Disinfection characteristics of Pseudomonas peli, a chlorine-resistant bacterium isolated from a water supply network
Graphical abstract
Introduction
Drinking water treatment processes include disinfection of harmful or otherwise objectionable organisms (Mir et al., 2010). The inactivation of pathogenic microorganisms is closely related to human health. Since early in the last century, chlorine has been the commonest disinfectant in drinking water treatment, and has vastly enhanced the biological safety of drinking water. Chlorine has strong oxidation ability that destroys microbial nucleic acids and causes membrane damage, is easy to handle, and requires low investment cost (Anastasi et al., 2013), so it is still the most widely used disinfection method. To guarantee the safety of drinking water, World Health Organization regulatory standards stipulate treatment with a free chlorine concentration of 0.5 mg/L for 30 min (WHO, 2001), and a free chlorine concentration of 0.05 mg/L has been specified as the safety limit of pipeline water in China. However, some bacteria survive and even reproduce in residual chlorine, and are designated “chlorine-resistant bacteria” (Langsrud et al., 2003).
The emergence of chlorine-resistant bacteria results in an immense but hidden danger to the safety of drinking water. Some chlorine-resistant bacteria are pathogenic or conditional pathogens. Once they enter the pipe network, they lead to risk of water-borne infectious diseases, such as Legionella infections in the United States in the 1970s (Pandey et al., 2014). Meanwhile, nonpathogenic chlorine-resistant bacteria that persist in the form of biofilms or suspensions in the pipe network reduce the biological stability of the water supply pipe network, and the proliferation of some microorganisms could affect the color, turbidity or smell of the drinking water (Lehtola et al., 2004; Vreeburg and Boxall, 2007). Removal of chlorine from the water by chlorine-resistant bacteria also causes a series of problems, such as corrosion of the pipeline, nitrification, and growth of pathogens in the water supply network (Zhang et al., 2008).
The composition and concentration of water disinfection methods exerts a selective pressure on the biofilm population, including the number of bacteria present and their diversity (Hong et al., 2013; Roeder et al., 2010). Different growth environments affect the ability of bacteria to resist disinfectants. Thus, the tolerance of chlorine-resistant bacteria is a relative concept rather than fully quantitative. Researchers have found that an ever-increasing number of bacteria in the water supply system have different degrees of resistance to chlorine. The minimal inhibitory concentration of sodium hypochlorite toward Escherichia coli isolated from mice by Ketyi was 10–32 times higher than that toward normal E. coli (Kétyi, 1991). However, the high doses of chlorine disinfectants in drinking water disinfection could easily produce disinfection byproducts (DBPs) (Tsolaki and Diamadopoulos, 2010), such as chlorite, chlorate, trihalomethane, and so on (Al-Otoum et al., 2016). Thus, overall, routine chlorine disinfection may not ensure the safety of drinking water.
The inactivation effect of chlorine dioxide on bacteria is similar to or better than that of chlorine, and it can inactivate bacteria in a relatively wide pH range (Huang et al., 1996). With the same CT value (the product of disinfectant concentration and contact time), the inactivation effect of chlorine dioxide on heterotrophic bacteria was better than that of free chlorine (Gagnon et al., 2004). Studies have confirmed that chlorine dioxide effectively inactivates bacteria (Huang et al., 1996), viruses (Min et al., 2013), fungal spores (Wen et al., 2017), chlorine-resistant protozoa (such as Giardia and Cryptosporidium) (Ruffell et al., 2000), and zooplankton (Tao et al., 2014)(Tao et al., 2014).
Ultraviolet (UV) radiation does not produce DBPs, and its inactivation effect on bacteria has also been verified (Werschkun et al., 2012). A highly-chlorine-resistant but UV sensitive bacterium, Sphingomonas TS001, was documented for the first time in 2013. The activity of TS001 was reduced by only approximately 5% by 4 mg/L chlorine with 240 min retention time. In contrast, 3 lg unit-inactivation (i.e. 99.9%) was obtained at a UV dose of 40 mJ/cm2 (Sun et al., 2013). However, as another example, the CT value for 99.9% inactivation of Mycobacterium fortuitum by chlorine was 600 times that for E. coli, and M. fortuitum was 2–10 times more resistant to UV radiation than E. coli (Eun-Sook et al., 2010).
Only 0.1% of microbes in the natural environment can be cultured by conventional methods (Hirano and Upper, 1991), the vast majority of bacteria are viable but non-cultivable. Incompleteness of conventional microbiological testing methods has led to the neglect of a certain degree of microbiological risk. Using traditional culturability analysis methods, the effectiveness of a sterilization process might be overestimated due to the uncured and suicide responses of bacteria (Aldsworth et al., 1999). The widespread use of flow cytometry (FCM) in microbiology solves this problem. FCM combined with different fluorescent dyes can help characterize a bacterial population (Berney et al., 2008). For instance, propidium iodide (PI) does not penetrate into viable cells with intact membranes, but leaks into cells with compromised membranes. Thiazole orange (TO) is a permeant dye that enters all cells in different conditions to varying degrees. The fluorescent signal from TO in living cells allows them to be counted. Thus, TO plus PI staining may be used to assess the integrity and permeability of the cell membrane.
Here, we obtained a strain with high chloride resistance from an urban water supply network in northern China and identified as Pseudomonas peli by 16S rDNA gene analysis. This study is the first investigation of the resistance of P. peli to free chlorine and chlorine dioxide. We examined the kinetics of P. peli inactivation by the two disinfectants and UV radiation. In addition, membrane integrity and permeability of the treated strain was evaluated by FCM. The results identify the effective concentration of disinfectant to inactivate this chlorine-resistant bacterium. The mechanism of chlorine resistance was also explored to provide a theoretical basis for the assessment and control of microbial safety in water supply networks.
