Jurnal Riset Biologi dan Aplikasinya, Volume 5, Issue 1, March 2023 Genetic Identification of Superbugs from River Streams in State University of Malang Reeno Al Hikmatus Sholekah, Dwi Listyorini* Department of Biology, Faculty of Mathematics and Natural Sciences, State University of Malang, Jl. Semarang No. 5 Malang, East Java, Indonesia *Corresponding author e-mail: listyorini.aljabari@um.ac.id Article History ABSTRACT Received : 9 November 2022 Superbugs revert to bacterial strains, which exhibit resistance to antibiotics. These bacteria could cause some problems in disease treatment and the environment. This research aimed to identify the presence of superbugs in the river stream that flows through State University of Malang campus area. Amoxicillin, tetracycline, and chloramphenicol were selected to test the possibly found resistant bacteria. Water samples taken from a decided spot was spread over Luria Bertani’s agar on both antibiotic supplemented and antibiotics-free LB agar media with no delay. Molecular identification was carried out using 16S rRNA gene in DNA barcoding approach, completed with morphological and Gram staining analyses. A total of 16 isolates of gram-negative colonies were found in the form of bacilli, diplobacilli, cocci, and diplococci. The genetic identification of eight resistant colonies led us to suggest that the isolates may belong to Aeromonas, Shigella, and Bacillus. Further studies are still required to get a clearer view of the correct taxonomical position of those resistant isolates. Revised : 4 February 2023 Approved : 18 March 2023 Published : 31 March 2023 Keywords Superbugs; antibiotic; 16S rRNA; phylogenetic How to cite: Sholekah, R.A.H & Listyorini, D. (2023). Genetic Identification of Superbugs from River Streams in State University of Malang. Jurnal Riset Biologi dan Aplikasinya, 5(1):27-36. DOI: 10.26740/jrba. v5n1.p.27-36. INTRODUCTION Superbug is a term used to describe strains of microorganisms, which are resistant to one or more antibiotics (Rajendran, 2018). Antibiotic resistance is a phenomenon that occurs when bacteria cannot be killed or their growth is not inhibited by the administration of antibiotics (World Health Organization, 2014). There are more than 1.5 million deaths worldwide yearly caused by superbug infection (Hall et al., 2018). Various pathogenic bacteria, including Escherichia coli, Mycobacterium tuberculosis, Clostridium perfringens, Legionella pneumophila, Pseudomonas aeruginosa, Shigella flexneri, Vibrio cholera, and Salmonella enterica have been detected in wastewater (Cai & Zhang, 2013). Among those pathogenic bacteria, several strains of P. aeruginosa, E. coli, Acinetobacter sp., and Enterobacteriaceae has been known to be resistant to antibiotics (Jia & Zhang, 2019). Reports on resistant bacteria are still very limited in Indonesia. Verawaty et al. (2020) reported the presence of E. coli that is resistant to ampicillin, tobramycin, and tetracycline in the waters of Palembang, meanwhile Suhartono et al. (2021) reported the presence of Salmonella spp. that are resistant to azithromycin, tetracycline, and streptomycin in the Lamnyong and Krueng waters, Aceh. Research on resistant bacteria in Indonesian waters is still limited, providing the basis for carrying out this research. The main cause of antibiotic resistance is the misuse of antibiotics, especially in humans and livestock (Parathon et al., 2017), due to the easy access of the public to antibiotics without a prescription either in drugstores or food stalls (Yarza et al., 2015). Amoxicillin was the most widely Jurnal Riset Biologi dan Aplikasinya https://journal.unesa.ac.id/index.php/risetbiologi mailto:listyorini.aljabari@um.ac.id https://doi.org/10.26740/jrba.v5n1.p27-36 Jurnal Riset Biologi dan Aplikasinya, 5(1): 27-36, March 2023 | 28 consumed antibiotic in Indonesia during 2008-2010, while tetracycline and chloramphenicol were ranked fifth and tenth, respectively (Pradipta et al., 2015). Amoxicillin is commonly used to treat respiratory infections (Akhavan et al., 2021) as well as tetracycline (Bidell et al., 2020), while chloramphenicol is used to treat typhoid fever (Hanekamp & Bast, 2015). Amoxicillin is a ß-lactam antibiotic of the penicillin class (amino-penicillin), which acts as a bactericide (Bernatová et al., 2013). Tetracycline is a tetracycline-class antibiotic that works as a bacteriostatic to inhibit bacteria replication (Shutter, 2022). Meanwhile, chloramphenicol works through both bacteriostatic and bactericidal mechanisms at high concentrations (Oong & Tadi, 