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Objective: Fluid shear stress is thought to be a regulator of endothelial cell behavior during angiogenesis. The link, however, requires an understanding of stress values at the capillary level in angiogenic microvascular networks. Critical questions remain. What are the stresses? Do capillaries experience similar stress magnitudes? Can variations explain vessel-specific behavior? The objective of this study was to estimate segment-specific shear stresses in angiogenic networks., Methods: Images of angiogenic networks characterized by increased vascular density were obtained from rat mesenteric tissues stimulated by compound 48/80-induced mast cell degranulation. Vessels were identified by perfusion of a 40 kDa fixable dextran prior to harvesting and immunolabeling for PECAM. Using a network flow-based segment model with physiologically relevant parameters, stresses were computed per vessel for regions across multiple networks., Results: Stresses ranged from 0.003 to 2328.1 dyne/cm 2 and varied dramatically at the capillary level. For all regions, the maximum segmental shear stresses were for capillary segments. Stresses along proximal capillaries branching from arteriole inlets were increased compared to stresses along capillaries in more distal regions., Conclusions: The results highlight the variability of shear stresses along angiogenic capillaries and motivate new discussions on how endothelial cells may respond in vivo to segment-specific microenvironment during angiogenesis., (© 2023 John Wiley & Sons Ltd.)

Stromal vascular fraction (SVF), isolated from adipose tissue, identifies as a rich cell source comprised of endothelial cells, endothelial progenitor cells, pericytes, smooth muscle cells, fibroblasts, and immune cells. SVF represents a promising therapeutic heterogonous cell source for growing new blood microvessels due to its rich niche of cells. However, the spatiotemporal dynamics of SVF within living tissues remain largely unknown. The objective of this chapter is to describe a protocol for culturing SVF on mouse mesentery tissues in order to aid in the discovery of SVF dynamics and associated vessel growth over time. SVF was isolated from the inguinal adipose from adult mice and seeded onto mesentery tissues. Tissues were then cultured for up to 5 days and labeled with endothelial cell and pericyte markers. Representative results demonstrate the observation of SVF-derived vasculogenesis characterized by de novo vessel formation and subsequent vessel connection., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Francisella tularensis is a fastidious, Gram-negative coccobacillus and is the causative agent of tularemia. To assess viability yet overcome lengthy incubation periods, a culture-based PCR method was used to detect early growth of the lowest possible number of F. tularensis cells. This method utilized a previously developed enhanced F. tularensis growth medium and is based on the change in PCR cycle threshold at the start and end of each incubation., Results: To test method robustness, a virulent Type A1 (Schu4) and B (IN99) strain and the avirulent Live Vaccine Strain (LVS) were incubated with inactivated target cells, humic acid, drinking and well water, and test dust at targeted starting concentrations of 1, 10, and 100 CFU mL - 1 (low, mid, and high, respectively). After 48 h, LVS growth was detected at all targeted concentrations in the presence of 10 6 inactivated LVS cells; while Schu4 and IN99 growth was detected in the presence of 10 4 Schu4 or IN99 inactivated cells at the mid and high targets. Early detection of F. tularensis growth was strain and concentration dependent in the presence of fast-growing well water and test dust organisms. In contrast, growth was detected at each targeted concentration by 24 h in humic acid and drinking water for all strains., Conclusions: Results indicated that the culture-based PCR assay is quick, sensitive, and specific while still utilizing growth as a measure of pathogen viability. This method can circumvent lengthy incubations required for Francisella identification, especially when swift answers are needed during epidemiological investigations, remediation efforts, and decontamination verification.

Inactivation of Bacillus globigii spores in wash water was studied to simulate chlorine inactivation of Bacillus anthracis spores in water generated during biological cleanups. Eight waters were studied, with six containing detergent. Chlorine levels were approximately 3000 mg/L. Results across different waters showed decreasing inactivation with increasing pH. Inactivation did not appear to be influenced by chemical oxygen demand, suspended solids, turbidity, or dissolved solids. Inactivation efficacy was expressed as the time calculated to yield 6 log10 inactivation at 3000 mg NaOCl/L. This time ranged from 5 to 51 minutes at ~21 °C and from 11 to 209 minutes at ~5 °C. For one wash water, inactivation was conducted when there was no pH adjustment, and when the pH was buffered at 7 and 8. Inactivation in these buffered waters was rapid, but inactivation decreased sharply at a pH above ~9.3.

