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Effect of disinfectant exposure on Legionella pneumophila associated with simulated drinking water biofilms: release, inactivation, and infectivity Yun Shen, Conghui Huang, Jie Lin, Wenjing Wu, Nicholas J. Ashbolt, Wen-Tso Liu, and Thanh H. Nguyen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04754 • Publication Date (Web): 13 Jan 2017 Downloaded from http://pubs.acs.org on January 15, 2017

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Environmental Science & Technology

Biofilms developed under disinfectant exposure Stiff

Biofilms developed under disinfectant lacking condition Biofilm Development

Soft

L. pneumophila Associated with Biofilms Disinfectant Flush

L. pneumophila Released from Biofilms under Flow

Disinfectant Flush

Released L. pneumophila: Lower inactivation and higher infectivity

Released L. pneumophila: Higher inactivation and lower infectivity ACS Paragon Plus Environment


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Effect of disinfectant exposure on Legionella pneumophila associated with simulated

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drinking water biofilms: release, inactivation, and infectivity

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Yun Shen1*, Conghui Huang1, Jie Lin1, Wenjing Wu1, Nicholas J. Ashbolt2, Wen-Tso Liu1,

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Thanh H. Nguyen1

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1

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Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign, School of Public Health, University of Alberta, AB T6G 2G7 Canada.

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

* Present Address: 1351 Beal Ave., 219 EWRE Bldg. Ann Arbor, MI 48109-2125. Phone: +1 (217) 898 1087. Email: [email protected]

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Abstract

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Legionella pneumophila, the most commonly identified causative agent in drinking water

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associated with disease outbreaks, can be harbored by and released from drinking water

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biofilms. In this study, the release of biofilm-associated L. pneumophila under simulated

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drinking water flow containing a disinfectant residual was examined. Meanwhile, the

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inactivation and infectivity (to amoebae) of the released L. pneumophila were studied. To

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simulate drinking water system conditions, biofilms were prepared under either

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disinfectant exposure (pre-disinfected biofilms) or disinfectant-free (untreated biofilms)

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conditions, respectively. For experiments with water flow containing a disinfectant to

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release the biofilm-associated L. pneumophila from these two types of biofilms, the L.

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pneumophila release kinetics from pre-disinfected and untreated biofilms under flow

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condition were not statistically different (One-way ANOVA, p>0.05). However,

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inactivation of the L. pneumophila released from pre-disinfected biofilms was 1-2 times

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higher and amoeba infectivity was 2-29 times lower than that from untreated biofilms. The

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higher disinfectant resistance of L. pneumophila released from untreated biofilms was

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presumably influenced by the detachment of a larger amount of biofilm material

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(determined by 16S rRNA qPCR) surrounding the released L. pneumophila. This study

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highlights the interaction among disinfectant residual, biofilms, and L. pneumophila, which

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provides guidelines to assess and control pathogen risk.

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Introduction

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From 2014-2015, a serious Legionnaires’ disease outbreak occurred in Flint, MI, causing

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87 illness cases and 11 deaths (data collected from June 2014 to March 2015 and May 2015

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to October 2015).1 At the same time, Legionella pneumophila, the causative agent of

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Legionnaires’ disease, was detected in half of the 60 premise plumbing water samples

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collected at two hospitals in Flint.2 Although the link between L. pneumophila in drinking

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water and the Legionnaires’ outbreak has not been identified yet in Flint, L. pneumophila

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was the most commonly reported pathogen causing drinking water disease outbreaks in the

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United States.3-6 From 2011 to 2012, L. pneumophila led to 21 drinking water outbreaks,

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contributing to 66% of the total reported disease outbreaks associated with drinking water

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in the United States.6 Although disinfectant residual is required in most drinking water

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systems by the U.S. Environmental Protection Agency (USEPA), L. pneumophila is known

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to be more resistant to disinfectants when associated with drinking water biofilms.7-13

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Biofilms in drinking water distribution or premise plumbing systems can accumulate L.

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pneumophila and provide nutrients for its growth.12,

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pneumophila cells can be protected from disinfection because biofilm extracellular

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polymeric substance (EPS) consumes disinfectant and reduces disinfectant permeation.9, 15

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Subsequently, L. pneumophila cells may be released to the drinking water with sloughed

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off biofilms and reach consumers. Therefore, biofilm protection and the efficacy of

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disinfectants are two key factors determining the health risk of L. pneumophila released

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into drinking water. Furthermore, mechanistic understanding of the interactions among

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biofilms, disinfectants, and L. pneumophila is critical for developing Legionnaires’ disease

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control strategies.

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These biofilm-associated L.


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Biofilm microbial composition and disinfectant types were reported to influence the

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disinfection efficacy of biofilm-associated L. pneumophila and other pathogens.16-22 For

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example, biofilms composed by single species (Microbacterium phyllosphaerae or L.