2021). Superbug have also reported to be found in the air environment of hospitals, livestock sector, and sewage treatment plants (Zhou et al., 2020). In the aquatic environment, superbugs can also be found due to the fact that in many areas, water bodies become the estuaries of domestic waste (Puspita et al., 2016), hospital waste (Sarkar et al., 2019), agricultural waste (Habibi et al., 2014), mining waste (Mulyadi et al., 2020), and industrial wastewater (Rahardjo & Prasetyaningsih, 2021). This causes adverse ecotoxicological effects (Smith et al., 2020). In many cases, the disposal of wastewater treatment plants located upstream is a major source of antibiotic-resistant bacteria in waters (Ren & Hongqiang, 2019). Surface and groundwater have been reported in several studies to have a role in causing the emergence of antibiotic-resistant organisms (Li et al., 2014; Hughes et al., 2013; Bradford et al., 2017). The river stream within State University of Malang Campus is an estuary of domestic waste disposal originating from densely populated residential areas upstream. Wastewater has been reported to be one of the reservoirs for superbugs and an important source for the spread of antibiotic resistance (Jia & Zhang, 2019). Taking into consideration the negative impact of resistant bacteria on the treatment of diseases and environment, we studied the possible existence of superbugs in the river flow within the campus. Molecular identification of superbugs was done using 16S rRNA gene sequence based on the report that 16S rRNA gene is highly conserved which only undergoes slight nucleotide changes in the evolutionary process (Lan et al., 2016). This research aimed to identify the presence of superbugs in the river stream that flows through Malang State University campus area. MATERIALS AND METHODS Location and time of observation This explorative descriptive research was carried out from September 2021 through August 2022 at Biology Laboratory of the State University of Malang. Sampling was taken at river flow of the State University of Malang, Jl. Semarang No.5, Malang, Indonesia. The location of sampling coordinates is located at 7°57'41.0"S, 112°37'06.9"E. Culture Medium Luria Bertani (LB) agar and Luria Bertani broth were used as a bacterial culture medium. All media and tools were sterilized in an autoclave at 121⁰C, 15 lbs. for 15 minutes. As much as 32 mg/L of amoxicillin, 16 mg/L of tetracycline, or 32 mg/L of chloramphenicol were added to LB agar media (Huang et al., 2012; Tahrani et al., 2015) to detect the possible existence of antibiotic resistant bacteria. The media were then stored in the refrigerator until being used. Bacteria Sample Bacterial samples were isolated from water taken at 7 a.m. from the river behind the Green House area of State University of Malang. A total of 15 mL of river water in a sterile centrifuge tube was brought to the laboratory in a cooler box. Water quality parameters were measured, including water pH using the Hanna Instrument pH 211 and water turbidity using a digital turbidity meter, SGZ- 200BS. Water sample was diluted gradually (10-1, 10- 2, 10-3, 10-4, 10-5, and 10-6) and homogenized in 0.1% peptone media. A total of 0.1 mL of water from each sample was inoculated on both LB agar and antibiotic added LB agar media in three repetitions. Incubation was carried out for 1 x 24 hours at 37⁰C (Bergeron et al., 2015). Colony Morphological Analysis and Bacterial Gram Staining Each colony was purified using the quadrant streak plate method to obtain a single colony. Each isolated colony was observed for colony morphology, which included: colony shape, colony edge, colony elevation, and colony color. The isolated colonies were also inoculated on slanted agar media for short- term stock and preserved in glycerin solution for extended time storage. Bacterial Gram and cell shape were microscopically observed. Gram staining was performed using ammonium oxalate crystal violet reagent, iodine, 96% alcohol, and safranin. 