Please cite this article as: Al-Jassim, N., Ansari, M.I., Harb, M., Hong, P.-Y., Removal of bacterial contaminants and antibiotic resistance genes by conventional wastewater treatment processes in Saudi Arabia: Is the treated wastewater safe to reuse for agricultural irrigation?, Water Research (2015Research ( ), doi: 10.1016Research ( /j.watres.2015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT Abstract 1This study aims to assess the removal efficiency of microbial contaminants in a local wastewater 2 treatment plant over the duration of one year, and to assess the microbial risk associated with 3 reusing treated wastewater in agricultural irrigation. The treatment process achieved 3.9 logs 4 removal of heterotrophic bacteria and up to 3.9 logs removal of fecal coliforms. The final 5 chlorinated effluent had 1.8 x 10 2 MPN/100 mL of fecal coliforms and fulfils the required quality 6 for restricted irrigation. 16S rRNA gene-based high-throughput sequencing showed that several 7 genera associated with opportunistic pathogens (e.g. Acinetobacter, Aeromonas, Arcobacter, 8 Legionella, Mycobacterium, Neisseria, Pseudomonas and Streptococcus) were detected at 9 relative abundance ranging from 0.010 to 21 % of the total microbial community in the influent. 10

Water scarcity is a global problem, and is particularly acute in certain regions like Africa, the Middle East, as well as the western states of America. A breakdown on water usage revealed that 70% of freshwater supplies are used for agricultural irrigation. The use of reclaimed water as an alternative water source for agricultural irrigation would greatly alleviate the demand on freshwater sources. This paradigm shift is gaining momentum in several water scarce countries like Saudi Arabia. However, microbial problems associated with reclaimed water may hinder the use of reclaimed water for agricultural irrigation. Of particular concern is that the occurrence of antibiotic residues in the reclaimed water can select for antibiotic resistance genes among the microbial community. Antibiotic resistance genes can be associated with mobile genetic elements, which in turn allow a promiscuous transfer of resistance traits from one bacterium to another. Together with the pathogens that are present in the reclaimed water, antibiotic resistant bacteria can potentially exchange mobile genetic elements to create the “perfect microbial storm”. Given the significance of this issue, a deeper understanding of the occurrence of antibiotics in reclaimed water, and their potential influence on the selection of resistant microorganisms would be essential. In this review paper, we collated literature over the past two decades to determine the occurrence of antibiotics in municipal wastewater and livestock manure. We then discuss how these antibiotic resistant bacteria may impose a potential microbial risk to the environment and public health, and the knowledge gaps that would have to be addressed in future studies. Overall, the collation of the literature in wastewater treatment and agriculture serves to frame and identify potential concerns with respect to antibiotics, antibiotic resistant bacteria, and antibiotic resistance genes in reclaimed water.

This study examined the decay kinetics and molecular responses of two Escherichia coli strains upon solar irradiation. The first is E. coli PI-7, a virulent and antibiotic-resistant strain that was isolated from wastewater and carries the emerging NDM-1 antibiotic resistance gene. The other strain, E. coli DSM1103, displayed lower virulence and antibiotic resistance than E. coli PI-7. In a buffer solution, E. coli PI-7 displayed a longer lag phase prior to decay and a longer half-life compared with E. coli DSM1103 (6.64 ± 0.63 h and 2.85 ± 0.46 min vs 1.33 ± 0.52 h and 2.04 ± 0.36 min). In wastewater, both E. coli strains decayed slower than they did in buffer. Although solar irradiation remained effective in reducing the numbers of both strains by more than 5-log in <24 h, comparative genomics and transcriptomics revealed differences in the genomes and overall regulation of genes between the two E. coli strains. A wider arsenal of genes related to oxidative stress, cellular repair and protective mechanisms were upregulated in E. coli PI-7. Subpopulations of E. coli PI-7 expressed genes related to dormancy and persister cell formation during the late decay phase, which may have accounted for its prolonged persistence. Upon prolonged solar irradiation, both E. coli strains displayed upregulation of genes related to horizontal gene transfer and antibiotic resistance. Virulence functions unique to E. coli PI-7 were also upregulated. Our findings collectively indicated that, whereas solar irradiation is able to reduce total cell numbers, viable E. coli remained and expressed genes that enable survival despite solar treatment. There remains a need for heightened levels of concern regarding risks arising from the dissemination of E. coli that may remain viable in wastewater after solar irradiation.

bThe emergence of resistance to last-resort antibiotics is a public health concern of global scale. Besides direct person-to-person propagation, environmental pathways might contribute to the dissemination of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). Here, we describe the incidence of bla NDM-1 , a gene conferring resistance to carbapenems, in the wastewater of the city of Jeddah, Saudi Arabia, over a 1-year period. bla NDM-1 was detected at concentrations ranging from 10 4 to 10 5 copies/m 3 of untreated wastewater during the entire monitoring period. These results indicate the ubiquity and high incidence of bla NDM-1 in the local wastewater. To track the bacteria carrying bla NDM-1 , we isolated Escherichia coli PI7, a strain of sequence type 101 (ST101), from wastewater around the Hajj event in October 2013. Genome sequencing of this strain revealed an extensive repertoire of ARGs as well as virulence and invasive traits. These traits were further confirmed by antibiotic resistance profiling and in vitro cell internalization in HeLa cell cultures. Given that this strain remains viable even after a certain duration in the sewerage, and that Jeddah lacks a robust sanitary infrastructure to fully capture all generated sewage, the presence of this bacterium in the untreated wastewater represents a potential hazard to the local public health. To the best of our knowledge, this is the first report of a bla NDM-1 -positive E. coli strain isolated from a nonnosocomial environment in Saudi Arabia and may set a priority concern for the need to establish improved surveillance for carbapenem-resistant E. coli in the country and nearby regions.

