Assessment of the Role of Wastewater Treatment Plant in Spread of Antibiotic Resistance and Bacterial Pathogens

Abstract

In current study, we performed a comparative study on bacterial load, total coliform counts and type of organisms present in pre- and post-treated wastewater samples from municipal wastewater treatment plant of Pune, India. In addition, we also studied the antibiotic resistance profiling and role of the selected treatment plant in spread of antibiotic resistance in the environment. Data showed that total 30 different bacterial species from 18-different genera were present in untreated wastewater while only 9 species from 6-different genera were present in post-treated effluent. Furthermore, pre-treated wastewater sample contains wide range of organisms with high levels of antibiotic resistance while bacterial load reduced drastically and pathogens were absent from post-treated effluent.

Electronic supplementary material

The online version of this article (10.1007/s12088-019-00793-2) contains supplementary material, which is available to authorized users.

Wastewater treatment plants (WWTP) receives wastewater from different sources and contains various kinds pollutants, toxic substances and a pool of bacteria [1, 2]. Discharge of untreated water into fresh water bodies, results into death of the flora and fauna. The aim of wastewater treatment plants (WWTP) is to minimize the adverse effect of untreated wastewater on ecology, environment and human health by its adequate treatment prior to release in ecosystem.

Different kinds of antibiotics have been used in human therapy, veterinary and animal farming and a huge load of antibiotics are being released into the municipal wastewater which ultimately finds its way into environment [3, 4]. Widespread use of antibiotics exerts selection pressure on bacterial population and leads to spread of antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARB) in environment [5, 6]. This process further augmented by transfer the resistant genes through horizontal gene transfer [7]. Urban WWTP are considered to be the hotspots for development of antibiotic resistant bacterial population because it receives residual antibiotics from various sources through wastewater and thus play an important role in spread of ARB and ARGs [8]. Therefore, a mechanistic understanding about the type of treatment plant, their source of wastewater, load and antibiotic profiling of microorganisms in influent and effluent is imperative to improve the treatment process and to mitigate the problem of spread of ARGs and ARB.

In current study, we tried to explore the cultivable bacterial population from pre- and post-treated wastewater samples collected from municipal wastewater treatment plant of Pune, India and studied their antibiotic susceptibility and resistance. We also enumerated bacterial load in pre- and post-treated wastewater sample by total viable count and most probable number (MPN) methods and studied the efficacy of current wastewater treatment plant in reduction of bacterial load and spread of antibiotics resistant bacteria in the environment. Result of the study indicated that the wastewater treatment plant was working effectively and efficiently by reducing the bacterial load in post-treated wastewater.

An influent and effluent water sample from wastewater treatment plant operating on Sequential Batch Reactor (SBR) process of Pune Municipal Corporation was collected using aseptic procedure. The capacity of selected sewage treatment plant was 20 million litres per day (20MLD Fig. 1). Samples were transported to the laboratory on ice and stored in cold room at 4 °C and bacterial isolation was started within 4-h of sampling. In order to capture the maximum culturable diversity, different media were used. A tenfold serial dilutions of influent and effluent water samples were prepared and spread on media plates. Plates were incubated at 30 °C for 72–96 h and checked for colonies. Morphologically distinct colonies were picked and purified by several re-streaking and preserved in glycerol stocks as earlier [9, 10]. Purified strains were identified using MALDI-TOF MS and 16S rRNA gene sequencing approach as discussed earlier [11, 12].

Fig. 1.

Image of influent sampling from sewage treatment plant of Pune Municipal Corporation operating on a sequential batch reactor model

For 16S rRNA gene sequencing, genomic DNA was extracted amplified using universal bacterial primers as described in previous reports [13, 14]. Sequencing was carried out using DNA Analyzer (Applied BioSystems, USA) and raw sequence files were manually edited and contigs were generated using ChromasPro (http://www.technelysium.com.au/ChromasPro.html). Similarity search was conducted using EzTaxon database (http://www.eztaxon.org) and phylogeny was constructed using MEGA-6 as discussed by Prakash et al. [8, 15]. The sequences were deposited in nucleotide database of NCBI under sequence accession numbers are MH972165-MH972193.

Bacterial load in untreated and treated wastewater samples, was enumerated using standard protocol of total viable count (TVC) and most probable number (MPN) methods [16]. To check the dominant organisms in pre- and post-treated water samples, we also cultured the bacteria from highest positive dilution and identified them. Antibiotic resistance was determined for all identified isolates using Kirby–Bauer antibiotic testing [17]. Twenty one antibiotics namely, cefpodoxime (CPD), chloramphenicol (C), vancomycin (VA), streptomycin (S), ritampicin (RIF), levofloxacin (LE), ceftriaxone (CTR), clindamycin (CD), amikacin (AK), cefixime (CFM), tetracycline (TE), co-trimoxazole (COT), colistin (CL), augentin (AMC), netillin (NET), norfloxacin (NX), ciprofloxacin (CIP), cephotaxime (CTX), Gentamicin (GEN), furazolidone (FR), amoxicillin (AMX) were used for testing procedure. Antibiotic discs (Hi media discs DE001, DE007 Dodeca Universal) were used for this purpose. Sensitivity and resistance was determined using standard table provided by the Hi-Media (Mumbai, India).

