Journal Pre-proof

Risk assessment of Enterococcus faecium, Klebsiella

pneumoniae, and Pseudomonas aeruginosa in environmental
water sources: Development of surrogate models for antibiotic
resistance genes

Julia Denissen, Brandon Reyneke, Tobias Barnard, Sehaam Khan,

Wesaal Khan

PII: S0048-9697(23)04842-8
DOI: https://doi.org/10.1016/j.scitotenv.2023.166217
Reference: STOTEN 166217

To appear in: Science of the Total Environment

Received date: 31 March 2023

Revised date: 8 August 2023
Accepted date: 8 August 2023

Please cite this article as: J. Denissen, B. Reyneke, T. Barnard, et al., Risk assessment
of Enterococcus faecium, Klebsiella pneumoniae, and Pseudomonas aeruginosa in
environmental water sources: Development of surrogate models for antibiotic resistance
genes, Science of the Total Environment (2023), https://doi.org/10.1016/
j.scitotenv.2023.166217

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© 2023 Published by Elsevier B.V.

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Risk assessment of Enterococcus faecium, Klebsiella pneumoniae, and Pseudomonas

aeruginosa in environmental water sources: Development of surrogate models for antibiotic

resistance genes

Julia Denissen1, Brandon Reyneke1, Tobias Barnard2, Sehaam Khan2 and Wesaal Khan1*

1
Department of Microbiology, Faculty of Science, Stellenbosch University, Private Bag X1,

Stellenbosch, 7602, South Africa

2
Water and Health Research Centre, Faculty of Health Sciences, University of Johannesburg, PO

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Box 17011, Doornfontein, 7305, South Africa

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*Corresponding Author: Wesaal Khan; Phone +27218085804; Email: wesaal@sun.ac.za

Abbreviations1

1
AAC - aminoglycoside N-acetyltransferase; A. baumannii – Acinetobacter baumannii; AR – Antibiotic resistance; ARB – Antibiotic
resistant bacteria; ARG – Antibiotic resistance gene; CA – Community-acquired; DNA – Deoxyribonucleic acid; E. coli – Escherichia
coli; E. faecium – Enterococcus faecium; EMA – Ethidium monoazide bromide; EMA-qPCR – Ethidium monoazide bromide quantitative
polymerase chain reaction; GC – Gene copies; HA – Hospital-acquired; HGT – Horizontal gene transfer; IF – Infective fraction;
K. pneumoniae – Klebsiella pneumoniae; LLOD – Lower limit of detection; P. aeruginosa – Pseudomonas aeruginosa; QMRA –
Quantitative microbial risk assessment; qPCR – Quantitative polymerase chain reaction; S. aureus – Staphylococcus aureus; UTI –
Urinary tract infection; WHO – World Health Organization; WWTP – Wastewater treatment plant; β – Beta; 16S rRNA – 16S ribosomal
ribonucleic acid.
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Abstract

The presence of Enterococcus faecium (E. faecium), Klebsiella pneumoniae (K. pneumoniae),

Pseudomonas aeruginosa (P. aeruginosa), and the aminoglycoside resistance genes, aac(6’)-Ib

and aac(6’)-aph(2’’), was investigated in environmental water sources obtained from informal

settlements in the Western Cape (South Africa). Using ethidium monoazide bromide quantitative

polymerase chain reaction (EMA-qPCR) analysis, E. faecium, K. pneumoniae, and P. aeruginosa

were detected in 88.9%, 100%, and 93.3% of the samples (n = 45), respectively, with a

significantly higher mean concentration recorded for K. pneumoniae (7.83 × 104 cells/100 mL) over

the sampling period. The aac(6’)-Ib gene was detected in 95.6% (43/45) of the environmental

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water samples [mean concentration of 7.07 × 106 gene copies (GC)/100 mL], while the aac(6’)-

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aph(2’’) gene was detected in 100% (n = 45) of the samples [mean concentration of 6.68 × 105
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GC/100 mL]. Quantitative microbial risk assessment (QMRA) subsequently indicated that the risks

posed by K. pneumoniae and P. aeruginosa were linked to intentional drinking, washing/bathing,

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cleaning of the home, and swimming, in the samples collected from the various sampling sites.
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Surrogate risk assessment models were then designed and applied for Gram-positive [aac(6’)-

aph(2’’) gene] and Gram-negative [aac(6’)-Ib gene] pathogens that may exhibit aminoglycoside
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resistance. The results indicated that only the Gram-negative pathogens posed a risk (> 10-4) in all

the samples for cleaning of the home and intentional drinking, as well as for washing laundry by
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hand, garden hosing, garden work, washing/bathing, accidental consumption, and swimming at the
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stream and marsh sites. Thus, while environmental waters may pose a health risk of exposure to

pathogenic bacteria, the results obtained indicate that screening for antibiotic resistant genes,

associated with multiple genera/species, could serve as a surrogate model for estimating risks with

the target group under investigation.

Keywords: Risk assessment; E. faecium; K. pneumoniae; P. aeruginosa; aac(6’)-aph(2’’) gene;

aac(6’)-Ib gene
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1. Introduction

Enterococcus faecium (E. faecium), Klebsiella pneumoniae (K. pneumoniae), and

Pseudomonas aeruginosa (P. aeruginosa), are notorious for their increased antibiotic

resistance (AR) and virulence, and role in the onset of hospital-acquired infections (Navidinia

et al., 2017). While extensive research has been conducted on the identification,

characterisation and risk associated with these species in clinical settings, E. faecium,

K. pneumoniae, and P. aeruginosa are also frequently associated with community-acquired

(CA) infections linked to the use of contaminated environmental water (John et al., 2017;

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Denissen et al., 2022). Epidemiological studies can be used to evaluate the health hazards

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associated with exposure to these CA-related pathogens in the environment, however, they
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tend to be both costly and time-consuming (Abia et al., 2016). Quantitative microbial risk

assessment (QMRA) has thus been employed as a useful tool to estimate the health risks
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posed by different pathogens, based on various exposure pathways (e.g., inhalation,

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intentional drinking, bathing, washing laundry etc.) in environmental samples (Zhang et al.,

2019; Owens et al., 2020).

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Specifically, QMRA has been successfully used for more than 40 years to estimate the
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health risks posed by various water sources such as drinking water, reclaimed water,

wastewater, irrigation water and recreational waters, to end-user communities (Zaneti et al.,
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2021). For example, Alsalah et al. (2015) applied a QMRA framework using an exponential

dose response model for both E. faecalis (k = 2.19 × 10-11) and P. aeruginosa (k = 1.87 × 10-
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), to determine the risk associated with the consumption of fruit that had been irrigated with

groundwater from two wells contaminated with these species. Overall, the calculated risks

from the worst-case scenario (highest exposure level) for E. faecalis was 5.98 × 10-6, while

the risk for P. aeruginosa was 9.55 × 10-4. While the results suggest that the accidental

consumption of fruit contaminated with E. faecalis during irrigation may not pose a human

health risk; the risk posed by P. aeruginosa was close to the acceptable benchmark limit

(1 × 10-4; one infection per 10 000 people per year). The authors therefore suggested that

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improved agricultural practices are required in the western area of Saudi Arabia (where the

samples were collected) in order to improve food safety. In another study, Al-Jassim et al.

(2015) performed QMRA to determine the risks associated with the dermal exposure of

agricultural workers to liquid particulates containing P. aeruginosa and Aeromonas

hydrophila, during the reuse of wastewater influent, effluent, and chlorinated effluent, for

agricultural irrigation. The annual calculated risks for primary influent (9.9 × 10-1) and non-

chlorinated effluent (4.9 × 10-2) containing P. aeruginosa, were above the 1 × 10-4

acceptable benchmark limit. However, the annual risk associated with the use of chlorinated

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effluent containing P. aeruginosa was zero, thus indicating that the chlorinated wastewater

effluent under investigation, may safely be used for agricultural irrigation purposes.

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It is however, also well known that antibiotic resistance genes (ARGs) can be easily
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transferred between bacteria and aquatic environments, via horizontal gene transfer (HGT)
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on plasmids, transposons, or insertion sequences (Nguyen et al., 2019). As antibiotics such
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as beta(β)-lactams, aminoglycosides, macrolides, sulphonamides, and tetracyclines are

commonly used in animal husbandry, genes encoding resistance to these antibiotic classes,
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could be disseminated into the environment (Lachmayr et al., 2009; Al Salah et al., 2019).

However, while the conceptual role played by the environment in the rise and spread of
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antibiotic resistance and ARGs is understood (e.g., it acts as a reservoir for ARGs and
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resistant bacteria and facilitates their transmission between different reservoirs, while also

playing an influential role in the development of novel resistance factors), there remains

uncertainty surrounding the importance and extent of its role in the development of antibiotic

resistance, due to a lack of quantitative data (Larsson et al., 2018; Bengtsson-Palme, 2019).

