Pola Resistensi Antibiotik Bakteri Penyebab Infeksi Saluran Kemih di Puskesmas Ibrahim Adjie Kota Bandung

Bakteri penyebab Infeksi saluran kemih (ISK) telah mengalami resistensi terhadap berbagai antibiotik. Pola resistensi bakteri tersebut perlu diperbaharui datanya untuk memastikan pemberian antibiotik yang tepat. Tujuan penelitian mengidentifikasi dan melihat pola resistensi bakteri penyebab ISK di Puskesmas Ibrahim Adjie Kota Bandung. Koloni bakteri yang berasal dari urin 9 pasien  suspect ISK ditumbuhkan dan hanya sampel yang berasal dari 3 pasien memenuhi persyaratan terindikasi  ISK karena adanya jumlah koloni di atas  105 cfu/ml. Selanjutnya dilakukan penentuan Gram bakteri, isolasi DNA total kromosom bakteri, amplifikasi gen pengkode 16S rRNA, sekuensing (penentuan urutan nukleotida) dan uji resistensi terhadap antibiotik golongan penisilin, penisilin+inhibitor β-lactamase, sefalosporin (generasi 1,2,3,4), fluorokuinolon, aminoglikosida dan karbapenem. Hasinya didapatkan tiga isolat (P1,P2,P3) yang teridentifikasi secara genetik Escherichia coli. Isolat P1 resisten terhadap penisilin, penisilin+inhibitor β-laktamse, sefalosporin generasi 1 dan fluorokuinolon. isolat P2 resisten terhadap penisilin. Isolat P3 resisten terhadap penisilin, penisilin+inhibitor β-laktamse, sefalosporin (generasi 1,2,3,4) dan fluorokuinolon. E. coli penyebab ISK telah mengalami resistensi terhadap antibiotik penisilin, penisilin+inhibitor β-lactamase, sefalosporin (generasi 1,2,3,4) dan fluorokuinolon. Aminoglikosida dan karbapenem masih memiliki aktivitas terhadap ketiga isolat E. coli resisten.Kata kunci: bakteri; ISK; isolasi; identifikasi; resistens

Review: Antibacterial Activities of Various Parts of Mengkudu (Morinda citrifolia L.) Plants on Some Species of Bacteria

The high incidence of infections and the increase in antibiotic resistance have become a global health concern. The emergence of strains of resistant bacteria has also encouraged the exploration of potential antibacterial agents, especially from natural sources. This review aims to provide information regarding the antibacterial activity of stems, leaves, fruits and seeds of mengkudu (M. citrifolia L,) in inhibiting various bacterial species. In this article review, online and offline literature were used. Online literature was obtained from local and international journals and scientific articles of the last 10 years, 2008-2018, which were obtained through direct search results online using online search engines namely Google and Google Scholar. Whereas the offline literature used is books and e-books. It is known that the root, stem, leaves, fruit, and seeds of mengkudu (M. citrifolia L,) with various solvents used can inhibit various species of bacteria with different diameter inhibition zones

IDENTIFICATION OF BACTERIA CAUSING NECROTIC PULP WITH 16S rRNA GENE POLYMERASE CHAIN REACTION AND ANTIBIOTIC RESISTANCE TESTING AT THE DENTAL HOSPITAL IN SEKELOA, BANDUNG, INDONESIA

