Disinfection characteristics of Pseudomonas peli, a chlorine-resistant bacterium isolated from a water supply network

https://doi.org/10.1016/j.envres.2020.109417Get rights and content

Highlights

  • A strain of chlorine-tolerant bacteria obtained from the drinking water distribution network system.

  • Pseudomonas peli have highly resistant to free chlorine but sensitive to UV radiation.

  • Describe the disinfection kinetics of disinfectant inactivated bacteria.

  • FCM evaluate the membrane integrity and permeability of strains.

Abstract

Lack of microbial contamination is crucial for drinking water quality and safety. Chlorine-resistant bacteria in drinking water distribution systems pose a threat to drinking water quality. A bacterium was isolated from an urban water supply network in northern China and identified as Pseudomonas peli by 16S rDNA gene analysis. This P. peli strain had high chlorine tolerance. The CT value (the product of disinfectant concentration and contact time) to achieve 3 lg unit (i.e. 99.9%)-inactivation of this P. peli isolate was 51.26–90.36 mg min/L, inversely proportional to the free chlorine concentration. Chlorine dioxide could inactivate the bacterium faster and more efficiently than free chlorine, as shown by flow cytometry. Thiazole orange plus propidium iodide staining indicated that free chlorine and chlorine dioxide inactivated P. peli primarily by disrupting the integrity and permeability of the cell membrane. The P. peli was also sensitive to ultraviolet (UV) radiation; a UV dose of 40 mJ/cm2 achieved 4 lg unit (99.99%)-inactivation. The Hom model was more suitable for analyzing the disinfection kinetics of P. peli than the Chick and Chick-Watson models.

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.

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