Nguyen et al. (2019) then investigated the prevalence of various ARGs in the Tama River

(Tokyo, Japan) and Lake Kasumigaura (Ibaraki, Japan). Quantitative polymerase chain

reaction (qPCR) analysis was used to determine the occurrence of the blaTEM (encodes for

extended-spectrum β-lactamase), tetA (encodes for tetracycline resistance), ereA (encodes

for macrolide-lincosamide-streptogramin resistance), and sul1 (encodes for sulphonamide

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resistance) genes, as well as the intI1 integron (a mobile genetic element) in the

environmental water samples. Overall, the ereA and blaTEM genes were below the

quantification level at the sampling site located furthest upstream of the wastewater

treatment plant (WWTP) discharge point, in the Tama River. However, at the sampling site

located at the point where the effluent of the WWTP is discharged into the river, the copies

of these two genes increased by an order of 106 – 107 copies/L. Similarly, the gene copies

(GC) of tetA and sul1 and the intI1 integron increased at sites located closer to the discharge

point of the wastewater effluent in the Tama River. For the samples obtained from Lake

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Kasumigaura, the highest copy numbers of the ereA, sul1, and intI1 genes were detected at

the site located closest to the wastewater effluent discharge point and the Sakura River

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mouth. As all the ARGs were detected at the furthest sampling point for Lake Kasumigaura,
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the authors hypothesised that the ARGs can persist in water environments. Thus, while it is
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well known that ARGs have augmented the threat posed by microorganisms on human

health through enhancing resistance; very few studies have assessed the relative health
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risks posed by ARGs. This is due to the complex nature of performing such research, as
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factors such as gene abundance, tendency of the ARGs to be transferred via HGT, and the

ability of the ARGs to be expressed in the receiving host, need to be considered (Zhang et
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al., 2022). There is thus a need to increase the surveillance of ARGs in environmental

reservoirs, as they may pose a health risk to humans and animals (Nguyen et al., 2019).
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The aim of this study was thus to determine the abundance of viable E. faecium,

K. pneumoniae, and P. aeruginosa cells, as well as GC of the aac(6’)-Ib (associated with

Gram-negative pathogens) and aac(6’)-aph(2’’) (associated with Gram-positive pathogens)

aminoglycoside resistance genes, in environmental water sources obtained from rural

(Vlottenburg informal settlement) and urban informal (Sir Lowry‟s Pass Village) settlements

in the Western Cape (South Africa), using ethidium monoazide bromide quantitative

polymerase chain reaction (EMA-qPCR). Basic sanitation and waste removal infrastructure

are however, generally lacking in rural and urban informal settlements, and access to

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potable water is scarce. Consequently, there may be health risks associated with the use of

non-potable water sources in these settings, which may lead to the onset of CA disease

outbreaks. The health risk associated with the use of the environmental water sources for

several potable and non-potable domestic activities (e.g., washing laundry by hand, garden

work and garden hosing, washing/bathing, intentional drinking, and accidental consumption,

amongst others), was thus estimated using QMRA. In addition, to improve the current state

of research regarding risk assessment analyses and the health risks posed by antibiotic

resistant pathogens, surrogate risk assessment models were designed for Gram-positive

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(E. faecium and S. aureus) and Gram-negative (K. pneumoniae, P. aeruginosa,

A. baumannii, and E. coli) bacterial species, using the aac(6’)-aph(2’’) (associated with

Gram-positive bacteria) and aac(6’)-Ib

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(associated with Gram-negative bacteria)
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aminoglycoside resistance genes, respectively.
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2. Materials and Methods
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2.1 Sample Collection

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Samples (1 - 5 L) were collected in sterile bottles from various environmental water sources

(stream, marsh, stormwater, and surface runoff) located in the Vlottenburg informal
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settlement (rural settlement in Cape Town, South Africa; GPS co-ordinates: 33°95'35'' S;
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18°79'75'' E), and Sir Lowry‟s Pass Village (urban settlement in Cape Town, South Africa;

GPS co-ordinates: 34°11'95'' S; 18°90'86'' E) (Figure 1). To account for seasonal variability,

eleven sampling sessions were conducted from 21 June 2021 to 31 May 2022 (Table A1;

Supplementary file). All the collected samples (n = 45) were utilised for the EMA-qPCR and

risk assessment analysis.

2.2 Environmental Water Concentration, EMA Treatment and DNA Extraction

To account for differences in sample composition (i.e., concentration of organic matter),

80 mL to 1 L of the collected water samples (n = 45) was subjected to flocculation as

described by Dobrowsky et al. (2015). Each of the flocculated samples were filtered through

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non-charged, mixed ester membrane filters with a pore size of 0.45 µm (Merck, Millipore,

Billerica, MA, United States), whereafter each filter was placed in a 9 cm petri dish

containing 1.5 mL citrate buffer (0.3 M, pH 3.5) and agitated gently using an orbital platform

shaker to remove the cells from the filter. Each 1.5 mL concentrated sample was then

centrifuged for 5 min at 16 000 × g, whereafter the remaining pellet was subjected to 6 µM

ethidium monoazide bromide (EMA) (Biotium, Hayward, CA, United States) treatment as

described by Reyneke et al. (2017). Ethidium monoazide bromide is a nucleic acid binding

dye which allows for the removal of extracellular deoxyribonucleic acid (DNA) or DNA from

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membrane-compromised cells. Upon photoactivation, EMA binds covalently to the DNA,

which subsequently inhibits the amplification of the bound DNA during quantification assays

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(e.g., qPCR) (Reyneke et al., 2017). Treatment of the samples with EMA was thus
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performed to allow for the detection of intact and potentially viable E. faecium,
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K. pneumoniae, and P. aeruginosa cells, as well as the aac(6’)-Ib and aac(6’)-aph(2’’) genes

from intact host bacterial cells, in the collected environmental water samples. Following EMA
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treatment, total genomic DNA extractions (i.e., DNA from intact and presumedly viable cells)

were performed using the Quick-DNA™ Fecal/Soil Microbe Miniprep Kit (Zymo Research,
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Irvine, CA, United States), as per the manufacturer‟s instructions.

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2.3 Quantitative Microbial Risk Assessment

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i. Hazard identification and quantification of target bacterial species/resistance genes

Quantitative polymerase chain reaction analysis was conducted on the EMA treated DNA

samples using a LightCycler® 96 Instrument (Roche Diagnostics, Mannheim, Germany) and

the FastStart Essential DNA Green Master (Roche Diagnostics) to quantify intact and viable

E. faecium, K. pneumoniae, and P. aeruginosa, in the environmental water samples. In order

to quantify intact bacterial cells containing the aac(6’)-aph(2’’) and aac(6’)-Ib aminoglycoside

resistance genes, qPCR analysis was conducted using the StepOnePlus™ Real-Time PCR

System (ThermoFisher Scientific, Waltham, Massachusetts, United States) and the FastStart

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Essential DNA Green Master (Roche Diagnostics). The primers and cycling parameters for

each of the target organisms and resistance genes are outlined in Table A2

(Supplementary File). All EMA-qPCR assays had a reaction mixture with a final volume of

20 µL, consisting of 10 µL FastStart Essential DNA Green Master (1X), 0.4 µL of the forward

and reverse primers (0.2 µM) and 5 µL template DNA. In addition, to minimise the level of

PCR inhibitors, each of the DNA samples were diluted 10-fold before EMA-qPCR analysis

(Reyneke et al., 2017). A negative control of sterile milliQ was included in the analysis for

each EMA-qPCR reaction, while the specificity of the primer sets was verified via the

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inclusion of melt curve analysis for all SYBR® Green EMA-qPCR assays (temperature

increase from 65 °C to 97 °C at 0.2 °C/s and continuous fluorescent signal acquisition at 15

readings/°C) (Reyneke et al., 2017).

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Standard curves were generated for the EMA-qPCR assays using the methodology outlined
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in Reyneke et al. (2017). Briefly, to obtain positive control DNA for the EMA-qPCR assays,
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conventional PCR assays (Table A2; Supplementary File) were conducted using the PCR

components and concentrations outlined in Table A3 (Supplementary file), to amplify the

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respective target genes using DNA obtained from E. faecium (clinical isolate; Ef CD1),

K. pneumoniae ATCC 13883, P. aeruginosa ATCC 27853, and wastewater collected from
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Tygerberg Hospital and various WWTPs (for the aminoglycoside resistance genes; Western
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Cape, South Africa). Sterile milliQ was utilised as a negative control for each PCR reaction.

Thereafter, the positive PCR products for each of the target species and resistance genes

were purified and concentrated using the Wizard® SV Gel and PCR Clean-up System

(Promega, Madison, Wisconsin). The purified, concentrated PCR products were sent for

sequencing using the BrilliantDye™ Terminator (v3.1) Cycle Sequencing Kit (ThermoFisher

Scientific) at Inqaba Biotechnical Industries (Pty) Ltd (Pretoria, South Africa), in order to

confirm the correct species identity, prior to use in the qPCR analyses. The DNA

concentration [determined using a NanoDrop® ND-1000 (Nanodrop Technologies Inc.)] and

gene product sizes were used to calculate the dilution required to obtain a final DNA

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concentration of 109 GC/μL. Serial 10-fold dilutions (109 to 100 GC/µL) of the purified positive

control PCR products were used to generate a standard curve for the EMA-qPCR assays.

