Update Artikelilmiah: 45313

INDAH SULISTIYAWATI

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Abstrak (Bhs. Indonesia)

Ciprofloxacin sebagai antibiotik berspektrum luas saat ini menunjukkan pola resistansi yang tinggi pada beberapa negara yang dilaporkan WHO. Ketidaktepatan dalam pemberian resep menjadi pemicu kasus multi drug resistance (MDR).  Klebsiella pneumoniae dikenal sebagai bakteri Gram negatif yang paling sering menjadi penyebab infeksi dan resistan terhadap antibiotik sehingga sangat sulit diobati. Bakteri memiliki beberapa mekanisme resistansi meliputi; produksi biofilm;  penurunan permeabilitas sel karena aktivitas porin dari ekspresi gen porin Omp K35 dan Omp K36; aktivitas efflux pump;  serta mutasi gen penyandi replikasi DNA yaitu gen gyrA dan parC. Ekspresi mRNA porin sangat berpengaruh terhadap permeabilitas porin yang berakibat pada penurunan permeasi antibiotik. K. pneumoniae dengan gen penyandi porin OmpK35 dan OmpK36 menunjukkan mekanisme resistansi antibiotik. Adanya penurunan ekspresi porin mengakibatkan hilangnya fungsional porin. Perubahan profil menyebabkan fungsi porin impermeabel sehingga bakteri resistan terhadap antibiotik.  Efflux pump berperan penting dalam resistansi antibiotik, karena secara fisiologis fungsinya sangat relevan pada bakteri selama infeksi. Aktivitasnya efflux pump dapat dihambat oleh efflux pump inhibitor (EPI). Profil resistansi K. pneumoniae akan terus berubah sepanjang waktu seiring dengan peningkatan penggunaan antibiotik di wilayah geografis yang berbeda. Kemunculan mutasi gen gyrA dan parC pada K. pneumoniae dilihat dari kuantitas mutasi, letak titik mutasi dan polanya yang menggambarkan perbedaan profil resistansi. 
Tujuan penelitian ini adalah menganalisis prevalensi K. pneumoniae resistan ciprofloxacin, dan menemukan pola mekanisme resistansi K. pneumoniae  berdasarkan produksi biofilm, permeabilitas sel aktivitas porin, karakteristik pola aktivitas efflux pump K. pneumoniae resistan ciprofloxacin, menganalisis ekspresi gen porin Omp K35 dan Omp K36, serta mendeteksi mutasi gen gyrA dan parC                K. pneumoniae dari spesimen klinis pasien di RSUD Prof. Dr. Margono Soekarjo Purwokerto Indonesia. Metode in vitro yang digunakan untuk mencapai tujuan,  dilakukan dengan pengujian spesimen klinis, tahapan awal isolasi dan identifikasi yang dilanjutkan pengujian resistansi antibiotik ciprofloxacin. Isolat K. pneumoniae resistan ciprofloxacin dianalisa secara simultan dengan meneliti pembentukan biofilm menggunakan metode mikrobiologi klinis, permeabilitas membran dalam aktivitas porin. Pola mekanisme resistansi K. pneumoniae aktivitas efflux pump diujikan menggunakan Ethidium Bromide (EtBr)-Agar Cartwheel dan pengujian pengaruh efflux pump inhibitor (EPI) menggunakan senyawa Phenylalanine-Arginine                                  β-Naphthylamide (PAN) dan chlorpromazine (CPZ). Analisis aktivitas ekspresi gen OmpK35 dan gen OmpK36 diuji dengan RT-qPCR. Analisis mutasi gen penyandi resistansi ciprofloxacin menggunakan uji PCR dan sekuensing DNA.
Analisis statistik frekuensi dan persentase digunakan untuk menggambarkan variabel dalam penelitian ini. Korelasi antara variabel pembentukan biofilm pada isolat K. pneumonieae yang resistan dengan jumlah sel bakteri berdasarkan nilai MIC menggambarkan permeabilitas porin, diuji menggunakan korelasi pearson dan analisis segresi korelasi logaritmik dengan perangkat lunak IMB SPSS Statistic 26. Hasil disajikan sebagai rasio prevalensi dengan interval kepercayaan 95%, signifikansi statistik ditetapkan jika p-value <0,05. 