29| Sholekah & Listyorini., Genetic Identification of Superbugs Genetic Identification (DNA Barcoding) For genetic identification purposes, each of antibiotic resistant isolates was cultured in 2 ml of liquid LB medium with respective antibiotic added in duplicate. Incubation was carried out for 2x24 hours. Total bacterial DNA isolation was carried out following the protocol provided by the manufacturer (Geneaid PrestoTM Mini gDNA Bacteria Kit). The quality and concentration of total DNA obtained were measured using a Spectrophotometer NanoDrop ND-2000. Target gene amplification was carried out by PCR technique using universal primers for 16S rRNA gene. The forward primer used was 27F, 5’- AGAGTTTGATCMTGGCTCAG-3’, and reverse primer used was R1492, 5’- TACGGYTACCTTGTTACGACT-3’ (Pertiwi et al., 2018). Pre-denaturation was carried out at 95°C for 3 minutes, followed by 40 amplification cycles consisting of denaturation at 95°C for 1 minute, annealing at 50°C for 1 minute, extension at 72°C for 1 minute, and the final extension was carried out at 72°C for 10 minutes. Electrophoresis was performed to visualize the PCR product on 1% agarose gel running in 50 volts electrical current for 60 minutes (Pertiwi et al., 2018). The results of electrophoresis are visualized using a UV trans illuminator. The sequence data was taken for eight isolates demonstrating antibiotic resistance. Sequencing process was carried out by First BASE Laboratories, Malaysia. Data Analysis Colony morphology, bacterial Gram, and sequence data were analyzed descriptively. The target gene sequences were read using Finch TV. Consensus sequences from forward and backward sequencing of each sample were constructed using ClustalX, then compared to the respective sequence databases available in the Gene Bank NCBI using online Basic Local Alignment Search Tool (BLAST). Phylogenetic tree reconstruction in order to analyze the taxonomic position of each bacterial isolate was carried out using MEGA-11 software for Neighbor Joining (NJ) model with the Kimura-2-Parameter in a 1000 bootstrap value (Prasetio et al., 2021). RESULTS AND DISCUSSION Water Parameter Water sample taken from a decided spot within State University of Malang was neutral (pH 7,33) and clear (0,1 NTU turbidity). Colony Morphology and Gram Staining Bacterial colonies were growing on both LB and antibiotic added LB media (Figure1 & 2). From the original inoculation plates, we subcultured in order to purify the distinct colonies. In LB media we found eight different colonies. Meanwhile, three different colonies were growing on both Amoxicillin and Chloramphenicol media, and only two different colonies were growing on Tetracycline media. The observed colonies were dominated by circular opaques with entire margin, and only a few showing other shapes of their edges and being translucent in color (Table 1). The observation through Gram staining showed that all colonies growing on LB media were bacilli, while those growing on antibiotic added media were bacilli, diplobacilli, cocci, or diplococci (Table 1, Figure 2). Figure 1. Initial inoculation on LB Agar media and antibiotic added LB Agar media. Scale bar = 2 cm Jurnal Riset Biologi dan Aplikasinya, 5(1): 27-36, March 2023 | 30 Table 1. Morphologies and Gram-staining of 16 distinct purified colonies No Control (LB) Gram LB + Antibiotic Gram 1 2.1 circular; entire; raised; opaque; milky white Negative bacilli Amoxicillin 2.1 circular; entire; raised; opaque; milky white Negative bacilli 2 2.2 circular; entire; flat translucent; clear white Negative bacilli Amoxicillin 2.2 circular; entire; flat; translucent; clear white Negative bacilli 3 2.3 circular; wooly; flat; opaque; murky white Negative bacilli Amoxicillin 2.3 circular; irregular; flat; opaque; murky white Negative diplo bacilli 4 2.4 circular; entire; flat; opaque edge; milky white Negative bacilli Chloramphenicol 2.1 circular; entire; raised; opaque; milky white Negative cocci 5 2.5 circular; irregular; flat; opaque; milky white Negative bacilli Chloramphenicol 2.2 circular; entire; flat; translucent; clear white Negative bacilli 6 2.6 circular; undulate; flat; opaque; milky white Negative bacilli Chloramphenicol 2.3 circular; entire; raised; opaque; murky white Negative bacilli 7 2.7 circular; wooly; flat; opaque; milky white Negative bacilli Tetracycline 2.1 circular; entire; raised; opaque; milky white Negative bacilli 8 2.8 circular; ciliate; flat; opaque; murky white Negative bacilli Tetracycline 2.2 circular; entire; flat; translucent; clear white Negative diplococci Among all subcultured colonies, we identified that there were colonies growing on two different antibiotic media, i.e: those which were growing on both Amoxicillin (AMX 2.1; Figure 2I) and Tetracycline (TET 2.1; Figure 2O) and those which were growing on both Amoxicillin (AMX 2.2; Fig. 2J) and Chloramphenicol (CAP 2.2; Figure 2M). A total of 16 bacterial isolates were found. On control media grew, eight isolates, on amoxicillin and chloramphenicol media, three isolates were grown, and on tetracycline media grew only two isolates. It can be understood that those number are less than