The lower limit of detection (LLOD) for each EMA-qPCR assay was described as the lowest

concentration at which 95% of positive samples were detected (Clinical and Laboratory

Standards Institute, 2022). The EMA-qPCR performance characteristics for each of the

target species and resistance gene EMA-qPCR assays were analysed using the Roche

LightCycler® 96 Software version 1.1 and the StepOne Software version 2.3, respectively.

Following the EMA-qPCR analyses, the GC for E. faecium, K. pneumoniae, and

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P. aeruginosa were converted to GC/100 mL of the original environmental water samples, as

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outlined by Waso et al. (2018). Thereafter, the number of copies of the target gene in each

of the relevant host species (Table A2; Supplementary File) was used to convert the
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GC/100 mL into cell equivalents (cells/100 mL). In contrast, the target ARGs could not be
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accurately converted to potential cell equivalents, as these genes are carried on plasmids in
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multiple Gram-positive and Gram-negative bacteria, and the copy number has been shown

to vary between species (Table A2; Supplementary File) (Casetta et al., 1998; Ruiz et al.,
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2012). As a result, the number of copies detected for the target ARGs, using EMA-qPCR

analysis, were reported as GC/100 mL.

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ii. Exposure assessment

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The primary exposure routes associated with the use of the water sources collected in the

Vlottenburg informal settlement and Sir Lowry‟s Pass Village, were identified through visual

observation by the research team in the settlements during sampling collection and personal

communication with community members. These exposure scenarios included cleaning of

the home, washing laundry by hand, washing/bathing, garden hosing, garden work, playing

in/near the water samples, swimming, intentional drinking, and accidental consumption (in

various cases). An outline of the various exposure scenarios and the formulas used to

calculate the ingestion dose for each scenario, as well as descriptions of each exposure

route, are outlined in Table A4 and Table A5 (Supplementary file).

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The concentration of each of the target species and resistance genes was obtained from the

respective EMA-qPCR analysis results. Based on literature, the infectious fraction (i.e., the

fraction of the detected bacterial population that is pathogenic/likely to cause infection) of

E. faecium, K. pneumoniae, and P. aeruginosa was set to 0.019 to 0.03 (Stone et al., 2008),

0.03 to 0.15 (Paczosa and Mecsas, 2016), and 0.38 to 1.0 (Mena and Gerba, 2009),

respectively (Table 1).

iii. Dose-response

Based on the available “best-fit” dose-response models for each target organism, the

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exponential dose-response model (Equation 1) was used to calculate the risk of infection

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linked to pathogenic E. faecium (ingestion exposure), K. pneumoniae (ingestion exposure),
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and P. aeruginosa (ingestion exposure) within the environmental water samples for various

exposure scenarios (Domenico et al., 1982; Alsalah et al., 2015; Toh et al., 2017).
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Pinf = 1 – exp(-kd) Equation 1

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This equation describes the probability of infection (Pinf) following an individual exposure

event, where the parameter k defines the likelihood of the infective agent surviving the host‟s
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defence and subsequently initiating infection, while d is the amount (dose) of the organism(s)
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that is ingested. All parameters associated with the dose-response model and target

organism concentration distributions (based on the EMA-qPCR data) are outlined in Table 1
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and Table A6 (Supplementary file), respectively.

As no literature exists regarding the use of ARGs, such as the aac(6’)-Ib and aac(6’)-aph(2’’)

aminoglycoside resistance genes, in QMRA models to estimate the health risk posed by

pathogens containing these genes, no specific infective fractions or dose-response models

were available. However, the aac(6’)-Ib gene is commonly found in Gram-negative bacteria,

including K. pneumoniae, P. aeruginosa, A. baumannii, and E. coli (Ruiz et al., 2012; De

Oliveira et al., 2020). Therefore, a surrogate dose-response model was designed for the

aac(6’)-Ib gene using a combination of the infective fraction (IF%) (to estimate dose) and

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available exponential dose-response models of K. pneumoniae, P. aeruginosa,

A. baumannii, and E. coli, respectively (k-values inserted as a range into the exponential

dose-response model; Table 1). Similarly, as the aac(6’)-aph(2’’) gene is associated with

Gram-positive bacteria, specifically enterococci and staphylococci (Boehr et al., 2004;

Emaneini et al., 2013), a surrogate dose-response model was designed for the aac(6’)-

aph(2’’) gene by using a combination of the IF% (to estimate dose) and available exponential

dose-response models of E. faecium and Staphylococcus aureus (S. aureus) (k-values

inserted as a range into the exponential dose-response model; Table 1). The use of

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surrogate dose-response models for the aac(6’)-aph(2’’) (associated with Gram-positive

bacteria) and aac(6’)-Ib (associated with Gram-negative bacteria) genes made it possible to

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predict the risk of CA-infection caused by aminoglycoside-resistant Gram-positive and Gram-
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negative pathogens within the target communities, associated with the use of contaminated
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environmental water sources for various potable and non-potable purposes in these settings.
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iv. Risk characterisation

Lastly, to characterise the risk associated with using the various water sources for the
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domestic activities described in Table A4 (Supplementary file), risk characterisation was

conducted. It must be noted that the data from the surface runoff sites (i.e., samples
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collected from the centre and periphery of the settlements, respectively) obtained from the
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Vlottenburg informal settlement (n = 7) and Sir Lowry‟s Pass Village (n = 15), were combined

during analysis (for each site respectively) to determine the risk associated with surface

runoff from these sites. The likelihood of infection for each of the exposure routes was

calculated for each of the target species and resistance genes. This was described as the

probable number of infections per 10 000 persons per year, as previously outlined by Haas

(1999) (Equation 2).

P = 1 – (1 – Pinf)n Equation 2

This equation describes the probability of infection (P) following n exposure events per year

(Table A4; Supplementary file), determined using the previously calculated exposure

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probability of infection (Pinf). Monte Carlo analysis in RStudio (version 1.0.153) was used to

simulate each exposure scenario using 500 000 iterations. The various dose parameters

[e.g., ingestion volumes (Table A4; Supplementary file) and pathogen concentrations

(Table 1)] and exposure events per year (Table A4; Supplementary file), were sampled

randomly throughout the analyses.

3. Results

3.1 Quantitative PCR Analyses for the Target Bacterial Species and Resistance Genes

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The qPCR performance characteristics for the E. faecium, K. pneumoniae, and

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P. aeruginosa, as well as the aac(6’)-aph(2’’) and aac(6’)-Ib genes are included in Table A7

(Supplementary file). However, as the target bacterial species and resistance genes were
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not always detected in 100% (n = 45) of the samples, Figure 2 and Figure 3 only illustrate
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the results obtained for the detected cells/100 mL (E. faecium, K. pneumoniae, and
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P. aeruginosa) or GC/100 mL [aac(6’)-Ib and aac(6’)-aph(2’’)] within the positive samples

collected at the respective sampling sites. Please note that the sampling points referenced in
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the results refer to the sampling points outlined in Figure 1.

Overall, E. faecium had a high detection frequency and was present in 88.9% (40/45) of the
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water samples collected throughout the sampling period (Figure 2A). For the respective
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sites, E. faecium was detected in 100% (n = 6) of the Sir Lowry‟s Pass Village stormwater

samples (Point 1; concentration range of 9 to 5.71 × 102 cells/100 mL), and in 90.9% (10/11)

of the Sir Lowry‟s Pass Village stream samples (Point 2; concentration range of 1.52 × 101

to 4.24 × 102 cells/100 mL) (Figure 2A). Similarly, 85.7% (6/7) of the surface runoff collected

at the centre of the Sir Lowry‟s Pass Village contained E. faecium (Point 3; concentration

range of 6 to 2.70 × 103 cells/100 mL). All (100%; n = 8) the surface runoff samples collected

from the periphery of the Sir Lowry‟s Pass Village also contained E. faecium (Point 4;

concentration range of 3.59 × 101 to 4.36 × 103 cells/100 mL) (Figure 2A). For the

Vlottenburg informal settlement marsh water, E. faecium was detected in 83.3% (5/6) of the

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samples (Point 5; concentration range of 6 to 6.79 × 102 cells/100 mL), while it was detected

in 100% (n = 2) of the surface runoff samples (Point 6; concentration range of 1.43 × 102 to

2.82 × 102 cells/100 mL) collected in the centre of the Vlottenburg informal settlement.

Lastly, it was detected in 60% (3/5) of the surface runoff samples (Point 7; concentration

range of 1.23 × 101 to 7.08 × 102 cells/100 mL) collected at the periphery of the Vlottenburg

informal settlement (Figure 2A).