Hasil penelitian spesimen klinis yang diisolasi sejumlah 155 isolat                   K. pneumoniae, mayoritas pasien laki-laki 86 (55,48%) dan perempuan 69 (44,52%). Spesimen sampel diperoleh dari pasien dengan perawatan di ruang ICU, PICCU, ICCU, HCU, Poli Bedah, dan perawatan inap, yang sebagian besar berasal dari kelompok usia pasien >60 tahun (56,77%). Berdasarkan kualifikasi sampel spesimen yang dikumpulkan diperoleh data sampel darah (10/ 6,45%), sputum (101/ 65,16%), pus (27/ 17,42%), urine (13/ 8,39%), feses (3/ 1,94%), dan cairan pleura (1/ 0,65%).   Pengujian sensitivitas isolat K. pneumoniae ciprofloxacin diperoleh 72 (46,5%) isolat resistan, 73 (47%) sensitif, dan 10 (6,5%) isolat intermediat. Nilai prevalensi yang dimasukkan dalam rumus perhitungan untuk kasus resistansi ciprofloxacin selama bulan Agustus-Oktober 2022 sebesar 46,5%. Analisa mekanisme resistansi berdasarkan jumlah spesimen klinis 58 sampel dari 72 sampel resistan hal ini dikarenakan terdapat 14 sampel drop out tidak memenuhi kriteria inklusi sehingga tidak dapat dilanjutkan. Pembentukan biofilm menghasilkan nilai OD rata-rata diperoleh dengan uji produksi biofilm secara kuantitatif, berdasarkan nilai cut-off ODc = 0,089459, 41,3% (24/58) isolat masuk dalam kategori produsen biofilm yang kuat (rata-rata OD = 0,9401),  37,9% (22/58) sebagai biofilm sedang (rata-rata OD= 0,2428) dan 20,7% (12/58) sebagai produsen biofilm yang lemah (rata-rata OD = 0,1176).  Permeabilitas porin ditunjukkan dengan nilai MIC ditemukan nilai MIC 1, 2, dan 4 mg/L dan jumlah sel bakteri rata-rata masing-masing 32x106 CFU/mL, 19x106 CFU/mL, dan 34x106 CFU/mL yang mengalami penurunan dari jumlah sel bakteri kontrol awal 10,15x107 CFU/mL. Terdapat hubungan korelasi signifikan produksi biofilm dengan jumlah sel bakteri nilai p=<0,05 yang mengidikasikan pembentukan biofilm berhubungan dengan jumlah sel bakteri.
Aktivitas fenotipik efflux pump K. pneumoniae resistan ciprofloxacin MIC                 1 mg/L, 2 mg/L dan 4 mg/L ketika diuji dengan metode Ethidium bromide (Et-Br)-Agar tidak menunjukkan fluoresensi pada konsentrasi Et-Br minimal 0,25 mg/L sampai maksimal 1,5 mg/L. Kombinasi EPI CPZ dan PAN dengan ciprofloxacin menunjukkan penurunan nilai MIC empat kali lipat dari nilai MIC awal. CPZ yang ditambahkan menurunkan Nilai MIC 1 mg/L menjadi 0,25 mg/L dengan jumlah sel bakteri 388 CFU/mL, lebih rendah dibandingkan nilai kontrol, dan pada nilai MIC 4 mg/L menjadi 1 mg/L tidak menunjukkan adanya pertumbuhan pada isolat bakteri uji. Senyawa PAN yang digunakan dapat menurunkan MIC 1 mg/L, 2 mg/L dan 4 mg/L masing-masing menjadi 0,25 mg/L, 0,5 mg/L, dan 1 mg/L dengan tidak ada pertumbahan sel bakteri. Nilai fold change ekspresi relatif mRNA OmpK35 (0,8925) dan OmpK36 (0,5877) menunjukkan ekspresi rendah pada isolat K. pneumoniae resistan Ciprofloxacin. Permeabilitas porin ekspresi gen OmpK35 dan gen OmpK36 berkorelasi positif dengan nilai MIC, masing-masing gen OmpK35 (nilai p=0,029*) dan gen OmpK36 (nilai p=0,016*). Mutasi gen gyrA dan gen parC sebagai indikator resistansi ciprofloxacin pada kinerja enzim DNA gyrase dan Topoisomerase IV. Hasil amplifikasi gen gyrA dan parC dari 58 isolat K. pneumoniae resistan ciprofloxacin masing-masing ∼626-630 bp, dan ∼460-480 bp. Hasil analisis sekuensing DNA ditemukan mutasi gyrA Ser21Asp (1), Ser21Ile(1), Ser21Arg (8), dan Ser83Tyr (10). Gen parC Trp10Tyr (1), Trp10Arg (6), Ser80Ile (2), Ser80Arg (8).
Isolat K. pneumoniae yang resistan teruji memiliki mekanisme resistansi dengan memproduksi biofilm kualifikasi kuat, sedang dan lemah, serta rendahnya kemampuan permeabilitas sel porin. Kolerasi signifikan kuat terbentuk pada produksi biofilm dengan jumlah pertumbuhan sel bakteri pada nilai MIC, yang dapat meningkatkan kasus prevalensi resistansi. Aktivitas fenotipik efflux pump                       K. pneumoniae resistan tidak menunjukkan fluorosensi pada konsentrasi Et-Br minimal. Penghambatan kombinasi EPI CPZ dan PAN dengan ciprofloxacin mampu menurunkan empat kali lipat dari MIC awal, yang disertai penurunan jumlah sel bakteri bahkan mematikan bakteri. Profil mekanisme ekspresi gen OmpK35 dan gen OmpK36 menurun  yang berkorelasi dengan peningkatan nilai MIC. Mutasi gen gyrA dan gen parC ditemukan pada isolat K. pneumoniae resistan yang ditandai dengan kemunculan pita (band) dari produk amplifikasi yang memiliki mutasi dengan perubahan posisi substitusi nukleotida pada gen gyrA dan gen parC.