the number of bacterial isolates that grew in LB media. The presence of antibiotic eliminates the non- resistant ones that grow naturally on the control media due to the absence of any agents that inhibit bacterial growth. The inhibition of bacterial growth in antibiotic supplemented media may be caused by the bacteriostatic effect, which caused disruption of bacterial replication (Bernatová et al., 2013; Nemeth et al., 2015) and/or bactericidal effect of given antibiotics that cause bacterial death (Baquero & Levin, 2021). The number of isolates grown on amoxicillin and chloramphenicol media was greater than that grown-on tetracycline media (Table 1; Figure 2). We suggest that this finding resulted from the high usage of amoxicillin and chloramphenicol in society in the area upstream to the river. Amoxicillin is widely used to treat various bacterial infections, such as pneumonia, tonsillitis, ear infections (Akhavan et al., 2021), sinusitis dermatitis (Sartelli et al., 2018), and urinary tract infections (Tan & Chlebicki, 2016). Chloramphenicol is used to be applied to treat typhoid fever (Hanekamp & Bast, 2015), bacterial conjunctivitis (eye infection) and otitis externa (Oong, 2021). Tetracyclines are common to be used to treat respiratory tract infections (Bidell et al., 2020) and certain skin inflammations (Orylska et al., 2022). As it has been well understood that the excessive metabolites will be removed from body through excretory system, either through feces, urine, or sweat. (Garza et al., 2020). Then, in turn, the disposal of house or hospital sewage, which may contain excess antibiotics, directly into any type of water body will not only pollute the water, but also induce the living organism including bacteria, to develop their resistance. 31| Sholekah & Listyorini., Genetic Identification of Superbugs Figure 2. Colony morphology and Gram type. Blue Scale bar = 10 μm Amoxicillin works by binding to penicillin- binding protein, activating autolytic enzymes, and thereby causing cell wall lysis (Akhavan et al., 2021). Certain antibiotic resistant bacteria, such as E. coli, produce ß-lactamase enzymes. This enzyme degrades ß-lactam ring of the antibiotic which resulting in the deactivation of its function (Wu et al., 2021). Chloramphenicol works by interfering the protein synthesis on the 50S ribosomal subunit and inhibiting aminoacyl-tRNA binding at the ribosomal site (Blanco & Antonio, 2017). Thus, the mechanism of bacterial resistance against chloramphenicol is different from the mechanism of resistance to amoxicillin. It can be through drug inactivation, inhibiting protein synthesis (Kapoor et al., 2017), or the existence of an efflux system mechanism (Reygaert, 2018). Tetracyclines work by binding to 16S rRNA and inhibiting tRNA from binding to the ribosome site (Chukwudi, 2016). It had been reported that tetracycline resistance in Gram-negative bacteria can occur through the efflux system mechanism. The efflux pump gene codes for a membrane protein that pumps tetracycline out of cells, rendering the drug ineffective (Amaral et al., 2014). The presence of superbugs in amoxicillin, chloramphenicol, and tetracycline media suggested Jurnal Riset Biologi dan Aplikasinya, 5(1): 27-36, March 2023 | 32 that the isolated bacteria in this study may develop resistance mechanisms. Further studies are required to fully understand this finding. Identification of bacteria based on the 16S rRNA gene From all antibiotic resistant isolates, we got 955 to 1460 bp length fragments. Reconstructed phylogenetic tree analysis revealed that two isolates of Amoxicillin resistant bacteria (AMX 2.1 and AMX 2.2) were sitting among Aeromonas species with low bootstrap value. A similar situation was shown by one Chloramphenicol-resistant isolate (CAP 2.2), which was sitting among Shigella species with relatively low bootstrap value. It means that position is not confidently accepted and needs more genetic analysis to clarify this result. On the other hand, TET 2.1, CAP 2.3, AMX 2.3, AND CAP 2.1 were forming a distinct clade splited from Bacillus spesies with high bootstrap value (Figure 3). The results of the phylogenetic tree reconstruction showed that AMX 2.1 and AMX 2.2 isolates were suspected to be Aeromonas caviae or Aeromonas dhakensis. A. dhakensis causes soft tissue infections, bloodstream infections in immunocompromised individuals with malignancy and cirrhosis. A higher mortality rate was observed in cases of bacteremia (Puah et al., 2022). It has been reported that A. veronii isolated from Lake Erie showed resistance