Klebsiella pneumoniae had a high prevalence and was detected in 100% (n = 45) of the

water samples collected throughout the sampling period (Figure 2B). For the respective

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sites, K. pneumoniae was detected at a concentration range of 1.01 × 102 to 1.15 × 104

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cells/100 mL in the Sir Lowry‟s Pass Village stormwater samples (Point 1), and at a

concentration range of 2.03 × 102 to 4.08 × 103 cells/100 mL in the Sir Lowry‟s Pass Village
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stream samples (Point 2; Figure 2B). Similarly, it was detected at a concentration range of
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1.02 × 102 to 6.23 × 105 cells/100 mL in the surface runoff collected at the centre of the Sir
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Lowry‟s Pass Village (Point 3), and at a concentration range of 4.29 × 102 to 2.42 × 106

cells/100 mL in the surface runoff samples collected from the periphery of the Sir Lowry‟s
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Pass Village (Point 4; Figure 2B). For the Vlottenburg informal settlement marsh water

(Point 5), K. pneumoniae was detected at a concentration range of 4.47 × 101 to 3.98 × 103
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cells/100 mL, while it was detected at a concentration range of 1.16 × 103 to 1.30 × 103
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cells/100 mL in the surface runoff samples collected in the centre of the Vlottenburg informal

settlement (Point 6). Lastly, it was detected at a concentration range of 1.06 × 101 to

6.09 × 103 cells/100 mL in the surface runoff samples collected at the periphery of the

Vlottenburg informal settlement (Point 7; Figure 2B).

Pseudomonas aeruginosa also had a high prevalence and was detected in 93.3% (42/45) of

the water samples collected throughout the sampling period (Figure 2C). For the respective

sites, P. aeruginosa was detected in 100% (n = 6) of the Sir Lowry‟s Pass Village stormwater

samples (Point 1; concentration range of 4.14 × 101 to 8.22 × 102 cells/100 mL), and in

100% (n = 11) of the Sir Lowry‟s Pass Village stream samples (Point 2; concentration range

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of 3 to 6.68 × 101 cells/100 mL) (Figure 2C). Additionally, 71.4% (5/7) of the surface runoff

collected at the centre of the Sir Lowry‟s Pass Village contained P. aeruginosa (Point 3;

concentration range of 4.24 × 101 to 1.20 × 102 cells/100 mL). All (100%; n = 8) the surface

runoff samples collected from the periphery of the Sir Lowry‟s Pass Village also contained

P. aeruginosa (Point 4; concentration range of 1.54 × 101 to 2.03 × 102cells/100 mL)

(Figure 2C). For the Vlottenburg informal settlement marsh water, P. aeruginosa was

detected in 100% (n = 6) of the samples (Point 5; concentration range of 7 to 3.11 × 102

cells/100 mL), while it was detected in 100% (n = 2) of the surface runoff samples (Point 6;

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concentration range of 5.67 × 101 to 8.79 × 101 cells/100 mL) collected in the centre of the

Vlottenburg informal settlement. Lastly, it was detected in 80% (4/5) of the surface runoff

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samples (Point 7; concentration range of 5 to 1.92 × 102 cells/100 mL) collected at the
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periphery of the Vlottenburg informal settlement (Figure 2C).
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To characterise the risk posed by aminoglycoside-resistant Gram-positive and Gram-
lP

negative pathogens in environmental reservoirs, the aac(6’)-aph(2’’) (associated with Gram-

positive species) and aac(6’)-Ib (associated with Gram-negative species) genes were also
na

quantified using EMA-qPCR. While the results of the qPCR analyses for the target bacterial

species were reported as cells/100 mL, the qPCR results obtained for the resistance genes
ur

in the current study were reported as GC/100 mL, as these genes are carried on plasmids
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where the copy number may vary, depending on the species in which they are found

(Casetta et al.,1998; Ruiz et al., 2012).

The aac(6’)-Ib gene had a high prevalence and was detected in 95.6% (43/45) of the water

samples collected throughout the sampling period. For the respective sites, the aac(6’)-Ib

gene was detected in 100% (n = 6) of the Sir Lowry‟s Pass Village stormwater samples

(Point 1; concentration range of 1.05 × 103 to 3.45 × 105 GC/100 mL), and in 90.9% (10/11)

of the Sir Lowry‟s Pass Village stream samples (Point 2; concentration range of 1.25 × 103

to 5.82 × 105 GC/100 mL) (Figure 3A). Similarly, 100% (n = 7) of the surface runoff collected

at the centre of the Sir Lowry‟s Pass Village contained the aac(6’)-Ib gene (Point 3;

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concentration range of 3.11 × 105 to 2.09 × 108 GC/100 mL). All (100%; n = 8) the surface

runoff samples collected from the periphery of the Sir Lowry‟s Pass Village also contained

the aac(6’)-Ib gene (Point 4; concentration range of 7.91 × 103 to 8.42 × 106 GC/100 mL)

(Figure 3A). For the Vlottenburg informal settlement marsh water, the aac(6’)-Ib gene was

detected in 83.3% (5/6) of the samples (Point 5; concentration range of 3.96 × 103 to

7.45 × 104 GC/100 mL), while it was detected in 100% (n = 2) of the surface runoff samples

(Point 6; concentration range of 9.38 × 103 to 7.87 × 104 GC/100 mL) collected in the centre

of the Vlottenburg informal settlement. Lastly, the aac(6’)-Ib gene was detected in 100%

of
(n = 5) of the surface runoff samples (Point 7; concentration range of 1.86 × 103 to

7.92 × 105 GC/100 mL) collected at the periphery of the Vlottenburg informal settlement

(Figure 3A).
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Similar to what was observed for K. pneumoniae, the aac(6’)-aph(2’’) gene was detected in
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100% (n = 45) of the environmental water samples. For the respective sites, the aac(6’)-
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aph(2’’) gene was detected at a concentration range of 3.99 × 102 to 3.35 × 104 GC/100 mL

in the Sir Lowry‟s Pass Village stormwater samples (Point 1), and at a concentration range
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of 1.84 × 101 to 2.70 × 104 GC/100 mL in the Sir Lowry‟s Pass Village stream samples

(Point 2; Figure 3B). Similarly, it was detected at a concentration range of 1.87 × 102 to
ur

2.69 × 105 GC/100 mL in the surface runoff collected at the centre of the Sir Lowry‟s Pass
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Village (Point 3), and at a concentration range of 4.52 × 102 to 1.70 × 105 GC/100 mL in the

surface runoff samples collected from the periphery of the Sir Lowry‟s Pass Village (Point 4;

Figure 3B). For the Vlottenburg informal settlement marsh water (Point 5), the aac(6’)-

aph(2’’) gene was detected at a concentration range of 3.52 × 102 to 5.57 × 105 GC/100 mL,

while it was detected at a concentration range of 3.75 × 104 to 2.94 × 106 GC/100 mL in the

surface runoff samples collected in the centre of the Vlottenburg informal settlement (Point

6). Lastly, the aac(6’)-aph(2’’) gene was detected at a concentration range of 2.34 × 102 to

2.54 × 107 GC/100 mL in the surface runoff samples collected at the periphery of the

Vlottenburg informal settlement (Point 7; Figure 3B).

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3.2 Health Risk Associated with the Environmental Water Sources

The annual risks of infection linked to the exposure of community members to the

environmental water sources, while performing the various activities outlined in Table A4

(Supplementary file), was determined based on the presence of pathogenic E. faecium,

K. pneumoniae, and P. aeruginosa. This was achieved by comparing all the calculated

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annual risks to the hypothetical annual benchmark limit for drinking water, which has a value

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of 1 × 10-4, representing one infection per 10 000 people per year.
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The mean annual risk of infection posed by E. faecium for accidental consumption (~10-11 to

~10-13), garden work (~10-12 to ~10-14), intentional drinking (~10-8 to ~10-10), washing laundry
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by hand (~10-12 to ~10-14), washing/bathing (~10-8 to ~10-10), garden hosing (~10-11 to ~10-13),
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cleaning of the home (~10-8 to ~10-10), swimming (~10-10 to ~10-12), and playing in/near the

water (~10-14 to ~10-15), was below the annual infection risk benchmark limit (1 × 10-4) for all
na

of the samples obtained from both the Vlottenburg informal settlement and Sir Lowry‟s Pass

Village (Figure A1; Supplementary file). The mean annual risk of infection posed by
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K. pneumoniae in all the samples collected from Sir Lowry‟s Pass Village and the
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Vlottenburg informal settlement, was then below the 1 × 10-4 benchmark limit for, garden

hosing (~10-6 to ~10-8), washing laundry by hand (~10-7 to ~10-9), accidental consumption

(~10-5 to ~10-8), garden work (~10-6 to ~10-8), and playing in/near the water sources (~10-9 to

~10-11). In contrast, in all the samples collected at the respective sites, the mean annual risk

of infection for K. pneumoniae exceeded the 1 × 10-4 risk limit for the intentional drinking

scenario, with the calculated risks ranging from ~10-2 to ~10-4 infections per person per year

(Figure 4A and 4B). Similarly, for the washing/bathing exposure scenario, 60% of the

samples exceeded (~10-3) the annual risk, except for the Sir Lowry‟s Pass Village surface

runoff (~10-5) and Sir Lowry‟s Pass Village stormwater (~10-5) samples. Results of the risk

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assessment analysis for cleaning of the home then indicated that while the Sir Lowry‟s Pass

Village surface runoff (~10-5) and Sir Lowry‟s Pass Village stormwater (~10-5) samples were

below the annual risk of infection, the remaining samples exceeded (~10-2 to ~10-3) the

annual benchmark limit (1 × 10-4) (Figure 4A and 4B). The risk associated with swimming in

the environmental water samples was then only exceeded for the Vlottenburg informal

settlement marsh (~10-4) and Vlottenburg informal settlement surface runoff (~10-4) samples

(Figure 4B), while the remaining samples were below (~10-5 to ~10-7) the benchmark limit.