Section snippets
Target strain
P. peli was obtained from a water supply pipe network system in a northern Chinese city. Here we call it P. peli-083992. To obtain P. peli-083992, water samples were concentrated by vacuum filtration using a polycarbonate membrane with pore size 0.22 μm. Bacteria were eluted with a 0.85% (w/v) sodium chloride solution and collected by centrifugation at 3500 rpm, and then added to a bouillon culture-medium at 37 °C for 24 h. The culture medium was centrifuged, then we prepared a bacterial
Chlorine tolerance of target strain
To test the tolerance of P. peli-083992 to chlorine, P. peli-083992 and P. peli BNCC:139697 were treated with 2.5 mg/L chlorine in the same environmental conditions. Fig. 1 shows the residual concentration of free chlorine and the bacterial viability.
The same dose of free chlorine has different inactivation effects on different strains, and the consumption of residual chlorine also varies. P. peli BNCC:139697 had less resistance to free chlorine than P. peli-083992; P. peli BNCC:139697 showed
Conclusions
Through the results of this study, it can be seen that P. peli-083992 has high chlorine tolerance. The presence of P. peli-083992 will consume a lot of disinfectants in the drinking water supply pipe network system, and the failure of chlorine disinfection will provide a favorable environment for the growth and reproduction of other microorganisms. This paper systematically studies the sterilization characteristics of P. peli-083992, which is of great significance for the assessment, prevention
Declaration of competing interest
The authors declare no financial/commercial conflicts of interest.
Acknowledgement
This study was supported by National Major Projects on Water Pollution Control and Management Technology (No. 2017ZX07501003, No. 2017ZX07502003-06) and the Special Project of Taishan Scholar Construction Engineering (ts201712084).
We thank Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
References (52)
- et al.
Disinfection by-products of chlorine dioxide (chlorite, chlorate, and trihalomethanes): occurrence in drinking water in Qatar
Chemosphere
(2016) - et al.
Survival of Escherichia coli in two sewage treatment plants using UV irradiation and chlorination for disinfection
Water Res.
(2013) - et al.
Impacts of water organic load on chlorine dioxide disinfection efficacy
J. Hazard Mater.
(2009) - et al.
Rapid, cultivation-independent assessment of microbial viability in drinking water
Water Res.
(2008) - et al.
Inactivation of resistant mycobacteria mucogenicum in water: chlorine resistance and mechanism analysis
Biomed. Environ. Sci.
(2012) Resistance mechanisms of bacteria to antimicrobial compounds
Int. Biodeterior. Biodegrad.
(2003)- et al.
Inactivation of cryptosporidium parvum oocysts using medium- and low-pressure ultraviolet radiation
Water Res.
(2001) - et al.
Free chlorine demand and cell survival of microbial suspensions
Water Res.
(2007) - et al.
Inactivation of bacteriophage MS2 upon exposure to very low concentrations of chlorine dioxide
Water Res.
(2011) - et al.
Bacterial disinfectant resistance—a challenge for the food industry
Int. Biodeterior. Biodegrad.
(2003)
Microbiology, chemistry and biofilm development in a pilot drinking water distribution system with copper and plastic pipes
Water Res.
The effect of inorganic precursors on disinfection byproduct formation during UV-chlorine/chloramine drinking water treatment
Water Res.
Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting
J. Microbiol. Methods
Long-term effects of disinfectants on the community composition of drinking water biofilms
Int. J. Hyg Environ. Health
Inactivation of Cryptosporidium parvum oocysts with chlorine dioxide
Water Res.
Characterization and identification of a chlorine-resistant bacterium, Sphingomonas TS001, from a model drinking water distribution system
Sci. Total Environ.
Inactivation of enteric adenovirus and feline calicivirus by ozone
Water Res.
Discolouration in potable water distribution systems: a review
Water Res.
Inactivation of three genera of dominant fungal spores in groundwater using chlorine dioxide: effectiveness, influencing factors, and mechanisms
Water Res.
Disinfection by-products in ballast water treatment: an evaluation of regulatory data
Water Res.
A novel model simulating reclaimed water disinfection by ozonation
Separ. Purif. Technol.
Effect of pipe corrosion scales on chlorine dioxide consumption in drinking water distribution systems
Water Res.
Chlorine dose determines bacterial community structure of subsequent regrowth in reclaimed water
Appl. Environ. Microbiol.
Bacterial suicide through stress
Cellular & Molecular Life Sciences Cmls
Kinetics and mechanism of bacterial disinfection by chlorine dioxide
Appl. Microbiol.
Effect of bacterial growth stage on resistance to chlorine disinfection
Water Science & Technology A Journal of the International Association on Water Pollution Research
Cited by (22)
Effect and influence mechanism of biofilm formation on the biological stability of reclaimed water
2024, Science of the Total EnvironmentIncidence of co-resistance to antibiotics and chlorine in bacterial biofilm of hospital water systems: Insights into the risk of nosocomial infections
2023, Journal of Infection and Public HealthChlorine-resistant bacteria in drinking water: Generation, identification and inactivation using ozone-based technologies
2023, Journal of Water Process EngineeringOccurrence of fungal spores in drinking water: A review of pathogenicity, odor, chlorine resistance and control strategies
2022, Science of the Total Environment