to tetracycline (Skwor et al., 2014). Aeromonas spp. isolates were susceptible to chloramphenicol found in freshwater fish (Fauzi et al., 2021). Resistance of A. dhakensis to erythromycin, amoxicillin, and ampicillin has also been reported (Soto-Rodriguez et al., 2018). This study also revealed that AMX 2.3 isolate, CAP 2.1 isolate, CAP 2.3 isolate, and TET 2.1 isolate were suspected to be species of the genus Bacillus. Some species of Bacillus are involved in food poisoning, except for B. anthracis which causes anthrax (Tarek et al., 2021). Furthermore, some Bacillus sp. was found to have high resistance to β- lactams, fluoroquinolones, and tetracyclines (Mbhele et al., 2021). Analyzing the position of CAP 2.2 isolate in the phylogenetic tree, we suggest that the isolate belongs to Shigella sp. Contamination of Shigella spp. to the water can be occurred through human and pet waste (Garcia et al., 2017). Shigella spp. is the most common bacterium that causes bacillary dysentery (shigellosis), a disease characterized by damage to the colonic mucosa caused by bacterial invasion (Garcia et al., 2017). This member of Shigella has also been reported to be resistant to several antibiotics, such as ampicillin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole (Meiyanti et al., 2016). Again, a thorough investigation is required to prove it. The TET 2.2 isolate was suspected to be Aeromonas veronii since this isolate stayed in the same clade with a convincing bootstrap value. A. veronii is a facultative anaerobic, gram-negative, and bacillus- shaped bacterium (Li et al., 2020). This species had been known to cause diarrhea and sepsis in humans (Fernández-Bravo et al., 2020). In particular, A. veronii is a common pathogen in aquaculture, which infects freshwater fish. Drug sensitivity tests showed that the A. veronii isolate strain 18BJ181 was resistant to against four antibacterial drugs, including amoxicillin, madinomycin, penicillin, and sulfamethoxazole, while quite sensitive to erythromycin and rifampin (Wang et al., 2021). Resistance to tetracyclines has also been reported in isolates of A. veronii which have been confirmed to have tetA and tetE genes (Kim et al., 2020). The AMX 2.3, CAP 2.1, CAP 2.3, and TET 2.1 isolates, which were found to be Gram negative, phylogenetically formed an exclusive clade inside Bacillus genus big cluster which well known as Gram positive bacteria. Considering carefully the finding that these isolates were multi-antibiotic resistant, thus we suggest that both morphological and physiological changes have been occurred. Those changes might be caused by certain significant mutations. On the other hand, changes in bacterial colony morphology and cell shape found in this study, might be possible as a result of antibiotic treatment and the concentration given in this experiment as of reported by Peach et al. (2013) and the length of exposure (Cushnie et al., 2016). The morphology of E. coli bacterial cells exposed to the antibiotics after three hours showed a change in cell shape to resemble to the diplobacilli-like, diplococci, or forming a typical intracellular inclusion body (Dufour et al., 2017). Overall, our findings in this study are still far from clear. More genetic studies including DNA Barcoding approaches using stronger markers, genetic analysis on responsible genes that may induce antibiotic resistance, and enzymatic bioassays are urgently required. Enzymatic analysis would confirm whether the isolates produce β-lactamase that makes it resistant. Meanwhile, genetic analysis which focusing on certain genes mutation may reveal how those isolates developed their resistance, especially those that became multi-antibiotic resistant while phylogenetically coexisting Gram- 33| Sholekah & Listyorini., Genetic Identification of Superbugs REFERENCES Figure 3. Isolates taxonomical position based on 16S rRNA in Neighbor Joining method positive bacteria, may help to determine the possible mutations which lead to the resistance to the given antibiotic. CONCLUSION This study found eight isolates grew on LB media, three isolates grew on amoxicillin media, three isolates grew on chloramphenicol media, and two isolates grew on tetracycline media. Based on colony morphology and gram staining, we found that one isolate was multi-resistant to amoxicillin and tetracycline, and the other was multi-resistant against amoxicillin and chloramphenicol. Genetically, the isolates found were suggested to be members of Aeromonas, Shigella, and Bacillus. 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