Differential Expression of Antioxidant Enzymes in Chlorine-Resistant Acinetobacter and Serratia spp. Isolated from Water Distribution Sites in Mumbai: A Study on Mechanisms of Chlorine Resistance for Sustainable Water Treatment Strategies

Abstract

Chlorination is a widely used process for disinfecting drinking water, but the emergence of chlorine-resistant bacteria has become a significant concern. While previous research has focused on identifying chlorine-resistant organisms, there has been limited investigation into the mechanisms behind chlorine resistance. Some bacterial isolates that display resistance to chlorine treatment may protect themselves using various mechanisms, including biofilm production, antibiotic resistance, horizontal transfer of antibiotic resistance genes, or producing antioxidant enzymes. Given that chlorination employs hypochlorous acid (HOCl), which is an extremely potent oxidizing agent, the most critical mechanism to investigate is antioxidant enzymes. This study investigated the antioxidant profile of eight chlorine-resistant isolates (three of the Serratia sp. and five of the Acinetobacter) after chlorine exposure. The profiles, both between and within species, were noticeably different. Among the isolates, Acinetobacter junii NA 3-2 showed a significant increase in the specific activity of superoxide dismutase, catalase, and ascorbate peroxidase after exposure to 20 ppm chlorine. In the guaiacol peroxidase (GPX) assay, only isolates belonging to Serratia marcescens showed GPX activity, and Serratia marcescens 3929-1 showed significant increase after exposure to 20 ppm of chlorine. None of the isolates belonging to Acinetobacter spp. showed GPX activity. Additionally, almost all control samples exhibited some enzyme activity, which may explain their survival against chlorine treatment in reservoirs. Principal component analysis revealed no strain-dependent similarities, while the balance of scavenging enzymes changed, as demonstrated in the heat map. Thus, this study suggests that antioxidant enzymes may be one mechanism of protection for some bacterial species against oxidative stress from chlorination, resulting in chlorine resistance. Understanding the mechanism of chlorine resistance is critical to identifying potential solutions. This study highlights the need to consider more modern approaches to disinfecting drinking water.

1. Introduction

Water quality is crucial for human health and environmental sustainability, but it has been extensively compromised due to increasing human activities. Poor water quality poses significant risks to public health, with waterborne diseases being a major cause of morbidity and mortality worldwide. Traditional water treatment methods, such as chlorination and others, have been employed for ages. Chlorination is the most widely used method of disinfecting public water systems globally due to its low cost, simplicity, and relatively slower decay rate in distribution systems or storage [1,2]. This ensures the maintenance of water hygiene over an extended period of time. However, the emergence of chlorine-resistant bacteria is a global concern. Luo et al. summarized the various risks associated with chlorine-resistant bacteria, highlighting the most significant risk as the development of antibiotic resistance among waterborne pathogens [3]. This is very worrying because antibiotic resistance can make standard treatments ineffective and is a serious threat to public health because it limits how infectious diseases can be treated.

Among the chlorine species, hypochlorous acid (HOCl) is the most potent in deactivating the microorganisms present in the water, as it is a strong oxidizing agent [4]. Reactive oxygen species (ROS) such as superoxides, peroxides, the hydroxyl radical, singlet oxygen, and alpha oxygen are generally produced, leading to cell damage. Oxidative stress caused by increased ROS can lead to damage to bacterial DNA, proteins, and lipids. In some cases, excessive ROS production can lead to the inactivation of ATP synthase or the apoptosis of bacteria [5]. Antioxidant enzymes play a vital role in preventing the oxidation of molecules and thus maintaining the balance of oxidants [6]. They help to eliminate the superoxide ions and hydrogen peroxide by converting them into nascent oxygen and water [7]. The important antioxidant enzymes that can be activated even in the presence of stress include superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), monohydroascorbate reductase (MDHAR), and glutathione reductase (GR).