In this study, we have fabricated robust patterned surfaces that contain biocompatible and antifouling stripes, which cause microorganisms to consolidate into bare silicon spaces. Copolymers of methacryloyloxyethyl phosphorylcholine (MPC) and a methacrylate-substituted dihydrolipoic acid (DHLA) were spin-coated onto silicon substrates. The MPC units contributed biocompatibility and antifouling properties, and the DHLA units enabled cross-linking and the formation of robust thin films. Photolithography enabled the formation of 200-μm-wide poly(MPC-DHLA) stripped patterns that were characterized using atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and rhodamine 6G staining. Regardless of the spacing between poly(MPC-DHLA) stripes (10, 50, or 100 μm), Escherichia coli rapidly adhered to the bare silicon gaps that lacked the copolymer, confirming the antifouling nature of MPC. Overall, this work provides a surface modification strategy for generating alternating biofouling and nonfouling surface structures that are potentially applicable for researchers studying cell biology, drug screening, and biosensor technology.

Francisella tularensis is a fastidious organism that requires a lengthy incubation time in liquid growth media for detection. The objective of this study was to develop a medium formulation using readily available supplements that enhanced early growth of F. tularensis. Francisella tularensis live vaccine strain was used to evaluate the growth responses for each of the media formulations tested. Growth in brain heart infusion broth supplemented with 2% Vitox, 10% Fildes and 1% histidine (BVFH) resulted in a significant increase in growth after 8 h incubation compared to other media formulations tested (P < 0·001). Virulent strains of F. tularensis grown in BVFH medium demonstrated similar enhanced early growth. Cell densities of 3·9-5·2 log 10 CFU per ml were obtained after 24 h of growth in BVFH from a 1-2 cell ml -1 starting inoculum of the virulent Type A Schu4 strain, indicating the suitability of this medium in rapidly amplifying low starting titres of F. tularensis. Collectively, these results indicate that the novel formulation of the BVFH medium was capable of producing enhanced growth response for F. tularensis., Significance and Impact of the Study: The need for rapid cultivation of Francisella tularensis is essential for detection and monitoring during natural outbreak events or intentional bioterrorism attacks. The addition of selected supplements into the base medium BHI (BVFH) developed in this study enhanced growth of F. tularensis Type A1, A2 and B strains compared to BHI alone. Growth of these organisms in BVFH will allow for improved response time should a natural or intentional contamination event occur., (© 2017 The Society for Applied Microbiology.)

Transmission of Francisella tularensis, the etiologic agent of tularemia, has been associated with various water sources. Survival of many waterborne pathogens within free-living amoeba (FLA) is well documented; however, the role of amoebae in the environmental persistence of F. tularensis is unclear. In this study, axenic FLA cultures of Acanthamoeba castellanii, Acanthamoeba polyphaga, and Vermamoeba vermiformis were each inoculated with virulent strains of F. tularensis (Types A and B), the attenuated live vaccine strain, and Francisella novicida. Experimental parameters included low and high multiplicity of infection and incubation temperatures of 25 and 30 °C for 0-10 days. Francisella spp. survival was enhanced by the presence of FLA; however, bacterial growth and protozoa infectivity were not observed. In contrast, co-infections of A. polyphaga and Legionella pneumophila, used as an amoeba pathogen control, resulted in bacterial proliferation, cytopathic effects, and amoebal lysis. Collectively, even though short-term incubation with FLA was beneficial, the long-term effects on Francisella survival are unknown, especially given the expenditure of available amoebal derived nutrients and the fastidious nature of Francisella spp. These factors have clear implications for the role of FLA in Francisella environmental persistence.

Decontamination of Bacillus spores adhered to common drinking water infrastructure surfaces was evaluated using a variety of disinfectants. Corroded iron and cement-mortar lined iron represented the infrastructure surfaces, and were conditioned in a 23 m long, 15 cm diameter (75 ft long, 6 in diameter) pilot-scale drinking water distribution pipe system. Decontamination was evaluated using increased water velocity (flushing) alone at 0.5 m s -1 (1.7 ft s -1 ), as well as free chlorine (5 and 25 mg L -1 ), monochloramine (25 mg L -1 ), chlorine dioxide (5 and 25 mg L -1 ), ozone (2.0 mg L -1 ), peracetic acid 25 mg L -1 ) and acidified nitrite (0.1 mol L -1 at pH 2 and 3), all followed by flushing at 0.3 m s -1 (1 ft s -1 ). Flushing alone reduced the adhered spores by 0.5 and 2.0 log 10 from iron and cement-mortar, respectively. Log 10 reduction on corroded iron pipe wall coupons ranged from 1.0 to 2.9 at respective chlorine dioxide concentrations of 5 and 25 mg L -1 , although spores were undetectable on the iron surface during disinfection at 25 mg L -1 . Acidified nitrite (pH 2, 0.1 mol L -1 ) yielded no detectable spores on the iron surface during the flushing phase after disinfection. Chlorine dioxide was the best performing disinfectant with >3.0 log 10 removal from cement-mortar at 5 and 25 mg L -1 . The data show that free chlorine, monochloramine, ozone and chlorine dioxide followed by flushing can reduce adhered spores by > 3.0 log 10 on cement-mortar., (Published by Elsevier Ltd.)