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pneumophila) were shown to be less tolerant to hydrogen peroxide or dendrimer exposure

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than mixed-species biofilms.21,

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distribution systems (DWDS) could shift their community composition to resist

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disinfectant exposure23,

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pathogens. Also, compared to free chlorine, monochloramine had slower reactivity but

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diffused deeper into biofilms, thus monochloramine could provide better biofilm

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disinfection efficiency.19,

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associated pathogen disinfection under quiescent conditions,16-22 ignoring possible effects

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associated with drinking water flow conditions. Flow is an important consideration for

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biofilm-associated pathogens disinfection, as shear stress brings disinfectants to the biofilm

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surface and may enhance the flux of disinfectants into biofilms.25 Meanwhile, shear stress

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may lead to higher release of biofilm-associated pathogens.14 Overall, disinfection efficacy

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during concurrent release of biofilm-associated pathogens under continuous flow

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conditions appears to be unreported. Moreover, in a real drinking water system, non-

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uniform exposure of biofilms to disinfectants may exist because of disinfectant

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consumption (by the biomass, natural organic matter, or pipe corrosion materials), altered

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temporal and spatial flow rate, and different water system operation. Furthermore, biofilms

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grown under different disinfectant exposure conditions could have different chemical

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compositions and mechanical properties, providing variation in the protection of biofilm-

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associated pathogens.26-28 How disinfectant residual exposure during biofilm development

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20

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These mixed-species biofilms in drinking water

and thus maintain their protection for biofilm-associated

These previous studies have largely investigated biofilm-



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influences the subsequent biofilm-associated pathogen release and disinfection is also

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unknown. What is known, however, is that planktonic L. pneumophila cells were still able

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to infect Acanthamoeba castellanii four months after the commencement of

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monochloramine treatment.29 Yet the infectivity of released biofilm-associated pathogens

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in the presence of residual disinfectants is poorly documented and confounded by methods

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available to detect viable versus active cells.30

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Here we present a comprehensive study to understand the interactions among biofilms,

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disinfectants (free chlorine vs. monochloramine), and L. pneumophila. The biofilms used

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in this study were developed under simulated DWDS conditions in the presence or absence

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of a disinfectant residual. The L. pneumophila release process in drinking water systems

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was simulated by continuously flowing the water containing disinfectant over the biofilms

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harbored with L. pneumophila. The released samples were then collected for further

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analysis of L. pneumophila release kinetics, inactivation, and infectivity. The effect of pre-

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disinfecting biofilms and disinfectant types was then identified, accordingly. The results of

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this study will provide guidelines to assess the potential health risks of L. pneumophila in

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drinking water.

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Materials and Methods

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Biofilm preparation

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Biofilms used in this study were developed from sand filtered groundwater, a source of

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drinking water in Urbana–Champaign, IL. This groundwater source has pH of 7.5 and

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hardness of 280 mg/L. The metals, TOC, and TDS in this groundwater source were shown

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in the Supporting Information (SI, Table S1). To remove excessive mineral precipitates


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formed when the groundwater was exposed to air, the groundwater was pre-treated by sand

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filtration. As described previously,14, 31 this groundwater was continuously introduced into

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CDC reactors (CBR 90-2, BioSurface Technologies Corporation, Bozeman, MT) that were

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stirring at 125 rpm or a Reynold number of 2384, so as to develop biofilms on PVC coupons

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(RD 128-PVC, BioSurface Technologies Corporation, Bozeman, MT). No external

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bacteria or nutrients were intentionally added to the CDC reactors. The biofilms were

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grown in the CDC reactor for one year before disinfection. The one-year old biofilms had

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the thickness of 120±8 m.26

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After one year of growing biofilms with disinfectant-free groundwater, PVC biofilm

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coupons were then distributed across three CDC reactors for a further six months of

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disinfectant/disinfectant-free exposure (Figure 1). Specifically, groundwater amended with

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monochloramine or free chlorine (4 mg Cl2·L-1) was continuously introduced to Reactors

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1 and 2, respectively. The pH of disinfectant-containing groundwater was 7.5. The total

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chlorine and free chlorine concentration was determined by a DR 2700™ Portable

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Spectrophotometer according to the DPD colorimetric method (Standard Method 4500-Cl

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G). Reactor 3 was only exposed to the disinfectant-free groundwater. Reactors 1 and 2 were

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used to simulate high disinfectant exposure conditions for DWDS, while Reactor 3 was

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used to simulate the situation when the disinfectant residual was lacking. The feed

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disinfectant solutions were prepared and replenished every second day. The biofilms from

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these three reactors were imaged by optical coherence tomography (OCT) and shown in

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Figure S1. No significant difference in biofilm thickness was observed (p>0.05).

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Disinfection and release of biofilm-associated L. pneumophila



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To simulate the association of pathogenic L. pneumophila to drinking water biofilms,

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L. pneumophila (ATCC 33152) was pre-adhered to monochloramine-treated (Reactor 1),

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free-chlorine-treated (Reactor 2), and untreated (Reactor 3) biofilms using a parallel plate

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flow chamber (FC 71, BioSurface Technologies Corporation, MT), as described

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previously.13 Before each experiment, one biofilm coupon was carefully removed from the

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CDC reactor and inserted into the flow chamber. Groundwater containing 1–5 × 107

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cells∙mL-1 of L. pneumophila was then pumped into the flow chamber at an average flow

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velocity of 0.007 m.s-1 for half an hour. The HRT for the L. pneumophila solution above

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the biofilm coupon was approximately 2s. These adhered cells were then allowed to

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incorporate into the biofilms under quiescent flow conditions for two days before the

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subsequent L. pneumophila release experiments.