In our previous study, we isolated chlorine-resistant bacteria from thirty-six reservoirs in the Municipal Corporation of Greater Mumbai (MCGM). Among the 89 isolates, eight isolates showing the highest (20 ppm) chlorine resistance were identified [8]. These isolates were found to belong to Acinetobacter and Serratia spp. The study also showed that the isolates were resistant to several broad-spectrum antibiotics and showed the ability to produce biofilms. In continuation of that previous work, the objective of this current study was to evaluate the activity levels of SOD, CAT, APX, and GPX in Acinetobacter and Serratia spp. isolates in the presence of high chlorine concentrations; in addition, to determine by enzyme assays how antioxidant enzymes, specifically SOD, CAT, APX, and GPX, offer protection to bacteria in chlorine-induced oxidative stress. This will help shed light on the potential mechanism(s) underlying the chlorine resistance of these bacterial isolates. By identifying the limitations and drawbacks of traditional water treatment methods such as chlorination and identifying potential new strategies, a holistic approach can be adopted to promote the development of more sustainable and efficient water treatment technologies that protect human health and the environment.

2. Materials and Methods

2.1. Bacterial Cultures

Eight bacterial isolates that were tolerant to 20 ppm of chlorine were isolated from chlorinated drinking water samples procured from 36 reservoirs of the Municipal Corporation of Greater Mumbai [8]. They were identified by 16s rRNA sequencing, and their sequences were deposited in the NCBI GenBank. The identified isolates are DB4 (Serratia marcescens, MW013141), 1750 (Serratia marcescens, MT974429), 3929-1 (Serratia marcescens, MW149491), IA2 (Acinetobacter baumannii, MT974422), KB1 (Acinetobacter baumannii, MT994588), NA3-2 (Acinetobacter junii, MT974430), NA4-1 (Acinetobacter junii, MT993631), and NA4-2 (Acinetobacter pittii, MT994257). The chemicals and reagents used throughout the study were purchased from Sisco Research Laboratories Pvt. Ltd. (SRL), Mumbai, India and Molychem, Mumbai, India. All microbiological media were procured from Hi-media Pvt. Ltd., Mumbai, India. Enzyme assays were done using a spectrophotometer from Shimadzu, Japan.

2.2. Chlorine Treatment

All 8 chlorine-resistant isolates were inoculated separately in Nutrient Broth and incubated at 37 °C for 24 h. The cell pellet was harvested by centrifugation (8000 rpm, 20 min, 4 °C). The pellet was resuspended in 0.85% saline, and the optical density was adjusted to 0.5 at 610 nm, followed by exposure to 20 ppm chlorine for 30 min (test). After exposure, the reaction was terminated using 1 M sodium thiosulphate [9]. An unexposed control was also maintained for all the isolates.

2.3. Enzyme Extraction

For studying the production of antioxidant enzymes by chlorine-resistant bacteria, enzyme extraction was carried out. Both test and control samples of all isolates were sonicated for 6 min (10 s on, 20 s off cycle) on ice. After sonication, 2 mL of the upper layer was collected in an Eppendorf tube and centrifuged at 12,000 rpm for 30 min at 4 °C. The supernatant was collected and stored at 4 °C for protein estimation (which was performed by the Bradford assay) and enzyme assays.

2.4. SOD Assay

The superoxide dismutase (SOD) (EC 1.15.1.1) assay was performed following the method described by Weydert and Cullen [10]. The assay reaction mixture consists of 770 µL sodium carbonate buffer (77 mM, pH 10.2), 2 µL ethylenediamine acetic acid (EDTA) (1 mM), 100 µL Triton X-100 (0.3%), freshly prepared 240 µL nitro blue tetrazolium chloride (NBT) (2400 µM) and 10 µL hydroxylamine hydrochloride (100 µM, pH 6), 30 µL distilled water, and 50 µL of the sample (as prepared in Section 2.3). Distilled water was used instead of samples in the blank. The absorbance was recorded spectrophotometrically at 560 nm. The percent inhibition of NBT was calculated using the below mentioned formula:

% NBT Inhibition = (Abs of Blank − Abs of Control/Abs Blank) × 100

2.5. Catalase (CAT) Assay

The catalase (CAT) (EC 1.11.1.6) assay was performed following the method described by Aebi [11]. The assay reaction mixture of 1 mL contained freshly prepared 970 µL phosphate buffer (0.05 M, pH 7), 20 µL H₂O₂ (0.02 M), and 10 µL of the sample (as prepared in Section 2.3). A decrease in absorbance was recorded spectrophotometrically at 240 nm after 5 min. Specific activity was expressed in U/mg of protein.

2.6. Guaiacol Peroxidase (GPX) Activity

Guaiacol peroxidase (GPX) (EC 1.11.1.7) activity was measured by following the method described by Uarrota et al. [12]. The assay reaction contained 50 mM phosphate buffer (pH 7), 9 mM guaiacol, 19 mM H₂O₂, and 50 µL of the sample (as prepared in Section 2.3). The absorbance was recorded spectrophotometrically at 470 nm for 1 min. The extinction coefficient (26.6 mM⁻¹ cm⁻¹ at 470 nm) was taken into consideration for calculating peroxidase activity. One unit of peroxidase was defined as the amount of enzyme that caused the formation of 1 mM of tetraguaiacol per minute.

2.7. Ascorbate Peroxidase (APX) Activity

Total APX (EC 1.11.1.11) activity was measured spectrophotometrically by taking the absorbance at 290 nm using a method described by Uarrota et al. [12]. The reaction mixture comprises 50 mM potassium phosphate buffer (pH 7.0), 2 mM ascorbate, 200 µL of the sample (as prepared in Section 2.3), and 0.1 mM H₂O₂ to start the reaction. A decrease in absorbance was noted as ascorbate (e = 2.8 mM⁻¹ cm⁻¹) was oxidized for 3 min. APX activity was expressed as mM ascorbate min⁻¹ mg⁻¹ of proteins.

2.8. Statistical Analysis

GraphPad Prism 8 software was used for the analysis of the data from all the experiments. All the experiments were performed in triplicate. The results are expressed as the mean ± SD. A two-dimensional heat map was generated after normalizing the data. Statistical significance was studied using one-way and two-way ANOVA. Values with p < 0.033 were considered significant, and statistical significance is indicated in the figure legends. Standardized data were used for multivariate principal component analysis using the Microsoft Excel extension XLSTAT.

3. Result

3.1. SOD Assay

Superoxide dismutase (SOD) is the only enzyme in the antioxidant family that catalyses the superoxide ion into less toxic molecules of hydrogen peroxide and molecular oxygen. Out of all the isolates, only Acinetobacter junii NA3-2 showed a significant increase (p < 0.001) in the percentage of NBT inhibition after exposure to chlorine. According to a previous report, a constant rise in SOD activity might promote resistance to stress and protect the bacterial cells from ROS attack [13]. However, Acinetobacter junii NA4-1 showed a significant decline (p < 0.001). In this case, the decline in the test sample can probably be attributed to an increase in H₂O₂ toxicity or the removal of metal ions from its active site [14]. Apart from these, the remaining isolates belonging to either the Serratia or Acinetobacter species showed some SOD activity with no significant difference between the control (0 ppm) and the test (20 ppm) samples (Figure 1). It is noteworthy that all control samples demonstrated basal SOD activity, which could potentially be adequate for the survival of bacteria under chlorine treatment in treatment plants and reservoirs.