Water supplies and water distribution systems have been identified as potential targets for contamination by bacterial biothreat agents. Since the 2001 Bacillus anthracis bioterrorist attacks, additional efforts have been aimed at research to characterize biothreat organisms in regards to their susceptibility to disinfectants and technologies currently in use for potable water. Here, we present a review of research relevant to disinfection of bacteria with the potential to pose a severe threat to public health and safety, and their potential surrogates. The efficacy of chlorine, monochloramine, chlorine dioxide, and ultraviolet light to inactivate each organism in suspension is described. The complexities of disinfection under varying water conditions and when the organisms are associated with biofilms in distribution systems are discussed.

Disinfecting water generated from a bioterrorism contamination event will require large amounts of disinfectant since the volume of water flushed from a drinking water distribution system or wash water collected from a contaminated outdoor area can accumulate quickly. Commonly used disinfectants may be unavailable in the necessary amounts, so evaluation of alternative disinfectants is needed. This study focuses on disinfection of Bacillus spores in water using acidified nitrite. The effect of varying pH (2 or 3), temperature (5°C or 24°C), nitrite concentration (0.01 or 0.1M), buffer (Butterfields or Phosphate Buffered Saline, PBS) and Bacillus species (B. globigii and B. anthracis Sterne) was evaluated. B. globigii was more resistant to disinfection under all water quality conditions. Disinfection was more effective for B. globigii and B. anthracis Sterne at 0.1M nitrite, pH 2, and 24°C. Disinfection of B. anthracis Sterne was enhanced in low ionic strength Butterfields buffer compared to PBS., (Published by Elsevier Ltd.)

The hydrophobic character and electrophoretic mobility (EPM) of microorganisms are vital aspects of understanding their interactions with the environment. These properties are fundamental in fate-and-transport, physiological, and virulence studies, and thus integral in surrogate selection. Hydrophobic and electrostatic forces are significant contributors to particle and microorganism mobility in the environment. Herein, the surface properties of commonly used Bacillus anthracis surrogate endospores were tested under comparable conditions with respect to culture, endospore purification, buffer type and strength. Additionally, data is presented of endospores suspended in dechlorinated tap water to evaluate the surrogates in regard to a breach of water infrastructure security. The surface properties of B. anthracis were found to be the most hydrophobic and least electronegative among the six Bacillus species tested across buffer strength. The effect of EPM on hydrophobicity varies in a species-specific manner. This study demonstrates that surrogate surface properties differ and care must be taken when choosing the most suitable surrogate. Moreover, it is shown that Bacillus thuringensis best represents Bacillus anthracis-Sterne with respect to both EPM and hydrophobicity across all test buffers.

Francisella tularensis, the etiologic agent of tularemia, can survive under acidic conditions. Tularemia can be acquired by several routes, including by ingestion of contaminated food or water. While acid resistance is usually associated with a low oral infective dose (ID), the ID for gastrointestinal illness is quite high. In this study, four strains of F. tularensis ssp. tularensis (type A) and four strains of F. tularensis ssp. holarctica (type B) were examined for innate acid resistance and the ability to survive in synthetic gastric fluid (SGF) under in vitro conditions similar to passage through the human stomach. Survival for all strains was significantly less in pH 2.5 SGF than in pH 2.5 phosphate-buffered saline and pH 4.0 SGF. Attenuated strains were consistently less resistant. Type B strains are most often associated with waterborne outbreaks and were examined after storage in natural water. Low-nutrient preadaptation resulted in increased resistance. Although F. tularensis can persist under certain acidic conditions, it is sensitive to conditions replicating the fasting human stomach. This may help explain the high ID required for gastrointestinal infections., (© 2013 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

The electrophoretic mobility (EPM) of endospores of Bacillus anthracis and surrogates was measured in aqueous solution across a broad pH range and several ionic strengths. EPM values trended around phylogenetic clustering based on the 16S rRNA gene. Measurements reported here provide new insight for Bacillus anthracis surrogate selection and for attachment/detachment and transport studies.

Germination was evaluated as an enhancement to decontamination methods for removing Bacillus spores from drinking water infrastructure. Germinating spores before chlorinating cement mortar or flushing corroded iron was more effective than chlorinating or flushing alone.

