ReseaRch PaPeR Journal of Agricultural and Marine Sciences 2022, 27(2): 10–18 DOI: 10.53541/jams.vol27iss2pp10-18 Received 25 July 2021 Accepted 11 January 2022 Can the irrigation of soils with Amoxicillin-enriched water cause the proliferation of Bacteria resistant to antibiotics among culturable heterotrophic aerobic soil bacteria? Adhari Al-Kalbani1, Daniel Menezes-Blackburn1,*, Buthaina Al-Siyabi1, Mohamed Al-Kasbi2, Ahmed Al-Busaidi2 Daniel Menezes-Blackburn1( ) danielblac@squ.edu.om, 1Depart- ment of Soil, Water and Agricultural Engineering, College of Agricul- tural and Marine Science, Sultan Qaboos University, Muscat, Sultanate of Oman, 2Environment Authority, Qurum, Muscat, Sultanate of Oman. Introduction The reuse of treated wastewater is one of the al-ternative methods for increasing water conserva-tion and sustainability, especially the use of treat- ed wastewater for irrigation can be highlighted. The use of treated wastewater for irrigation may positively af- fect soil fertility and increase crop yields (Asano, 1988). There are nonetheless safety issues associated with soil irrigation with treated wastewater, namely the presence of microbial pathogens, heavy metals, organic pollutants (Asano, 1988; EPA, 2012), and pharmaceuticals such as antibiotics (Wu et al., 2014). Antibiotics are bioactive compounds secreted by certain microbial species, which directly inhibit the growth of other competing bacteria (Negreanu et al., 2012). Amoxicillin belongs to the β-lac- هل ميكن أن يؤدي ري الرتبة ابملياه الغنية ابملضاد احليوي األموكسيسيلني )Amoxicillin( إىل انتشار البكترياي املقاومة لألموكسيسيلني بني بكترياي الرتبة اهلوائية غري ذاتية التغذية )البكترياي العضوية(؟ عذاري الكلباين1، دانيال مينيزيس - بالكبرين1 *، بثينة السيايب1، حممد الكاسيب2 ، أمحد البوسعيدي2 Abstract. This study investigated the short-term impact of irrigation with Amoxicillin solutions on the presence of the amoxicillin-resistance trait among culturable soil heterotrophic aerobic bacteria. The microcosm experimental design consisted of 15 days of incubation of 10 g soil samples irrigated daily with distilled water containing increasing doses of amoxicillin (0, 0.1, 1, 10, 100, 1000 µg g-1 of soil day-1). The hypothesis was that continuous daily addition of antibiotics would increase the proportion of antibiotic-resistant bacteria in soils. After the incubation period, the total and antibiotic resistance heterotrophic aerobic bacteria communities were assessed through serial dilution of soil sus- pensions, followed by agar plate culture enumeration, isolation, identification and microscopy observation. The pres- ence of antibiotic-resistant bacteria was also evaluated directly on treated wastewater used for field irrigation before this microcosm study to assess the amoxicillin-resistant bacteria bioaugmentation hypothesis. Results indicated that the Amoxicillin resistance was widespread among bacteria present in both treated wastewater used for irrigation and in the receiving soil. A microcosm experiment was attempted as a ‘proof of concept’ to demonstrate that irrigation with treated wastewater containing antibiotics would exert selective pressure and promote the proliferation of antibiotic re- sistance bacteria. Unexpectedly, the results from the microcosm incubations indicated the daily addition of amoxicillin did not increase bacterial antibiotic resistance trait abundance in soils, which even significantly decreased for all tested doses. The antibiotic-resistant species identified among the isolates were Pseudomonas mosselii, P. otitidis, P. men- docina, P. flavescens, Stenotrophomnas maltophilia, Bacillus thuringiensis, Aeromonas veronii, Candida parapsilosis, Streptomyces violaceoruber and Microbacterium barkeri. Keywords: Treated wastewater, Amoxicillin Antibiotic, Antibiotic Resistance Bacteria. امللخص:حبثــت هــذه الدراســة يف التأثــري قصــري املــدى لــري الرتبــة ابمليــاه املعاجلــة الغنيــة ابملضــاد احليــوي أموكسيســيلني )Amoxicillin( علــى وجــود مســة مقاومــة للمضــاد احليــوي أموكسيســيلني بــني البكتــرياي اهلوائيــة غــري ذاتيــة التغذيــة )Amoxicillin resistance heterotrophic bacteria( يف الرتبــة القابلــة للزراعــة. مت تصميــم الدراســة التجريبيــة ابســتخدام 10 جــرام مــن الرتبــة املرويــة يوميــا ابملــاء املقطــر الــي حتتــوي علــى جرعــات متزايــدة مــن املضــاد احليــوي أموكسيســيلني )0، 0.1، 1، 