3.2. CAT Assay

Catalase enzyme is a tetramer associated with a prosthetic group. It is the foremost defense enzyme against ROS by breaking down hydrogen peroxide into water and nascent oxygen. Out of all the isolates (Figure 2), only Acinetobacter junii NA 3-2 showed significant increase (p < 0.001) in the specific activity of catalase enzyme under a chlorine stress of 20 ppm concentration. All other Acinetobacter isolates showed much less activity, while all isolates belonging to Serratia marcescens showed no significant difference between the control (0 ppm) and the test (20 ppm) samples. Catalase activity is regulated by H₂O₂, and greater deposition of the same may reduce the activity of the enzyme [15].

3.3. GPX Activity

Guaiacol peroxidases are called ‘stress’ enzymes, as they act as a defense enzyme to protect the bacterial cell from any kind of oxidant stress [16]. As seen in Figure 3, Acinetobacter spp. showed the least amount of GPX activity compared to Serratia spp. This could be because many Acinetobacter spp. can degrade phenolic substances or aromatic hydrocarbons, and guaiacol is phenolic in nature [17,18]. Several reports also depict that the Acinetobacter lwoffii NCIB 10553 species was able to utilize aromatic and aliphatic carboxylic esters as carbon sources [17]. Among the isolates belonging to Serratia marcescens, only 3929-1 showed a significant increase in the specific activity (p < 0.001) of the test sample under chlorine stress, depicting that GPX enzymes probably play a role in combating chlorine stress. DB4 showed the highest specific activity, but there was no significant difference in the control and the test samples for GPX, which might still be sufficient for surviving chlorine treatment in the reservoir.

3.4. APX Activity

Ascorbate peroxidase is also one of the stress enzymes and generally reduces hydrogen peroxide into water. Out of all the isolates, only Acinetobacter junii NA3-2 showed a significant increase (p < 0.033) in the specific APX activity under a chlorine stress of 20 ppm concentration. On the other hand, Serratia marcescens 1750 showed a very significant decrease (p < 0.001) in the specific activity of the test sample as compared to the control. Other isolates showed no significant decrease in the specific activity of the control and test samples (Figure 4) because APX is rarely found in all bacteria and is abundant in plants to protect them from stress [19]. However, the presence of even a small amount of the APX enzyme in the control might be sufficient for its survival against chlorine stress in drinking water samples.

3.5. Analysis of Antioxidant Enzyme Activities Using a Heat Map and PCA

To overcome H₂O₂-related cell damage, bacteria possess various antioxidant mechanisms. Figure 5 presents a data matrix that shows the numeric differences in the form of a heat map. A sequential color scheme has been provided with the highest value in dark blue and the lowest values in white. The heat map provides a notable difference in the antioxidant activities checked as well as between the bacterial strains exposed to 0 ppm and 20 ppm chlorine. Isolate Acinetobacter junii NA3-2 (20 ppm) showed the highest SOD activity and showed similar activities when exposed to chlorine stress (comparing 0 ppm and 20 ppm). To counteract the higher SOD, the catalase activity of isolate NA3-2 was also high. In the case of catalase activity, the strains DB4, 3929, 1750, and NA4-1 exposed to 20 ppm chlorine showed similar activities as their unexposed strains. The result is further supported by statistical analysis where SOD and catalase had a positive correlation (R² 0.609, data not included). APX and GPX enzymes are known to increase under various stress conditions [20]. Our results presented a large degree of difference between the results of these two activities. In the case of GPX, most of the isolates showed little or no activity. The decreased GPX activity could be attributed to defects in antioxidant enzymes [20]. However, isolates DB4, 3929, and 1750 (all Serratia marcescens) were the only isolates that showed higher GPX activity.

The PCA results focused on the observation tables and the correlation tables obtained during analysis. In Figure 6A, the red vectors represent the investigated variables. SOD, APX, and catalase were positively linked variables, which can be inferred from the angles between the vectors. Additionally, catalase and GPX were unrelated variables presented in right angled vectors. Figure 6B shows a scatterplot having axes that correspond to two different principal components. F1 (54.45%) and F2 (25.05%) were the first two components showing the largest variance. This chart (Figure 6B) relates the isolates to variables and to one another. As observed in the scatterplot, there were no strain-dependent similarities in the results obtained (No distinct pattern was seen in the Acinetobacter or Serratia species), although isolate DB4 was detected to be an outlier. However, there were similarities in the antioxidant activities of the isolates obtained for different exposure conditions.

