Vol.1 , No. , 202 , p -6 2 December 2 24 30 DOI: 10.5454/mi.1 . .6 2 24-30 The qPCR Assay for Detecting the Presence and Relative Abundance of Pseudomonas aeruginosa aadA2and Antibiotic Resistance Gene in Hospital Wastewater of National Reference Hospital RIDA TIFFARENT , ROSDIANA IRAWATI , CONNY RIANA TJAMPAKSARI , 1* 2 3 FITRIYAH SJATHA , WINDI MUZIASARI , ANIS KARUNIAWATI 3 4 3 AND 1 National Research and Innovation Agency (BRIN), Master Programme in Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jakarta, DKI Jakarta, Indonesia; 2 Departement of Waste Water Treatment Plants of RSCM. Jakarta, DKI Jakarta, Indonesia; 3 Departement of Microbiology, Faculty of Medicine, Universitas Indonesia, Jakarta, DKI Jakarta, Indonesia; 4 Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, University of Helsinki, Viikinkaari 9, Helsinki, 00014, Finland; Resistomap Oy, Helsinki, Finland. Antimicrobial resistance is one of the top 10 global health threats. The hospital wastewater (HWW) potentially becomes the reservoir and dissemination of antibiotic resistance gene (ARG) and bacterial pathogens. In Indonesia, the protocol to monitor the ARGs form HWW has not been established. This study aimed to detect the presence and find the relative abundance of and gene from National Reference HospitalP. aeruginosa aadA2 (NRH) inlet and outlet wastewater through qPCR assay. The primers used were supported by Resistomap. The study revealed that the qPCR assay was able to detect the Ct value of and . The geneP. aeruginosa aadA2 aadA2 was found in all waste water samples, meanwhile was only found in some of inlet samples.P. aeruginosa aadA2 had the highest relative abundance and this gene's mobility uses plasmids and integrons that potentially enhance the acquired antimicrobial resistance (AMR) mechanism. This study implicated that qPCR assay was capable to detect pathogenic bacteria and ARG, and ARG could be released to the environment even though the wastewater samples have been proceeded in wastewater treatment plants (WWTP). The qPCR assay can be used as the method to monitor the AMR status in a hospital and the spreading potency to the environment using the HWW. Key words: antibiotic resistance genes, antimicrobial resistance, environment, extrinsic resistance mechanism, wastewater Resistensi antimikroba adalah salah satu dari 10 ancaman terbesar untuk kesehatan global. Air limbah rumah sakit (HWW) berpotensi menjadi reservoir dan penyebaran gen resistensi antibiotik (ARG) dan bakteri patogen. Di Indonesia, protokol untuk memantau ARG dari HWW belum ditetapkan. Penelitian ini bertujuan untuk mendeteksi keberadaan dan nilai gen dan dari air limbah inlet dan outletrelative abundance P. aeruginosa aadA2 Rumah Sakit Rujukan Nasional (NRH) melalui uji qPCR. Primer yang digunakan dalam studi didapatkan dari Resistomap. Studi ini mengungkapkan bahwa uji qPCR mampu mendeteksi nilai Ct dan .P. aeruginosa aadA2 Gen ditemukan pada semua sampel air limbah, sedangkan hanya ditemukan pada beberapaaadA2 P. aeruginosa sampel inlet. memiliki nilai tertinggi dan mobilitas gen ini menggunakan plasmid danaadA2 relative abundance integron yang dapat berpotensi meningkatkan mekanisme resistensi antimikroba (AMR) dapatan. Penelitian ini mengimplikasikan bahwa uji qPCR mampu mendeteksi bakteri patogen dan ARG, serta kemungkinan dilepaskannya ARG ke lingkungan meskipun sampel air limbah telah diproses di instalasi pengolahan air limbah (IPAL) sebelumnya. Uji qPCR dapat digunakan sebagai metode untuk memantau status AMR di Rumah Sakit dan potensi penyebarannya ke lingkungan menggunakan HWW. Kata kunci: air limbah, gen resisten antibiotik, lingkungan, mekanisme resisten ekstrinsik, resistensi antimikroba MICROBIOLOGY INDONESIA Available online at http://jurnal.permi.or.id/index.php/mionline ISSN 1978-3477, eISSN 2087-8575 * C o r r e s p o n d i n g a u t h o r : P h o n e ;: + 6 2 E - m a i l :- rida003@brin.go.id 2019). Antibiotic resistance in bacteria can occur because these bacteria have or overexpress the genes that encode the resistance characteristics. Mechanisms of antibiotic resistance can occur