10، 100، 1000 ميكروغــرام /اليــوم/ الرتبــة( ملــدة حضانــة 15 يومــا. كانــت الفرضيــة املبدئيــة هــي أن اإلضافــة اليوميــة املســتمرة للمضــادات احليويــة مــن احملتمــل أن تزيــد نســبة البكتــرياي املقاومــة للمضــادات احليويــة يف الرتبــة. بعــد انتهــاء فــرتة احلضانــة، مت تقييــم جممــل جممــوع اجملتمعــات البكتــرياي اهلوائيــة غــري ذاتيــة التغذيــة املقاومــة للمضــادات احليويــة مــن خــالل التخفيــف املتسلســل لعينــات الرتبــة، تليهــا حســاب صفيحــة قطعــة هــالم اآلجــار، والعــزل البكتــريي، والتعــرف والكشــف عــن األنــواع البكترييــة، واملالحظــة اجملهريــة. مت أيًضــا تقييــم وجــود البكتــرياي املقاومــة للمضــادات احليويــة مباشــرة علــى ميــاه الصــرف الصحــي املعاجلــة املســتخدمة للــري Amoxicillin resistance heterotrophic( امليــداين قبــل هــذه الدراســة املصغــرة لتقييــم فرضيــة الــزايدة احليويــة للبكتــرياي املقاومــة لألموكسيســيلني bacteria(. أشــارت النتائــج إىل انتشــار البكتــرياي املقاومــة لألموكسيســيلني يف ميــاه الصــرف الصحــي املعاجلــة املســتخدمة يف الــري ويف الرتبــة املســتقبلة للميــاه. مت تطبيــق الدراســة املصغــرة »كدليــل علــى املفهــوم« إلثبــات أن الــري مبيــاه الصــرف الصحــي املعاجلــة احملتويــة علــى مضــادات حيويــة مــن شــأهنا أن متــارس ضغطًــا انتقائًيــا وتعــزز مــن تكاثــر ومنــو البكتــرياي املقاومــة للمضــادات احليويــة. وبشــكل غــري متوقــع، أشــارت النتائــج أيضــا إىل أن اإلضافــة اليوميــة ملضــادات األموكسيســيلني ال تزيــد مــن مقاومــة البكتــرياي للمضــادات احليويــة يف الرتبــة، والــي اخنفضــت بشــكل ملحــوظ جلميــع اجلرعــات املختــرة. العــزالت البكترييــة املقاومــة الــي مت تعريفهــا فصائلهــا Pseudomonas mosselii, P. otitidis, P. mendocina, P. flavescens, Stenotrophomnas maltophilia :هــي ,Bacillus thuringiensis, Aeromonas veronii, Candida parapsilosis, Streptomyces violaceoruber Microbacterium barkeri. الكلمات املفتاحية: مياه الصرف الصحي املعاجلة، املضاد احليوي أموكسيسيلني )Amoxicillin( ، البكترياي املقاومة للمضادات احليوية. 11Research Paper Al-Kalbani, Blackburn, Al-Siyabi, Al-Kasbi, Al-Busaidi tam group of antibiotics, and it is one of the most wide- ly used antibiotics in human and veterinary medicine (Diz Dios et al., 2006). Amoxicillin is an antibiotic of the amino-penicillin group used against a broad spectrum of Gram-positive and Gram-negative bacteria (Croydon and Sutherland 1970). Clavulanic acid is a natural sub- stance produced by Streptomyces clavuligerus and has slight antibacterial activity but a very high inhibition of many β-lactamases. It is usually added to amoxicillin to increase its half-life. Low concentrations of clavulanic acid (0.05-2 µg mL-1) cause irreversible time-dependent inactivation of many of the β-lactamases, including pen- icillinases produced by Staphylococcus aureus (Reading and Cole, 1977). There is increasing societal concern about the evolving antibiotic resistance of natural bac- teria, with special concerns expected to occur when antibiotics are continuously released into soil and water environments (Bisht et al., 2009). Therefore, leading to the continuous search for new generations of antibiotic compounds (Czekalski et al., 2012). In this short-term study, amoxicillin was used because it has been reported to have a short half-life compared to other antibiotics (Diz Dios et al., 2006). Many antibiotics are poorly absorbed in the human digestive tract, and it is estimated that 25% to 75% of the ingested antibiotics are excreted in faeces (Ash et al., 2002). In both animals and humans, up to 95% of anti- biotics can be excreted in an unaltered state (Elmund et al. 1971). Most of the antibiotics used for animals and humans are not entirely removed in wastewater treat- ment plants (WWTPs). As a result, treated wastewater are a natural reservoir of residual concentrations of an- tibiotic and antibiotic-resistant bacteria (AR). Waste- water treatment plants are not designed to remove or monitor pharmaceuticals (Pruden et al., 2013). Due to this low removal efficiency, multiple antibiotics in mu- nicipal wastewater and treatment plants were frequently detected in previous studies, e.g. Zhang and Li (2011). The combination of antibiotics, nutrients, and bacteria in treated domestic sewage wastewater could potential- ly result in the selection of antibiotic resistance among bacterial populations present in the environment (Ne- greanu et al., 2012). Soil organic matter