The prevalence of selected emerging pathogenic species in wastewater and receiving water bodies

Abstract

Antibiotic resistance is one of the biggest threats to global health, due to the excessive use of antibiotics, among other factors. Aquatic environments are considered hotspots for antibiotic-resistant bacteria and genes due to pollution caused by various anthropogenic activities. In this study, four emerging opportunistic pathogens viz., Acinetobacter spp., Pseudomonas spp., Aeromonas spp., and Stenotrophomonas maltophilia were investigated to understand their distribution, source, and resistance patterns in wastewater and surface water. Among these, Acinetobacter baumannii and Pseudomonas aeruginosa have been listed by the World Health Organization (WHO) in 2017 as priority bacteria for further research and development. This study focused on the Umhlangane River, located in the north of Durban, in KwaZulu Natal, South Africa. The possible effect of anthropogenic activities such as discharges from wastewater treatment plants (WWTPs), hospitals, informal settlements, and veterinary clinics on the occurrence of antibiotic-resistance, and virulence signatures of the targeted organisms, was investigated. Sixty samples (12 wastewater, 48 surface water) were collected monthly (November 2016 to April 2017). This included influent and effluent of a wastewater treatment plant (WWTP) and four additional sampling sites (upstream and downstream of the WWTP, a hospital, an informal settlement, and a veterinary clinic). In addition, to the sixty samples, further samplings of aquatic plants (n=16) and sediments (n=16) were done in October 2017, specifically for the isolation of Stenotrophomonas maltophilia. The isolation and enumeration were carried out on selective media for each bacterium. The PCR positive isolates were identified using Matrix-Assisted Laser Desorption Ionization -Time of Flight Mass Spectrometry (MALDI-TOF MS) and 16S rRNA sequencing. In addition, advanced methods such as Flow Cytometry (FCM) and Droplet Digital PCR (ddPCR) were used to detect and quantify the bacteria, in comparison to conventional methods. The multiple antibiotic resistance (MAR) index was calculated to ascertain the contribution of these pollution sources to the proliferation of antibiotic-resistant bacteria in surface water. Varying counts (log10 CFU/mL) of Aeromonas spp. (2.5±0.8 to 3.3±0.4), Pseudomonas spp. (0.6±1.0 to 1.8±1.0) and Acinetobacter spp. (2.0±1.5 to 2.6±1.2) were obtained. S. maltophilia was found in the water column only at two sites and ranged from 2.7±0.3 to 4.1±1.0 log10 CFU/mL. However, it was found abundantly in the plant rhizosphere (3.6±0.1 to 4.2±0.6 log10 CFU/mL) and sediment (3.8±0.1 to 5.0±0.1 log10 CFU/mL) samples. The major Aeromonas species identified by MALDI-TOF MS was A. hydrophila / caviae (58%) whilst P. putida (51%) was common amongst the Pseudomonas isolates. The Acinetobacter genus was dominated by the Acinetobacter baumannii complex (26%), in contrast, all Stenotrophomonas maltophilia identities were confirmed via Polymerase Chain Reaction (PCR) and MALDI-TOF MS. Aeromonas (71%) and Pseudomonas (94%) isolates displayed resistance to three or more antibiotics. Aeromonas isolates displayed high resistance against ampicillin and had higher MAR indices, downstream of the hospital. The virulence gene, aer in Aeromonas was positively associated with the antibiotic resistance gene blaOXA (χ 2=6.657, p<0.05) and the antibiotic ceftazidime (χ 2=7.537, p<0.05). Pseudomonas exhibited high resistance against third-generation cephalosporins in comparison to carbapenems. Some Pseudomonas and Aeromonas isolates were extended-spectrum β-lactamase producing bacteria as the blaTEM gene was detected in Aeromonas spp. (33%) and Pseudomonas spp. (22%). All S. maltophilia isolates were resistant to the antibiotic’s trimethoprim-sulphamethoxazole, meropenem, imipenem, ampicillin, and cefixime. Acinetobacter isolates were resistant to trimethoprimsulphamethoxazole (96%) and polymyxin (86%). The genes coding for resistance against these antibiotics were detected in both S. maltophilia and Acinetobacter. Efflux pump genes were detected in all isolates of S. maltophilia. High MAR indices were observed in isolates of Pseudomonas, S. maltophilia, and Acinetobacter at the hospital site. However, Aeromonas spp. had the highest MAR in isolates from the WWTP effluents. A comparative analysis of three different methods was performed to understand their applicability and accuracy in detecting these pathogens from wastewater samples. The total viable count using the LIVE/DEAD Baclight bacterial viability kit measured an average count (log10 bacteria per mL) of 7.8±0.03 (influent) and 6.7±0.07 (effluent) using the Flow Cytometer. The total viable count using the BacLight kit was higher than the total plate count, which was 6.46±0.02 and 4.63±0.07 log10 CFU/mLfor influent and effluent, respectively. Similarly, the concentration for each of the target bacteria determined using Flow Cytometry combined with Fluorescent-In situ hybridization (Flow-FISH) method ranged from 5.41±0.07 to 5.92±0.02 (influent) and 3.43±0.2 to 4.31±0.15 (effluent) log10 bacteria per mL which was higher than the selective plate counts (3.81±0.35 to 4.17±0.1 and 3.16±0.17 to 3.7±0.20 log10 CFU/mL, for influent and effluent respectively). The ddPCR results obtained showed the highest concentration of bacteria from both influent and effluent samples in comparison to the Flow-FISH and the plate count methods, indicating the sensitivity of this method in detecting both live and dead cells. Pseudomonas was observed to be dominant and was found in the concentration of 7.19±0.24 copies per mL (influent) and 6.48±0.20 copies per mL (effluent) while S. maltophilia (influent: 5.4 ± 0.90 copies per mL effluent: 4.53±0.57 copies per mL) was detected in the lowest concentration. A similar trend was observed in comparison to the data from the plate counts, albeit at lower concentrations. This study, therefore, makes significant contributions in several areas; firstly, it shows the abundance of opportunistic, antibiotic-resistant, and virulent bacteria in wastewater and surface water within Durban. It further demonstrates that these bacteria are mainly from anthropogenic sources such as hospitals and WWTPs. Additionally, the findings indicate the potential for community-acquired infections with these bacteria, necessitating the need for risk reduction interventions aimed at reducing environmental pollution and exposure.