Francisella tularensis has been associated with naturally occurring waterborne outbreaks and is also of interest as a potential biological weapon. Recovery of this pathogen from water using cultural methods is challenging due to the organism's fastidious growth requirements and interference by indigenous bacteria. A 15-min acid treatment procedure prior to culture on a selective agar was evaluated for recovery of F. tularensis from seeded water samples. Mean levels of reduction of virulent strains of F. tularensis subsp. holarctica and subsp. tularensis were less than 20% following acid treatment. The attenuated live vaccine strain (LVS) was less resistant to acid exposure. The acid treatment procedure coupled with plating on cystine heart agar with rabbit blood and antibiotics (CHARBab) allowed the isolation of F. tularensis seeded into five natural water samples.

A sensitive and specific method that also demonstrates viability is of interest for detection of E. coli O157:H7 in drinking water. A combination of culture and qPCR was investigated. Two triplex qPCRs, one from a commercial source and another designed for this study were optimized from 5 different assays to be run on a single qPCR plate. The qPCR assays were specific for 33 E. coli O157:H7 strains tested and detected 500 cells spiked in a background of 10(8) nontarget bacterial cells. The qPCR detection was combined with an enrichment process using Presence Absence (P/A) broth to detect chlorine and starvation stressed cells. qPCR analysis performed post-enrichment allowed the detection of 3-4 cells/L as indicated by a sharp increase in fluorescence (lowering of Ct values) from pre-enrichment levels, demonstrating a 5-6 log increase in the number of cells. When six vulnerable untreated surface water samples were examined, only one was positive for viable E. coli O157:H7 cells. These results suggest that the culture-PCR procedure can be used for rapid detection of E. coli O157:H7 in drinking water.

Aims: To determine the range of free available chlorine (FAC) required for disinfection of the live vaccine strain (LVS) and wild-type strains of Francisella tularensis., Methods and Results: Seven strains of planktonic F. tularensis were exposed to 0·5 mg·l(-1) FAC for two pH values, 7 and 8, at 5 and 25°C. LVS was inactivated 2 to 4 times more quickly than any of the wild-type F. tularensis strains at pH 8 and 5°C., Conclusions: Free available chlorine residual concentrations routinely maintained in drinking water distribution systems would require up to two hours to reduce all F. tularensis strains by 4 log10. LVS was inactivated most quickly of the tested strains., Significance and Impact of the Study: This work provides contact time (CT) values that are useful for drinking water risk assessment and also suggests that LVS may not be a good surrogate in disinfection studies., (© 2010 The Authors. Letters in Applied Microbiology © 2010 The Society for Applied Microbiology.)

Two bacteriophages, phi6 and phi8, were investigated as potential surrogates for H5N1 highly pathogenic avian influenza virus in persistence and chlorine inactivation studies in water. In the persistence studies, phi6 and phi8 remained infectious at least as long as the H5N1 viruses at both 17 and 28 degrees C in fresh water, but results varied in salinated water. The bacteriophage phi6 also exhibited a slightly higher chlorine resistance than that of the H5N1 viruses. Based upon these findings, the bacteriophages may have potential for use as surrogates in persistence and inactivation studies in fresh water.

Burkholderia pseudomallei is a select agent and the causative agent of melioidosis. Variations in previously reported chlorine and monochloramine concentration time (Ct) values for disinfection of this organism make decisions regarding the appropriate levels of chlorine in water treatment systems difficult. This study identified the variation in Ct values for 2-, 3-, and 4-log(10) reductions of eight environmental and clinical isolates of B. pseudomallei in phosphate-buffered water. The greatest calculated Ct values for a 4-log(10) inactivation were 7.8 mg.min/liter for free available chlorine (FAC) at pH 8 and 5 degrees C and 550 mg.min/liter for monochloramine at pH 8 and 5 degrees C. Ionic strength of test solutions, culture hold times in water, and cell washing were ruled out as sources of the differences in prior observations. Tolerance to FAC was correlated with the relative amount of extracellular material produced by each isolate. Solid-phase cytometry analysis using an esterase-cleaved fluorochrome assay detected a 2-log(10)-higher level of organisms based upon metabolic activity than did culture, which in some cases increased Ct values by fivefold. Despite strain-to-strain variations in Ct values of 17-fold for FAC and 2.5-fold for monochloramine, standard FAC disinfection practices utilized in the United States should disinfect planktonic populations of these B. pseudomallei strains by 4 orders of magnitude in less than 10 min at the tested temperatures and pH levels.

To determine resistance of highly pathogenic avian influenza (H5N1) virus to chlorination, we exposed allantoic fluid containing 2 virus strains to chlorinated buffer at pH 7 and 8, at 5 degrees C. Free chlorine concentrations typically used in drinking water treatment are sufficient to inactivate the virus by >3 orders of magnitude.