represents an important source of energy and nutrients for microorganisms and plants (Fahrenfeld et al., 2013; Menezes-Blackburn et al., 2013). Soil microbial biomass, activity, taxonomic and func- tional diversity have strong correlation with soil health and have been considered as good biological indexes (Menezes-Blackburn et al., 2020). The application of irrigation water containing antibiotic resistant bacteria to soil represents a possible contamination source for crops, groundwater and receiving water bodies. Many environmental factors such as temperature, pH, mois- ture, organic matter content, and others, may enhance the survival of pathogenic microbes on agricultural lands (Trevisan et al., 2002). Antibiotic-resistant pathogenic bacteria may spread across the environment by several different routes, including treated and untreated sewage effluents, sludge and agricultural runoff (Al-Bahry et al., 2007). However, antibiotic resistances are often formed when antibiotics are misused or overused; new bacteria strains arise that are no longer sensitive to these drugs. The presence of selective pressure of antimicrobial drug usage is responsible for the natural development of bac- terial antibiotic resistance (Al-Bahry et al. 2015). Under optimal conditions, bacteria multiply very rapidly, with generation times ranging from minutes to hours. The high number and short generation times allow for a high likelihood of spontaneous mutations to occur, allowing any given population of bacteria a significantly high ge- netic diversity to naturally occur (Al-Bahry et al., 2015). Treated and untreated sewage effluents may pose risks for groundwater pollution. In Oman, E. coli isolat- ed from contaminated wells were found to be resistant to multiple antibiotics, possibly due to the contamina- tion with sewage effluents (Al-Bahry et al., 2014). Intro- ducing antimicrobial agents into the environment might influence the change in the physiological traits of bacte- ria through the selection pressure over naturally occur- ring bacterial communities (Martinez, 2009). The bac- tericidal or bacteriostatic agents (AB) inhibit microbial growth either by: a) targeting membranes or cell walls; b) targeting protein synthesis through the ribosomal subunits as tetracycline, and fluoroquinolones; or c) in- terfering with the nucleic acid synthesis (Sengupta et al., 2013). Nowadays, the overuse of antibiotics is consid- ered the main cause of the emergence of antibiotic-resis- tant pathogens, resulting in direct risk for human health (McArdell et al., 2003). This short-term study investigated the scenario in which irrigating soil with antibiotic containing treat- ed wastewater could propagate antibiotic resistance in soils. The specific objectives were to: a) Evaluate the response soil bacteria enumeration for increasing dos- es of amoxicillin and the relative proportion of amox- icillin antibiotic resistant strains among the culturable heterotrophic aerobic bacterial community; and b) Evaluate the abundance and proportion of amoxicillin antibiotic resistance within the heterotrophic aerobic bacterial community present on the treated wastewater currently used for irrigation, to verify if this is a possible source of antibiotic-resistant bacterial inoculum to soils. Methodology Site of Soil Sample and Treated Wastewater Collection The soil sample used from the microcosm incubation study was collected from Sultan Qaboos University agricultural experiment station from a site irrigated with treated wastewater for five months (23°35’52.7”N 58°09’50.4”E) during spring 2019. The sampling location 12 SQU Journal of Agricultural and Marine Sciences, 2022, Volume 27, Issue 2 Can the irrigation of soils with Amoxicillin-enriched water cause the proliferation of Bacteria resistant to antibiotics among culturable hetero- trophic aerobic soil bacteria? is 49 mamsl and 9.27 km from the coastline, free from the influence of seawater intrusion. Approximately one kg of soil was collected from the top 5 cm of and sieved to below 2 mm particle size. The soil background phys- icochemical properties were 44.24% sand, 54.84 % silt, 0.92 % clay, electrical conductivity on the saturated paste extract (ECe) of 8.3 dS m-1, pH 8 and 0.33 % soil organic matter by loss on ignition. The water holding capacity was measured as the soil moisture content (g