The risk assessment for P. aeruginosa then revealed that the annual risk of infection for

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accidental consumption (~10-7 to ~10-8), garden work (~10-8 to ~10-9), swimming (~10-6 to

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~10-7), washing laundry by hand (~10-8 to ~10-9), playing in/near the water sources (~10-10 to

~10-11), and garden hosing (~10-7 to ~10-8) was below the benchmark limit for all the samples
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collected from Sir Lowry‟s Pass Village and the Vlottenburg informal settlement (Figure 5A
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and 5B). Similarly, all the samples were below the benchmark limit for washing/bathing (~10 -
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5
), except for the Sir Lowry‟s Pass Village stormwater samples, which exceeded (~10-4) the

annual risk of infection (Figure 5A). For the intentional drinking scenario, the annual risk of
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infection for P. aeruginosa in the Sir Lowry‟s Pass Village stream (~10-5) fell below the

benchmark limit, while the remaining samples (80%) exceeded (~10-3 to ~10-4) the annual
ur

infection risk benchmark limit (Figure 5A and 5B). Risk assessment for cleaning of the home
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then revealed that all the samples were within the acceptable risk limit (~10-5), except for the

Sir Lowry's Pass Village stormwater (~10-4) and Vlottenburg informal settlement marsh (~10-
4
), which exceeded the benchmark limit.

Using a combination of available dose-response models and input parameters which have

been previously outlined in literature for the individual risk assessment of K. pneumoniae,

P. aeruginosa, A. baumannii, and E. coli (Table 1), it was possible to estimate the potential

health risks posed by the presence of aminoglycoside-resistant Gram-negative bacteria in

the environmental water samples, based on the detected levels (using EMA-qPCR analysis)

of the aac(6’)-Ib gene (Figure 6). Overall, for the aac(6’)-Ib gene, the annual risk of infection

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associated with playing in/near the water sources was below the benchmark limit (~10 -6 to

~10-10) for all the samples collected from Sir Lowry‟s Pass Village and the Vlottenburg

informal settlement (Figure 6A and 6B). All the samples from both sampling sites then

exceeded the annual infection risk benchmark limit (1 × 10-4) for cleaning of the home (> 10-
4
) and intentional drinking (> 10-3) (Figure 6A and 6B). The annual risk of infection was also

exceeded for washing laundry by hand (~10-4), garden work (~10-4 to ~10-3), garden hosing

(~10-4 to ~10-3), washing/bathing (~10-4), accidental consumption (~10-3), and swimming

(~10-2), for both the Sir Lowry‟s Pass Village stream and Vlottenburg informal settlement

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marsh (Figure 6A and 6B). In contrast, the risks associated with washing laundry by hand

(~10-8), swimming (~10-6), garden hosing (~10-7), washing/bathing (~10-8), garden work (~10-

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7
to ~10-8), and accidental consumption (~10-7) were below the annual infection risk
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benchmark limit (1 × 10-4) for the Sir Lowry‟s Pass Village stormwater, Sir Lowry‟s Pass
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Village surface runoff, and Vlottenburg informal settlement surface runoff samples (Figure

6A and 6B).
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Similarly, using a combination of available dose-response models and input parameters

na

which have been outlined in literature for the individual risk assessment of E. faecium, and

S. aureus (Table 1), it was possible to estimate the potential health risks associated with the
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presence of aminoglycoside-resistant Gram-positive bacteria in the environmental water

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samples, based on the prevalence of the aac(6’)-aph(2’’) gene (determined using EMA-

qPCR analysis) (Figure 7). Overall, the annual risk of infection associated with the aac(6’)-

aph(2’’) gene in all the sample types collected from Sir Lowry‟s Pass Village and the

Vlottenburg informal settlement was below the benchmark limit for washing laundry by hand

(~10-10), garden work (~10-9 to ~10-10), cleaning of the home (~10-6), washing/bathing (~10-6),

swimming (~10-8), accidental consumption (~10-9), garden hosing (~10-9), intentional drinking

(~10-5 to ~10-6), and playing in/near the water samples (~10-10) (Figure 7A and 7B).

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4. Discussion

Ethidium monoazide bromide was incorporated in the current study as a nucleic acid binding

dye, to ensure that the amplification of any DNA from non-viable target species was

potentially eliminated during the detection and quantification (using EMA-qPCR) analyses

conducted for the environmental water samples (Reyneke et al., 2020). This is crucial,

particularly if the results are to be used to inform policy makers about the risks associated

with contaminated water and will aid in improving the health and safety of community

members by providing a possible solution to mitigate the risks that these water sources may

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pose. Overall, the EMA-qPCR analysis of the environmental water samples (n = 45)

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indicated that intact (viable) cells of the bacterial pathogens E. faecium, K. pneumoniae, and
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P. aeruginosa were present in 88.9%, 100%, and 93.3% of the total samples (n = 45)

analysed, respectively. The highest mean concentration of cells/100 mL for all the
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environmental water samples, collected from Sir Lowry‟s Pass Village and the Vlottenburg
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informal settlement, was recorded for K. pneumoniae (7.83 × 104 cells/100 mL), followed by

E. faecium (3.53 × 102 cells/100 mL), and P. aeruginosa (9.04 × 101 cells/100 mL).
na

Enterococcus faecium, K. pneumoniae, and P. aeruginosa are distributed ubiquitously in the

ur

environment, amongst soil, food (vegetables, fruit, and animal products), various water

sources (surface runoff, drinking water, streams, dams, and rivers, amongst others), plants,
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and sewage or municipal waste (Klockgether and Tümmler, 2017; Havenga et al., 2019;

Pachori et al., 2019). The detection of these species in the environment may also be

attributed to anthropogenic activities and general living conditions within the informal

settlement communities (Amarasiri et al., 2020). For example, communal ablution facilities

are intermittently dispersed throughout the informal settlements and are located adjacent to

several of the sites sampled in the current study. These facilities were often observed to be

in need of repair and free-flowing sewage contaminated the surface runoff streams flowing

through the settlements. Additionally, due to a lack of waste removal infrastructure within the

communities, household greywater is commonly discarded in the surface runoff streams or

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other environmental water sources, while municipal household waste is discarded on

communal garbage piles located throughout the informal settlements. Subsequently, waste

from human activities (e.g., food, an old toilet, general household waste etc.) was often

observed to accumulate within and surrounding the environmental water sources from which

samples were collected. Moreover, various domestic animals (e.g., dogs) were observed

throughout the settlement, while certain households located on the periphery of the

settlements housed pigs, goats, and chickens. It is thus hypothesised that all the preceding

factors may have contributed to the contamination of the environmental water sources with

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the target pathogens, as particularly E. faecium and K. pneumoniae (part of the coliform

bacteria group) commonly inhabit the gastrointestinal tract of humans and animals (Boehm

and Sassoubre, 2014; Harwood et al., 2014).

ro
-p
The widespread distribution of E. faecium, K. pneumoniae, and P. aeruginosa in the
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environmental reservoirs, and their ability to persist under unfavourable conditions (Navidinia
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et al., 2017), thus implies that when favourable conditions arise for bacteria, these

opportunistic pathogens can cause severe infections and disease, such as wound and soft
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tissue infections, urinary tract infections (UTIs), gastrointestinal illness, and pneumonia,

amongst many others (Denissen et al., 2022). The detection of K. pneumoniae and
ur

P. aeruginosa in the environmental water samples analysed in the current study is however,
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of particular concern, as these two species are notorious for their role in the onset of both

hospital-acquired (HA) and CA pneumonia outbreaks (Holt et al., 2015; John et al., 2017;

Martin and Bachman, 2018). In fact, Imai et al. (2016) reported on a case of CA pneumonia

with septicaemia in a 53-year-old woman, for which the exposure/causative scenario was not

known. The patient had no previous history of medical ailments or recent international travel

but admitted to being an active smoker for 30 years prior to the infection. Upon clinical

investigation, the patient was diagnosed with septic shock due to lobar pneumonia, with the

causative pathogen identified as P. aeruginosa. The crowded living conditions and lack of

adequate water, sanitation, and waste removal infrastructure in the informal settlements from

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which the samples were collected in the current study, implies that these communities may

be at a higher risk and that a single case of pneumonia (or any other infection or disease),

caused by exposure to these contaminated waters, may instigate a cluster outbreak. This is

concerning for the health and safety of community members in these settings as individuals

residing in lower income communities may be at greater risk of contracting life-threatening

infections, which in many cases, may lead to significant mortality and morbidity rates (Foster

et al., 2015; Ferreira-Coimbra et al., 2020).

