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Title: The prevalence of selected emerging pathogenic species in wastewater and receiving water bodies
Authors: Govender, Reshme 
Keywords: Antibiotic resistance;Antibiotic-resistant bacteria;Wastewater treatment plants
Issue Date: 16-Mar-2022
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

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.
Submitted in fulfillment of the requirements for the degree of Doctor of Philosophy (Ph.D.):
Biotechnology, Durban University of Technology, Durban, South Africa, 2022.
Appears in Collections:Research Publications (Applied Sciences)

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