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The process of L. pneumophila release from biofilms by the drinking water flow

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containing or lacking disinfectant was simulated in the flow chamber to resemble the varied

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disinfectant residual concentrations. Briefly, the groundwater containing disinfectant or

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groundwater free of disinfectant was introduced into the flow chamber at a flow velocity

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of 0.4 m·s-1 to detach the pre-adhered L. pneumophila from the biofilms (Figure 1). This

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flow velocity is within the range of real drinking water flow velocity.32 Specifically, for

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the monochloramine-treated biofilms, the adhered L. pneumophila in biofilms was

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detached by groundwater containing 0.5 mg Cl2·L-1 monochloramine. Similarly,

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groundwater containing 0.5 mg Cl2·L-1 free chlorine was used to release the L.

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pneumophila from free-chlorine-treated biofilms. As a control experiment, groundwater

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free of disinfectant was also used to release L. pneumophila from the monochloramine-

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treated or free-chlorine-treated biofilms. For the groundwater biofilms without disinfection


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treatment (untreated biofilms), the pre-adhered L. pneumophila cells were released from

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biofilms using groundwater, groundwater containing monochloramine, and groundwater

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containing free chlorine, respectively. For all of the above conditions, the groundwater

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containing or lacking disinfectant was introduced to the flow chamber for thirty minutes.

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The detached samples (containing released L. pneumophila and sloughed off biofilm

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materials) were collected at 1, 2, 3, 4, 5, 10, 20, and 30 min for further analysis of

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L. pneumophila inactivation and infectivity. The disinfectant in collected samples were

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immediately quenched using 5% (w/v) sodium thiosulfate. The L. pneumophila release

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experiment under each condition was repeated three times using biofilm coupons from the

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same reactor. Each L. pneumophila adhesion-release experiment lasted for two days, and

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all the L. pneumophila release experiments were conducted within two and a half weeks.

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Determination of total released L. pneumophila and detached biofilm materials by

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DNA extraction and qPCR

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Sample preparation and DNA extraction

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For determining the number of detached L. pneumophila in the collected samples, DNA of

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the detached sample collected at each time point was extracted for subsequent qPCR

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analysis. Specifically, 2 mL of each detached sample was concentrated to 50 L by

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centrifuging at 17,000 RCF for two minutes and then carefully removing the supernatant.

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DNA was then extracted from these concentrated samples using the Qiagen DNA

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extraction kits (DNeasy Blood & Tissue Kit, Hilden, Germany) following manufacturer’s

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protocol. After extraction, the DNA was stored in the elution solution provided by the

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Qiagen DNA extraction kit at -20℃ until used.



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Determination of total released L. pneumophila

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The total concentration of L. pneumophila (including all of the live cells and disinfected

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injured cells) in the detached samples were then enumerated by qPCR, following the

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previous studies.33, 34 The qPCR reactions were performed in the Applied Biosystems

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7900HT Fast Real-Time PCR system. The mixture used in each qPCR reaction (15 L)

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contained 2 L extracted DNA, 200 nM reverse primer, 200 nM forward primer, and 7.5

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L PowerUp™ SYBR® Green Master Mix (Austin, TX). The reverse and forward primers

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(mip_99F:

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CCGGATTAACATCTATGCCTTG-3’) were targeted in the macrophage infectivity

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potentiator (mip) gene of L. pneumophila, which were designed in a previous study.34 The

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PCR condition used in this study included an initial denaturing step at 95oC for 10 min

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followed by 40 cycles of 95oC for 10s and 60oC for 1 min. In each qPCR run, 10-fold serial

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dilutions of DNA standards (5.8×101-5.8×107 copy number·mL-1) were also amplified to

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create a standard curve. The slope and interception of each standard curve was then used

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to calculate the copy numbers of the target genes in the extracted DNA samples. The

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possible effect of qPCR inhibitors in the extracted DNA sample was excluded by running

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qPCR for a series of diluted extracted DNA samples (Figure S2). The qPCR efficiency was

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ranged from 0.98 to 1.02. The detection limit was from 101 to 107 copy numbers. Since a

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loss of DNA may occur during the DNA extraction process, directly using qPCR results

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(copy numbers) to represent the real concentration of L. pneumophila in the detached

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samples is not accurate. A linear correlation between the qPCR results and the colony-

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forming unit (CFU) of L. pneumophila was determined based on the qPCR results for

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untreated L. pneumophila samples with known CFU (Figure S3). The final concentration

5’-GGATAAGTTGTCTTATAGCATTGGTG-3’


and

mip_172R:

5’-

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of L. pneumophila in each detached sample was reported as CTotal

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(CFUqPCR∙mL-1). The details of the method converting between qPCR and L. pneumophila

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CFU was described previously.33

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Determination of total released bacteria

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The number of total detached (released) biofilm bacteria were quantified by qPCR

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following similar procedures described above. The V3 region of the 16S rRNA gene was

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amplified by forward primer 341F (5’-CCTACGGGAGGCAGCAG-3’) and reverse

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primer 518R (5’-ATTACCGCGGCTGCTGG-3’), as described in a previous study.5 The

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standard curve was generated by serial dilutions (10-fold, 7.7×101-7.7×108 copy

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number·mL-1) of 16S rRNA standards (540bp) through qPCR. To avoid degradation of the

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standards, each set of dilutions was thawed and used immediately after loading all the

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samples. The dilutions of standards were discarded after each thaw. The efficiency of the

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qPCR was in the range of 0.95 to 1.00. The detection limit was from 101 to 108 copy

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numbers.