Isolates of Escherichia coli belonging to clonal group A (CGA), a recently described disseminated cause of drug-resistant urinary tract infections in humans, were present in four of seven sewage effluents collected from geographically dispersed areas of the United States. All 15 CGA isolates (1% of the 1,484 isolates analyzed) exhibited resistance to trimethoprim-sulfamethoxazole (TMP-SMZ), accounting for 19.5% of the 77 TMP-SMZ-resistant isolates. Antimicrobial resistance patterns, virulence traits, O:H serotypes, and phylogenetic groupings were compared for CGA and selected non-CGA isolates. The CGA isolates exhibited a wider diversity of resistance profiles and somatic antigens than that found in most previous characterizations of this clonal group. This is the first report of recovery from outside a human host of E. coli CGA isolates with virulence factor and antibiotic resistance profiles typical of CGA isolates from a human source. The occurrence of "human-type" CGA in wastewater effluents demonstrates a potential mode for the dissemination of this clonal group in the environment, with possible secondary transmission to new human or animal hosts.

Seven species of bacterial select agents were tested for susceptibility to monochloramine. Under test conditions, the monochloramine routinely maintained in potable water would reduce six of the species by 2 orders of magnitude within 4.2 h. Bacillus anthracis spores would require up to 3.5 days for the same inactivation with monochloramine.

Persistence of Bacillus atrophaeus subsp. globigii spores on corroded iron coupons in drinking water was studied using a biofilm annular reactor. Spores were inoculated at 10(6) CFU/ml in the dechlorinated reactor bulk water. The dechlorination allowed for observation of the effects of hydraulic shear and biofilm sloughing on persistence. Approximately 50% of the spores initially adhered to the corroded iron surface were not detected after 1 month. Addition of a stable 10 mg/liter free chlorine residual after 1 month led to a 2-log(10) reduction of adhered B. atrophaeus subsp. globigii, but levels on the coupons quickly stabilized thereafter. Increasing the free chlorine concentration to 25 or 70 mg/liter had no additional effect on inactivation. B. atrophaeus subsp. globigii spores injected in the presence of a typical distribution system chlorine residual (approximately 0.75 mg/liter) resulted in a steady reduction of adhered B. atrophaeus subsp. globigii over 1 month, but levels on the coupons eventually stabilized. Adding elevated chlorine levels (10, 25, and 70 mg/liter) after 1 month had no effect on the rate of inactivation. Decontamination with elevated free chlorine levels immediately after spore injection resulted in a 3-log(10) reduction within 2 weeks, but the rate of inactivation leveled off afterward. This indicates that free chlorine did not reach portions of the corroded iron surface where B. atrophaeus subsp. globigii spores had adhered. B. atrophaeus subsp. globigii spores are capable of persisting for an extended time in the presence of high levels of free chlorine.

There is evidence that drinking water may be a source of infections with pathogenic nontuberculous mycobacteria (NTM) in humans. One method by which NTM are believed to enter drinking water distribution systems is by their intracellular colonization of protozoa. Our goal was to determine whether we could detect a reduction in the prevalence of NTM recovered from an unfiltered surface drinking water system after the addition of ozonation and filtration treatment and to characterize NTM isolates by using molecular methods. We sampled water from two initially unfiltered surface drinking water treatment plants over a 29-month period. One plant received the addition of filtration and ozonation after 6 months of sampling. Sample sites included those at treatment plant effluents, distributed water, and cold water taps (point-of-use [POU] sites) in public or commercial buildings located within each distribution system. NTM were recovered from 27% of the sites. POU sites yielded the majority of NTM, with >50% recovery despite the addition of ozonation and filtration. Closely related electrophoretic groups of Mycobacterium avium were found to persist at POU sites for up to 26 months. Water collected from POU cold water outlets was persistently colonized with NTM despite the addition of ozonation and filtration to a drinking water system. This suggests that cold water POU outlets need to be considered as a potential source of chronic human exposure to NTM.

Persistence of Klebsiella pneumoniae on corroded iron surfaces in drinking water was studied using biofilm annular reactors operated under oligotrophic conditions. Reactors were inoculated with K. pneumoniae, and persistence was monitored in the bulk and biofilm phases. Initial cell concentration of 10(6) MPN/mL in the bulkwater phase resulted in significantly longer adhesion than initial concentrations 1 and 2 orders of magnitude lower. K. pneumoniae cultured in low nutrient growth medium persisted longer in dechlorinated tap water than those cultured in full strength medium. Cell surface charge was more negative under low nutrient conditions, and this influenced electrostatic attraction between the cells and the oxidized iron surface. Cells grown in full strength media persisted longer in water with both low (<0.2 mg/L) and high (>0.5 mg/L) free chlorine residuals. Growth media injected with the cells dechlorinated the water allowing adhesion without inactivation. Microelectrode measurements showed a 40-70% drop in free chlorine from the bulk to the coupon surface, which decreased disinfectant potency against adhered cells. Growth and injection conditions clearly influenced cell adhesion and persistence, but permanent colonization of the corroded iron surface by K. pneumoniae was not observed.

