolated from a contaminated ground water source. These organisms, previously designated as aerotolerant Campylobacter, were capable of surviving in the ground water environment. Specific DNA probes were used to characterize the isolates in the initial identification and survival studies. Arcobacter butzleri was found to be sensitive to chlorine inactivation.

Chlorine inactivation of Escherichia coli O157:H7.

We analyzed isolates of Escherichia coli O157:H7 (which has recently caused waterborne outbreaks) and wild-type E. coli to determine their sensitivity to chlorination. Both pathogenic and nonpathogenic strains were significantly reduced within 1 minute of exposure to free chlorine. Results indicate that chlorine levels typically maintained in water systems are sufficient to inactivate these organisms.

Survival of Escherichia coli O157:H7 in drinking water associated with a waterborne disease outbreak of hemorrhagic colitis

Survival characteristics were similar for two strains of Escherichia coli O157: H7 and a typical indicator strain of E. coli in a portable ground water source. Die‐off was more rapid at 20°C than at 5°C. There was no significant difference among the rates of survival for the strains examined.

A systematic comparison of the electrokinetic properties of environmentally important microorganisms in water

The surface charge of microorganisms is an important factor controlling their stability in aqueous environments and removal during water treatment practices. The electrophoretic mobility (EPM) of a number of environmentally important microorganisms in phosphate buffers was measured. The effect of pH, ionic strength, and cation type (valence) and cation concentration on the EPM was investigated. The results show that microorganisms have significantly different charge properties in water. The surface charge of all microorganisms was impacted by pH, ionic strength and ionic make-up of the water. As a result, direct comparisons of reported surface charge values should be approached with caution. Increasing the pH increased the EPM in the negative direction. Increasing the ionic strength of the suspension by increasing the phosphate buffer concentration or adding simple salts generally decreased the EPM. The EPM was more neutral as the valence state of the cation in the salt increased.

A Spore Counting Method and Cell Culture Model for Chlorine Disinfection Studies of Encephalitozoon syn.Septata intestinalis

ABSTRACT The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth ofEncephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID50) and a minimal infective dose (MID) for E. intestinalis. The TCID50 is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a systems permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID50 have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25°C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log10 reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data suggest that chlorine treatment may be an effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies.

Pilot-Scale Ozone Inactivation of Cryptosporidium and Other Microorganisms in Natural Water

Abstract A pilot-scale study was conducted to evaluate the inactivation by ozone against Cryptosporidium oocysts, Giardia cysts, poliovirus, and B. subtilis endospores spiked into Ohio River water. The indigenous Ohio River populations of total coliform bacteria, heterotrophic plate count bacteria and endospores of aerobic spore forming bacteria were also evaluated. Endospores were the only organisms found to be more resistant to ozone than Cryptosporidium oocysts. Endospores may serve as an indicator of microbial treatment efficiency. Cryptosporidium oocysts were more resistant than Giardia cysts or poliovirus. Although HPC bacteria were less resistant than Cryptosporidium oocysts, variability limits their usefulness as an indicator of treatment efficiency. Ozone inactivation data generated in a pilot-scale study employing natural surface waters were comparable to inactivation data derived from previously published bench-scale studies using laboratory waters. The ozone requirements for inactivation of Cryptosporidium oocysts may produce elevated levels of bromate and ozone byproducts.

Vibrio cholerae 01 can assume a ‘rugose’ survival form that resists killing by chlorine, yet retains virulence

Vibrio cholerae 01 is able to shift between smooth and rugose colonial morphologies. Cultures of smooth V. cholerae strains were inactivated in less than 20 s at a concentration of 1.0 mg l‐1 free chlorine. In contrast, cultures of rugose variants exposed to this concentration of chlorine showed an initial rapid drop in viable counts, followed by persistence of a protected subpopulation of cells. Viable V. cholerae could still be recovered from rugose cultures even after exposure to 2.0 mg l‐1 free chlorine for 30 min. Preliminary studies suggest that resistance to killing by chlorine was due to formation of cell aggregates enclosed in a gelatinous mucoid material. Rugose strains appeared to be fully virulent, based on their ability to adhere to Caco‐2 cells and elicit fluid accumulation in rabbit ileal loops. Our data suggest that the V. cholerae rugose phenotype represents a fully virulent survival form of the organism that can persist in the presence of free chlorine.

Chlorine Inactivation of Spores of Encephalitozoon spp.

ABSTRACT This report is an extension of a preliminary investigation on the use of chlorine to inactivate spores of Encephalitozoonintestinalis and to investigate the effect of chlorine on two other species, Ecuniculi and E. hellem, associated with human infection. The 50% tissue culture infective doses of these three species were also determined. On the basis of the results obtained, it appears that chlorination of water is an effective means of controlling spores of these organisms in the aquatic environment.

Propidium monoazide reverse transcriptase PCR and RT-qPCR for detecting infectious enterovirus and norovirus

Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the public health significance of positive findings are limited. In this study, PMA RT-PCR and RT-qPCR assays were evaluated for selective detection of infectious poliovirus, murine norovirus (MNV-1), and Norwalk virus. Viruses were inactivated using heat, chlorine, and ultraviolet light (UV). Infectious and non-infectious viruses were treated with PMA before RT-PCR and RT-qPCR. PMA RT-PCR was able to differentiate selectively between infectious and heat and chlorine inactivated poliovirus. PMA RT-PCR was able to differentiate selectively between infectious and noninfectious murine norovirus only when inactivated by chlorine. However, PMA RT-PCR could not differentiate infectious Norwalk virus from virus particles rendered non-infectious by any treatment. PMA RT-PCR assay was not able to differentiate between infectious and UV inactivated viruses suggesting that viral capsid damage may be necessary for PMA to enter and bind to the viral genome. PMA RT-PCR on naked MNV-1 and Norwalk virus RNA suggest that PMA RT-PCR can be used to detect intact, potentially infectious MNV-1 and Norwalk viruses and can be used to exclude the detection of free viral RNA by PCR assay.

Detection of intrinsic vancomycin resistant enterococci in animal and human feces.

Fecal samples from animal species and humans were analyzed by quantitative culture for enterococci and vancomycin resistant enterococci (VRE). Each host species carried enterococci which exhibited intrinsic intermediate resistance to vancomycin and sensitivity to teicoplanin (Van C phenotype). The carriage rate in humans was 9%. Carriage rates varied among animal species with the highest percentages being found in deer, duck, goose, horse and turkey.