Analisis Gen Resisten Antibiotik Bakteri Asam Laktat Kandidat Probiotik Dan Kemampuannya Dalam Menekan Aktivitas Quorum Sensing Salmonella enterica sv Typhimurium ATCC 14028

Nurfazila Di'a Rifannisaa, Prof. Dr. Apt. Mustofa, M.Kes; Dr. Ema Damayanti, S.Si., M.Biotech

2025 | Tesis | S2 Ilmu Kedokteran Dasar dan Biomedis

Latar Belakang: Salmonella typhi adalah bakteri penyebab demam tifoid, yang menyebabkan banyak infeksi dan kematian di seluruh dunia. Resistensi antibiotik terhadap S. typhi semakin meningkat, sehingga diperlukan pengobatan alternatif. Bakteri asam laktat (BAL) berpotensi menjadi probiotik yang dapat menghambat pertumbuhan patogen, termasuk S. typhi. Gen resistensi antibiotik harus diidentifikasi sebelum dapat digunakan sebagai probiotik dengan aman. Selain itu, perkembangan S. typhi juga dapat disebabkan oleh aktivitas quorum sensing (QS). Tujuan Penelitian: Menganalisis gen resisten antibiotik pada isolat bakteri asam laktat (BAL), mengetahui resistensi BAL, kemampuan BAL dalam menghambat pertumbuhan Salmonella, menekan aktivitas QS dan mengkaji energi ikatan senyawa BAL terhadap protein target QS Salmonella. Metode: Penelitian ini menggunakan strain Lacticaseibacillus rhamnosus TS-4, TB-3 dan HI-1 serta Pediococcus acidilactici HI-5 dan LT-3. Aktivitas antibakteri diuji terhadap Salmonella enterica sv Typhimurium ATCC 14028 dengan metode mikrodilusi 96-well dan uji resistensi antibiotik memakai metode Kirby-Bauer. Data Whole Genome Sequencing dianalisis dengan RAST, MUSCLE, dan CARD. Uji anti quorum sensing (QS) menggunakan Chromobacterium violaceum. Analisis doking molekuler dilakukan pada 5 metabolit dari BAL terhadap SdiA dan lrsB menggunakan Autodock VINA, PyMOL, dan BioVia Discovery Studio. Hasil: Isolat BAL menunjukkan aktivitas antibakteri dengan MIC 50?n 70%. Uji kerentanan antibiotik mengungkapkan resistensi pada beberapa isolat. Isolat TS-4, TB-3, dan HI-1 memiliki gen resistensi beta-laktamase dan fluorokuinolon, sedangkan HI-5 dan LT-3 membawa gen resistensi fluorokuinolon dan multidrug resisten pompa efluks. Semua strain tidak memiliki elemen transposabel, agen pemindah gen, atau plasmid, sehingga transfer gen horizontal kemungkinan tidak terjadi. Konfirmasi gen resistensi melalui MUSCLE menunjukkan identitas ?81%, dan data CARD relevan dengan RAST (identitas 21-59%). Uji aktivitas anti QS menunjukkan penghambatan violacein >70?n hasil doking molekuler mengindikasikan beberapa metabolit BAL memiliki energi ikatan yang tinggi pada protein pengatur QS. Kesimpulan: Beberapa isolat BAL, seperti L. rhamnosus (TS-4, TB-3, HI-1) dan P. acidilactici (HI-5, LT-3), efektif sebagai antibakteri terhadap Salmonella. Meskipun beberapa isolat resisten terhadap antibiotik demam tifoid, gen resistensi mereka tidak terdapat pada plasmid atau elemen transposable, sehingga aman sebagai probiotik untuk manusia. Metabolit BAL juga menunjukkan energi ikatan yang baik terhadap protein pengatur QS menurut hasil doking molekuler.

Background: Salmonella typhi is a bacterium that causes typhoid fever, which leads to numerous infections and deaths worldwide. Antibiotic resistance in S. typhi has been increasing, creating a need for alternative treatments. Lactic acid bacteria (LAB) have the potential to act as probiotics that can inhibit the growth of pathogens, including S. typhi. Antibiotic resistance genes must be identified before these bacteria can be safely used as probiotics. In addition, the development of S. typhi can also be influenced by quorum sensing (QS) activity. Objectives: To analyze antibiotic resistance genes in lactic acid bacteria (LAB) isolates, determine LAB resistance profiles, evaluate the ability of LAB to inhibit the growth of Salmonella, suppress quorum sensing (QS) activity, and assess the binding energy of LAB compounds to QS target proteins of Salmonella. Methods: This study used the strains Lacticaseibacillus rhamnosus TS-4, TB-3, and HI-1, as well as Pediococcus acidilactici HI-5 and LT-3. Antibacterial activity was tested against Salmonella enterica sv. Typhimurium ATCC 14028 using the 96-well microdilution method, and antibiotic resistance testing was performed using the Kirby–Bauer method. Whole Genome Sequencing data were analyzed using RAST, MUSCLE, and CARD. Anti–quorum sensing (QS) activity was evaluated using Chromobacterium violaceum. Molecular docking analysis was conducted on five LAB metabolites against SdiA and LrsB using AutoDock VINA, PyMOL, and BioVia Discovery Studio. Results:  LAB isolates showed antibacterial activity with MIC values of 50% and 70%. Antibiotic susceptibility testing revealed resistance in several isolates. Isolates TS-4, TB-3, and HI-1 carried ?-lactamase and fluoroquinolone resistance genes, while HI-5 and LT-3 carried fluoroquinolone resistance genes and multidrug-resistant efflux pump genes. All strains lacked transposable elements, gene transfer agents, and plasmids, indicating that horizontal gene transfer is unlikely. Confirmation of resistance genes through MUSCLE showed identity values ?81%, and CARD data were consistent with RAST results (identity 21–59%). Anti-QS activity tests showed violacein inhibition of >70%, and molecular docking results indicated that several LAB metabolites had high binding affinity to QS regulatory proteins. Conclusion: Several LAB isolates, such as L. rhamnosus (TS-4, TB-3, HI-1) and P. acidilactici (HI-5, LT-3), were effective as antibacterial agents against Salmonella. Although some isolates were resistant to antibiotics commonly used to treat typhoid fever, their resistance genes were not located on plasmids or transposable elements, suggesting that they are safe as probiotics for human use. LAB metabolites also showed favorable binding energies with QS regulatory proteins according to molecular docking results.

Kata Kunci : Bakteri asam laktat, Salmonella typhi, Gen resisten, Quorum sensing, Doking molekuler