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Determination of L. pneumophila inactivation

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When the biofilm-associated L. pneumophila were exposed to the flowing groundwater

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containing disinfectant, some released L. pneumophila cells would be injured by the

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disinfectant and become non-culturable. In this study, we defined this process of L.

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pneumophila losing cultivability during disinfection by using the term “inactivation”. The

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amount of culturable L. pneumophila in the detached samples was determined by colony

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counting on the buffered charcoal yeast extract (BCYE) agar plates with 10 g·L-1

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chloramphenicol. The L. pneumophila strain used in our study was introduced with a


released L. pneumophila


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chloramphenicol resistance plasmid (pBG307).14, 35 Therefore, only the L. pneumophila

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cells in the detached samples were likely to grow on the chloramphenicol-contained BCYE

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plates, while other detached microorganisms from the biofilms were unlikely to grow. The

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chloramphenicol concentration of 10 g·L-1 was selected because this concentration did

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not inhibit the growth of L. pneumophila but could still prevent the growth of other bacteria

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that did not carry the chloramphenicol resistance plasmids (based on the results of the

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experiments described in SI). After each L. pneumophila release experiment, serial 10-fold

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dilutions were prepared for each detached sample collected at different time points.

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Aliquots (100 L) of each dilution were then spread-plated on the duplicated BCYE plates.

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After incubating these plates at 37 ℃ for five days, the number of characteristic colonies

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on each plate were counted. The concentration of culturable L. pneumophila in each

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detached sample (CCulturable L. pneumophila) was then determined by averaging these colony

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counting results. In this study, the inactivation of L. pneumophila was represented by the

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ratio of inactivated L. pneumophila over the concentration of total released L. pneumophila,

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as shown below:

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Inactivation ratio = 1 −

𝐶𝐶𝑢𝑙𝑡𝑢𝑟𝑎𝑏𝑙𝑒 𝐿.𝑝𝑛𝑒𝑢𝑚𝑜𝑝ℎ𝑖𝑙𝑎 𝐶𝑇𝑜𝑡𝑎𝑙 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑 𝐿.𝑝𝑛𝑒𝑢𝑚𝑜𝑝ℎ𝑖𝑙𝑎

(1)

(CFU·mL-1) is the concentration of the culturable

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where CCulturable

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L. pneumophila in each detached sample determined by colony counting; CTotal released L.

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-1 pneumophila (CFU·mL )

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and non-culturable L. pneumophila cells) in each detached sample determined by qPCR,

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as aforementioned. As a previous study has shown that some bacterial cells injured by

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disinfection may recover after staying in a disinfectant-free condition for some time,36 the

L.

pneumophila

is the concentration of the total L. pneumophila (including culturable


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possible fluctuation of colony counting results with time was tested for L. pneumophila at

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0, 1, and 2 days after disinfection (see the SI).

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Qualitative estimation of L. pneumophila infectivity

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The infectivity of released L. pneumophila cells was estimated by using Acanthamoeba

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castellanii (A. castellanii, ATCC 30234), an amoeba host of L. pneumophila commonly

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found in natural aquatic environments and water supply systems.37 The method of culturing

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A. castellanii was described previously.38 After each L. pneumophila release experiment,

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500 L of the detached sample was added into 4.5 mL of A. castellanii liquid culture at a

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concentration of 8×104 cells·mL-1 and incubated at 30 ℃ for three days. The total amount

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of L. pneumophila in the mixture was then determined by DNA extraction and qPCR

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analysis. The qPCR results were also converted to a concentration of L. pneumophila

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(CFUqPCR·mL-1) using the linear model shown in Figure S3. The infectivity of L.

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pneumophila was then qualitatively evaluated by the L. pneumophila population (including

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both viable and non-viable L. pneumophila cells) growth through infecting A. castellanii.

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Plate counting was not used in this step. The ratio of L. pneumophila concentration after

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incubating with A. castellanii over the concentration of total released L. pneumophila from

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biofilms was calculated to represent L. pneumophila infectivity:

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Infectivity =

𝐶𝑇𝑜𝑡𝑎𝑙 𝐿.𝑝𝑛𝑒𝑢𝑚𝑜𝑝ℎ𝑖𝑙𝑎 𝑎𝑓𝑡𝑒𝑟 𝑖𝑛𝑓𝑒𝑐𝑡𝑖𝑜𝑛 𝐶𝑇𝑜𝑡𝑎𝑙 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑 𝐿.𝑝𝑛𝑒𝑢𝑚𝑜𝑝ℎ𝑖𝑙𝑎

(2)

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where CTotal L.pneumophila after infection is the final concentration of the L. pneumophila after

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incubating with A. castellanii.

