Establishing a marine monitoring programme to assess antibiotic resistance: a case study from the Gulf Cooperation Council (GCC) region

The World Health Organization considers antimicrobial resistance as one of the most pressing global issues which poses a fundamental threat to human health, development, and security. Due to demographic and environmental factors, the marine environment of the Gulf Cooperation Council (GCC) region may be particularly susceptible to the threat of antimicrobial resistance. However, there is currently little information on the presence of AMR in the GCC marine environment to inform the design of appropriate targeted surveillance activities. The objective of this study was to develop, implement and conduct a rapid regional baseline monitoring survey of the presence of AMR in the GCC marine environment, through the analysis of seawater collected from high-risk areas across four GCC states: (Bahrain, Oman, Kuwait, and the United Arab Emirates). 560 Escherichia coli strains were analysed as part of this monitoring programme between December 2018 and May 2019. Multi-drug resistance (resistance to three or more structural classes of antimicrobials) was observed in 32.5% of tested isolates. High levels of reduced susceptibility to ampicillin (29.6%), nalidixic acid (27.9%), tetracycline (27.5%), sulfamethoxazole (22.5%) and trimethoprim (22.5%) were observed. Reduced susceptibility to the high priority critically important antimicrobials: azithromycin (9.3%), ceftazidime (12.7%), cefotaxime (12.7%), ciprofloxacin (44.6%), gentamicin (2.7%) and tigecycline (0.5%), was also noted. A subset of 173 isolates was whole genome sequenced, and high carriage rates of qnrS1 (60/173) and bla CTX-M-15 (45/173) were observed, correlating with reduced susceptibility to the fluoroquinolones and third generation cephalosporins, respectively. This study is important because of the resistance patterns observed, the demonstrated utility in applying genomic-based approaches to routine microbiological monitoring, and the overall establishment of a transnational AMR surveillance framework focussed on coastal and marine environments

Antimicrobial Resistance in Escherichia coli Isolated from Marine Sediment Samples from Kuwait Bay

This study presents antimicrobial resistance (AMR) in Escherichia coli derived from marine sediment in Kuwait. In total, 395 isolates of E. coli obtained were screened for their potential resistance to five commonly deployed frontline antibiotics by using the disk diffusion method. The results demonstrated widespread resistance across all the sites and in 98% of isolates. The highest counts were recorded in the sediment collected from sites near outfalls associated with local hospitals, where 58% of isolates screened displayed resistance to different antibiotic classes. The resistance was highest to ampicilin (beta-lactame class) > cefpodoxime (3rd generation cephalosporin class) > ciprofloxacin (flouroqunolone class) with AMR observance at 95%, 67% and 50% respectively. The latter two are wide spectrum antibiotics heavily used in Kuwait. This study demonstrates the presence of AMR bacteria in Kuwait’s marine environment, suggesting a need for environmental surveillance for AMR to be adopted as part of a One Health approach to Kuwait’s developing AMR national action plan

Antibiotic Resistance Mediated by <i>Escherichia coli</i> in Kuwait Marine Environment as Revealed through Genomic Analysis

Antibiotic-resistance gene elements (ARGEs) such as antibiotic-resistance genes (ARGs), integrons, and plasmids are key to the spread of antimicrobial resistance (AMR) in marine environments. Kuwait’s marine area is vulnerable to sewage contaminants introduced by numerous storm outlets and indiscriminate waste disposal near recreational beaches. Therefore, it has become a significant public health issue and warrants immediate investigation. Coliforms, especially Gram-negative Escherichia coli, have been regarded as significant indicators of recent fecal pollution and carriers of ARGEs. In this study, we applied a genome-based approach to identify ARGs’ prevalence in E. coli isolated from mollusks and coastal water samples collected in a previous study. In addition, we investigated the plasmids and intl1 (class 1 integron) genes coupled with the ARGs, mediating their spread within the Kuwait marine area. Whole-genome sequencing (WGS) identified genes resistant to the drug classes of beta-lactams (blaCMY-150, blaCMY-42, blaCTX-M-15, blaDHA-1, blaMIR-1, blaOKP-B-15, blaOXA-1, blaOXA-48, blaTEM-1B, blaTEM-35), trimethoprim (dfrA14, dfrA15, dfrA16, dfrA1, dfrA5, dfrA7), fluroquinolone (oqxA, oqxB, qnrB38, qnrB4, qnrS1), aminoglycoside (aadA2, ant(3’’)-Ia, aph(3’’)-Ib, aph(3’)-Ia, aph(6)-Id), fosfomycin (fosA7, fosA_6, fosA, fosB1), sulfonamide (sul1, sul2, sul3), tetracycline (tet-A, tet-B), and macrolide (mph-A). The MFS-type drug efflux gene mdf-A is also quite common in E. coli isolates (80%). The plasmid ColRNAI was also found to be prevalent in E. coli. The integron gene intI1 and gene cassettes (GC) were reported to be in 36% and 33%, respectively, of total E. coli isolates. A positive and significant (p intl1 (r = 0.311) and phenotypic AMR-GC (r = 0.188). These findings are useful for the surveillance of horizontal gene transfer of AMR in the marine environments of Kuwait