Kata kunci: biofilm, ciprofloxacin, efflux pump, Klebsiella pneumoniae, porin.

Abtrak (Bhs. Inggris)

Ciprofloxacin as a broad spectrum antibiotic currently shows a high resistance pattern in several countries reported by WHO. Inaccuracy in prescribing can trigger cases of multidrug resistance (MDR). Klebsiella pneumoniae is known as a Gram-negative bacterium that most often causes infections and is resistant to antibiotics so it is very difficult to treat. Bacteria have several resistance mechanisms including; biofilm production; decreased cell permeability due to porin activity from the expression of the porin genes Omp K35 and Omp K36; efflux pump activity; as well as mutations in genes encoding DNA replication, namely the gyrA and parC genes. Porin mRNA expression greatly influences porin permeability which results in decreased antibiotic permeation. K. pneumoniae with the porin-encoding genes OmpK35 and OmpK36 shows an antibiotic resistance mechanism. A decrease in porin expression results in loss of porin function. Changes in profile cause impermeable porin function so that bacteria are resistant to antibiotics. Efflux pumps play an important role in antibiotic resistance, because their function is physiologically very relevant in bacteria during infection. The activity of the efflux pump can be inhibited by efflux pump inhibitors (EPI). The resistance profile of               K. pneumoniae will continue to change over time as antibiotic use increases in different geographic regions. The emergence of gyrA and parC gene mutations in             K. pneumoniae can be seen from the quantity of mutations, the location of the mutation points and the patterns that describe differences in resistance profiles.
The aim of this study was to analyze the prevalence of ciprofloxacin-resistant K. pneumoniae, and find patterns of K. pneumoniae resistance mechanisms based on biofilm production, cell permeability, porin activity, characteristics of efflux pump activity patterns of ciprofloxacin-resistant K. pneumoniae, analyzing the expression of the porin genes Omp K35 and Omp K36, and detecting K. pneumoniae gyrA and parC gene mutations from patient clinical specimens at Prof. Hospital. Dr. Margono Soekarjo Purwokerto Indonesia. The in vitro method used to achieve the goal was carried out by testing clinical specimens, the initial stages of isolation and identification followed by testing for resistance to the antibiotic ciprofloxacin. Ciprofloxacin-resistant K. pneumoniae isolates were analyzed simultaneously by examining biofilm formation using clinical microbiology methods, membrane permeability and porin activity. The resistance mechanism pattern of K. pneumoniae efflux pump activity was tested using Ethidium Bromide (EtBr)-Agar Cartwheel and the effect of efflux pump inhibitor (EPI) was tested using the compounds Phenylalanine-Arginine β-Naphthylamide (PAN) and chlorpromazine (CPZ). Analysis of the expression activity of the OmpK35 gene and OmpK36 gene was tested using RT-qPCR. Analysis of mutations in the ciprofloxacin resistance gene using PCR testing and DNA sequencing.
Frequency and percentage statistical analysis was used to describe the variables in this study. The correlation between the variables of biofilm formation in resistant K. pneumonieae isolates and the number of bacterial cells based on MIC values describing porin permeability, was tested using Pearson correlation and logarithmic correlation regression analysis with IMB SPSS Statistics 26 software. Results are presented as prevalence ratios with 95% confidence intervals, statistical significance was established if p-value <0.05.