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Results and Discussion 12 ACS Paragon Plus Environment


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L. pneumophila and biofilm material release kinetics from biofilms

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To estimate L. pneumophila release kinetics under continuous flow conditions, the

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concentrations of total released L. pneumophila were normalized by the initial density of

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L. pneumophila on biofilm (SI) and plotted as a function of release time (Figure 2). Figure

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2a shows the normalized concentration of total L. pneumophila (including all live cells and

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disinfected injured cells) released from monochloramine-treated, free-chlorine-treated, and

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untreated biofilms during thirty minutes of groundwater flow. The pre-disinfected biofilms

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(monochloramine- and free-chlorine-treated) used in this study were grown to mimic

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biofilms that develop in the presence of disinfectant-containing fresh drinking water in a

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distribution or premise plumbing system. Conversely, the untreated biofilms simulated

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drinking water biofilms in locations where disinfectant residual is lacking or low, such as

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in stagnant or corroded-pipe zones.39 From all the studied pre-disinfected and untreated

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biofilms, the release of L. pneumophila rapidly reduced at the beginning of release process

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and then leveled off. For example, from the untreated biofilms, the release of

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L. pneumophila decreased from 0.06±0.02 mm-1 to 0.004±0.003 mm-1 in the first five

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minutes. However, from the fifth to the thirtieth minute, the released L. pneumophila was

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not significantly different (0.004±0.003 mm-1 to 0.003±0.003 mm-1, t-test, p=0.54). Also,

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the normalized concentrations of L. pneumophila released from monochloramine-treated,

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free-chlorine-treated, and untreated biofilms had comparable values. For example, at the

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second minute, the total L. pneumophila released from untreated, monochloramine-treated,

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and free-chlorine-treated biofilms was 0.02±0.009 mm-1, 0.03±0.01 mm-1, and

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0.01±0.0005 mm-1, respectively (one-way ANOVA, p=0.24). In summary, pre-

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disinfecting biofilms did not change the L. pneumophila release kinetics.


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In addition to determining the total released L. pneumophila from biofilms, the total

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bacterial 16S rRNA gene copies in the detached samples, which represent the released

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biofilm materials, were also determined (Figure 2b). The change of total released bacterial

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16S rRNA gene copies as a function of time showed similar trend with the change of total

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released L. pneumophila. Specifically, the release of biofilm bacteria decreased

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dramatically at the beginning of release process and then became stable. The total released

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L. pneumophila and released bacteria as a function of time showed similar trends,

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suggesting that the release of L. pneumophila always accompanied the detachment of

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biofilm materials. Previous studies also suggested that sloughing off biofilm materials can

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release the pathogens into drinking water.12, 40 Moreover, by using the disinfectant-free

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groundwater to wash the biofilms, biofilm materials released from monochloramine-

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treated (red triangles) and free-chlorine-treated (green triangles) biofilms were similar.

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However, higher biofilm materials release was observed for the untreated biofilms (grey

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circles) compared with the monochloramine- or free-chlorine-treated biofilms (e.g., at 3

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min, p=0.02, one-way ANOVA). A previous study compared the stiffness of long-term

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disinfected and untreated simulated drinking water biofilms and reported that the biofilms

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were stiffer after long-term disinfectant exposure.26 The stiffer biofilms were expected to

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detach (release) less because of better resistance to flow shear stress.41 Therefore, under

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the same flow velocity (0.4 ms-1), the biofilm materials released from monochloramine-

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or free-chlorine-treated biofilms were lower than those from untreated biofilms.

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Moreover, the release kinetics of L. pneumophila and biofilm materials under different

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types of disinfectant exposures were compared. Figure 3a shows the normalized

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concentration of total L. pneumophila released from untreated biofilms by flowing 14 ACS Paragon Plus Environment


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groundwater, groundwater containing monochloramine, and groundwater containing free

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chlorine as a function of time. Under all the studied disinfectant/disinfectant-free exposure

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conditions, the fatest L. pneumophila release occurred within the first five minutes of

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release process. At most of the time points (1, 2, 5, 10, 15, 20, 30 min), the release of L.

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pneumophila from untreated biofilms under all studied exposure conditions was similar

326

(e.g., at 1 min, p=0.52, one-way ANOVA). However, at the third and the fourth minute,

327

the release of L. pneumophila under monochloramine exposure (e.g., 0.02±0.003 mm-1 at

328

4 min) was higher than those observed under disinfectant-free (e.g., 0.008±0.004 mm-1 at

329

4 min) or free chlorine (e.g., 0.007±0.005 mm-1 at 4 min) exposure conditions. In summary,

330

the exposure to disinfectant did not significantly alter the release kinetics of L.

331

pneumophila from same biofilms.

332

The number of total bacterial 16S rRNA gene copies in the detached samples under

333

different types of disinfectant exposures was also compared (Figure 3b). While the total

334

released bacteria under disinfectant-free and free chlorine exposure conditions was similar,

335

the total released bacteria under monochloramine exposure condition was higher than that

336

under the other two conditions at the third and fourth minute. For example, at the third

337

minute, the total released bacteria under monochloramine exposure was (4.35±0.76)×106

338

copy number·mL-1, which is statistically higher than that under disinfectant-free ((1.55±

339

0.53)×106 copy number·mL-1) and free chlorine exposure ((1.66 ± 0.41)×106 copy

340

number·mL-1)

341

monochloramine exposure led to higher L. pneumophila and biofilm material detachment

342

was presumably due to the higher detachment of biofilm clusters occurring at three to four

343

minutes. Previous studies showed that monochloramine can better penetrate into biofilms

conditions

(one-way

ANOVA,

p=0.03).