g-1) remaining after one day of free drainage of a saturated 20 g soil sample covered with a plastic film to prevent evaporation. Seventy per cent of this moisture content was used during initial microcosm incubation conditions. The treated wastewater was col- lected from the irrigation system at the same site and moved to the laboratory for immediate microbial analyses. Soil microcosm Incubation with Amoxicillin Amoxicillin-clavulanic acid (AM-CL) was chosen for being the most commonly used antibiotic, due to its common occurrence in treated wastewater effluents, and as a result of its short half-life and high soil deg- radation (up to 80%) (Braschi et al., 2013). The human adult’s standard dosage of amoxicillin (0.5 mg/kg/day) was taken as a reference maximum dosage treatment. To simulate a constant addition of Amoxicillin through daily irrigation with treated wastewater, a logarithmic increase dosage was designed as 0, 0.001, 0.01, 0.1, 1 and 10 mg kg-1 day-1. These antibiotic solutions (0.1 mL 10 g-1 soil) were added daily for 15 days in triplicate incubations. Each of the 10 g incubation replicates was kept at open-air room temperature (25±1.34 °C) to avoid moisture buildup and anoxic conditions and were daily mixed after Amoxicillin application. Enumeration of Amoxicillin-resistant Hetero- trophic Aerobic Bacteria Soil bacterial colonies were examined from soil matri- ces by suspending 10 g of freshly collected soil samples (kept at 4 oC) in 95 mL of saline solution (0.85% NaCl2). Samples were shaken at 100 rpm for 10 min, and soil suspensions were then subjected to four sequential 10× dilutions. For each sample, four replicates were used to increase accuracy. Luria Bertani (LB) agar was used a basal general-bacteria-growth-media in three condi- tions, either: a) unamended (control); (b) amended with 0.5 mg mL-1, or (c) 20 mg mL-1 of (AM-CL) , and were used to cultivate heterotrophic aerobic bacteria without and with antibiotic resistance respectively. The LB agar media plates were inoculated with 0.1 mL of the freshly prepared soils suspensions and incubated at room tem- perature for 72 hours, to ensure enough time for bac- terial growth. The bacteria colonies were enumerated (abundance) and calculated as Colony-Forming Units per gram of soil (CFU g-1). The relative proportion of antibiotic-resistant culturable bacteria was calculated by comparing the number of CFUs growing on plates with antibiotics compared to the number of CFUs growing on control plates without antibiotics. The same agar me- dium and growth conditions were used for soil suspen- sions and the fresh treated wastewater sample. Bacteria Strain Isolation, Identification and Mi- croscopy Antibiotic-resistant bacterial colonies able to grow on the amoxicillin-supplemented LB-Agar medium were iso- lated in pure culture by standard streak-clean procedure on fresh LB-Agar medium supplemented with 20 mg ml-1 (AM-CL). The bacterial colonies’ morphology was examined both through direct naked eye observation of Figure 1. Average culturable bacteria enumeration in Luria Bertani Agar (CFU mL-1) medium inoculated of Treated Waste- water used for soil irrigation at the agricultural experiment station at Sultan Qaboos University. Columns labelled with different letters are statistically different (Tukey p≤0.05). 13Research Paper Al-Kalbani, Blackburn, Al-Siyabi, Al-Kasbi, Al-Busaidi colony colour, shape and texture, and through brightfield microscopy with gram-staining for distinguishing be- tween the different isolates. Bacterial species identifica- tion was performed by using a MALDI Bio-typer (Bruker Co., UK) based on proteomic fingerprinting analysed by high-throughput MALDI-TOF mass spectrometry. Statistical Analysis The data obtained were subjected to analysis of variance (one way ANOVA) and subsequently, Tukey’s HSD test was used to separate the means of the selected soil prop- erties. The significance level was defined as p≤0.05. Results Amoxicillin-resistant Heterotrophic Aerobic Bacteria in Treated Waste Water A considerable fraction of wastewater bacteria were able to grow in LB medium supplemented Amoxicillin-clavu- lanic acid (0.5 or 20 µg mL-1) as shown in Figure S1. The average bacterial cells present in the control LB medium (without Amoxicillin-clavulanic acid) was 240 CFU mL-1 (Figure 1). In the plates containing 0.5 and 20 