Objectives: This study was designed to determine the role of flushing dental water lines for the removal of heterotrophic plate count bacteria, Legionella spp., and free-living protozoa., Methods: Forty dental offices were surveyed in the study. An initial sample and a sample taken after three minutes of flushing were obtained from the air/water syringe at each location. All samples were quantitatively analyzed for heterotrophic bacteria using three bacteriological procedures. The samples were analyzed for the presence of Legionella spp. using cultural, immunological, and molecular procedures and for the occurrence of free-living protozoa using a killed bacteria plate procedure., Results: The flushing process reduced the level of heterotrophic plate count bacteria by 1.1 to 1.5 log10 CFU/ml. Compliance with recommendations for bacterial levels varied depending on the methodology employed in the analysis. The flushing process did not reduce the occurrence of Legionella spp. or free-living protozoa., Conclusion: The results support recent U.S. Centers for Disease Control and Prevention recommendations that the process of flushing dental water lines cannot be relied upon as a sole means of reliably improving the quality of water used in dental treatment.

The putative virulence factor enterohemolysin, encoded by the ehlyA gene, has been closely associated with the pathogenic enterohemorrhagic Escherichia coli (EHEC) group. Escherichia coli isolates from effluents from seven geographically dispersed municipal wastewater treatment plants were screened for the presence of enterohemolysin. A total of 338 E. coli isolates were found to express the ehlyA gene. However, none of the isolates contained the toxin-encoding genes (stxA or stxB) associated with EHEC. Two of the 338 isolates possessed the virulence factor intimin, encoded by the eae gene. These findings suggest that the ehlyA gene may be widely distributed among non-EHEC isolates in the environment.

Three species of Bacillus were evaluated as potential surrogates for Bacillus anthracis for determining the sporicidal activity of chlorination as commonly used in drinking water treatment. Spores of Bacillus thuringiensis subsp. israelensis were found to be an appropriate surrogate for spores of B. anthracis for use in chlorine inactivation studies.

Seven species of bacterial select agents were tested for susceptibility to free available chlorine (FAC). Under test conditions, the FAC routinely maintained in potable water would be sufficient to reduce six species by 2 orders of magnitude within 10 min. Water contaminated with spores of Bacillus anthracis spores would require further treatment.

The bioassays assimilable organic carbon (AOC) and coliform growth response are better indexes than biological oxygen demand to determine water quality and water's ability to support the growth of bacteria. Ozonated (5 mg/liter) and chlorinated tap water were used to wash alfalfa seeds for 30 min. After washing in the ozonated tap water, the AOC concentration increased 25-fold, whereas the dissolved ozone decreased to undetectable levels. The AOC levels for the chlorinated water after washing the seeds also increased. These increases are due to ozone's strong oxidizing ability to break down refractory, large-molecular-weight compounds, forming smaller ones, which are readily used as nutrient sources for microorganisms. This same phenomenon was observed when using ozone in the treatment of drinking water. The AOC value increased from 1,176 to 1,758 micrograms C-eq/liter after the reconditioned wastewater was ozonated. When the ozonated wastewater was inoculated with Salmonella serotypes, the cells survived and increased generation times were observed. The increased nutrients would now become more readily available to any pathogenic microorganisms located on alfalfa seed surface as seen with the increase in the inoculated levels of Salmonella in the ozonated wastewater. If the washing process using ozonated water is not followed by the recommended hypochlorite treatment or continually purged with ozone, pathogen growth is still possible.

The main objective of this paper is to use Bayesian methods to estimate the kinetic parameters for the inactivation kinetics of Cryptosporidium parvum oocysts with chlorine dioxide or ozone which are characterized by the delayed Chick-Watson model, i.e., a lag phase or shoulder followed by pseudo-first-order rate of inactivation. As the length of the lag phase (CT(lag)) is not known, Bayesian statistics provides a more accurate approach than traditional statistical methods to fitting the delayed Chick-Watson kinetics. Markov Chain Monte Carlo method is used to estimate CT(lag) and first-order rate constant values. This method is also used to estimate the minimum CT requirement (with safety factor) for 99% inactivation of C. parvum oocysts.