The

observation

that

Page 17 of 32


344

and thus may lead to higher cell inactivation/release.42,

345

disinfectant-free and free chlorine exposure conditions, higher bacteria release under

346

monochloramine exposure was observed at three to four minutes.

347

Inactivation of L. pneumophila released from biofilms

348

By using the groundwater flow containing disinfectant to release the biofilm-associated L.

349

pneumophila from biofilms, some L. pneumophila cells were inactivated. The inactivation

350

of L. pneumophila released from biofilms under disinfectant exposure was qualitatively

351

assessed by the ratio of non-culturable L. pneumophila to the total released L. pneumophila

352

(Figure 4). Under all examined conditions, the L. pneumophila inactivation as a function

353

of time revealed two regimes. At the beginning of L. pneumophila release process, the L.

354

pneumophila inactivation ratio increased rapidly. For example, under monochloramine

355

exposure, the ratio of inactivated L. pneumophila released from untreated biofilms (red

356

circles in Figures 4a and 4b) increased from 0.43±0.11 to 0.90±0.05 in the first five minutes.

357

However, with longer disinfectant exposure, the change of inactivation ratio leveled off.

358

The ratio of inactivated L. pneumophila under monochloramine exposure changed from

359

0.90±0.05 to 0.95±0.06 during the fifth to thirtieth minute. The observation of these two

360

regimes could be explained by the L. pneumophila and biofilm materials detachment

361

process. During the first five minutes, superficial L. pneumophila on the biofilm surface

362

was continuously exposed to disinfectant and detached, thus a rapid increase of the

363

inactivation ratio was observed. However, with longer exposures to the flowing

364

groundwater containing monochloramine, the L. pneumophila incorporated into the inner

365

layer biofilms started to be exposed to disinfectant and detached. Those newly exposed L.


43

Therefore, compared to


366

pneumophila cells were not significantly inactivated, thus the inactivation ratio did not

367

dramatically increase from the fifth to the thirtieth minute.

368

By using the flowing groundwater containing same disinfectant to release biofilm-

369

associated L. pneumophila, the inactivation ratios of L. pneumophila released from pre-

370

disinfected and untreated biofilms were compared. As shown in Figure 4a, the L.

371

pneumophila released from monochloramine-treated biofilms showed higher inactivation

372

ratios than that from untreated biofilms. For example, at the first minute, the ratios of

373

inactivated L. pneumophila released from untreated biofilms was 0.43±0.11, which was

374

statistically lower (t-test, p=0.04) than that from monochloramine-treated biofilms

375

(0.83±0.03). Comparison on the inactivation of L. pneumophila released from untreated

376

biofilms and free-chlorine-treated biofilms also showed a similar trend (Figure S4). The

377

lower inactivation ratios for L. pneumophila released from untreated biofilms suggested

378

that L. pneumophila associated with untreated biofilms had better resistance to the

379

disinfectants. As shown in Figure 2b, higher detachment of biofilm materials was observed

380

for the untreated biofilms than the pre-disinfected biofilms. For those L. pneumophila

381

released together with other detached biofilm materials, these biofilm materials could

382

protect L. pneumophila from disinfection by limiting the penetration of disinfectant.44, 45

383

Therefore, higher biofilm materials detachment from untreated biofilms caused the lower

384

ratios of inactivated L. pneumophila released from untreated biofilms. Although

385

disinfectant residual was reported to take limited effect on biofilm removal in distribution

386

or premise plumbing systems,26 exposing biofilms to disinfectants could reduce the risk of

387

L. pneumophila release when L. pneumophila spike occurs.


Page 18 of 32

Page 19 of 32


388

In addition to comparing the L. pneumophila inactivation associated with different

389

types of biofilms, the inactivation ratios of L. pneumophila released under different species

390

of disinfectant exposure were compared. The inactivation ratio of released L. pneumophila

391

from untreated biofilms under monochloramine and free chlorine exposures were not

392

significantly different (Figure 4b, t-test at each time point, all p>0.05). Whereas previous

393

studies reported monochloramine as a stronger biofilm disinfectant, by penetrating

394

biofilms more than free chlorine,42 free chlorine inactivated non-biofilm-associated

395

bacteria more effectively than monochloramine.46 In this study, our qualitative assessment

396

of inactivation and similar effects of either disinfectant may be explained by the overall

397

effect of biofilm-associated and non-biofilm-associated L. pneumophila inactivation.

398

Infectivity of L. pneumophila released from biofilms

399

The infectivity of L. pneumophila was represented by the amplification of L.

400

pneumophila population after infecting A. castellanii. The infectivity of L. pneumophila

401

released from untreated biofilms and pre-disinfected biofilms was compared in Figures 5a

402

and S4. By using monochloramine-contained groundwater to release the L. pneumophila

403

cells from biofilms, the infectivity of L. pneumophila released from untreated biofilms was

404

significantly higher than that released from the monochloramine-treated biofilms (Figure

405

5a). For example, at the end of the L. pneumophila release experiment, the infectivity of L.

