1. Introduction
South Africa is a water stressed country due to the low average received rainfall (465 mm), which is below the global average of 860 mm [
1]. Demand for this important scarce resource is expected to increase due to rapid industrial development, increasing human population,
per capita consumption increases and the resulting impact of human activities on the environment [
2,
3]. High water demand and consumption also leads to increases in the volume of wastewater generated [
4]. The availability of good quality water is of paramount importance bringing to the fore the consequence of contamination of water bodies with pathogenic microorganisms [
5]. Water bodies such as rivers are subject to dramatic changes in microbial and physicochemical qualities as a result of a variety of anthropogenic activities in the watershed. These changes are caused by discharges of municipal raw waters or treated effluent at a specific point source into the receiving surface waters [
6,
7]. Point-source pollution problems will not only increase treatment costs considerably, but may also introduce a wide range of pathogens and harmful chemicals into surface waters that may be supplied to many rural and urban communities, thus resulting in incidences of waterborne diseases with far reaching socio-economic implications [
8,
9]. Although a vast majority of microorganisms present in wastewater are not pathogenic [
10], some pathogenic bacteria possibly originating from discharge of inadequately treated wastewater effluent have been implicated in the outbreak of waterborne diseases over the years [
11].
Salmonella spp. are important Gram-negative bacilli, which infect both humans and animals causing a wide range of diseases such as gastroenteritis, typhoid fever, osteomyelitis, septicemia and meningitis. This genus comprises of over 2000 recognized serotypes and is divided into two species namely
S. bongori and
S. enterica.
Salmonella enterica consist of six subspecies, namely
enterica, arizonae, salamae, diarizonae, houtenae and indica [
12,
13]. It is estimated that 93.8 million cases of gastroenteritis due to
Salmonella spp. occur globally each year, with some 155,000 deaths [
14]. This high number of infections emphasizes the importance of this intracellular pathogen and indicates a considerable worldwide disease burden. Mortality rate of
Salmonella infections is a health problem mainly in developing countries [
15], while morbidity due to acute
Salmonella infection can also have socio-economic impacts in developed countries [
16]. Added to this disease burden are the complications arising from the inefficacy and failures of antimicrobial chemotherapies applied in clinical practice to remedy these diseases. Bacterial resistance to antibiotics has increased globally in recent years and poses threats to human health [
17]. Antimicrobial resistance in
Salmonella has been associated with an increase in the number of adverse events following infection such as higher levels of hospitalization, longer illness, and higher risk of invasive illness as well as treatment failures [
18]. The World Health Organization (WHO), the European Commission and the United States Centre for Disease Control and prevention (CDC) have recognized the importance of studying the emergence of resistance genes as well as the need for control strategies [
19]. In most countries, the microbial quality of final treated effluent is estimated based on the level of indicator organisms present [
20,
21]. However, several studies have shown that the presence of indicator organisms does not always correlate with the presence of pathogens [
22,
23]. This study therefore aimed to determine the prevalence of antibiotic resistant and virulent
Salmonella spp. in treated effluents and receiving surface water of two wastewater treatment plants located in Durban, South Africa.
4. Discussion and Conclusions
The impact of substandard quality effluent or untreated wastewater discharged into receiving water bodies can be detrimental to water availability and security. Wastewater management is the first barrier in a multi-barrier system to ensure safe drinking water, public health and environmental sustainability [
32]. Physicochemical analysis of the wastewater gives an indication of the quality of effluents being discharge into the environment. The temperature regime varied depending on the season but was still within the acceptable limit of 25 °C [
33] and did not pose any threat to the receiving watershed. The temperature is a very important parameter because of its effect on the chemical reaction and reaction rates, aquatic life and suitability of the water for beneficial uses. Increase in temperature can lead to the disruption of the ecosystem in the receiving watershed resulting in high mortality of aquatic life and encouraged growth of undesirable algae and wastewater fungi [
34]. At both plants, the pH ranged between 6.41–8.08 and 6.37–7.88. The neutral to alkaline pH values recorded in this study are similar to previous reports [
7]. Very low or high pH is known to be toxic to aquatic life and alters the solubility of chemicals in water [
35]. The pH of most natural waters is in the range of 4−9 and the target limit set by the South African Department of Water Affairs is between 5.5 and 9.5 [
33]. The pH of water can provide important information about many chemical and biological processes and provides indirect correlations to a number of different impairments in the wastewater treatment processes [
36]. Changes in pH can also be indicative of industrial pollution, photosynthesis or the decomposition of organic matter by microorganisms [
37]. Hence, the pH values recorded in this study fell within the acceptable range indicating that discharge of the treated wastewater may have no negative impact on the river water with respect to pH.