The U.S. Environmental Protection Agency currently recommends Ct (disinfectant concentration multiplied by the exposure time) values to achieve required levels of inactivation of Giardia lamblia cysts by different disinfectants including free chlorine. Current guidance covers inactivation levels in the pH range between 6 and 9. Subsequent to the publication of these criteria, studies on the inactivation kinetics of Giardia cysts by chlorine have been performed at pH levels greater than 9. It has been shown that the chlorine inactivation process for Giardia cysts is pH dependent. The objective of this paper is to extend the current Ct values for higher pH levels. The Chick-Watson first order kinetics model was used to develop Ct values for pH levels from 6 to 11.5. A formula is provided to calculate a 100(1 - alpha)% upper confidence bound for these values.

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.

Cryptosporidium parvum, a protozoan parasite, has been implicated in a number of waterborne disease outbreaks and is difficult to inactivate using free chlorine. It appears, however, to be inactivated more easily by other oxidants such as chlorine dioxide or ozone. A major element of the EPA (US EPA) strategy for controlling C. parvum (oocysts) in drinking water is the possible use of Ct (concentration of disinfectant in mg/L times time in minutes) values. To support this strategy a Ct equation, based on first-order kinetics, is proposed to provide guidance to drinking water utilities for the application of chlorine dioxide for controlling C. parvum oocysts. The equation is based on standard statistical techniques using available bench scale data. It can predict mean inactivation levels as well as a statistically conservative upper bound Ct value. This upper bound could be used to insure an appropriate safety factor for the protection of public health.

Waste wash waters from chicken egg-processing facilities can harbor high densities of bacteria, including salmonellae. For this study, we enumerated total coliforms, Escherichia coli, and Salmonella spp. in the egg wash waters of a large egg producer. We then determined how long these organisms would survive at temperatures of 5, 15, and 25 degrees C. We found that the fraction of salmonellae surviving over time at a given temperature was comparable to the fraction of indicator organisms that survived. We also found that the survival of these organisms varied with temperature, with 16, 8, and < 2 days being required for a 90% reduction of Salmonella in waste wash water held at 5, 15, and 25 degrees C, respectively. Finally, we noted that the response of laboratory-derived cultures to environmental stresses mimics the response of the indigenous microbial population, but individual cells within that population may survive for longer periods than laboratory-cultured strains.

Aims: The research was initiated to confirm earlier ultraviolet (u.v.) light inactivation studies performed on Giardia cysts using excystation as the viability indicator. Following this, a comparison of in vitro excystation and animal infectivity was performed for assessing cyst viability after exposure to low-pressure u.v. irradiation., Methods and Results: Cysts of Giardia muris were inactivated using a low-pressure u.v. light source. Giardia muris was employed as a surrogate for the human pathogen Giardia lamblia. Cyst viability was determined by both in vitro excystation and animal infectivity. Cyst doses were counted using a flow cytometer for the animal infectivity experiments. Using in vitro excystation as the viability indicator, fluences as high as approximately 200 mJ cm(-2) did not prevent some cysts from excysting, thus verifying earlier work. Using animal infectivity, u.v. fluences of 1.4, 1.9 and 2.3 mJ cm(-2) yielded log10 reductions ranging from 0.3 to >or= 4.4., Conclusions: Results indicate that in vitro excystation is not a reliable indicator of G. muris cyst viability after u.v. disinfection. Very low doses of u.v. light rendered G. muris cysts non-infective in the mouse model employed., Significance and Impact of the Study: Data presented represent the only complete u.v. inactivation curve for G. muris. This research provides evidence that u.v. can be an effective barrier against Giardia spp. in the treatment of drinking water supplies.

Cryptosporidium parvum, a protozoan parasite, has been implicated in a number of waterborne disease outbreaks. It is difficult to inactivate using free chlorine, but appears to be easily inactivated by ozone. Therefore, the US EPA has promulgated the Interim Enhanced Surface Water Treatment Rule, which for the first time, addresses the control of C. parvum in drinking water. The use of Ct (concentration of disinfectant in mg/L times, time in minutes) values is being considered as one of the options for controlling this organism. This paper proposes a Ct equation, based on first order kinetics, to provide guidance to drinking water utilities for the application of ozone for controlling C parvum oocysts in drinking water. The equation, which provides mean estimates of inactivation, was developed using standard statistical techniques and currently available field and bench scale data. In addition, the possibility of using a statistically conservative upper bound Ct value in order to insure an appropriate safety factor is explored.