This study examines the groundwater quality in wells situated near agricultural fields in Saudi Arabia. Fruits (e.g., tomato and green pepper) irrigated with groundwater were also assessed for the occurrence of opportunistic pathogens to determine if food safety was compromised by the groundwater. The amount of total nitrogen in most of the groundwater samples exceeded the 15 mg/L permissible limit for agricultural irrigation. Fecal coliforms in densities > 12 MPN/100 mL were detected in three of the groundwater wells that were in close proximity to a chicken farm. These findings, coupled with qPCR-based fecal source tracking, show that groundwater in wells D and E, which were nearest to the chicken farm, had compromised quality. Anthropogenic contamination resulted in a shift in the predominant bacterial phyla within the groundwater microbial communities. For example, there was an elevated presence of Proteobacteria and Cyanobacteria in wells D and E but a lower overall microbial richness in the groundwater perturbed by anthropogenic contamination. In the remaining wells, the genus Acinetobacter was detected at high relative abundance ranging from 1.5% to 48% of the total groundwater microbial community. However, culture-based analysis did not recover any antibiotic-resistant bacteria or opportunistic pathogens from these groundwater samples. In contrast, opportunistic pathogenic Enterococcus faecalis and Pseudomonas aeruginosa were isolated from the fruits irrigated with the groundwater from wells B and F. Although the groundwater was compromised, quantitative microbial risk assessment suggests that the annual risk incurred from accidental consumption of E. faecalis on these fruits was within the acceptable limit of 10−4. However, the annual risk arising from P. aeruginosa was 9.55 × 10−4, slightly above the acceptable limit. Our findings highlight that the groundwater quality at this agricultural site in western Saudi Arabia is not pristine and that better agricultural management practices are needed alongside groundwater treatment strategies to improve food safety.

Bacteriophages active against a New Delhi metallo beta lactamase (NDM)-positive E. coli PI-7 were isolated from municipal wastewater and tested for their lytic effect against the bacterial host. Bacteriophages were highly specific to E. coli PI-7 when tested for host-range. After determining host-specificity, bacteriophages were tested for their ability to sensitize E. coli PI-7 to solar irradiation. Solar irradiation coupled with bacteriophages successfully reduced the length of the lag-phase for E. coli PI-7 from 4 h to 2 h in buffer solution. The reduction of lag-phase length was also observed in filtered wastewater effluent and chlorinated effluent. Previously, we found through gene expression analysis that cell wall, oxidative stress, and DNA repair functions played a large role in protecting E. coli PI-7 against solar damage. Here, gene expression analysis of bacteriophage-supplemented solar-irradiated E. coli PI-7 revealed downregulation of cell wall functions. Downregulation of functions implicated in scavenging and detoxifying reactive oxygen species, as well as DNA repair genes, was also observed in bacteriophage-supplemented solar-irradiated E. coli PI-7. Moreover, solar irradiation activates recA, which can induce lytic activity of bacteriophages. Overall, the combined treatment led to gene responses that appeared to make E. coli PI-7 more susceptible to solar disinfection and bacteriophage infection. Our findings suggest that bacteriophages show good potential to be used as a biocontrol tool to complement solar irradiation in mitigating the persistence of antibiotic-resistant bacteria in reuse waters.

A membrane bioreactor (MBR)-based wastewater treatment plant (WWTP) in Saudi Arabia is assessed over a five-month period in 2015 and once in 2017 for bacterial diversity and transcriptional activity using metagenomics, metatranscriptomics and real time quantitative polymerase chain reaction (RT-qPCR). Acinetobacter spp. are shown to be enriched in the chlorinated effluent. Members of the Acinetobacter genus are the most abundant in the effluent and chlorinated effluent. At the species level, Acinetobacter junii have higher relative abundances post MBR and chlorination. RNA-seq analysis show that, in A. junii, 288 genes and 378 genes are significantly upregulated in the effluent and chlorinated effluent, respectively, with 98 genes being upregulated in both. RT-qPCR of samples in 2015 and 2017 confirm the upregulation observed in RNA-seq. Analysis of the 98 genes show that majority of the upregulated genes are involved in cellular repair and metabolism followed by resistance, virulence, and signaling. Additionally, two different subpopulations of A. junii are observed in the effluent and chlorinated effluent. The upregulation of cellular repair and metabolism genes, and the formation of different subpopulations of A. junii in both effluents provide insights into the mechanisms employed by A. junii to persist in the conditions of a WWTP.

Resistance to antibiotics is increasingly being recognized as an emerging contaminant posing great risks to effective treatment of infections and to public health. Pristine soils or even soils that predate the antibiotic era naturally contain ARB and ARG. This book chapter explores the native resistome of soils, and collates information on whether soil perturbation through wastewater reuse can lead to accumulation of ARB and ARGs in agricultural soils. Special emphasis was given to ARGs, particularly the blaNDM gene that confers resistance against carbapenem. The fate and persistence of these emerging ARGs have not been studied in depth, however this book chapter reviews available information on other ARGs to gain insight into the possibility of horizontal gene transfer events in wastewater-irrigated soils and plant surfaces and tissues. Lastly, this book chapter visits solar irradiation and bacteriophage treatment as intervention options to limit dissemination of emerging contaminant threats.