The turbidity of the water samples in this study ranged between 1.42 NTU to 76.43 NTU and varied seasonally (
Table 2 and
Table 3). Higher turbidity values recorded in some months at the D.P could be the result of storm runoff and inadequate treatment leading to the high variation in turbidity. This high variation has been reported in previous studies in the Eastern Cape province of South Africa [
7]. The turbidity values recorded in this study was higher than the <5 NTU set by the World Health Organization (WHO) for effluent discharge into the environment [
7]. Turbidity is caused by small particles that may be organic or inorganic and can provide food and shelter for pathogens providing a possible explanation of its strong positive correlation with
Salmonella count at the NWWTW (
Table 4). If not removed, turbidity can promote the regrowth of pathogens in the final effluent of receiving water body into which the effluent is discharged [
38]. Turbidity also limits the bactericidal effect of chlorine in the wastewater during disinfection [
35] and may react with organic compounds in the water to form micro-contaminants such as trihalomethane (THM). THMs are carcinogenic and render the water unfit for recreational activities to consumers who may depend on the receiving water shed for such [
9,
39].
Although the BOD
5 values recorded was stable across each sampling point in each month, the values varied significantly in the course of the study ranging from 1.03 mg/L to 11.04 mg/L. There is no South African guideline for BOD
5 levels in the final effluent of wastewater; however, the EU recommends a discharge limit of 3 to 6 mg/L for aquatic ecosystems [
6]. Discharge of effluent high in BOD into water bodies would result in rapid depletion of dissolved oxygen leading to anoxic conditions, and consequent disruption of balance in the aquatic ecosystem [
6]. On most occasions the recorded BOD
5 values at the D.P were within the recommended EU limit. The COD of the water samples varied remarkably throughout the study period. High COD values were recorded at U.S while, the average recorded values (212 mg/L) at the D.P greatly exceeded the South African limit of 30 mg/L [
33]. High levels of COD observed U.S could be attributed to runoff, agricultural activities and anthropogenic activities. Igbinosa and Okoh [
7] reported a similar observation and attributed the increased COD to addition of organic and inorganic substances from the environment as well as organic contaminants entering the system from municipal sewage treatment plants or non-point sources of pollution. Higher average COD values varying from 512 to 698.11 mg/L were reported in a study on river quality in India and was attributed to the presence of inorganic chemicals in the wastewater of a nearby chemical industry [
40].
Tertiary treatment of final sewage effluent with chlorine at the wastewater treatment plants (WWTPs) under investigation reduced the number of viable presumptive
Salmonella spp. at the discharge point during the sampling period but failed to totally eliminate them (
Figure 1). Presumptive
Salmonella spp. were also recovered downstream of the receiving rivers indicating the negative impact of the wastewater treatment plants on the microbial quality of the river. This indicates that treated wastewater effluents discharged from these treatment plants are a source of contamination of receiving surface waters with this potential pathogen. Upstream of the river at the NGWTP is an informal settlement with poor sanitation and inadequate sewage disposal system, which contaminates the river with human and animal wastes while the bank of the Umgeni River downstream, is littered with feces. Storm runoff from this informal settlement and riverbanks explains the high count of presumptive
Salmonella spp. observed upstream. In contrast to findings from this study, Olaniran, Naidoo and Pillay [
24] reported lower counts of
Salmonella spp. from treated wastewater of same plants under investigation, which may be due to the short duration of the study or an improvement in the operational conditions of the treatment plants. Elsewhere, Momba, Osode and Sibewu [
6] reported recovery of pathogenic microorganisms including
Salmonella spp. in the final effluents of four WWTPs in the Eastern Cape province of South Africa and concluded that WWTPs serve as a point source of microbial pollution of water bodies. Recent reports have also suggested that WWTPs plants in South Africa are either dysfunctional or non-functional [
41], inefficient in removing microbial pathogens from wastewater and produce wastewater effluent of unacceptable standard that does not meet discharge guidelines set by the department of water affairs, South Africa [
42,
43,
44,
45]. The issue of treatment efficiency is of major importance if the reclaimed water is intended for recreational or potable reuse or is to be discharged into water bodies because disposal of inadequately treated wastewater into surface water recipient is one of the major sources of pathogens in the environment [
42,
46]. Though
Salmonella is isolated from water in lower numbers than indicator bacteria such as fecal coliform, fecal streptococci and enterococci; counts in the range of 15−1000 cfu/mL may pose public health risks [
47]. Thus, the presence of this organism in the final effluent of wastewater and receiving surface water is a serious cause for concern especially where the contaminated water is depended on for irrigation and rural socio-economic activities.