The results of research on clinical specimens isolated 155 isolates of                       K. pneumoniae, the majority of patients were male 86 (55.48%) and female 69 (44.52%). Sample specimens were obtained from patients treated in the ICU, PICCU, ICCU, HCU, Surgical Clinic, and inpatient care, most of whom came from the patient age group >60 years (56.77%). Based on the qualifications of the specimen samples collected, data on blood samples (10/ 6.45%), sputum (101/ 65.16%), pus (27/ 17.42%), urine (13/ 8.39%), feces were obtained. (3/ 1.94%), and pleural fluid (1/ 0.65%). Testing the sensitivity of K. pneumoniae ciprofloxacin isolates showed that 72 (46.5%) isolates were resistant, 73 (47%) were sensitive, and 10 (6.5%) were intermediate isolates. The prevalence value included in the calculation formula for ciprofloxacin resistance cases during August-October 2022 is 46.5%. Analysis of resistance mechanisms based on the number of clinical specimens, 58 samples out of 72 samples were resistant, this was because 14 drop out samples did not meet the inclusion criteria so they could not be continued. Biofilm formation resulted in an average OD value obtained by quantitative biofilm production tests, based on the cut-off value ODc = 0.089459, 41.3% (24/58) of isolates fell into the category of strong biofilm producers (average OD = 0.9401), 37.9% (22/58) as moderate biofilm producers (mean OD = 0.2428) and 20.7% (12/58) as weak biofilm producers (mean OD = 0, 1176). Porin permeability is indicated by the MIC value, MIC values were found to be 1, 2, and 4 mg/L and the average number of bacterial cells was 32x106 CFU/mL, 19x106 CFU/mL, and 34x106 CFU/mL respectively, which experienced a decrease in the number of bacterial cells. initial control 10.15x107 CFU/mL. There is a significant correlation between biofilm production and the number of bacterial cells, p = <0.05, which indicates that biofilm formation is related to the number of bacterial cells. 
Phenotypic activity of efflux pump ciprofloxacin-resistant K. pneumoniae MIC 1 mg/L, 2 mg/L and 4 mg/L when tested by the Ethidium bromide (Et-Br)-Agar method showed no fluorescence at a minimum Et-Br concentration of 0.25 mg /L to a maximum of 1.5 mg/L. The combination of EPI CPZ and PAN with ciprofloxacin showed a fourfold decrease in MIC values from the initial MIC values. The added CPZ reduced the MIC value of 1 mg/L to 0.25 mg/L with a bacterial cell count of 388 CFU/mL, lower than the control value, and at an MIC value of 4 mg/L to 1 mg/L it did not show any growth in test bacterial isolates. The PAN compound used can reduce the MIC of 1 mg/L, 2 mg/L and 4 mg/L to 0.25 mg/L, 0.5 mg/L and 1 mg/L respectively with no bacterial cell growth. . The fold change value of the relative expression of OmpK35 (0.8925) and OmpK36 (0.5877) mRNA showed low expression in Ciprofloxacin-resistant K. pneumoniae isolates. The porin permeability of the OmpK35 gene and OmpK36 gene expression was positively correlated with the MIC value of the OmpK35 gene (p value=0.029*) and the OmpK36 gene (p value=0.016*), respectively. Mutations of the gyrA gene and parC gene as indicators of ciprofloxacin resistance on the performance of the DNA gyrase and Topoisomerase IV enzymes. The results of amplification of the gyrA and parC genes from 58 ciprofloxacin-resistant K. pneumoniae isolates were ∼626-630 bp, and ∼460-480 bp, respectively. The results of DNA sequencing analysis found gyrA mutations Ser21Asp (1), Ser21Ile (1), Ser21Arg (8), and Ser83Tyr (10). parC genes Trp10Tyr (1), Trp10Arg (6), Ser80Ile (2), Ser80Arg (8). 
The tested resistant K. pneumoniae isolates had a resistance mechanism by producing strong, medium and weak qualifying biofilms, as well as low porin cell permeability. A strong significant correlation is formed in biofilm production with the number of bacterial cell growth at the MIC value, which can increase the prevalence of resistance. The phenotypic activity of the efflux pump of resistant               K. pneumoniae showed no fluorescence at minimal Et-Br concentrations. Combination inhibition of EPI CPZ and PAN with ciprofloxacin was able to reduce the initial MIC fourfold, which was accompanied by a decrease in the number of bacterial cells and even killed the bacteria. The expression mechanism profile of the OmpK35 gene and OmpK36 gene decreased which correlated with an increase in the MIC value. Mutations in the gyrA gene and parC gene were found in resistant   K. pneumoniae isolates which were characterized by the appearance of a band from the amplification product which had a mutation with a change in the position of nucleotide substitution in the gyrA gene and parC gene.

Key words: biofilm, ciprofloxacin, efflux pump, Klebsiella pneumoniae, porin.

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