Monochloramine for controlling Legionella in biofilms: how much we know?
Maria Anna Coniglio1, Stefano Melada2, Mohamed H Yassin3
¡°G.F. Ingrassia¡± ¨C Hygiene and Public Health,
of Legionella in water system is a
challenge especially when biofilm is present. Hospitals, in particular, deal
with vulnerable population requiring additional protection against Legionella. Monochloramine (MC) has been
used for small-scale hospital systems in Europe and the
Legionella | monochloramine | biofilm control | disinfection
On surfaces exposed to water a majority of microorganisms tend to form a ¡®biofilm¡¯, which is composed of extracellular polymeric substances (EPS) excreted by the microorganisms themselves. In a long distribution system, with wide variations in flow rates, biofilm would eventually be produced due to the sedimentation of organic particles. Because biofilms may contain enough bacteria to give an infective dose, they represent a potential health risk especially in hospitals, where patients constitute a vulnerable population. Furthermore, biofilms on distribution pipes contribute to bacterial regrowth and disinfectant decay in water.
Legionellae are ubiquitous and they could live as free-living planktonic forms or as intracellular parasites of protozoans (Hsu et al. 2011). The organisms are also able to survive chlorination and thus enter water supply systems and proliferate in humid thermal habitats, including air conditioners, water systems, cooling towers, water fountains and hospital equipments. Legionellae are frequently found in biofilms on the surfaces of these systems, where they are more resistant to eradication compared to their free-living planktonic counterparts (Lin et al. 2011).
One of the key issues for controlling the growth of legionellae in biofilms is to recommend an effective disinfection method. Two main factors interfere with the activity of antimicrobial agents: (i) they may fail to penetrate through the EPS or bind to it before they reach legionellae (Simões et al. 2010); (ii) legionellae may become resistant to conventional chemical antimicrobials (Cooper and Hanlon 2010).
present, chlorination is the most commonly used treatment for Legionella control in water systems.
Lapses in chlorination or discontinuous chlorination with chlorine or chlorine
dioxide can lead to an increased resistance of
biofilm bacteria to chlorine (Casini et
al. 2008). Copper-silver ionization is currently used for Legionella control in water distribution
systems and there is evidence that accumulation of copper and silver inside the
biofilms is responsible for the prolonged bactericidal effect (Liu et al. 1998). Nevertheless, in
Among the antimicrobial agents of relatively most recent application in the disinfection of water, monochloramine (MC) seems to be more effective for decreasing Legionella within the biofilms in vitro (Lee et al. 2010) and in hospital plumbing systems in the US (Kandiah et al. 2012) as well as in Italy (Marchesi et al. 2012; Casini et al. 2014). The aim of this review is to assess the available literature that supports the effectiveness of MC for controlling Legionella growth in biofilms.
Search for literature was carried out using terms: ¡®Legionella¡¯ OR ¡®Legionellosis¡¯ OR ¡®Legionnaires¡¯ Disease¡¯ AND ¡®Biofilm¡¯ AND ¡®Disinfection NEAR Legionella¡¯ AND ¡®Monochloramine¡¯. The search, including published papers between 1980 and 2013, was conducted in relevant chemical and biomedical databases: ACS Publications, Elsevier, JSTOR, Nature Publishing Group, PubMed, SDOS and Wiley Online Library. Considering the relatively new application of MC, non published research, letters and conference communications were also included. Inclusion criteria were: studies about the effectiveness of MC under in vitro conditions, and studies with more than three months of follow up in real conditions. The literature review has been divided in five main parts to evaluate the following aspects of the action of MC: (i) biofilm penetration, residual concentrations and production of disinfection by-products; (ii) influence of pipe material on biofilm formation and disinfectant penetration; (iii) effect of nitrification on decomposition rate; (iv) MC treatment and viable but nonculturable (VBNC) L. pneumophila in biofilms; (v) effect of protozoa on MC disinfection of biofilm.
After this initial search, 125 articles were identified for further review. Each article was evaluated with respect to the use of MC, if the technology was adequately tested for validity and/or accuracy against biofilm, and if the management of the water distribution system was associated with the control of Legionella. After this initial process, 32 articles were considered. Of those, 1 was a review article (Simões et al. 2010) and the others were surveys.