Due to the excessive use and exploitation of antimicrobial compounds in the agricultural and

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clinical sectors (for therapeutic, growth promoting, and prophylactic purposes), antibiotic

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resistant bacteria (ARB) and ARGs are also becoming increasingly prevalent in natural

environmental reservoirs (Rodriguez-Mozaz et al., 2015; Khan et al., 2019). However, as

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most research focuses on reservoirs that have been highly contaminated with waste from
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anthropogenic and pharmaceutical activities, limited information is available on the
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occurrence, distribution patterns, transmission capabilities, and health risks of ARGs in

natural environments or surface runoff in informal settlement communities (Wright, 2010;

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Khan et al., 2019). Thus, as aminoglycosides are considered first-line antibiotics for the

treatment of enterococcal infections and are also frequently used to treat infections caused
ur

by Gram-negative pathogens such as K. pneumoniae and P. aeruginosa (Tsai et al., 2012;

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Krause et al., 2016), the aac(6’)-Ib and aac(6’)-aph(2’’) aminoglycoside resistance genes

were quantified in the environmental water samples collected from Sir Lowry‟s Pass Village

and the Vlottenburg informal settlement, using EMA-qPCR analysis.

Overall, the aac(6’)-Ib (95.6%; n = 43/45) and aac(6’)-aph(2’’) (100%; n = 45) genes were

detected at a high frequency in the environmental water samples collected from the various

sites in the current study. The aac(6’)-Ib gene is an aminoglycoside N-acetyltransferase

(AAC), which codes for resistance to several aminoglycosides in Gram-negative species

(Ramirez et al., 2013). This gene, and its variants [e.g., aac(6’)-Ib7; aac(6’)-Ib8; aac(6’)-Ib-

cr], are generally located on gene cassettes associated with disrupted or truncated

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integrons, insertion sequences such as IS26, and class 1 integrons (Sarno et al., 2002;

Woodford et al., 2009; Ramirez and Tolmasky, 2010). Similarly, the aac(6’)-aph(2’’) gene is

a plasmid-borne aminoglycoside resistance gene that is situated on Tn5281-like transposons

in various Gram-positive species such as enterococci, streptococci, and staphylococci

(Zhang et al., 2018). The aac(6’)-aph(2’’) gene encodes for the bifunctional 6‟-

acetyltransferase-2‟‟-phosphotransferase enzyme which induces high-level gentamicin

resistance in enterococci and was formed due to the fusion of genes encoding the individual

enzymes aac(6‟) and aph(2‟‟) (Zhang et al., 2018). As the genetic elements on which the

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aac(6’)-Ib and aac(6’)-aph(2’’) genes are found, commonly form part of genomic islands,

plasmids, and transposons, the probability for the dissemination of these genes at cellular

ro
and molecular levels, is high (Tolmasky, 2007; Rice et al., 2008). Subsequently, the number
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of microbial pathogens exhibiting aminoglycoside resistance has increased due to the
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widespread dissemination of the aac(6’)-Ib and aac(6’)-aph(2’’) genes amongst various

bacterial species, effectively limiting the use of antibiotics such as amikacin and gentamicin,
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to treat life-threating infections (Tolmasky, 2007; Dupont et al., 2011; Khani et al., 2016).
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Moreover, as aminoglycosides are commonly used in the agricultural sector due to their low

cost, growth promoting properties, and ability to prevent disease outbreaks (i.e., prophylactic
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benefits) (Glinka et al., 2020), there is great potential for the spread of these genes amongst
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clinical and environmental reservoirs, thereby enhancing aminoglycoside resistance in these

settings. For example, variants of the aac(6’) gene have been detected in wastewater (He et

al., 2019), surface soil from agricultural fields (Wu et al., 2020), surface water and hospital

sewage (Hamiwe et al., 2019), and urban sewage samples (Hendriksen et al., 2019),

amongst other reservoirs. The high detection levels obtained for the two aminoglycoside

resistance genes in the current study may thus indicate an increased persistence of

aminoglycoside-resistant pathogens in the environmental waters, which may be attributed to

their broad distribution in both clinical and environmental settings, as well as their prevalence

in multiple bacterial species. Furthermore, the high mean concentration detected for aac(6’)-

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Ib (7.07 × 106 GC/100 mL) and aac(6’)-aph(2’’) (6.68 × 105 GC/100 mL), is hypothesised to

be due to the presence of multiple copy numbers on a single plasmid, depending on which

species they are associated with (Woodford et al., 1992; Ramirez et al., 2013). These results

are concerning and suggest that the environmental water sources may pose a health risk to

community members in these settings, due to the possibility of contracting MDR infections

caused by Gram-positive and Gram-negative pathogens which may harbour the aac(6’)-

aph(2’’) and aac(6’)-Ib genes, respectively.

Subsequently, a QMRA framework was applied to assess the health risks associated with

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the utilisation of the environmental water sources containing potentially pathogenic

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E. faecium, K. pneumoniae, and P. aeruginosa, for various potable and non-potable

purposes (i.e., washing laundry by hand, cleaning of the home, garden hosing, garden work,
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washing/bathing, intentional drinking, swimming, accidental consumption, and playing
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in/near the surface runoff). Results for the QMRA then showed that none of the calculated
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risk values for E. faecium were above the 1 × 10-4 benchmark limit for any of the exposure

scenarios assessed in the current study (Figure A1; Supplementary file). It is thus
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hypothesised that the presence of this pathogen in the water sources would most likely not

pose a health threat to the residents of the target communities, as it was also detected at a
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low mean cell concentration (3.53 × 102 cells/100 mL) within the samples and has the lowest
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infective fraction of all the target bacterial species analysed (Table 1). Similar results were

reported by Stone et al. (2008), who determined the risk of gastrointestinal illness amongst

surfers at six Oregon (United States) beaches, for the ingestion of sea water contaminated

with enterococci. The average ingestion volumes for the surfers, along with other appropriate

risk assessment input parameters (e.g., frequency of occurrence, duration of the activity,

route of exposure etc.) were determined using a web-based survey. The likelihood of

exceeding an exposure equal to the onset of illness or infection in 19 people per 1000, then

ranged from ~2% at Otter Rock Beach, to 23% at Humbug Mountain Beach, indicating that

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the annual risk of gastrointestinal infection associated with the ingestion of enterococci-

contaminated sea water at these beaches was not significant.

In contrast to the results obtained for E. faecium, the risks posed by P. aeruginosa were

exceeded for washing/bathing in the Sir Lowry‟s Pass Village stormwater (~10-4), cleaning of

the home using Vlottenburg informal settlement marsh water (~10-4) and Sir Lowry‟s Pass

Village stormwater (~10-4), as well as for intentional drinking (~10-3 to ~10-4) in all the water

samples, except for the Sir Lowry‟s Pass Village stream (~10-5) (Figure 5). These results are

comparable to results reported by Reyneke et al. (2020) who observed that the health risk

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posed by P. aeruginosa in roof-harvested rainwater, was exceeded for the intentional

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drinking (~10-2) scenario. While tap water and recreational waters have been linked to

outbreaks caused by Pseudomonas, the exact role that water plays in the transmission of
-p
this bacterium to humans, and the subsequent onset of disease, remains unclear (Mena and
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Gerba, 2009). The results of the QMRA for P. aeruginosa in the current study, however,
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highlights the potential for an increased occurrence of P. aeruginosa infection to occur in the

Vlottenburg informal settlement and Sir Lowry‟s Pass Village, as children were often
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observed to play near the sites where the water samples were collected, while members of

the Sir Lowry‟s Pass Village were also observed to regularly wash their laundry in the
ur

stream.
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To date, limited research has however, been conducted on the health risks associated with

K. pneumoniae (Harb and Hong, 2017; Toh et al., 2017), and there is also currently limited

literature on the quantitative risk posed by this species in environmental water sources. Risk

assessment analysis conducted in the current study subsequently indicated that the risk

associated with K. pneumoniae for the intentional drinking scenario was exceeded for all the

environmental water samples (~10-2 to ~10-4). All samples, except for the Sir Lowry‟s Pass

Village surface runoff (~10-5) and Sir Lowry‟s Pass Village stormwater (~10-5), were then

found to exceed the annual infection risk benchmark limit for cleaning of the home (~10-
2
to ~10-3) and washing/bathing (~10-2 to ~10-3) (Figure 4). Furthermore, the risk associated

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with swimming was exceeded for the Vlottenburg informal settlement marsh (~10-4) and

Vlottenburg informal settlement surface runoff (~10-4) (Figure 4B). Thus, as the annual

infection risk was exceeded for many of the samples, collected at both sampling sites, for the

intentional drinking, washing/bathing, swimming, and cleaning of the home exposure

scenarios, it is hypothesised that there may be an increased risk associated with the

presence of pathogenic K. pneumoniae in these environmental water samples.