µg mL-1 of Amoxicillin, the abundance of bacteria colonies was 140 and 90 CFU mL-1, respectively. This corresponds to a very high proportion of the total CFU in control plates, approximately 58.3% and 37.5% for plates containing 0.5 µg mL-1 and 20 µg mL-1 of Amoxicillin, respectively. Considering a hypothetical irrigation with 5 L m-2 day-1, a total of 1.20×106 CFU m-2 would be the daily inocu- lation of these soils with highly Amoxicillin-clavulanic acid-resistant bacteria. Average culturable bacteria enumeration in Luria Bertani Agar (CFU mL-1) medium inoculated of Treated Wastewater used for soil irrigation at the agricultural ex- periment station at Sultan Qaboos University. Columns labelled with different letters are statistically different (Tukey p≤0.05). In soils, the average soil bacterial CFU g-1 enumer- ation in LB medium supplemented with 0.5 µg mL-1 was not significantly reduced with regards to the con- trol (Figure 2). Whereas in LB medium supplemented with 20 µg mL-1 Amoxicillin the reduction in CFU g-1 was significantly decreased by two orders of magnitude. Therefore, the LB medium supplemented with 20 µg mL-1 was used for further soil studies. Curiously, in the soils irrigated with treated wastewater for five months before the experiment, the proportion of bacterial cells culturable in LB medium supplemented with 20 µg mL-1 was only one per cent of the control LB-agar medium; much lower than the 37.5% observed directly on the treated wastewater. It is worth noticing that Amoxicil- lin-resistant bacteria were found to be present in the soil even when no antibiotic or wastewater was applied. The visual aspect LB agar plates of heterotrophic aero- bic showing total bacterial cell growth with much low- er abundance at 20 µg mL-1 of Amoxicillin can be seen in figure S2. Average culturable bacteria enumeration in Luria Bertani Agar (CFU mL-1) medium inoculated suspensions of soils irrigated with treated wastewater at the agricultural station at Sultan Qaboos University. Columns labelled with different letters are statistically different (Tukey p≤0.05). Amoxicillin-resistant Heterotrophic Aerobic Bacteria in Soil Irrigated with Amoxicillin solu- tions - Microcosm assays In this microcosm experiment, the abundance of anti- biotic resistance bacteria was evaluated after 15 days of soil incubation with Amoxicillin-clavulanic acid added daily with the irrigation water (0, 0.001, 0.01, 0.1, 1 and 10 mg Amoxicillin kg-1 soil day-1). The same LB medium Figure 2. Average culturable bacteria enumeration in Luria Bertani Agar (CFU mL-1) medium inoculated suspensions of soils irrigated with treated wastewater at the agricultural station at Sultan Qaboos University. Columns labelled with differ- ent letters are statistically different (Tukey p≤0.05). 14 SQU Journal of Agricultural and Marine Sciences, 2022, Volume 27, Issue 2 Can the irrigation of soils with Amoxicillin-enriched water cause the proliferation of Bacteria resistant to antibiotics among culturable hetero- trophic aerobic soil bacteria? cultivation conditions described herein was followed for the microcosm soil samples (0.5 and 20 µg Amoxicillin mL-1). Amoxicillin only negatively affected the total cul- turable bacteria CFU g-1 soil at the 1 mg kg-1 soil day-1 dose, and at 10 mg kg-1 soil day-1, the CFU g-1 soil even significantly increased. There was an intensive and sig- nificant decrease in the total and relative abundance of antibiotic-resistant bacteria in soils treated with higher doses of amoxicillin (Figure 3). This effect was contrary to our central hypothesis that the addition of AM-CL would increase the relative abundance of this trait. The total bacterial CFU g-1 soil in all samples where Amoxi- cillin-clavulanic acid was added was below 105 CFU g-1. On the other hand, the average bacterial CFU g-1 soil was 2.42x106 CFU g-1 at incubation conditions without Amoxicillin addition. Enumeration of bacteria colony forming units (CFU g-1 soil) in Luria Bertani Agar medium LB medium with or without Amoxicillin supplementation (0 and 20 µg amoxicillin mL-1). Soil samples treated with 0, 0.1, 1, 10, 100, 1000 mg kg-1 of soil sample day-1 for 15 days. Col- umns labelled with different letters are statistically dif- ferent (Tukey p≤0.05). The relative proportion (%) of antibiotic resistance bacteria was dramatically and significantly