406

pneumophila released from untreated biofilms was 6.0±0.6, statistically higher than the

407

infectivity of 0.2±0.1 for L. pneumophila released from the monochloramine-treated

408

biofilms (t-test, p=0.04). Comparison on the infectivity of L. pneumophila released from

409

untreated biofilms and free-chlorine-treated biofilms also showed a similar trend (Figure

410

S5). The higher infectivity of L. pneumophila released from untreated biofilms agreed with 18 ACS Paragon Plus Environment


411

the lower inactivation ratio of L. pneumophila released from same biofilms, further

412

suggesting that untreated biofilms can better protect L. pneumophila from disinfection.

413

The infectivity of L. pneumophila released from untreated biofilms under

414

monochloramine, free chlorine, and disinfectant-free exposure conditions was compared

415

in Figure 5b. Under the disinfectant-free exposure condition, the L. pneumophila infectivity

416

at each time point was statistically the same (one-way ANOVA, p=0.98), suggesting that

417

the infectivity of L. pneumophila released from biofilms did not change significantly with

418

time. Specifically, the infectivity was 20±6 at the thirtieth minute, which was constant with

419

the infectivity of 19±5 at the first minute. Compared with the infectivity of released L.

420

pneumophila under disinfectant-free exposure, the L. pneumophila infectivity under

421

disinfectant exposures was significantly lower (t-test, p0.05) and showed a similar trend as a function of time.

424

Specifically, under both monochloramine and free chlorine exposure conditions, the

425

infectivity showed a decrease in the first five minutes, indicating that the overall L.

426

pneumophila population amplification after infecting A. castellanii gradually decreased.

427

The infectivity then became stable from the fifth to the thirtieth minute under

428

monochloramine or free chlorine exposure. In addition, at the thirtieth minute of the

429

monochloramine and free chlorine exposure, (4±2) % and (7±2) % of the total L.

430

pneumophila cells released from untreated biofilms were still culturable, respectively

431

(Figure 4b). Thus, compared with the L. pneumophila released under non-disinfectant

432

condition, the ratio of culturable L. pneumophila cells was twenty five and seventeen times

433

lower under monochloramine and free chlorine exposure, respectively. However,


Page 20 of 32

Page 21 of 32


434

infectivity of L. pneumophila released under monochloramine and free chlorine exposure

435

was only four and three times lower, respectively, than that without disinfectant present

436

(Figure 5b). Therefore, some non-culturable L. pneumophila cells also contributed to the

437

L. pneumophila population growth after infecting A. castellanii. In other words, part of the

438

non-culturable L. pneumophila cells were viable but non-culturable (VBNC) and still had

439

the ability of infecting A. castellanii. The VBNC state was also reported for the planktonic

440

L. pneumophila treated with monochloramine, free chlorine, and heat shock.47-49 The

441

existence of VBNC L. pneumophila released from biofilms under disinfectant exposure

442

suggested that the infectivity of L. pneumophila released from biofilms may not be

443

sufficiently reduced even under disinfectant exposures. When the drinking water

444

disinfectant does not inactivate the biofilm-associated L. pneumophila, some of the L.

445

pneumophila cells would still be healthy, and other cells would turn to a non-viable state

446

and VBNC state (patricianly injured). If amoebae are present in the DWDS system, both

447

healthy and VBNC L. pneumophila cells can infect their host amoebae and replicate inside

448

the amoebae. This L. pneumophila replication process will produce more L. pneumophila

449

with higher virulence, and thus increase L. pneumophila risk and cause more human health

450

concerns.

451

In summary, disinfectant type (free chlorine vs. monochloramine) had no apparent

452

effect on L. pneumophila release nor disinfection. Real drinking water biofilms would be

453

expected to develop with and without disinfectant exposure, with released pathogens

454

becoming inactivated in flowing drinking water containing disinfectant. Our study

455

indicated that compared with the biofilms developed under a disinfectant-free condition,

456

the long-term disinfected biofilms provided less protection for the biofilm-associated



457

pathogens. Although the long-term disinfectant exposure was shown to not effectively

458

remove biofilm biomass,15, 26 the pre-exposing of biofilms to disinfectant could alter the

459

biofilm properties, thus reducing the regrowth and infection risk of pathogens released

460

from biofilms. Secondly, the exposure to free chlorine and monochloramine did not show

461

any obvious difference on inactivating biofilm-associated L. pneumophila. While free

462

chlorine is a stronger and faster-acting oxidizer, it diffuses less into biofilms than

463

monochloramine,42, 43 thus the inactivation efficiency for biofilm-associated pathogens in

464

drinking water distribution systems is a combined effect of disinfectant oxidation strength

465

and diffusion ability. Finally, the results of this study revealed that part of the inactivated

466

L. pneumophila cells could still infect and propagate within amoebae. Therefore, even

467

though the L. pneumophila was inactivated by disinfectant in drinking water, the risk of L.

468

pneumophila reviving in drinking water distribution systems and infecting the human body

469

should be further studied.

470

Supporting Information

471

In support of the methods and results for this study, the SI mainly includes: method

472

description for normalization of L. pneumophila release, the OCT image of biofilms used

473

in this study (Figure S1), qPCR inhibition test (Figure S2), correlation between qPCR and

474

CFU counting (Figure S3), inactivation and infectivity of biofilm-associated L.

