5th International Seminar on Pharmaceutical Science and Technology (ISPST)-3rd International Seminar and Expo on Jamu-13th Annual ISCC 2022. | 40 Β-LACTAMASES INHIBITOR-PRODUCING SOIL BACTERIA FOR AMPICILLIN- RESISTANT UROPATHOGENIC ESCHERICHIA COLI ISOLATE Original Article SRI AGUNG FITRI KUSUMA 1* , VALENTINA YURINA 2 , DEBBIE S. RETNONINGRUM 3 , INDAH LAILY HILMI 4 , SUSI AFRIANTI RAHAYU 5 , YUNI NOER ANGGRAINI 1 1 Department of Biology Pharmacy, Faculty of Pharmacy, Padjadjaran University, Sumedang, West Java, Indonesia, 2 Department of Pharmacy, Faculty of Medicine, Brawijaya University, Malang, East Java, Indonesia, 3 School of Pharmacy, Bandung of Institute Technology, Bandung, West Java, Indonesia, 4 Department of Pharmacy, Singaperbangsa University, Karawang, West Java, Indonesia, 5 Department of Pharmacy, Bumi Siliwangi Academic, Bandung, West Java, Indonesia * Email: s.a.f.kusuma@unpad.ac.id Received: 03 Sep 2022, Revised and Accepted: 05 Nov 2022 ABSTRACT Objective: The goals of this investigation were to identify the species of the producers and ascertain the dose-dependent effect of extracellular products of Indonesian bacteria that generate β-lactamases inhibitors. Methods: An agar diffusion technique for the lactamase inhibitor activity assay was performed. Observation of bacteria using phenotypic analysis was performed by observing colony color and cell shape morphology, biochemical assays and a series of carbohydrate fermentation tests. Bacterial identification was performed by comparing the nucleotide sequence of the 16S rDNA gene of target bacteria with available nucleotide sequences in Gene Library (NCBI). Combining data from phenotypic and genotypic analyses allowed for the identification of the producers. Results: According to our findings, none of the bacteria's extracellular products, which contain β-lactamase inhibitors in a range of concentrations, showed a discernible impact on the values of the inhibition zone. The producers are Aeromonas popoffii, Alcaligenes faecalis, Streptomyces brasiliensis, Staphylococcus equorum, Pseudomonas putida, Pseudomonas fluorescens, Salmonella typhi, Enterobacter hormaechei, Serratia marcescens and Enterobacter sp. The highest potency of β-lactamase inhibitor was provided by the extracellular product of VR3 isolate bacteria which was identified as Serratia marcescens. Conclusion: In conclusion, this study clearly showed that our isolated bacteria have the potential to be further investigated in order to maximize the recovery of β-lactamase inhibitor compounds. Keywords: ®-lactamase, Soil, Inhibitor, Escherichia coli, Ampicillin, Serratia marcescens © 2022 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/) DOI: https://dx.doi.org/10.22159/ijap.2022.v14s5.03 Journal homepage: https://innovareacademics.in/journals/index.php/ijap INTRODUCTION Limiting the use of beta-lactam antibiotics or their combination with beta-lactamase inhibitors for the initial treatment of cystitis is reported due to facts that these drugs are resistant to the uropathogenic bacteria, cystitis-causing bacteria [1]. As in Indonesia, antibiotic resistance is an obstacle in the treatment of cystitis. Based on medical record information from a hospital in Indonesia, urine culture examination results showed that the majority of bacteria found in the samples were Gram-negative bacteria with characteristics of multidrug resistance to several antibiotics, including cephalotin, cephazolin, ampicillin, sulphametoxazole, and trimethoprim. Escherichia coli was the most prevalent pathogen identified in urine culture among Gram-negative bacteria [2]. According to earlier research, E. coli is the main cause of cystitis, and ampicillin resistance was the highest case in isolated E. coli [3-5]. In order to combat bacterial resistance, serine ®-lactamases produced by E. coli, which inactivate the beta-lactam ring, are predominantly blocked by combinations of ®-lactamase inhibitors [6-8]. Since the inhibitor and beta-lactam antibiotics are competing for the same binding site on the beta-lactamase enzyme, evolutionarily, the beta-lactamase enzyme's gene may become mutated, making the enzyme more likely to be vulnerable to inhibitors [9]. Clavulanic acid, for example, is a ®- lactamase inhibitor that is beneficial in boosting the activity of ®- lactam antibiotics in the treatment of severe Enterobacteriaceae infections. For the treatment of severe Enterobacteriaceae infections, a number of ®-lactamase inhibitors are useful for repurposing the activity of ®-lactam antibiotics, including clavulanic acid, sulbactam and tazobactam. The development of resistance to the combination of ®-lactam-®-lactamase inhibitors is accelerated by the misuse of antibiotics. Additionally, there is a substantial rise in the occurrence of clinically significant inhibitor-resistant types of ®-lactamases. As a result, there is an urgent need for efficient inhibitors that can revive ®-lactam action [10]. Our earlier research demonstrated that a number of soil bacteria's extracellular products have ®-lactamase inhibitory action, which can improve the effectiveness of ampicillin from the ®-lactamases inactivation made by clinical isolates of ampicillin-resistant E. coli. Since ampicillin-resistant bacteria that produce ®-lactamases can be killed by the extracellular products of soil microorganisms, compounds of microbial products may contain new ®-lactamase inhibitors. The development of drugs will be significantly impacted by the discovery of novel ®-lactamase inhibitors. Because the potential sources of ®-lactamase inhibitors were found in Indonesian soil, this study has the potential to expand our understanding of microbial biodiversity. The objectives of this investigation were to determine the species of ®-lactamase and investigate the dose-dependent inhibition of bacterial extracellular products with possible ®-lactamase inhibitory activity and to determine the species of microbes that produce ®-lactamase inhibitors using phenotypic and genotypic methods. MATERIALS AND METHODS Materials Co-amoxiclav (Indofarma), Sodium chloride (Merck), Potassium Chloride (Merck), Sodium Hydrophosphate (Merck), Potassium Hydrophosphate (Merck), Distilled water, Agarose (Boehringer Mannheim), forward primer (5'GGTTAC(G/C)TTGTACGACTT3') (Proligo), and reverse primer (5'AGAGTTTGATC(A/C)TGGCTCAG 3') (Proligo), deoxynucleotide triphosphate (dNTP) (MD Bio-Korea), Taq DNA polymerase (Stratagene), Marka 100 bp. Wizard Genomic DNA Purification Kit (Promega), absolute ethanol, 70% ethanol, distilled water, double distilled water, Tris-base 10 mmol, EDTA 1 mmol, pH 8.0, magnesium chloride (Merck), loading buffer (sucrose and 0.25% bromphenol blue (Merck)), ethidium bromide, Mueller Hinton Broth (MHB-Pronadisa), Mueller Hinton Agar (MHA-Pronadisa) and Luria Bertani (LB) medium. International Journal of Applied Pharmaceutics ISSN- 0975-7058 Vol 14, Special Issue 5, 2022