decreased nearly ten folds for soils receiving amoxicillin (Figure 4). . The daily application of AM-CL reduced the % of amoxicillin-tolerant heterotrophic aerobic bacterial Figure 3. Enumeration of bacteria colony forming units (CFU g-1 soil) in Luria Bertani Agar medium LB medium with or without Amoxicillin supplementation (0 and 20 µg amoxicillin mL-1, unshaded and unshaded bars respectively). Soil sam- ples treated with 0, 0.1, 1, 10, 100, 1000 mg kg-1 of soil sample day-1 for 15 days. Columns labelled with different letters are statistically different (Tukey p≤0.05). Figure 4. Relative abundance (%) of bacteria colony forming units (CFU g-1 soil) able to grow in Luria Bertani Agar medium LB medium containing 20 µg Amoxicillin mL-1 medium with regards to 0 µg Amoxicillin mL-1 controls. Soil samples were treated with 0, 0.1, 1, 10, 100, 1000 mg kg-1 of soil sample day-1 for 15 days. Columns labelled with different letters are statis- tically different (Tukey p≤0.05). 15Research Paper Al-Kalbani, Blackburn, Al-Siyabi, Al-Kasbi, Al-Busaidi abundance in all treatments. The percentage differenc- es in antibiotic resistant bacteria were not statistically significant among the AM-CL treated soil samples. Moreover, the relative bacterial abundance in LB-agar supplemented with 20 µg AM-CL mL-1 was on average 0.5% for Amoxicillin treated soils with respect to control LB plates. Whereas it was over 4% for the control soils without Amoxicillin. Relative abundance (%) of bacteria colony forming units (CFU g-1 soil) in Luria Bertani Agar medium LB medium containing 20 µg Amoxicillin mL-1 medium compared to 0 µg Amoxicillin mL-1 controls. Columns labelled with different letters are statistically different (Tukey p≤0.05). Antibiotic-resistant Bacteria Isolation, Identifi- cation and Microscopy The isolated AM-CL resistant microorganisms were identified at the species level using MALDI Bio-typer (Brooker-UK) and observed by bright light microsco- py. Based on these tests, the majority of the AM-CL re- sistant bacteria isolated from treated wastewater were gram-negative, and in soil, most were gram-positive. The bacterial shape, arrangement, and structure of pure isolated colonies were also recorded. Among isolates, the positively identified strains were Pseudomonas mosselii, Pseudomonas otitidis, Pseudomonas mendocina, Steno- trophomnas maltophilia (Pseudomonas beteli), Pseudo- monas flavescens, Bacillus thuringiensis Aeromonas ve- ronii, Candida parapsilosis, Streptomyces violaceoruber, Microbacterium barkeri. Figure 5. Petri dish LB-agar-streaked colonies image and gram-stained bright light microscopy (1000x amplification) image of the microbial strains isolated in this study. The species identification was performed using Maldi-Biotyper. 16 SQU Journal of Agricultural and Marine Sciences, 2022, Volume 27, Issue 2 Can the irrigation of soils with Amoxicillin-enriched water cause the proliferation of Bacteria resistant to antibiotics among culturable hetero- trophic aerobic soil bacteria? Amoxicillin-clavulanic acid-resistant strains isolated from treated wastewater (a to f ) and from soils (g to l) irrigated with the same treated wastewater. Isolation, purification and continuous propagation of these strains were performed using Luria Bertani-Agar medium sup- plemented with 20 µg Amoxicillin mL-1. Species identifi- cation was performed with MALDI Bio-typer (ID>97%). Discussion Hundreds of different bacteria species can be isolated even from tertiary treated wastewater (Wu et al., 2014). Based on the work from Karthikeyan et al. (2006), during the sewage treatment process, only partial antibiotic re- moval is achieved, and treated wastewater contains a trace amount of a variety of antibiotics. Bacteria exposed to antibiotic compounds may be selected for their anti- biotic resistance traits. Nevertheless, minimal inhibitory concentrations may not be exceeded in treated waste- waters (Martinez, 2009). Bacterial communities exposed to inhibitory antibiotic concentrations can be enriched for antibiotic resistance, and this is related to an overall higher antibiotic concentration in natural environments (Gullberg et al., 2011). Contrary to our hypothesis and the current literature, in our study, less antibiotic resis- tance was obtained with the addition of AM-CL through irrigation. Hereby, we cannot suggest