475

pneumophila released under free chlorine (Figures S4 and S5), fluorescence microscope

476

image of L. pneumophila adhered on biofilms (Figure S6), groundwater and tap water

477

quality parameters (Tables S1 and S2).

478

Acknowledgements


Page 22 of 32

Page 23 of 32


479

This publication was made possible by research supported by grant R834870 (agreement

480

number RD-83487001) from the U.S. Environmental Protection Agency (EPA). Its

481

contents are solely the responsibility of the grantee and do not necessarily represent the

482

official views of the EPA. Further, the EPA does not endorse the purchase of any

483

commercial products or services mentioned in the publication. We also want to

484

acknowledge Guillermo L. Monroy and Stephen A. Boppart (University of Illinois at Urbana

485

Champaign) for their help in OCT imaging.

486 487



488

References

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42. Lee, W. H.; Wahman, D. G.; Bishop, P. L.; Pressman, J. G., Free chlorine and monochloramine application to nitrifying biofilm: comparison of biofilm penetration, activity, and viability. Environ. Sci. Technol. 2011, 45, (4), 1412-1419. 43. Türetgen, I., Comparison of the efficacy of free residual chlorine and monochloramine against biofilms in model and full scale cooling towers. Biofouling 2004, 20, (2), 81-85. 44. Chen, X.; Stewart, P. S., Chlorine penetration into artificial biofilm is limited by a reaction-diffusion interaction. Environ. Sci. Technol. 1996, 30, (6), 2078-2083. 45. Miller, H. C.; Wylie, J.; Dejean, G.; Kaksonen, A. H.; Sutton, D.; Braun, K.; Puzon, G. J., Reduced efficiency of chlorine disinfection of Naegleria Fowleri in a drinking water distribution biofilm. Environ. Sci. Technol. 2015, 49, (18), 11125-11131. 46. Au, K.-K., Water treatment and pathogen control: Process efficiency in achieving safe drinking-water. IWA Publishing: 2004. 47. Ducret, A.; Chabalier, M.; Dukan, S., Characterization and resuscitation of ‘nonculturable’cells of Legionella pneumophila. BMC Microbiol. 2014, 14, (1), 1. 48. Alleron, L.; Khemiri, A.; Koubar, M.; Lacombe, C.; Coquet, L.; Cosette, P.; Jouenne, T.; Frere, J., VBNC Legionella pneumophila cells are still able to produce virulence proteins. Water Res. 2013, 47, (17), 6606-6617. 49. Epalle, T.; Girardot, F.; Allegra, S.; Maurice-Blanc, C.; Garraud, O.; Riffard, S., Viable but not culturable forms of Legionella pneumophila generated after heat shock treatment are infectious for macrophage-like and alveolar epithelial cells after resuscitation on Acanthamoeba polyphaga. Microb. Ecol. 2015, 69, (1), 215-224.

647 648 649



650 651 652 653 654 655 656

Figure 1 Biofilm preparation and L. pneumophila release experiment process. Biofilms were developed from groundwater for 1 year, and then distributed to three reactors for subsequent six months of disinfectant exposures. Next, the L. pneumophila cells were allowed to adhere and incorporate into biofilms. Finally, these biofilm-associated L. pneumophila cells were released using the groundwater flow containing or lacking disinfectant.

657


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658 659 660 661 662 663 664

Figure 2 Total a) L. pneumophila and b) bacteria released from untreated biofilms, monochloramine-treated biofilms, and free-chlorine-treated biofilms, respectively. The total released L. pneumophila in the y axis of a) was calculated by normalizing the concentration of released L. pneumophila (#/mL) by the initially adhered L. pneumophila on biofilms (#/mm2). The total released biofilm bacteria in the y axis of b) was represented by 16S rRNA qPCR results (copy number·mL-1).

665



666 667 668 669 670 671 672 673 674

Figure 3 Total a) L. pneumophila and b) bacteria released from untreated biofilms as a function of time. The groundwater, groundwater containing monochloramine, and groundwater containing free chlorine were continuously flowing through biofilms and detached L. pneumophila and other bacteria from biofilms, respectively. The total released L. pneumophila in the y axis of a) was calculated by normalizing the concentration of released L. pneumophila (#/mL) by the initially adhered L. pneumophila on biofilms (#/mm2). The total released bacteria in the y axis of b) was represented by 16S rRNA qPCR results (copy number·mL-1).

675 676


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677 678 679 680 681 682

Figure 4 a) Inactivation ratio of L. pneumophila released from untreated and monochloramine-treated biofilms under monochloramine exposure. b) Inactivation ratio of L. pneumophila released from untreated biofilms under monochloramine and free chlorine exposures. The inactivation ratio was defined by the ratio of non-cultivable L. pneumophila to total released L. pneumophila.

683



684 685 686 687 688 689

Figure 5 a) Infectivity of L. pneumophila released from untreated biofilms and monochloramine-treated biofilms under monochloramine exposure. b) Infectivity of L. pneumophila released from untreated biofilms by monochloramine and free chlorine. The infectivity was defined by the ratio of L. pneumophila population after infecting amoeba to L. pneumophila population before infecting amoebae.

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