4. Discussion

Antioxidant enzymes are crucial for the survival of organisms under stressful conditions, including exposure to oxidants like chlorine. To better understand the mechanisms by which antioxidant enzymes protect bacterial cells from chlorine-induced stress, this study aimed to investigate the activity of these enzymes in bacterial isolates obtained from chlorinated drinking water during three seasons, namely, winter (Nov–Feb), summer (Mar–May), and monsoon (July–Oct). The isolates were exposed to high concentrations of chlorine to assess their survival potential. In a prior study conducted by Jathar et al. [8], only eight out of 89 isolates were found to survive exposure to 20 ppm of chlorine, highlighting the importance of investigating the role of antioxidant enzymes in the survival of bacterial isolates under chlorine stress. The primary objective of this study was to explore the relationship between antioxidant enzyme activity and the survival of bacterial isolates under chlorine stress. Through this investigation, the study aimed to gain insights into the antioxidant defense mechanisms of the isolates and shed light on the factors that contribute to their survival under adverse conditions.

In this study, all the isolates that were studied showed the presence of at least one or two antioxidant enzymes in the non-treated control sample. This suggests that the presence of these enzymes may contribute to their survival in chlorinated drinking water. Apart from that, Acinetobacter junii NA 3-2 showed significantly high specific activities for SOD, catalase, and APX, whereas Serratia marcescens 3929-1 showed significantly high activity for GPX in the test sample treated with 20 ppm of chlorine, showing the resistant nature of the species against chlorine stress. In addition, Serratia marcescens 1750 showed a significant decrease in APX activity. Mohamd et al. (2020) attributed the inactivation of APX activity to the formation of H₂O₂ as a result of oxidative stress, which may lead to reduced ascorbate depletion [21]. It was also observed that the Acinetobacter species showed no GPX activity. This may be a result of HOCl, which has the ability to oxidize both the enzyme’s protein component and the heme group of the protoporphyrin IX prosthetic group. According to a number of studies, protein oxidation in bacteria under chlorine stress may lead to a reduction in a specific activity but not a total loss of enzyme function [22]. The results of this study were able to show that antioxidant activities varied between and within species. This finding is in line with the literature, which reports that bacteria exposed to salinity stress exhibit strain-specific antioxidant responses [23]. Interestingly, Serratia marcescens DB4 presented an extreme deviation from the other isolates, and a possible explanation for this might be that it was the only organism isolated in the monsoon season. This can be due to the inbuilt potential of the organism against super-chlorination, which is employed during monsoon season.

According to this study, the antioxidant enzymes SOD, catalase, APX, and GPX were expressed differently in the Serratia and Acinetobacter species. SOD is a ubiquitous enzyme found in all living organisms, and different species of microorganisms possess distinct types of SOD enzymes, while some microorganisms lack SOD enzymes altogether [7,24]. The primary function of SOD is to break down harmful superoxide radicals into molecular oxygen and H₂0₂, which can be further broken down by the catalase enzyme [25]. OxyR protein is activated in the presence of H₂0₂, which helps to activate the catalase enzyme [26]. An increase in GPX and APX enzyme activity is observed under environmental stress, such as osmotic stress and dehydration [27]. Researchers have previously stated that an increased production of APX enzyme activity indicates that the bacteria are able to fight against pollutants like antimony (Sb) [28]. In this study, the stress can be attributed to chlorine, resulting in excessive production of H₂0₂. It is noteworthy that soil, water, and animals serve as the natural habitat for Acinetobacter and Serratia species, which enables them to survive environmental stress more effectively [29].