Objective: This study aims to identify the bacteria that cause necrotic pulp in teeth of dental patients and test resistance of the bacteria found to antibiotics. Methods: Bacteria were taken with paper points that were inserted into the root canal of patients at the Dental Hospital of the Faculty of Dentistry, Universitas Padjadjaran, Sekeloa, Bandung. Bacteria were cultured and Gram-stained. Bacterial DNA was isolated to be identified by polymerase chain reactions 16S rRNA method against known sequences of bacterial DNA. Then, identified bacteria were tested for antibiotic resistance to tetracycline (30 μg), clindamycin (2 μg), amoxicillin (10 μg), and ampicillin (10 μg). Results: The 16s rRNA gene fragment of the main bacterium found was in 98% homology with 16S rRNA gene database in http://blast.ncbi.nlm. nih.gov, i.e., Pseudomonas aeruginosa and Acinetobacter schindleri. The results from inhibition zone of each antibiotic were 20.12 mm, 8.97 mm, 8.12 mm, and 8.03 mm for tetracycline, clindamycin, amoxicillin, and ampicillin, respectively, to P. aeruginosa. While inhibition zone of tetracycline to A. schindleri was 37.7 mm. Conclusion: Based on the study results, P. aeruginosa from patients with necrotic pulp samples were resistant to clindamycin, amoxicillin, ampicillin, and decreased activity to tetracycline. While Acinetobacter schindleri was still sensitive to tetracycline

PREPARATION AND CHARACTERIZATION OF SONNERATIA ALBA LEAF EXTRACT MICROCAPSULES BY SOLVENT EVAPORATION TECHNIQUE

Objective: Sonneratia alba leaves were used by the community for traditional medicine to cure muscle pain, back pain, antioxidants, rheumatism, malaria, wounds, tuberculosis (TB) and as a spermicide. S. alba leaves extract was easy to damage because of the light exposure, change of pH, weather and a long period of storage time. The problem can be solved by coating the extract with a microencapsulation technique. The purpose of this research was to formulate the microcapsules of S. alba leaves extract with solvent evaporation technique using Ethocel 10 cP and Eudragit E100 as a matrix. Methods: S. alba leaves were extracted using ethanol 96%. This extract was dried by a rotary evaporator. The microencapsulation process of S. alba leaves extract was done by solvent evaporation technique (O/W: oil in water). The formula of S. alba leaves extract microcapsules was designed into six formulas (Eudragit E100: EA1, EA2, EA3 and Ethocel 10 cP: EB1, EB2, EB3). Microcapsules of S. alba leaves extract were characterized for particle size in terms of surface morphology by scanning electron microscope (SEM) and encapsulation efficiency. Antioxidant activity of the formulation have been evaluated by DPPH method. Physical characterization on microparticles was performed by conducting entrapment efficiency and SEM picture. Results: In this research, the microparticles containing S. alba extract has been developed by using ethyl cellulose (Ethocel 10 cP) and eudragit (Eudragit E100) as the polymer matrix. The results showed that a high concentration of polymer (Ethocel 10 cP and Eudragit E100) used in microencapsulation resulted in better S. alba leaves extract microcapsules in terms of physical characteristics. Particle size of microcapsules containing S. alba leaves extract were in the range of 0.701 to 1.163 μm. Encapsulation efficiency (% EE) was categorized as poor because the value were ≤ 80% to which 74.386% (EB3) and 75.248% (EA1). SEM picture of EA1 (Eudragit E100) revealed that the surface of microcapsule were rough and porous. When Ethocel 10 cP was used as a polymer, a smoother surface and less visible pores of microcapsule were obtained. The antioxidant ability of S. alba leaves extract microcapsule showed that IC50 values were 53.26 ppm. Conclusion: It can be concluded that microcapsules of S. alba leaves extract can be prepared by solvent evaporation technique using Eudragit E100 and Ethocel 10 cP as polymer. S. alba leaves has potent antioxidant activity either as an extract or after being formulated into microcapsules

VALIDATION OF WARFARIN ANALYSIS METHOD IN HUMAN BLOOD PLASMA USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY WITH FLUORESCENCE DETECTION