through both intrinsic and acquired mechanisms. The mechanism of intrinsic resistance is related to the genetics of bacteria that are normally present in these bacteria. The mechanism of acquired resistance is related to the horizontal transfer of antibiotic resistance genes so that resistance In 2019, the World Health Organization (WHO) declared antimicrobial resistance as one of the top 10 global health threats (Holmes 2016; WHO Worldet al. Health Organization 2021). Overuse of antimicrobials is a major driver of resistance of infectious pathogens (CDC Centers for Disease Control and Prevention characteristic from one bacteria can be transferred to another (Holmes 2016; Peterson and Kaur 2018).et al. Pseudomonas aeruginosa is a rod shaped Gram- negative bacteria with a genome length of 5.5 – 7 Mbps. It is known as an opportunistic pathogen associated with nosocomial infections and ventilator- associated pneumonia (Pachori 2019; Panget al. et al. 2019). is one of the ESKAPE groupP. aeruginosa (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa Enterobacter, and species) which is a group of pathogenic bacteria that responsible for infections in hospitals with their ability to escape from antibiotics (Helen W Boucher, 2020). The results of the latest surveillance conducted by the European Center for Disease Prevention and Control (ECDC) showed that nearly 31% of P. aeruginosa isolates were resistant to at least one group of tested antibiotics (European Centre for Disease Prevention and Control. 2018). The multidrug resistance (MDR) P. aeruginosa arises concern because of the nature of the bacteria which is easy to adapt to various environmental conditions and can spread widely (Helen W Bouche, 2020). detection canP. aeruginosa be conducted by conventional bacterial culture methods, immunological assays, and molecular assays (Tang 2017). In this study, we conducted theet al. detection of through molecular assay, theP. aeruginosa qPCR, targeting the complete genome at certain region of as referred to Stedtfeld et al.'s studyP. aeruginosa (2018). Wastewater Treatment Plants (WWTP) are the important reservoir of antimicrobial resistance issue (Pärnänen 2019). Study from Oliveiraet al. et al.(2018) reported the finding of 11% (from total 27 isolates) resistant isolated from WWTPP. aeruginosa Rio Para located in the City of Divinópolis, Southern Brazil. Data from Brazil showed a high profile of antibiotic resistance in isolated fromP. aeruginosa hospital wastewater (HWW) treatment (HWWTP), the discovery of MDR were 85.4% in PassoP. aeruginosa Fundo, 82% in Rio de Janeiro, and 60% in Manaus. The P. aeruginosa isolates (15 isolates) from clinical specimens in National Reference Hospital Dr. Cipto Mangunkusumo (RSCM) from April to November 2015 were resistant to ceftazidime, ciprofloxacin, amikacin, and carbapenems thorough in vitro Vitek 2 compact et al.test (Prasetyo 2022). Amikacin is one of the aminoglycosides antibiotic group and this group is also one of drug of choice for treatment P. aeruginosa infection (CLSI Clinical and Laboratory Standards Institute, 2020). However, there have been no studies reporting and analyzing the profiles of pathogenic bacteria, resistance genes, and antibiotic residues contained in hospitals wastewater especially National Reference Hospital (NRH). This research aimed to detect and find the relative abundance of and antibiotic resistanceP. aeruginosa gene (ARG) , for the antimicrobial resistanceaadA2 monitoring study in hospital wastewater (HWW). MATERIALS AND METHODS The research design is a descriptive study to detect certain gene of bacteria and ARG in HWW and approved by RSCM Ethic Committee No. 21-09-0905. Hospital Wastewater (HWW) Samples Collection. Samples were collected from Oct 25th – Nov 27th, 2021 from NRH's wastewater area inlet and outlet every 3 days. Total samples were 24 (12 samples each inlet and outlet). The inlet and outlet wastewater area were collected each 1 liter and placed in alcohol 70%-disinfected bottle. The wastewater samples were transported to laboratory and filtrated immediately using Polyethersulfone (PES) 