For the risk assessment analysis conducted in the current study, it is important to note that

while the use of a viability dye, to ensure the detection of only the viable bacterial community

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using molecular analysis, was implemented, and samples were regularly collected over

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multiple seasons, certain limitations are noted. For example, due to the continuous

discarding of household greywater by community members in the surface runoff streams, the
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samples collected on a specific day, may provide a snapshot overview of the concentration
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of the bacterial contaminants at that point in time and may have been an under- or over-
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estimation of the risk posed, compared to other sections of the surface runoff stream.

However, due to the continuous use of the surface runoff stream by community members for
na

greywater disposal, it can be concluded that this water source poses a significant health risk

to community members, especially as children often played near this water source.
ur

Additionally, over time, the surface runoff could contribute to the continuous contamination of
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the marsh or stream/river and increase the risks posed by these sources for domestic

activities; highlighting the need for intervention strategies to mitigate the risks posed by the

surface runoff (i.e., surface runoff/grey water removal systems in the community).

While we implemented published dose-response models, they did not account for

differences pertaining to the increased susceptibility that immunocompromised individuals or

children may have. The current reported health risks may thus be underestimated as census

data for the regions indicate that a high proportion of the population within the informal

settlement communities are immunocompromised and “diarrheal disease” and “lower

respiratory infections” are consistently included amongst the top ten contributors of mortality

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in children below 5 years of age (Health Systems Trust, 2018). However, while various

exposure scenarios were assessed in the current study, it is important to highlight that not all

of them are directly applicable to each waster source and were included as possible “worst-

case” scenarios. For example, the “intentional drinking” scenario would not be applicable to

the surface runoff in the communities; however, due to the abundance of the surface runoff

throughout the settlement there is a high probability that it could cross-contaminate

household water supplies (e.g., household stored water in buckets being contaminated

through splashing, putting a lid on the ground etc.)

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Furthermore, while risk assessment is beneficial for addressing risk management questions

ro
and allows options for risk management to be compared using both qualitative

(e.g., community surveys, interviews with community members or operators) and

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quantitative (e.g., qPCR data) approaches [World Health Organization (WHO), 2016], there
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remain several pitfalls associated with currently available QMRA information. For example,
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while several studies have investigated the risk associated with K. pneumoniae and

P. aeruginosa in different environmental reservoirs (Al-Jassim et al., 2015; Toh et al., 2017;
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Reyneke et al., 2020), the exposure scenarios and dose-response parameters that are

available for these species are primarily for dermal and ocular contact, resulting in skin or
ur

eye infections (P. aeruginosa), or gastroenteritis (K. pneumoniae). However, these scenarios
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do not highlight the more serious diseases or infections that K. pneumoniae and

P. aeruginosa are notoriously associated with, such as pneumonia (Denissen et al., 2022).

In the South African context, where a high proportion of the population living in informal

settlements are either immunocompromised or have tuberculosis, dose-response models

that may be used to estimate the health risk associated with respiratory infections are

required and would contribute significantly to the implementation of intervention strategies by

policy makers. Therefore, specific dose-response models should be established for these

particular infection endpoints, as they pose the greatest risk to communities in low-to-middle

income countries. Additionally, if these specific dose-response models are not available,

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suitable surrogate models (i.e., a similar infection endpoint caused by another closely related

bacterium) need to be identified and used to generate risk assessment data (Schoen et al.,

2021).

A large contributing factor to the issue of antimicrobial resistance is, however, the transfer of

ARGs and other resistance determinants amongst bacteria, as well as across the human-

animal-environment interface (Ahmed et al., 2021; Zhang et al., 2022). While a large body of

research has investigated the influence of ARGs on human, animal, and environmental

health, using a One Health approach, limited literature is available regarding the potential

of
health risks posed by these genes in the environment, based on quantitative data and

ro
predictive modelling or risk assessment approaches (Bengtsson-Palme et al., 2021). As

indicated, due to their broad-spectrum of activity, aminoglycosides are commonly used in

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clinical settings for the treatment of infections such as pneumonia, sepsis, neonatal sepsis,
re
and UTIs (Avent et al., 2011; Xie et al., 2011; Serio et al., 2018), as well as in the agricultural
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sector for pest control, the treatment and prevention of disease, and as a feed supplement

(Arsand et al., 2016; Glinka et al., 2020). There is thus increased opportunity for residues of
na

genes encoding aminoglycoside resistance, or bacteria harbouring these genes, to

contaminate environmental reservoirs via incorrectly discarded clinical or agricultural waste,

ur

amongst other sources (Garneau-Tsodikova and Labby, 2016; Glinka et al., 2020), and
Jo

subsequently, they may pose a risk to human health. Therefore, surrogate risk assessment

models were applied for aminoglycoside-resistant Gram-positive [E. faecium and S. aureus;

aac(6’)-aph(2’’) gene], and Gram-negative [K. pneumoniae, P. aeruginosa, A. baumannii,

and E. coli; aac(6’)-Ib gene] species, to predict the human health risks and potential for CA

outbreaks to occur, if the environmental water samples are used for various potable and

non-potable purposes in the informal settlement communities.

Overall, for the aac(6’)-Ib gene, the risks associated with cleaning of the home (> 10-4) and

intentional drinking (> 10-3) were exceeded for all of the environmental water samples, while

the risks associated with washing laundry by hand (~10-4), garden hosing (~10-4 to ~10-3),

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garden work (~10-4 to ~10-3), washing/bathing (~10-4), accidental consumption (~10-3), and

swimming (~10-2) were also exceeded for the Sir Lowry‟s Pass Village stream and

Vlottenburg informal settlement marsh samples (Figure 6). The aac(6’)-Ib gene was used as

a representative for the risk posed by aminoglycoside-resistant Gram-negative bacteria in

the environmental water samples. Therefore, a range was used for the k-value, with an

upper limit of 6.93 × 10-6 (A. baumannii) and a lower limit of 9.70 × 10-9 (E. coli). Similarly, a

range was used for the infective fraction of the aac(6’)-Ib gene, using 0.5% (IF% 0.005;

E. coli) as the lower limit, and 100% (IF% 1.00; P. aeruginosa) as the upper limit. A high

of
infective fraction of P. aeruginosa was used as the upper limit for the aac(6’)-Ib gene, as this

ro
infective fraction (IF% 1.00; P. aeruginosa) has been used in previous studies (Reyneke et

al., 2020) where the calculated risks were not significantly high and did not exceed the
-p
benchmark limit (1 × 10-4) for most exposure scenarios. Further research is however,
re
required to corroborate the results obtained and confirm that, based on the high infective

fraction of P. aeruginosa applied, the risks associated with the presence of this gene in the
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environment was not overestimated. In contrast, and in correlation with the results obtained
na

for the E. faecium risk assessment, there were no risks associated with the presence of the

aac(6’)-aph(2’’) gene for any of the exposure scenarios at any of the sampling sites (Figure
ur

7). Thus, based on the comparable QMRA results obtained for E. faecium and the aac(6’)-

aph(2’’) gene, as well as for K. pneumoniae, P. aeruginosa and the aac(6’)-Ib gene,
Jo

screening environmental waters for ARGs associated with a specific genus or species [e.g.,

the aac(6’)-aph(2’’) gene is associated with enterococci and staphylococci, while the aac(6’)-

Ib gene is associated with a variety of Gram-negative species], could potentially serve as a

surrogate model for estimating the risks associated with the corresponding genus/species

under investigation.

5. Conclusions

The detection of viable and intact E. faecium, K. pneumoniae, and P. aeruginosa within the

environmental water samples indicates that nosocomial-associated pathogens can survive

28
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and flourish in environmental reservoirs outside of clinical settings and may pose a serious

health threat to community members in low-to-middle income countries. High levels of

detection were also recorded for the aac(6’)-Ib and aac(6’)-aph(2’’) genes in the samples,

signifying the persistence of these genes within the environment. Subsequently, there is a

high probability for the transfer of these genes between different bacterial species via HGT,

further compounding the issue of antibiotic resistance. This is concerning as many of the

environmental water samples assessed in the current study are used by community

members residing in these areas for domestic activities such as washing laundry, home

of
cleaning, and washing/bathing, amongst others.

ro
Furthermore, while QMRA indicated that no risks were posed by E. faecium or the aac(6’)-

aph(2’’) gene for any of the exposure scenarios, K. pneumoniae was found to pose a threat if
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water collected from all the sites in the Vlottenburg informal settlement and Sir Lowry‟s Pass
re
Village, is used for intentional drinking (~10-2 to ~10-4). Klebsiella pneumoniae then posed a
lP

health risk for washing/bathing (~10-3), swimming (~10-4) and cleaning of the home (~10-2 to

~10-3) in several samples, while P. aeruginosa posed a risk for intentional drinking, (~10-3 to
na

~10-4), washing/bathing (~10-4), and cleaning of the home (~10-4). The aac(6’)-Ib gene posed

a risk for intentional drinking (> 10-3) and cleaning the home (> 10-4) in all the samples, as
ur

well as posed risks in the Sir Lowry‟s Pass Village stream and Vlottenburg informal
Jo

settlement marsh for most of the remaining exposure scenarios (i.e., washing/bathing,

garden hosing, garden work, accidental consumption, and swimming).