A total 30 species belonging to 18-different genera and only 9 different species from 6-different genera were cultivated from pre- and post-treated wastewater sample respectively (Supplementary Table 1). Result of similarity search indicated that untreated wastewater sample harbour diverse group of bacteria from wide range of habitats. Phylogenetic relatedness of the strains with their closest relative retrieved from diverse ecological niches is given in Fig. 2. Data on cultivable microbial community was dominated from clinical setup, and most of them are facultative anaerobes and reported as opportunistic pathogens. Several organisms of clinical significance like Acinetobacter septicus, Citrobacter farmeri, Klebsiella oxytoca, Raoultellao rnithinolytica etc. were also reported in pre-treated wastewater sample (Supplementary Table 1). It indicated that discharge of untreated wastewater sample in fresh water ecosystem or its use for crop irrigation is highly contagious and hazardous [17]. It can lead to the spread of serious infection through drinking water or food and can be alarming to human health and environment. We did not report any clinically significant organisms from post-treated water samples.

Fig. 2.

16S rRNA based phylogenetic tree of the cultivated strains from pre- and post-treated wastewater sample showing their phylogenetic relationship with previously described strains from diverse ecological habitats. Tree was constructed using neighbour-joining method using MEGA-6 Software package. Values on node represent the bootstrap support for clustering

Data on water chemistry, bacterial population load and enumeration of total coliform population from pre- and post-treated wastewater are presented in Table 1. TVC data indicated that microbial load in post-treated water sample reduced by an order of magnitude. Although the counting using MPN method showed one order of magnitude higher microbial population than data obtained by TVC method but the trend of reduction of microbial population in post treated water sample is similar and findings was substantiated by both the experiments. Result of total coliform count also indicated that population of coliforms reduced in post-treated water sample. Overall data indicated that the bacterial load and total coliform counts decreased after treatment which indicated that the treatment plant was able to reduce bacterial load from influent but only up to a certain extent.

Table 1.

Microbiological and chemical analysis of pre- and post-treated wastewater used in this study

Parameter	Pre-treated	Post-treated
Total viable count (cells/ml)	3.4 × 107	3.5 × 106
Total coliforms	5.6 × 105	5.3 × 104
Most probable number (MPN)	1.1 × 108	2.9 × 107
BOD	723.0	73.0
COD	290.0	30.0
TSS	544.8	17.8
pH	6.3	7.2

COD chemical oxygen demand, BOD biological oxygen demand, TSS total suspended solid

Kirby–Bauer antibiotic test indicated that Citrobacter farmeri showed maximum multi drug resistance against 6 antibiotics followed by Acinetobacter septicus, Uruburellasuis and Escherichia fergusonii. Most of the isolates were resistant towards cefpodoxime, amoxicillin and augmentin (Supplementary Table 3). Among the cultivated strains, total six i.e. Brevundimonasolei, Acinetobactersepticus, Escherichia fergusonii, Citrobacterfarmeri, Klebsiella pneumonia, Uruburuellasuis showed resistance against 4 or more antibiotics. They were only found in pre-treated wastewater and absent in post-treated effluents (Supplementary Table 2).In order to distinguish from each other, they were further selected for physico-chemical analysis. Bacterial load enumeration and antibiotic profiling data indicated that post-treated water contain less microbial load and few microbial species with low antibiotic resistance. Comparative features of all the selected isolates that showed resistance against 4 or more antibiotics are given in Supplementary Table 4. Data indicated that they are phenotypically different, able to survive in wide range of temperature and salinity and did not belong to same group, and if released as such they have capacity to disseminate the antibiotic resistance gene among the organisms residing in different physicochemical conditions. It showed that traits of multidrug resistance in wastewater treatment plant bacteria is not limited to a particular group and widely distributed among the different group of organism.

In conclusion, present work demonstrates the comparison among culturable bacterial population present in influent and effluent of municipal wastewater treatment plant of Pune, India. Escherichia coli, Bacillus sp., Enterobactertabaci and Enterococcus hirae were prevalent. It was observed that the bacterial diversity decreased sufficiently (fourfolds) after the treatment. Populations of pathogenic and opportunistic pathogens with wide range of antibiotic resistance completely vanished and effluent mainly contained Bacillus and Escherichia coli. Furthermore, all the 6 strains which showed multi drug resistance against 4 or more antibiotics in influent were completely absent from effluent. It indicates that sewage treatment plant not only reduces BOD, COD and TSS of the wastewater it receives but also reduces the load of bacterial pathogens in the wastewater and most importantly, it brings down the load of antibiotic resistant pathogens. Results indicated that the selected wastewater treatment plant was working efficiently in terms of reduction of bacterial load, population of pathogenic and opportunistic pathogens with wide range of antibiotic resistance and it is safe to release the treated effluent in natural ecosystem from environmental and human health point of view. It also contributes in mitigating the risk of spread of antibiotic resistance in environment and thereby reduces the possibilities of spread of ARB and ARGs.

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