Objective: Validation of analysis method is important, especially in analyzing narrow-index drugs such as warfarin (WF). This study aimed to obtain a validated method of analyzing warfarin in human plasma according to European Medicine Agency guidelines. Methods: The optimum conditions for the analysis of warfarin in human plasma using fluorescence detector HPLC with Chiralcel OD-RH column (4.6 x 150 mm i.d., 5μm); Chiralcel OD-RH guard column (4.0 x 10 mm, 5μm), column temperature 45 °C. The mobile phase used was acetonitrile: phosphate buffer pH 2 (40:60), with an isocratic flow rate of 1 ml/min and an injection volume of 20 μl. Excitation and emission wavelengths were 310 and 350 nm (warfarin) and 300 and 400 nm (griseofulvin). The retention time of griseofulvin was 6-7.5 min; R-warfarin was 10-11.5 min; S-warfarin was 14-16 min. Results: The result of this validation obtained the optimum condition. This method yielded LOD values of 0.0674 ppm (R-warfarin) and 0.0897 ppm (S-warfarin). LOQ values were 0.225 ppm (R-warfarin) and 0.298 ppm (S-warfarin). Linearity at concentrations of 0.2-3 ppm with the line equation y = 0.0705x+0.0704 with R2 = 0.978 for R-warfarin and y = 0.0513x+0.0297 with R² = 0.9924 for S-warfarin. 75% of the seven concentrations met the reverse concentration requirements, which were below±15%. This method met the requirements of accuracy and precision within and between runs, selectivity and carryover where the %RSD and %diff values were below±15% Conclusion: This analytical method can be declared valid and can be used for sample measurement in warfarin pharmacokinetic studies

Improvement of extracellular secretion efficiency of recombinant protein from Escherichia coli: signal peptide fusion, surfactants addition, and phospholipase

The secretion of heterologous proteins into Escherichia coli cell culture media offers significant advantages for downstream processing, which can avoid the production of inclusion bodies, cost and time savings, and endotoxin reduction. However, E. coli does not secrete proteins into the extracellular medium naturally or under standard laboratory conditions. For this reason, several recombinant protein secretion strategies are carried out for targeting the protein to translocate into the extracellular environment to obtain the target proteins in an optimal amount. One important component of these strategies for E. coli is the secretion of proteins across phospholipid membranes. Thus, improving its secretion efficiency in E. coli is a main challenge that must first be solved. The generated efficient strategies that have been studied for improving extracellular protein secretion in E. coli are the use of signal peptides to translocate the target proteins across the cytoplasmic membrane into the periplasmic space and release the target protein into the culture media during the secretion process, the mechanical disruption by the addition of surfactants in growth media to chemically permeabilize the cell and the coexpression systems using phospholipase C to increase membrane permeability through its phospholipid hydrolysis activity

Teknik Long Polymerase Chain Reaction (LPCR) Untuk Perbanyakan Kerangka Baca Terbuka Gen Pengkode Polimerase Virus Hepatitis B

Teknik PCR dapat dikembangkan untuk mengamplifikasi potongan DNA dengan ukuran panjang. Salah satu teknik yang digunakan adalah Long PCR (LPCR). Teknik LPCR sering digunakan untuk mengamplifikasi gen atau potongan DNA yang berukuran lebih panjang ketika PCR standar tidak dapat diaplikasikan untuk amplifikasi tersebut. Tujuan dari penelitian ini adalah untuk memperoleh kerangka baca terbuka gen pengkode polimerase virus hepatitis B (PolHBV) menggunakan metode LPCR, dan menentukan urutan nukleotida potongan DNA tersebut. Amplifikasi dilakukan menggunakan Taq polimerase. Kerangka baca terbuka gen pengkode PolHBV telah berhasil diamplifikasi dengan LPCR yang telah dimodifikasi. Hal tersebut diindikasikan dengan keberadaan pita DNA berukuran pasangan basa (bp) pada elektroforesis gel agarosa yang mendekati ukuran teoritisnya yaitu 2532 bp. Hasil penentuan urutan nukleotida parsial menunjukkan bahwa potongan DNA yang diperoleh memiliki homologi 98% dengan gen PolHBV yang dideposit di GenBank.Technique of PCR can be improved to permit the amplification of longer DNA fragment. One of this technique is Long PCR (LPCR). LPCR is often used to amplify larger genes or large segment of DNA which standard PCR is not applicable. The objective of this study is to obtain the open reading frame of gene encoding polymerase of HBV (polHBV) using LPCR method, and to determine the nucleotide sequence of DNA fragment encoding polHBV. The amplification was conducted using Taq polymerase. The open reading frame of gene encoding polHBV was successfully amplified using modified LPCR method. It was identified by a band between 2000 dan 3000 base pairs (bp) DNA marker. The determination of nucleotide sequence informed that DNA fragment obtained was 98% homologue to the gene encoding PolHBV deposited on GenBank.