 The application of QMRA proved useful for monitoring and estimating the potential

human health risks associated with the use of the environmental water sources,

using a site-specific approach.

 As this is one of the first studies to investigate the use of surrogate risk assessment

models to estimate the risks associated with aminoglycoside-resistant Gram-positive

[aac(6’)-aph(2’’) gene] and Gram-negative [aac(6’)-Ib gene] bacteria, future studies

29
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can benefit from and use this information to design more comprehensive models for

ARGs and ARB in environmental reservoirs.

 It is uncertain whether the calculated risks may be an over- or under-estimation of the

risks posed by bacterial species harbouring the aac(6’)-Ib and aac(6’)-aph(2’’) genes

in environmental waters, due to the combined use of multiple k-values and infective

fractions. Factors such as i) the impact of environmental parameters on AMR, and ii)

the role played by AMR traits in influencing morbidity and mortality, should be

considered prior to the dose-response selection stage.

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Author Contributions

ro
JD, BR and WK conceived and designed the experiments. JD performed the experiments.
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JD and BR analysed the data. SK, TB and WK contributed reagents, materials, and analysis

tools. JD, BR and WK compiled the manuscript. SK and TB edited the manuscript. All
re
authors contributed to the article and approved the submitted version.
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Declaration of competing interest

na

The authors declare that the research was conducted in the absence of any commercial or

financial relationships that could be construed as a potential conflict of interest.

ur

Acknowledgements
Jo

The authors acknowledge the financial assistance provided by the Postgraduate Scholarship

Programme (PSP) at Stellenbosch University towards the postgraduate student bursary.

Opinions communicated and all conclusions arrived at are those of the authors and are not

necessarily attributed to Stellenbosch University.

Funding

This project has received funding from the Water Research Commission of South Africa

(Project No 2021/2023-00449) and National Research Foundation of South Africa (Grant no:

137962). Opinions expressed and conclusions arrived at are those of the authors and are

not necessarily to be attributed to the National Research Foundation.

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A B

2 4

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5
6

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[1] Stormwater Samples River [5] Marsh/Stream Samples Marsh

[2] River/Stream Samples Stormwater Canal [6] Surface Runoff Samples Settlement Border
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[3] Surface Runoff Samples Settlement Border [7] Surface Runoff Samples

[4] Surface Runoff Samples

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Figure 1. Sampling sites for the collection of various environmental water samples (i.e.,
streams/rivers, stormwater runoff, surface runoff, marsh) in (A) Sir Lowry‟s Pass Village and
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(B) Vlottenburg informal settlement (Western Cape, South Africa).

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Figure 2. Box and whiskers plot illustrating the concentration (cells/100 mL) for (A)
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E. faecium, (B) K. pneumoniae, and (C) P. aeruginosa detected in the different

environmental water samples [surface runoff and marsh water (Vlottenburg informal
settlement) and surface runoff, stream, and stormwater (Sir Lowry‟s Pass Village)]. The
st rd
outer box illustrates the 1 and 3 quartiles, the inner line represents the median, and the
whiskers illustrate the minimum and maximum detected cells/100 mL. The red dotted line
represents the lower limit of detection. C - centre of settlement; P - periphery of settlement.

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Figure 3. Box and whiskers plot illustrating the concentration (GC/100 mL) of the (A)

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aac(6’)-Ib and (B) aac(6’)-aph(2’’) resistance genes in the different environmental water
samples [surface runoff and marsh water (Vlottenburg informal settlement) and surface

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st
runoff, stream, and stormwater (Sir Lowry‟s Pass Village)]. The outer box illustrates the 1
rd
and 3 quartiles, the inner line represents the median, and the whiskers illustrate the
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minimum and maximum detected GC/100 mL. The red dotted line represents the lower limit
of detection. C - centre of settlement; P - periphery of settlement.
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Figure 4. Annual health risk associated with K. pneumoniae when using the various
environmental water sources in the (A) Sir Lowry‟s Pass Village and (B) Vlottenburg informal
st rd
settlement (Western Cape, South Africa). The outer whiskers represent the 1 and 3
quartiles, and the inner shape represents the median. The red dashed line represents the
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annual benchmark limit (1 × 10 ; one infection per 10 000 people per year).

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Figure 5. Annual health risk associated with P. aeruginosa when using the various
environmental water sources in the (A) Sir Lowry‟s Pass Village and (B) Vlottenburg
informal settlement (Western Cape, South Africa). The outer whiskers represent the
st rd
1 and 3 quartiles, and the inner shape represents the median. The red dashed
-4
line represents the annual benchmark limit (1 × 10 ; one infection per 10 000 people
per year).

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Figure 6. Annual health risk associated with the aac(6’)-Ib gene when using the
various environmental water sources in the (A) Sir Lowry‟s Pass Village and (B)
Vlottenburg informal settlement (Western Cape, South Africa). The outer whiskers
st rd
represent the 1 and 3 quartiles, and the inner shape represents the median. The
-4
red dashed line represents the annual benchmark limit (1 × 10 ; one infection per 10
000 people per year).

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Figure 7. Annual health risk associated with the aac(6’)-aph(2’’) gene when using the
various environmental water sources in the (A) Sir Lowry‟s Pass Village and (B) Vlottenburg
st
informal settlement (Western Cape, South Africa). The outer whiskers represent the 1 and
rd
3 quartiles, and the inner shape represents the median. The red dashed line represents the
-4
annual benchmark limit (1 × 10 ; one infection per 10 000 people per year).

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Table 1. Monte Carlo simulation dose-response input parameters for the target bacterial
species and antibiotic resistance genes.
Dose-
Infective
Organism/Gene Response Background Reference
Fraction
Model
Kay et al.
(1994);
Ottoson
Model: Human and
IF% Exponential
Stenström
E. faecium 0.019 to k = 2.19 x Exposure: Ingestion (2003);
0.03 10-11 Response: Infection/gastroenteritis Stone et
al. (2008);
Alsalah et

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Al-Jassim
Model: Mouse

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Exponential et al.
IF% 0.38 (2015);
P. aeruginosa k = 1.87 x Exposure: Ingestion
to 1.0
10-8 Response: Infection Alsalah et
-p al. (2015)
Model: Rats Domenico
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Exponential et al.
IF% 0.03 Exposure: Transtracheal (1982);
K. pneumoniae k = 7.68 x
to 0.15 instillation into lungs
10-7 Toh et al.
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Response: Lobar pneumonia (2017)

Reyneke
Model: Human et al.
IF% Exponential
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E. coli* (2020);
0.005 to k = 9.7 x Exposure: Ingestion
[aac(6’)-Ib] QMRA
0.1 10-9 Response: Infection Wiki
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(2022)
López-
Rojas et
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Model: Mouse al. (2011);

Exponential
A. baumannii* Exposure: Intraperitoneal Toh et al.
IF% 0.45 k = 6.93 x
[aac(6’)-Ib] inoculation (2017);
10-6
Response: Peritoneal sepsis Colquhoun
and Rather
(2020)
Model: Human Rose and
Exponential
Haas
S. aureus* IF% 0.15 k = 6.46 x Exposure: Subcutaneous (1999);
[aac(6’)-aph(2’’)] to 0.20 10-8 to application on forearm
Chen et al.
1.00 x 10-7 Response: Infection (2021)
IF% - infectious fraction of the organism (i.e., the percentage of strains in that species that
can cause human infection); k – probability of the pathogen surviving the host‟s defence to
initiate infection. * - additional dose-response model input parameters that were utilised for
the antibiotic resistance genes as they are present within the specific organisms.

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Declaration of interests

☐The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

☒The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:

Wesaal Khan reports financial support was provided by Water Research Commission.
Wesaal Khan reports financial support was provided by National Research Foundation.

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Graphical abstract

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Highlights

 E. faecium, K. pneumoniae, and P. aeruginosa persist in extra-hospital reservoirs.

 High gene copies of ABR aac(6’)-Ib and aac(6’)-aph(2’’) detected in environmental
waters.
 K. pneumoniae and P. aeruginosa posed a health risk for drinking and other
domestic activities.
 aac(6’)-Ib gene indicated health risk for drinking and selected domestic activities.
 Surrogate models for ARGs may be suitable to assess risks of related genera.

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