Persistence of surrogates for high consequence viral and bacterial pathogens in a pilot-scale activated sludge treatment system.

The persistence of high consequence public health pathogens in a wastewater treatment system can significantly impact worker safety, as well as the public and downstream water bodies, particularly if the system is forced to shut down the treatment processes. This study utilizes organism viability to compare the persistence of three pathogen surrogates in wastewater using a pilot-scale activated sludge treatment (AST) system, operated to mimic treatment processes of large-scale plants. Bacillus globigii spores, surrogate for Bacillus anthracis, persisted in the AST system for at least a 50-day observation period leading to a possible steady condition far beyond the solid retention time for sludge particles. MS2 bacteriophage, surrogate for Poliovirus and other non-enveloped enteric viruses, was observed for up to 35 days after introduction, which largely and expectedly correlated to the measured solid retention time. Phi-6 bacteriophage, a surrogate for Ebola virus and other enveloped viruses, was detected for no more than 4 days after introduction, even though the AST system was operated to provide three times slower solids removal than for the other surrogates. This suggests Phi-6 is subject to inactivation under AST conditions rather than physical removal. These results may suggest similar persistence for the surrogated pathogens, leading to appropriate consequence management actions.

Legionella Diversity and Spatiotemporal Variation in The Occurrence of Opportunistic Pathogens within a Large Building Water System.

Understanding Legionella survival mechanisms within building water systems (BWSs) is challenging due to varying engineering, operational, and water quality characteristics unique to each system. This study aimed to evaluate Legionella, mycobacteria, and free-living amoebae occurrence within a BWS over 18-28 months at six locations differing in plumbing material and potable water age, quality, and usage. A total of 114 bulk water and 57 biofilm samples were analyzed. Legionella culturability fluctuated seasonally with most culture-positive samples being collected during the winter compared to the spring, summer, and fall months. Positive and negative correlations between Legionella and L. pneumophila occurrence and other physiochemical and microbial water quality parameters varied between location and sample types. Whole genome sequencing of 19 presumptive Legionella isolates, from four locations across three time points, identified nine isolates as L. pneumophila serogroup (sg) 1 sequence-type (ST) 1; three as L. pneumophila sg5 ST1950 and ST2037; six as L. feeleii; and one as Ochrobactrum. Results showed the presence of a diverse Legionella population with consistent and sporadic occurrence at four and two locations, respectively. Viewed collectively with similar studies, this information will enable a better understanding of the engineering, operational, and water quality parameters supporting Legionella growth within BWSs.

Chlorine and Monochloramine Disinfection of Legionella pneumophila Colonizing Copper and Polyvinyl Chloride Drinking Water Biofilms.

Building water systems promote the regrowth and survival of opportunistic pathogens, such as Legionella pneumophila, especially within biofilms, where most drinking water microbes reside. However, compared to their planktonic form, disinfection efficacy for the biofilm-associated forms of water-based pathogens is unclear. The aim of this study was to determine the effectiveness of free chlorine and monochloramine in the inactivation of biofilm-associated L. pneumophila strain Philadelphia-1 serogroup 1 (LpP1s1). Mature (1.5- to 2-year-old) drinking water biofilms were developed on copper (Cu) and polyvinyl chloride (PVC) slides within biofilm annular reactors, then colonized with LpP1s1 at approximately 4 log10 CFU cm-2 and exposed to 2 mg liter-1 of free chlorine or monochloramine. Ct (disinfectant concentration × time, expressed as mg min liter-1) inactivation values for 2-, 3-, and 4-log10 reductions of planktonic and biofilm LpP1s1 were determined. For planktonic LpP1s1, free chlorine was more effective at inactivation than was monochloramine treatment, and for biofilm-associated LpP1s1, monochloramine was more effective on Cu biofilms while free chlorine was more effective on PVC biofilms. In contrast to monochloramine, free chlorine treatment of Cu and PVC biofilms, negatively impacted LpP1s1 16S rRNA gene transcript levels and may act synergistically with Cu surfaces to further reduce transcript levels. Moreover, LpP1s1 cells shed from biofilms into the bulk water were more resistant to disinfection than were prepared planktonic LpP1s1 cells. Results from this study indicate that biofilm association, disinfectant type, and substratum play an important role in the survival of Legionella pneumophila in building water systems.IMPORTANCE Microbial regrowth within building water systems are promoted by water stagnation, low disinfectant residual, high surface-to-volume ratio, amenable growth temperatures, and colonization of drinking water biofilms. Moreover, biofilms provide protection from environmental stresses, access to higher levels of nutrients, and opportunities for symbiotic interactions with other microbes. Disinfectant efficacy information is historically based on inactivation of pathogens in their planktonic, free-floating forms. However, due to the ecological importance of drinking water biofilms for pathogen survival, this study evaluated the efficacy of two common disinfectants, free chlorine and monochloramine, on Legionella pneumophila colonizing mature, drinking water biofilms established on copper and PVC surfaces. Results showed that inactivation was dependent on the disinfectant type and biofilm substratum. Overall, this, and other related research, will provide a better understanding of Legionella ecological stability and survival and aid policy makers in the management of exposure risks to water-based pathogens within building water systems.