IDENTIFIKASI MUTASI PADA DAERAH DNA POLIMERASE DAN HBsAg VIRUS HEPATITIS B

Infeksi virus hepatitis B (HBV) menyebabkan hepatitis akut dan kronis, dengan angka kematian 1,2 juta per tahun di seluruh dunia. Antivirus analog nukleosida dan vaksinHBV dapat menekan perkembangan infeksi HBV namun penggunaan untuk terapi jangkapanjang dilaporkan menginduksi terjadinya mutasi. Mutasi yang menyebabkan timbulmutan resisten-antivirus dan mutan lolos-vaksin menjadi kendala utama dalam pengobatandan pencegahan infeksi HBV. Telah dilakukan penelitian untuk mengidentifikasi mutasipada gen pengkode reverse transcriptase (RT) DNA polimerase dan HBsAg virus hepatitisB. Penelitian menggunakan 24 sampel cetakan HBV yang berasal dari penderita hepatitisB dari Medan (3), Jakarta (10), Bandung (9), Yogyakarta (1), dan Surabaya (1). Metodeyang dilakukan adalah amplifikasi fragmen gen pengkode DNA polimerase dan HBsAg,konfirmasi produk PCR dengan elektroforesis gel agarosa, pemurnian produk PCR dengan GFX column kit, penentuan urutan nukleotida, dan analisis hasil penentuan urutan nukleotida. Hasil penelitian menunjukkan sampel 12273 dari Jakarta mengalami mutasi di daerah gen pengkode RT DNA polimerase. Mutasi tersebut menyebabkan substitusi asam amino M475L, V519L, L526M, dan M550V. Sampel 12273 juga mengalami mutasi pada gen pengkode HBsAg yang menyebabkan substitusi asam amino M120L, V164L, L171M, dan M195V. Mutasi tersebut terjadi di daerah yang tumpang tindih dengan gen pengkode DNA polimerase, dan di luar determinan a. Mutan diklasifikasikan sebagai mutan resistenantivirus dengan substitusi asam amino ganda L526M dan M550V, dan tidak ditemukan mutasi pada daerah DNA pengkode determinan a.Kata kunci : HBV, RT DNA polimerase, HBsAg, Mutas

Cloning and Extracellular Expression of Glargine in Pichia pastoris

Patients with diabetes mellitus increase significantly every year. The increasing number of people with diabetes mellitus results in increased insulin requirements. There are two types of insulin used for diabetes mellitus treatment: human insulin and insulin analogues. Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, or Hansenula polymorpaha has been used to produce human insulin and insulin analogues. Pichia pastoris can produce glargine in large quantities, and the insulin protein produced will be secreted outside the cell to facilitate the purification process. The advantage of glargine has a long working time of up to 24 hours. Hence, glargine is more effective because patients with diabetes receive glargine injections only once daily. The research started with cloning the glargine gene, transforming pPICZαA-G plasmid into Pichia pastoris, and testing glargine production. 20 recombinant Pichia pastoris colonies were selected and regenerated. Eight recombinant Pichia pastoris colonies were tested for glargine production, and six colonies were detected producing glargine by electrophoresis SDS-PAGE gel stained with Coomassie blue. This study aims to produce glargine using Pichia pastoris as an expression system capable of producing glargine extracellularly, thus simplifying the purification process