Amoxicillin-clavu- lanic acid in treated wastewater would increase the abun- dance of antibiotic resistance bacterial traits in the soils. Amoxicillin-clavulanic acid stored at room temperature is known to be labile to bacterial degradation, and more than 30% of Amoxicillin-clavulanic acid degraded can be degraded after seven days (Gullberg et al. 2011). The dis- solved antibiotic in water is highly affected by environ- mental conditions, such as temperature ad pH (Gullberg et al., 2011). Amoxicillin clavulanic may be degraded and become a nutrient source to bacterial cells. We specu- late here that this may have caused an overgrowth of low generation time bacteria sensitive to Amoxicillin that benefited from the rapid degradation of this antibiotic and were stimulated by the freshly added carbon source. Moreover, several studies demonstrated that other compounds other than antibiotics could cause the pro- liferation of antibiotic-resistant bacteria in a process known as cross-resistance. These are usually due to the presence of a high concentration of quaternary ammo- nium compounds (QACs) (Sidhu et al. 2001), triclosan and triclocarban (used in soaps and other household compounds) (Aiello et al., 2005), or even heavy metals (Baker-Austin et al., 2006). The average bacteria biomass is about four times higher in the treated wastewater samples than in fresh- water samples, and their diversity can be ten folds higher (Aiello et al., 2005; Diz Dios et al., 2006). Chlorination, ozonation, and UV radiation post-treatment disinfec- tion processes were successful treatments against mi- crobial agents, and pharmaceutical ingredients can sig- nificantly reduce the risks associated with soils irrigated with treated wastewater (EPA, 2012). Using classical methods to quantify antibiotic-resis- tant bacteria can only evidence a small fraction (<1%) of the total community, and due to this fact, serial dilution and agar culturing methods are often inaccurate to rep- resent the whole soil system (Amann et al., 1995). The data presented reflect changes in the culturable antibiot- ic-resistant aerobic heterotrophic bacteria, and may not describe trends in the rest of the soil community. Among the isolated Amoxicillin-resistant strains, two were unidentifiable by Maldi Biotyper. Four were Pseu- domonas strains, gram-negative, rod-shaped, and mo- tile by a single polar flagellum. Pseudomonas mosselii, Pseudomonas otitidis, Pseudomonas mendocina, Pseu- domonas mendocina, Pseudomonas flavescens. Some of them are known opportunistic pathogens, but mostly these are commonly found in soil, water, plants, sewage and animals. Little is known about the antimicrobial mechanisms (Rapsinski et al. 2016). Other gram-neg- ative antibiotic-resistant bacteria found were Stenotro- phomnas maltophilia (Pseudomonas beteli), Aeromonas veronii and Streptomyces violaceoruber. Two Amoxicil- lin-resistant Gram-positive bacteria were isolated: Mi- crobacterium barkeri, common in sewage samples, and Bacillus thuringiensis used to control insects for agri- culture and public health purposes (Mizuki et al., 1999). Surprisingly not all isolated microbes were bacteria. One isolate from a soil sample treated with 0.1 µg Amox- icillin g-1 soil day-1 and cultivated/propagated in LB plates containing 20 µg mL-1 was identified as Rhodotorula mucilaginosa. A common environmental yeast found in air, soil, lakes, and ocean water. It produces pink to red colonies that are unicellular lacking pseudohyphae and hyphae. It is the most common microorganism isolat- ed from the hands of hospital employees and patients (Strausbaugh et al., 1996). Conclusion The effluent of treated wastewater used for irrigation carry antibiotic-resistant bacteria that may be an inoc- ulum source to soil environments. A significant frac- tion of bacterial colonies isolated from soils (4%) resists Amoxicillin and clavulanic acid antibiotics. The total antibiotic-resistant bacteria abundance was significantly decreased when the soil was incubated with increasing dose of antibiotics. This study sets an important prece- dent that irrigation with wastewater containing Amox- icillin-clavulanic acid does not propagate this trait in soils by directed evolution. This is likely due to the quick degradation of this antibiotic in soil environments. 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