Decontamination of Bacillus spores adhered to iron and cement-mortar drinking water infrastructure in a model system using disinfectants.

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 log10 from iron and cement-mortar, respectively. Log10 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 log10 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 log10 on cement-mortar.

Disinfection of Bacillus spores with acidified nitrite.

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.

Germinant-enhanced decontamination of Bacillus spores adhered to iron and cement-mortar drinking water infrastructures.

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.

Persistence of non-native spore forming bacteria in drinking water biofilm and evaluation of decontamination methods.

Persistence of Bacillus globigii spores, a surrogate for Bacillus anthracis, was studied on biofouled concrete-lined slides in drinking water using biofilm annular reactors. Reactors were inoculated with B. globigii spores and persistence was monitored in the bulk and biofilm phases, first in dechlorinated water and later with free chlorine concentrations of 1 and 5 mg/L. In the dechlorinated study, a steady state population of spores developed on the slides. The addition of free chlorine at 5 mg/L decreased the adhered spore density by 2-logs within 4 hours and spores were not detected after 67 and 49 hours in the presence of 1 and 5 mg/L free chlorine, respectively. This suggests that adhered spores can persist in non-chlorinated conditions, but detach and/or are inactivated upon addition of free chlorine. When injected into a chlorinated reactor, adhered spore density continually decreased and spores were either undetectable or unquantifiable by 48 hours for both 1 and 5 mg/L chlorine concentrations. Results from these experiments suggest that the presence of a free chlorine residual limits adherence of viable spores to biofouled concrete-lined pipe walls by inactivating spores before they have attached. Both free chlorine concentrations (1 and 5 mg/L) were equally effective at inactivating spores in terms of log reduction, but the higher concentrations yielded faster rates of log reduction.

Persistence and decontamination of surrogate radioisotopes in a model drinking water distribution system.

Contamination of a model drinking water system with surrogate radioisotopes was examined with respect to persistence on and decontamination of infrastructure surfaces. Cesium and cobalt chloride salts were used as surrogates for cesium-137 and cobalt-60. Studies were conducted in biofilm annular reactors containing heavily corroded iron surfaces formed under shear and constantly submerged in drinking water. Cesium was not detected on the corroded iron surface after equilibration with 10 and 100mgL(-1) solutions of cesium chloride, but cobalt was detected on corroded iron coupons at both initial concentrations. The amount of adhered cobalt decreased over the next six weeks, but was still present when monitoring stopped. X-ray absorption near-edge spectroscopy (XANES) showed that adhered cobalt was in the III oxidation state. The adsorbed cobalt was strongly resistant to decontamination by various physicochemical methods. Simulated flushing, use of free chlorine and dilute ammonia were found to be ineffective whereas use of aggressive methods like 14.5M ammonia and 0.36M sulfuric acid removed 37 and 92% of the sorbed cobalt, respectively.

Persistence and decontamination of Bacillus atrophaeus subsp. globigii spores on corroded iron in a model drinking water system.

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.

Persistence of Klebsiella pneumoniae on simulated biofilm in a model drinking water system.

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.