Previous reports indicate that Escherichia coli O157:H7, Salmonella spp., and Vibrio cholerae can grow in nutrient-limited, reconditioned wastewater over the temperature range of 4 to 46 degrees C when the biological oxygen demand of this water is <2, while its coliform growth response (CGR) is >2. In the current study, we investigated the growth response of Vibrio parahaemolyticus, Shigella spp., Vibrio vulnificus, and Pseudomonas aeruginosa in water samples with a CGR of >2 over the temperature range of 4 to 50 degrees C. Both the nonselective media, tryptic soy agar, and the selective media used to identify the pathogen were used for their recovery. The selective media were thiosulfate-citrate-bile-sucrose (TCBS), MacConkey agar (MAC), and Pseudomonas isolation agar (PIA) for the Vibrio, Shigella, and Pseudomonas spp., respectively. V. parahaemolyticus numbers declined rapidly after surviving for 6 days under the nutrient-limiting growth conditions. Shigella spp. did not grow but survived for >28 days at 4 to 25 degrees C. V. vulnificus grew over the narrow temperature range of 12 to 21 degrees C and survived for >21 days at the higher and lower temperature ranges. P. aeruginosa survived and grew during the 14-day test period at 13 to 35 degrees C. Recovery on the nonselective agar gave statistically (P > 0.05) higher numbers than the respective selective media commonly used for these pathogens. These results indicate that caution should be used in attempting direct recoveries using selective media of the four gram-negative bacteria species used in this study from the nutrient-limited water environment.

Cattle drinking water from two dairy farms was used in a study to determine the survival characteristics of the bacterial pathogen Escherichia coli O157:H7 and wild-type E. coli. The E. coli O157:H7 inoculum consisted of a consortium of isolates obtained from dairy cattle. Fresh manure was used as the source for the wild-type E. coli. In the water source from farm 1 the pathogens were present at both 5 and 15 degrees C during the 16-d duration of the study. In the water source from farm 2, the pathogens were detected at 5 degrees C through d 8 and through d 4 at 15 degrees C. The fecal indicator, wild-type E. coli, was always present when the pathogens were present.

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 of Encephalitozoon 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 (TCID(50)) and a minimal infective dose (MID) for E. intestinalis. The TCID(50) 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 system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID(50) 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 degrees 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 log(10) 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.

Public health protection requires an indicator of fecal pollution. It is not necessary to analyse drinking water for all pathogens. Escherichia coli is found in all mammal faeces at concentrations of 10 log 9(-1), but it does not multiply appreciably in the environment. In the 1890s, it was chosen as the biological indicator of water treatment safety. Because of method deficiencies, E. coli surrogates such as the 'fecal coliform' and total coliforms tests were developed and became part of drinking water regulations. With the advent of the Defined Substrate Technology in the late 1980s, it became possible to analyse drinking water directly for E. coli (and, simultaneously, total coliforms) inexpensively and simply. Accordingly, E. coli was re-inserted in the drinking water regulations. E. coli survives in drinking water for between 4 and 12 weeks, depending on environmental conditions (temperature, microflora, etc.). Bacteria and viruses are approximately equally oxidant-sensitive, but parasites are less so. Under the conditions in distribution systems, E. coli will be much more long-lived. Therefore, under most circumstances it is possible to design a monitoring program that permits public health protection at a modest cost. Drinking water regulations currently require infrequent monitoring which may not adequately detect intermittent contamination events; however, it is cost-effective to markedly increase testing with E. coli to better protect the public's health. Comparison with other practical candidate fecal indicators shows that E. coli is far superior overall.

The pathogen Escherichia coli O157:H7 has been recovered from various water sources and food samples. The growth potential of this bacterium in nutrient-limited, reconditioned wastewater from a pork-processing plant was determined over a temperature range of 4 to 46 degrees C. Even though the biological oxygen demand of the wastewater was <2 mg/liter, results of bioassays for assimilable organic carbon and the coliform growth response of the water suggested that sufficient nutrients were present to support limited bacterial growth. A three-strain mixture of E. coli O157:H7 grew over the temperature range of 10.2 to 29.4 degrees C. Bioassays appear to be a good indicator of the ability of this wastewater to support growth of this pathogen. Statistically higher levels of bacterial growth (P < 0.05) were detected on a nonselective medium (tryptic soy agar) than on a selective medium (sorbitol-MacConkey agar), suggesting that stress or injury of the bacterium occurs when the organism is exposed to the nutrient-limited conditions of the wastewater. These results indicate that E. coli O157:H7 can survive and grow in this particular nutrient-limited wastewater, suggesting a potential hazard if this water becomes contaminated with this pathogen.

The electrophoretic mobilities (EPMs) of a number of Escherichia coli O157:H7 and wild-type E. coli strains were measured. The effects of pH and ionic strength on the EPMs were investigated. The EPMs of E. coli O157:H7 strains differed from those of wild-type strains. As the suspension pH decreased, the EPMs of both types of strains increased.

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.

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.

[This corrects the article on p. 3350 in vol. 62, PMID: 8795225.].

Three strains of Helicobacter pylori were studied to determine their resistance to chlorination. The organisms were readily inactivated by free chlorine and should therefore be controlled by disinfection practices normally employed in the treatment of drinking water.