Table 1 shows a summary of the main studies on the effectiveness of MC against the biofilm.
Table 1. Description of noteworthy studies on effectiveness obtained through the application of MC
Table 2. Description of noteworthy studies comparing the effects of MC vs. other oxidative disinfectants
Biofilm penetration, residual concentrations and production of disinfection by-products
Despite its relatively low oxidative activity, MC is more effective than the other oxidative disinfectants (e.g. chlorine and chlorine dioxide). The ability of MC to better penetrate biofilms follows a dose-dependent effect. Table 2 compares MC to other disinfectants in controlling Legionella.It has been recently demonstrated that MC is able to penetrate biofilms 170 times faster than free chlorine (Pressman et al. 2012) and that even at concentrations as low as 1 ppm it is able to penetrate complex biofilm matrixes like that in cooling towers (van Schalkwyk et al. 2010).
Compared to the other oxidative disinfectants, MC has a longer residual effect in biofilm also at neutral pH (Chen and Stewart 2000). Maintenance of MC residual above 3 mg/L is needed to effectively control biofilm in cooling systems employing secondary-treated municipal wastewater as the only source of makeup water (Chien et al. 2012). Moreover, MC minimally reacts with organic matter but react specifically with bacterial membrane at high oxidant exposures (Ramseier et al. 2011).
The application of chloramines, as well as chlorine, may cause increased formation of highly carcinogenic nitrosamines and other disinfection by-products (DBPs) (Chang et al. 2011). Table 3 summarizes studies describing the effect of MC on DBPs. A recent study investigating the effects of corrosion products of copper in plumbing systems on N-nitrosodimethylamine (NDMA) formation from DMA found that the transformation of MC to dichloramine and complexation of copper with DMA were involved in elevating the formation of NDMA by copper at pH 7.0 (Zhang and Andrews 2013). Anyway, MC generally results in lower concentrations of DBPs compared to the other oxidative disinfectants. It has been recently demonstrated that DBPs levels in filtered river waters, as well as in coagulated surface waters collected from water treatment plants are generally higher after chlorination than after chloramination (Farr¨¦ et al. 2013).
Table 3. Description of noteworthy studies on the production of DBPs through the application of MC*
* DBP:Disinfection byproducts, NDMA: N-nitrosodimethylamine, DMA:Dimethylamine, THM: Trihalomethanes , HAA: halogenated acetic acid
Influence of pipe material on biofilm formation and monochloramine effectiveness
Another factor on formation and persistence within the biofilms of Legionella is the influence of pipe material. Copper piping has been in use in water systems to minimize the risk of Legionellosis because copper may prevent colonization of the pipe and could inhibit the biofilm growth. Anyway, a model system of copper can temporarily reduce Legionella colonization but, after 2 years, biofilm colonization on copper, stainless steel and cross-linked polyethylene (PEX) pipes was very similar (van der Kooij et al. 2005).
The decomposition rate of MC may be enhanced by copper due to the formation of a Cu(II)-humic acid complex (Fu et al. 2009). A laboratory experiment has confirmed that MC could decay rapidly only in the presence of new copper pipes, providing a possible explanation for the rapid disinfectant loss in the new buildings (Nguyen et al. 2012). Nevertheless accumulation of Cu(II) ions could occur also after years of use of copper/silver ionization. Figure 1 shows a longitudinal section of a water pipe after more than 10 years of use of copper/silver ionization. Note the severe corrosion within the pipe wall. Copper corrosion products can thus affect the MC disinfection by affecting its decomposition rate. An adequate chloramination system should be put in place to reduce MC decomposition rate and to avoid accumulation of DBPs.
Figure 1. Longitudinal section of a water pipe after more than 10 years of use of copper/silver ionization. (Picture from Boffardi BP, Hannigan JM. A limited evaluation of pitting corrosion of copper piping in a hospital domestic hot water system using copper-silver ionization for Legionella control. AWT, Mohegan, 2013.)