Microplastics in Kuwait’s Wastewater Streams

The wastewater stream is the most significant contributor of microplastics (MPs) to the environment. There are five wastewater treatment plants (WWTPs) in Kuwait. This baseline study provides an overview of MP removal in three major WWTPs in Kuwait that treat some 81.31% of the wastewater produced. The Sulabiya WWTP was the most efficient in MP removal, followed by the Kabd and Umm Al-Haiman WWTPs. The MP removal efficiency of plants in Kuwait is very high for Sulabiya WWTP and Kabd WWTP with an average of 2.5 MP L−1 in treated effluent comparable to the WWTPs in Australia, the United States, and Europe. The standard methodology of sample collection, preparation, and identification using microscopic examination and micro-Raman spectrometry was followed. Over 94.5 billion MPs enter the three WWTPs daily; 92.3 billion MPs are retained in sludge, while 2.2 billion are passed into the environment due to the use of treated effluent. The influent, effluent, and sludge MP inventories ranged between 119 and 230 MP L−1, 1 and 12 MP L−1, and 72 and 103 MP 10 g−1 respectively. The fiber was the dominant shape, and white, transparent, and black were prevalent colors. Currently, sludge is not used in Kuwait for any terrestrial or agricultural application; however, sludge is routinely used in many countries as a soil additive in agricultural farms. Using effluent water in irrigation leads to MP dissemination in the terrestrial environment. It is necessary to assess how far these MPs move in the soil profile and if they can contaminate the shallow aquifers. The observation of MP retention in sludge and effluent is empirical, and the use of these matrixes in agriculture is likely to raise an issue of food safety

A Comparative Assessment of High-Throughput Quantitative Polymerase Chain Reaction versus Shotgun Metagenomic Sequencing in Sediment Resistome Profiling

Prolonged and excessive use of antibiotics has resulted in the development of antimicrobial resistance (AMR), which is considered an emerging global challenge that warrants a deeper understanding of the antibiotic-resistant gene elements (ARGEs/resistomes) involved in its rapid dissemination. Currently, advanced molecular methods such as high-throughput quantitative polymerase chain reaction (HT-qPCR) and shotgun metagenomic sequencing (SMS) are commonly applied for the surveillance and monitoring of AMR in the environment. Although both methods are considered complementary to each other, there are some appreciable differences that we wish to highlight in this communication. We compared both these approaches to map the ARGEs in the coastal sediments of Kuwait. The study area represents an excellent model as it receives recurrent emergency waste and other anthropogenic contaminants. The HT-qPCR identified about 100 ARGs, 5 integrons, and 18 MGEs (total—122). These ARGs coded for resistance against the drug classes of beta-lactams > aminoglycoside > tetracycline, macrolide lincosamide streptogramin B (MLSB) > phenicol > trimethoprim, quinolone, and sulfonamide. The SMS picked a greater number of ARGs (402), plasmid sequences (1567), and integrons (168). Based on the evidence, we feel the SMS is a better method to undertake ARG assessment to fulfil the WHO mandate of “One Health Approach.” This manuscript is a useful resource for environmental scientists involved in AMR monitoring

Antibiotic Resistance Genes Associated with Marine Surface Sediments: A Baseline from the Shores of Kuwait

Marine sediments are a sink for antibiotic resistance genes (ARGs) and antibiotic-resistant microbes (ARMs). Wastewater discharge into the aquatic environment is the dominant pathway for pharmaceuticals reaching aquatic organisms. Hence, the characterization of ARGs is a priority research area. This baseline study reports the presence of ARGs in 12 coastal sediment samples covering the urban coastline of Kuwait through whole-genome metagenomic sequencing. The presence of 402 antibiotic resistance genes (ARGs) were recorded in these samples; the most prevalent were patA, adeF, ErmE, ErmF, TaeA, tetX, mphD, bcrC, srmB, mtrD, baeS, Erm30, vanTE, VIM-7, AcrF, ANT4-1a, tet33, adeB, efmA, and rpsL, which showed resistance against 34 drug classes. Maximum resistance was detected against the beta-lactams (cephalosporins and penam), and 46% of genes originated from the phylum Proteobacteria. Low abundances of ESKAPEE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterobacter sps., and Escherichia coli) were also recorded. Approximately 42% of ARGs exhibited multiple drug resistance. All the ARGs exhibited spatial variations. The major mode of action was antibiotic efflux, followed by antibiotic inactivation, antibiotic target alteration, antibiotic target protection, and antibiotic target replacement. Our findings supported the occurrence of ARGs in coastal marine sediments and the possibility of their dissemination to surrounding ecosystems

Implication of MAPK, Lipocalin-2, and Fas in the protective action of liposomal resveratrol against isoproterenol-induced kidney injury

Background and Objective: Isoproterenol (ISO) is a non-selective β-adrenergic receptor agonist. It can be used to treat bradycardia and cardiogenic shock. Despite its usefulness, the overstimulation of β-receptors by ISO can cause “cardiorenal syndrome,” a term used to describe heart and kidney damage. Resveratrol (RES), a natural polyphenol, has marked anti-inflammatory and antioxidant activities. The present work was designed to study the protective efficacy of liposomal resveratrol (L-RES) against ISO-induced kidney injury. Materials and Methods: The kidney injury was induced in rats by administering ISO (50 mg/kg, s.c.) twice a week for 2 weeks. RES and L-RES were administered at a dose (20 mg/kg/ day, p.o.) along with ISO for 2 weeks. Inflammatory and apoptotic biomarkers were analyzed, which were validated using histochemical analysis. Results: ISO caused renal dysfunction, which manifested as elevated urea, creatinine and uric acid, besides cystatin c and MAPK protein overexpression. In addition, ISO induced gene expression of Fas and lipocalin-2 and provoked genomic DNA fragmentation in renal tissues as compared with the control group. Histological examination confirmed morphological alterations of the kidney tissues obtained from the ISO group. Concurrent treatment of either RES or L-RES with ISO significantly ameliorated kidney damage as demonstrated by the improvement of all measured parameters with the best results for L-RES. The histopathological findings were correlated with the above biochemical parameters. Conclusion: L-RES could be a promising approach for the prevention of kidney injury induced by ISO, most likely via the downregulation of MAPK, cystatin c, Fas, and lipocalin-2