The enzymatic activity required for antioxidant defense is seen in the heat map results, where the balance of scavenging enzymes appears to be changing. As seen in the heat map, to compensate for reduced activity by catalase, APX, or GPX, activity has been induced and vice-versa [30]. This indicates that the bacteria are employing a multi-faceted approach to antioxidant defense, which could be crucial for their survival in environments with high oxidative stress. Interestingly, in our previous study, it was observed that the bacteria showed resistance to a broad spectrum of antibiotics and had the ability to produce biofilm, which is also a potential survival mechanism [8]. The ability to produce antioxidant enzymes, along with these other survival mechanisms, could be part of a multi-mechanistic approach that the bacteria employ to survive under challenging conditions.

One of the key findings is that relying solely on chlorine for water disinfection may not be sustainable in the long run, due to the emergence of chlorine-resistant bacteria. As such, this study highlights the need to consider more modern approaches to disinfecting drinking water that are less prone to resistance, such as ozone or ultraviolet light. By reducing the use of harmful disinfectants like chlorine, we can help protect aquatic ecosystems and ensure the availability of safe drinking water for human consumption, which is a fundamental component of sustainable development.

It is noteworthy to highlight the potential for horizontal transfer of antibiotic resistance genes from chlorine-resistant bacteria to non-chlorine bacteria. Antibiotic resistance is a major global public health concern, and reducing the spread of resistance is critical for sustainable healthcare. Understanding the mechanisms of resistance in bacteria, such as the production of antioxidant enzymes, can help inform the development of new strategies for addressing antibiotic resistance. In addition, this study demonstrates the importance of understanding the mechanisms underlying environmental challenges such as bacterial resistance to disinfectants, in order to develop more sustainable solutions. By identifying the antioxidant profile of chlorine-resistant bacteria, this study provides insights that could help inform the development of more effective and sustainable water treatment strategies.

Additionally, the presence of chlorine-resistant organisms in water can potentially indicate that the water is polluted or contaminated. Their presence suggests that the water has been exposed to sub-lethal levels of disinfectants, which could be due to a variety of factors such as inadequate treatment, equipment malfunctions, or high organic matter levels in the water. While chlorine-resistant organisms are not specific to fecal contamination like E. coli, their presence in water can still suggest a problem with water quality and warrant further investigation to identify potential sources of contamination. Therefore, monitoring for the presence of chlorine-resistant organisms can be an important component of water quality management and control.

5. Conclusions

It is a serious problem that chlorine-treated drinking water from different reservoirs of MCGM has been found to contain unfit water samples and organisms that are resistant to chlorine. The emergence of chlorine-resistant bacteria is a major concern because chlorination is widely regarded as the conventional method of disinfection. Understanding the mechanisms leading to resistance is a priority so that immediate resolutions can be made. Our present study mainly focuses on how chlorine-resistant isolates combat chlorine stress with the help of various antioxidant enzymes: SOD, catalase, APX, and GPX. The data from our previous study showed that all the highest chlorine-resistant (20 ppm) organisms belonged to Acinetobacter sp. and Serratia sp. Among the isolates, Acinetobacter junii NA3-2 was the only isolate to show significantly increased antioxidant activity after chlorine exposure in terms of SOD, catalase, and GPX. All other isolates had an inherent high antioxidant activity even without chlorine exposure. These findings indicated that antioxidant activity varies between genera and species. Moreover, the inherent antioxidant activity indicates a probable adaptability mechanism for these chlorine-resistant isolates. The most noteworthy fact is that, as per the literature, Serratia sp. and Acinetobacter sp. both belong to a pathogenic, drug-resistant group. The presence of these genera in drinking water is alarming. It is important to get these isolates identified further for their pathogenicity and drug resistance. Further, this study expands the consideration of a new indicator organism group in potable water safety. Even though chlorination when combined with UVC radiation is the standard method worldwide, the sustainability of this method in the future is questionable, and further new strategies for drinking water safety must be implemented.