Effect of nitrification on decomposition rate of monochloramine into biofilm
Chloramination provides a source of ammonia promoting the growth of nitrifying bacteria within the biofilms. Nitrifying bacteria can grow in the presence of MC due to their ammonia and nitrite oxidizing characteristics. Biofilm grown on pipe surfaces can harbor nitrifiers, belonging primarily to Nitrosomonas, Nitrobacter and Nitrosospira (Lipponen et al. 2004). Nitrification in drinking water distribution systems may result in water quality degradation and subsequent noncompliance with existing regulations. Accelerated chloramine decay is related to high levels of nitrification and when nitrification is absent chloramine is expected to better penetrate iron biofilms (Lee et al. 2011). A possible explanation for these evidences is that the presence of high free ammonia concentration due to MC decomposition allows the microorganisms deeper within the biofilm to remain active during MC application. Moreover, once initiated, nitrification is very hard to stop because CT value (product of disinfectant concentration and contact time) is too low in biofilm where the right residual MC cannot be maintained. In fact, limiting the free ammonia concentration during MC application could slow the onset of nitrification episodes by maintaining the biofilm biomass at a state of lower activity (Pressman et al. 2012).
Nonetheless, although there is evidence that maintaining a chloramine residual of at least 1-2 mg/L could be sufficient to limit nitrifier growth in drinking water (Wolfe et al. 1990), it has been shown that greater MC residuals may be required to inactivate bacteria inside the biofilms (Park and Kim 2008).
The degradation of the hypochlorite used to produce MC can lead to an increased level of free ammonia and this can affect the disinfection effectiveness of the in situ produced MC. Increased levels of Legionella occurred for a severe degradation of the hypochlorite reagent despite MC levels were in the desired range and, only after draining and cleaning of the reagent tank, free ammonia and Legionella were reduced to negligible levels. A major US University-based medical system experienced similar issue as high temperature in the storage room caused degradation of hypochlorite (personal data).
Monochloramine treatment and viable but nonculturable (VBNC) Legionella pneumophila in biofilms
After disinfections with chlorine compounds, L. pneumophila can completely lose its cultivability but do not lose viability entering the viable but nonculturable (VBNC) state. Decrease of cell cultivability significantly begins at 1 ppm dosage of MC, while at 1.5-2 ppm environmental L. pneumophila enters VBNC state (T¨¹retgen 2008).
It has also been shown that up to 20 ppm MC concentration, Legionellae enter VBNC state and are still able to synthesize virulence factors. Nonetheless, at this concentration, attempts to resuscitate VBNC cells with amoebas failed (Alleron et al. 2013). This suggests that the accumulation of virulence factors by VBNC cells may not be sufficient to maintain their virulence.
Anyway, disinfectants¡¯ in vitro activity is less effective in field applications and at the moment there is a lack of studies evaluating the effect of long exposures to MC in real water systems.
Effect of protozoa on monochloramine disinfection of biofilm
free-living amoebae (
Up to date, only a few studies have been focused on the effectiveness of MC against pathogenic FLA. Under laboratory conditions the biocidal activity of MC against Naegleria lovaniensis was 8x weaker than that of hypochlorite but 2x stronger than that of peracetic acid (Ercken et al. 2003). This evidence makes MC a good candidate for inactivation of pathogenic Neaegleria species and an ecologically less harmful alternative to hypochlorite.
Moreover, comparison of the efficacy of chlorine, MC and chlorine dioxide against trophozoites of three different Acanthamoeba strains showed that MC was more efficient than chlorine and chlorine dioxide at the same level towards free or co-cultured L. pneumophila (Dupuy et al. 2011). Nonetheless, despite its better effectiveness against amoeba trophozoites, MC appears less effective than chlorine dioxide against Acanthamoeba cysts (Dupuy et al. 2014).
The presence of Legionella within a biofilm makes eradication from water system very difficult. Among the antimicrobial agents, MC seems to be more effective for decreasing Legionella within the biofilms in vitro as well as in model plumbing systems. As of to date there are no published reviews on this topic, a critical and comprehensive update on the progress in the field is necessary.
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Conflict of interest: No conflicts declared.
Correspondence author: Dr. Maria Anna Coniglio
Department ¡°G.F. Ingrassia¡± ¨C Hygiene and Public Health
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