In this study, 200
Salmonella spp. were recovered and found to contain the
invA gene (
Figure 2), which led to their confirmation as
Salmonella spp. in agreement with previous studies [
48,
49]. The
invA gene is conserved in all
Salmonella spp. and encodes for a protein in the inner and outer membrane which is essential for virulence and is thought to trigger the internalization required for invasion into deeper tissues [
29,
30]. At the NWWTW, confirmed
Salmonella spp. were only recovered at the B.C point (6.5%) but not at the D.P indicating the plant was efficient at removing
Salmonella spp. from the wastewater during the sampling period. This is also corroborated by the fact that the NWWTW has a high Greendrop status rating [
50]. The Greendrop is an index developed by the Department of Water Affairs in South Africa to rate wastewater treatment plants in the country based on the quality of their final effluent. In the Eastern Cape province of South Africa, Momba, Osode and Sibewu [
6] observed the presence of
Salmonella spp. in 50% of final wastewater effluent and 35% in the receiving river samples. Poor sanitation, lack of access to proper sewage disposal systems, malnutrition and poverty have been described as some of the leading factors contributing to the high prevalence of salmonellosis and other diarrheal diseases in developing countries [
51,
52,
53].
Amplification of virulence genes (
Figure 2) revealed that the isolates contained one or more virulence genes present in the
Salmonella pathogenicity island (SPI). This can pose serious health threats to consumers who depend on the river water for daily and recreational activities. Pathogenicity islands are found on genomes of pathogenic bacteria but are absent in non-pathogenic strains of the same or related species [
54]. The presence of all four virulence genes were reported to be present in 87.2% of
Salmonella isolated from patients with systemic infection [
55] while 12.8% of
Salmonella spp. isolated from stool samples lacked the
misL and
orfL gene. The
spiC gene is found in the SPI-2 and is essential for systemic pathogenesis because it encodes a type III secretion system (T3SS) that is activated after invasion [
56,
57]. The T3SS system is used by the pathogen to deliver virulence factors to the host cell and interfere with or subvert normal host cell signaling pathways [
58]. The
misL gene located on SPI-3 encodes an autotransporter protein involved in intestinal colonization and essential for survival in macrophages [
55,
59]. The SPI-4 is a 25 kb pathogenicity island containing the
orfL gene thought to encode a type 1 secretion system (an autotransporter protein) that mediate the secretion of toxins and is necessary for macrophage survival [
55,
59], while the
pipD gene encodes effector proteins for the T3SS transport protein [
29,
30] and is mainly associated with enteropathogenesis [
58]. The presence of these virulence genes in
Salmonella spp. isolated from treated wastewater effluent and receiving surface water indicate the capabilities of these isolates in causing infections in susceptible hosts. Recently, there was report of an outbreak of acute gastroenteritis in KwaZulu-Natal, which was linked to food, contaminated with
Salmonella enterica serovar Enteritidis resulting in the hospitalization of 216 people [
60]. The report suggested a point source outbreak with a possibility of continued transmission. The true burden of
Salmonella disease in Africa is unclear thus a comprehensive epidemiological study is needed to elucidate it. The
Salmonella serotypes that most commonly cause invasive non-typhoidal
Salmonella in Africa are
S. typhimurium and
S. enteritidis, which are usually associated with a broad host range and with enteric diseases [
11].
Resistance against sulfamethoxazole (100%), nalidixic acid (27%) and streptomycin (14%) was observed among these isolates. Intermediate resistance was observed against streptomycin (74%), nalidixic acid (44%) and fosfomycin (8.5%). Resistance to nalidixic acid suggests possible resistance to third generation quinolones CLSI [
31]. However, this was not observed in this study, as isolates resistant to nalidixic acid were also susceptible to the third generation quinolones tested. Previous studies have suggested that quinolones should not be used in the treatment of invasive Salmonellosis due to strains with decreased sensitivity to fluoroquinolones and possible risk of treatment failure [
29,
61]. The results obtained in this study further emphasize the need for prudent use of fluoroquinolones and other commonly used antibiotics to prevent the emergence of resistant phenotypes [
62]. Consistent with this study,
Salmonella spp. were reported to be highly sensitive to third generation β-lactams [
29,
59] but resistant to sulfamethoxazole, nalidixic acid and streptomycin. Data from the National Antimicrobial Resistance Monitoring Systems (NARMS) in the US from 1996 to 2004 showed increase in resistance of clinical isolates of
Salmonella against antibiotics [
63]. The upsurge in multidrug resistant strains of
Salmonella over the past decade is threatening successful treatment of diseases caused by this organism especially in developing countries where disease burden is high [
64].
In conclusion, this study revealed that treated effluents from WWTPs investigated are reservoirs of antibiotic resistant and virulent Salmonella spp. The isolates were susceptible to most third generation β lactams tested whilst exhibiting resistance to other antibiotics. The presence of virulence genes is indicative of their capabilities to cause infection in susceptible hosts. Though certain physicochemical parameters were within the stipulated guidelines, the overall quality of the treated effluent is still low. Thus, in order to protect the valuable surface water resources, urgent intervention is required by the regulatory authorities and workers in these treatment plants to optimize treatment efficiency. Constant surveillance of the treatment processes and final effluent, infrastructural upgrade of the wastewater treatment works and provision of adequate sanitation and sewage disposal systems to rural communities on the banks of rivers is recommended.