0.22 µm membrane with 47 mm diameter. After the filtration, filter membranes were stored in -20 °C freezer until the DNA extraction step performed. The volume of filtered HWW samples were 50 mL for inlet and 100 mL for outlet. The reset of HWW samples were stored in -30 °C. DNA Extraction. DNA extraction from filter membrane was using kit DNeasy PowerWater Kit™ (Qiagen) and following manufacture instructions. At the final step, the pellet from extraction was homogenized using elution buffer and measured for DNA concentration and purity using NanoDrop with 260/280 nm wavelength. qPCR Assay. qPCR quantification was using SensiFAST™ SYBR ® No-ROX Kit (Bioline) with 10 μL total volume each reaction. One reaction consists of: 5 μL 2X Master mix, 0.4 μM forward primer, 0.4 μM reverse primer and 1 μL DNA (0.2 ng μL ) as -1 template. The primers were used in this study can be seen in Table 1. The primer pair targeting the P. aeruginosa was designed to detect the species-specific complete genome of P. aeruginosa from NCBI Reference Sequence, NC_002516.2 region 1410332- 1410412, “AGCGTTCGTCCTGCACAAGTTC GACGGCCTGTCCCAGGTCGAAGTGGCCGAGC GCATGGGAATCTCCCTGAGCATGGTGGA”. Real-time cycling conditions included 2 min enzyme activation at 95 °C followed by 40 cycles of Volume 1 , 2026 2 Microbiol Indones 25 denaturation at 95 °C for 5 sec and annealing 60 °C for 10 sec and elongation at 72°C for 10 sec. Each sample was tested duplo in qPCR assay. Relative Abundance. The relative abundance was obtained from normalize each gene with 16s rRNA gene using Livak formulation Relative abundance = 2 −ΔCt (ΔCt = Ct of gene – Ct of 16S rRNA gene). RESULTS Waste Water Samples Collection and DNA Extraction. The HWW treatment used in NRH was activated sludge with extended aeration system. The wastewater samples were collected from two spots as can be seen in HWW treatment diagram in Figure 1. The DNA concentrations from extraction process were 31.00-162.30 ng µL and 1.90-25.20 ng µL from inlet -1 -1 and outlet HWW samples, respectively (Table 2). qPCR Assay and Relative Abundance. Based on qPCR assay (Table 3) the was detected atP. aeruginosa certain date in inlet wastewater samples and not detected at all in outlet wastewater samples. The aadA2 gene was detected in all inlet and outlet samples with cycle threshold (Ct) values 17.650 to 35.285. The relative abundance value can be seen in Table 4. Gene Forward Primer Reverse Primer P. aeruginosa PAO1, complete genome region 1410332- 1410412 AGCGTTCGTCCTGCACAAGT TCCACCATGCTCAGGGAGAT aadA2 CAATGACATTCTTGCGGGTATC GACCTACCAAGGCAACGCTATG 16s rRNA GGGTTGCGCTCGTTGC ATGGYTGTCGTCAGCTCGTG 1 Sampling DNA Concentration (ng µL-1) Purity Inlet 25-Oct 81.90 1.91 28-Oct 96.80 1.95 31-Oct 75.90 2.01 3-Nov 142.90 1.98 6-Nov 31.00 1.99 9-Nov 47.00 1.95 12-Nov 162.30 1.93 15-Nov 123.90 1.96 18-Nov 57.30 1.97 21-Nov 46.80 1.90 24-Nov 150.30 1.93 27-Nov 57.20 1.95 Outlet 25-Oct 25.20 1.83 28-Oct 4.80 1.22 31-Oct 4.40 1.88 3-Nov 1.90 1.87 6-Nov 4.20 1.66 9-Nov 3.20 1.76 12-Nov 4.00 1.75 15-Nov 3.00 1.55 18-Nov 7.60 1.68 21-Nov 2.00 1.38 24-Nov 2.70 2.00 27-Nov 3.41 2.41 1 Table DNA concentration from HWW samples2 Table 1 sp. prevalence in 2017-2021Staphylococcus 26 TIFFARENT ET AL. Microbiol Indones Generally has higher relative abundance thanaadA2 P. aeruginosa. The relative abundance was visualized in heat map (Table 5). The darker colour shows higher value of relative abundance. DISCUSSION The gene was found constantly inaadA2 wastewater samples inlet and outlet, meanwhile the P. Table 3 Cycle threshold (Ct) values and standard deviation (Stdev) of and in qPCRP. aeruginosa aadA2 assay Sampling P. aeruginosa aadA2 16s rRNA Ct Value Stdev Ct Ct Value Stdev Ct Ct Value Stdev Ct Inlet 25-Oct 36.245 0.460 19.885 0.163 17.095 0.021 28-Oct 35.625 2.029 19.265 0.035 17.015 0.078 31-Oct - - 22.170 0.339 17.400 0.057 3-Nov - - 21.675 0.502 17.690 0.209 6-Nov - - 22.740 0.495 18.085 0.007 9-Nov - - 22.050 0.071 17.585 0.262 12-Nov - - 17.650 0.368 17.380 0.028 15-Nov 34.945 0.615 21.080 0.042 17.295 0.095 18-Nov - - 17.740 0.523 17.505 0.191 21-Nov 36.160 0.948 17.650 0.028 17.240 0.338 24-Nov 36.335 0.021 20.925 0.163 17.205 0.021 27-Nov 33.983 0.508 21.350 0.269 17.508 0.365 Outlet 25-Oct - - 22.305 0.304 19.755 0.187 28-Oct - - 25.140 0.014 22.425 0.064 31-Oct - - 24.335 0.163 21.725 0.148 3-Nov - - 25.570 0.085 25.395 0.007 6-Nov - - 32.640 0.170 25.628 0.321 9-Nov - - 27.588 0.484 23.615 0.247 12-Nov - - 35.285 1.549 25.640 0.354 15-Nov - - 32.485 0.021 25.425 0.007 18-Nov - - 23.330 0.099 21.105 0.064 21-Nov - - 27.495 0.361 27.880 0.071 24-Nov - - 24.330 0.085 21.035 0.078 27-Nov - - 25.115 0.035 21.515 0.332 1 Fig 1 The diagram of NRH HWW treatment, red asterisk signs were the sampling spots. Volume 1 , 2026 2 Microbiol Indones 27 Sampling P. aeruginosa aadA2 Inlet 25-Oct 0.000002 0.144586 28-Oct 0.000002 0.210224 31-Oct 0.036651 3-Nov 0.063153 6-Nov 0.039692 9-Nov 0.045279 12-Nov 0.82932 15-Nov 0.000005 0.072544 18-Nov 0.849685 21-Nov 0.000002 0.752623 24-Nov 0.000002 0.075887 27-Nov 0.000011 0.06971 Outlet 25-Oct 0.170755 28-Oct 0.152301 31-Oct 0.163799 3-Nov 0.885768 6-Nov 0.007745 9-Nov 0.063703 12-Nov 0.001249 15-Nov 0.007494 18-Nov 0.213899 21-Nov 1.30586 24-Nov 0.101884 27-Nov 0.082469 1 Table 4 Relative abundance and geneP. aeruginosa aadA2 Table 5 . Heat map relative abundance and geneP. aeruginosa aadA2 Sampling P. aeruginosa aadA2 Inlet 25-Oct 28-Oct 31-Oct 3-Nov 6-Nov 9-Nov 12-Nov 15-Nov 18-Nov 21-Nov 24-Nov 27-Nov Outlet 25-Oct 28-Oct 31-Oct 3-Nov 6-Nov 9-Nov 12-Nov 15-Nov 18-Nov 21-Nov 24-Nov 27-Nov Value:1 0.000002 - 1.30586 2 28 TIFFARENT ET AL. Microbiol Indones aeruginosa was found only in inlet samples. This indicated that the system of wastewater treatment has not completely eliminated the ARG. The ARG in outlet has the potency to contaminate the environment when it was released. The ARGs in studies that have been reported found in HWW were aadA25, dfrA16, dfrA5, macB, MexF, mexW, smeE, blaVEB, aadA2, blaGES, blaVIM, AAC.6…30/AAC.6…Ib., baeR, cpxA, CRP, dfrA1, emrA, qacH, tet36, ugd et al.(Majlander 2021; Cai 2021). This should be a concern because theet al. mobility characteristic of gene. According toaadA2 The Comprehensive Antibiotic Resistance Database (CARD) (Alcock 2020), is anet al. aadA2 aminoglycoside nucleotidyltransferase gene encoded by plasmids and integrons in ,K. pneumoniae Salmonella Corynebacterium glutamicum C.spp., , freundii Aeromonas, and spp. This gene expresses enzyme aminoglycosides 3'-adenyltransferase and causes the resistance to streptomycin and spectinomycin. The mobility of this gene uses plasmids and integrons that means this gene can be transferred intraspecies or interspecies (horizontally) in acquired resistance mechanism. is also one ofP. aeruginosa bacteria that was reported carrying the gene.aadA2 The isolates from patients in 3 GeneralP. aeruginosa Hospitals in Tehran, Iran were found carrying aadA2 gene with prevalence 47.6% (Salimizadeh 2018).et al. This study shows that the qPCR assay is capable to detect the presence of potential pathogen bacteria and ARG in HWW. The method can be used as the monitoring of antimicrobial resistance (AMR) status in hospital and the prevention and controlling of AMR spreading to environment. Further study can be developed to optimize the controlling of AMR such as the pathogenic bacteria isolation from HWW and then the bacteria will be assessed phenotypically (such as antibiotic susceptibility) and genotypically (PCR followed by sequencing) so the ARGs can be confirmed to be expressed in bacteria and can be traced back to see the potency of horizontal transfer. In conclusion, the qPCR assay can be a method for monitoring pathogen bacteria and ARGs to describe the AMR status in hospital using wastewater samples. ACKNOWLEDGMENTS We thank The Resistomap for funding and supplying some of the laboratory materials and primers in this study. REFERENCES Alcock BP, Raphenya AR, Lau TTY, Tsang KK, Bouchard M, Edalatmand A, Huynh W, Nguyen ALv, Cheng AA, Liu S. 2020. CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic r e s i s t a n c e d a t a b a s e . N u c l e i c A c i d s R e s . 48:D517–D525. 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