Ni Nengah Dwi Fatmawati, Ni Nengah Dwi
Department of Clinical Microbiology, Medical School, Faculty of Medicine, Udayana University

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MULTIPLEX PCR FOR DETECTION OF CAPSULAR POLYSACCHARIDES TYPES OF STREPTOCOCCUS PNEUMONIAE CLINICAL ISOLATES IN BALI Fatmawati, Ni Nengah Dwi; Tarini, Ni Made Adi; Mayura, I Putu Bayu
International Journal of Biosciences and Biotechnology Vol 2 No 2 (2015)
Publisher : Faculty of Agriculture, Udayana University in cooperation with Asia-Oceania Bioscience and Biotechnology Consortium (AOBBC)

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Abstract

Streptococcus pneumoniae is causative agent of non-invasive and Invasive Pneumococcal Diseases(IPD). One of the major virulence factors is capsular polysaccharides (CPS). The CPS is known as thepneumococcal vaccine component. Several types of S. pneumoniae CPS are dominant in Indonesiasuch as types 6, 23, 15, 33 and 12 in West Nusa Tenggara, type 7F in Jakarta, and types 6A/B dan15B/C in Central Java. No data is reported from Bali related to S.pneumoniae CPS typing. Therefore,the aim of this study was to determine CPS types of S. pneumoniae isolates in Clinical MicrobiologyLaboratory, Sanglah General Hospital, Denpasar, Bali by using Multiplex PCR. Twenty-one isolatesthat were isolated from blood (11/52.4%), sputum (5/23.8%), and other clinical specimens (5/23.8%)were included in this study. Identification of S. pneumoniae was based on optochin test and presenceof pneumolysin gene (ply). Uniplex PCR was conducted to determine capsular type of each isolates,and then continued with Multiplex PCR 1 and 2, which used in-house positive controls. All isolateswere positive for the presence of ply, confirming the isolates were S. pneumoniae. Moreover, thisstudy showed that type 19F was the predominant type (7 isolates (66.7%)); 2 isolates (9.5%) werepositive for each type 23F and also for type 6A/B; and, there was only 1 isolate (4.8%) for each type7F and 15B/C. Total of 8 isolates (38.1%) were found to be nontypeable isolates. Multiplex PCR wassuccessfully identified different types of CPS. Development of Multiplex PCR could help indiagnosing and identifying capsular type of S. pneumoniae simultaneously.
OPTIMASI DUPLEX PCR UNTUK DETEKSI SIMULTAN GEN PENYANDI FAKTOR VIRULENSI OMPW DAN CTXA VIBRIO CHOLERAE Praja, Rian Ka; Sukrama, I Dewa Made; Fatmawati, Ni Nengah Dwi; Hidayati, Wahyu
Indonesia Medicus Veterinus Vol 5 (5) 2016
Publisher : Faculty of Veterinary Medicine, Udayana University

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Abstract

Vibrio cholerae merupakan salah satu agen foodborne disease yang dapat ditularkan melalui seafood. Penelitian ini bertujuan untuk optimasi gen penyandi faktor virulensi outer membrane protein W (ompW) dan cholerae toxin subunit A (ctxA) menggunakan teknik Duplex Polymerase Chain Reaction (dPCR). Dua bakteri V. cholerae O1 serotipe Ogawa dan Inaba digunakan pada penelitian ini. Proses isolasi DNA dilakukan menggunakan metode Boil Cell Extraction (BCE). dPCR dilakukan menggunakan dua pasang primer (forward dan reverse) ompW-F, ompW-R dan ctxA-F, ctxA-R dengan panjang produk masing-masing 588 bp dan 302 bp. Tahap optimasi yang dilakukan dalam proses dPCR ini meliputi variasi suhu annealing, variasi konsentrasi primer serta variasi volume DNA template kemudian deteksi produk dPCR dilakukan dengan elektroforesis pada gel agarosa 1,5% dan divisualisasi menggunakan alat Gel DocTM XR (Bio-Rad). Hasil penelitian menunjukkan komposisi reaksi dPCR yang terbaik untuk mendeteksi gen ompW dan ctxA secara simultan terdiri dari PCR mix (Promega) 12,5 ?L, primer ompW-F, ompW-R 0,5 ?M, primer ctxA-F, ctxA-R 0,3 ?M, nuclease free water 6,5 ?L dan DNA template 2 ?L sehingga volume total menjadi 25 ?L. Kondisi mesin PCR terdiri dari pre-denaturasi 95oC selama 2 menit (1 siklus) diikuti oleh denaturasi 95oC selama 1 menit, annealing 53oC selama 1 menit, extension 72oC selama 1 menit (35 siklus), dan post-extension 72oC selama 5 menit (1 siklus). Dapat disimpulkan bahwa dPCR dapat digunakan untuk deteksi simultan gen penyandi faktor virulensi ompW dan ctxA V. cholerae.
DETECTION METALLO-BETA-LACTAMASE GENE IMP-1 AND IMP-2 OF PSEUDOMONAS AERUGINOSA CLINICAL ISOLATES IN SANGLAH HOSPITAL BALI Tarini, Ni Made Adi; Fatmawati, Ni Nengah Dwi; Mayura, I Putu Bayu
International Journal of Biosciences and Biotechnology Vol 3 No 1 (2015)
Publisher : Faculty of Agriculture, Udayana University in cooperation with Asia-Oceania Bioscience and Biotechnology Consortium (AOBBC)

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Abstract

Pseudomonas aeruginosa is a pathogen frequently found as an agent of Hospital Acquired infections. This bacterium is very easy to be resistant to several types of antibiotics through various mechanisms. Carbapenem such as Imipenem and Meropenem is a potential option for the therapy of this bacterium, but unfortunately P. aeruginosa has ability in hydrolyzing these antibiotics through enzyme metallo-?-lactamases (MBLs). Recently, IMP and VIM, MBLs enzyme group are reported common from various countries, but no data is reported for these enzymes in Indonesia especially in Bali. In fact, the resistant data of P. aeruginosa against carbapenem group antibiotics such as meropenem and imipenem is quite high in Sanglah General Hospital in 2014 was 35% and 45% respectively. Therefore, the aim of this study was to detect IMP-1 and IMP-2 genes of MDR P. aeruginosa, which are phenotypically resistant to the antibiotic Imipenem and Meropenem disks based on CLSI standards in Clinical Microbiology Laboratory, Sanglah General Hospital, Denpasar, Bali. Eighty-six isolates were isolated from sputum (25 / 29.1%), wound (25 / 29.1%), urine (15 / 17.4%),endotracheal Tube (11 / 12.8), pus (6/7% ), blood (3 / 3.5%) and tissue (1 / 1.1%). In this study, all isolates were subjected to PCR for detection of IMP-1 and IMP-2. The result showed that 9 isolates were positive IMP-1 gene (10.5%), but there was no isolate positive for IMP-2 gene. The result was similar with that of the other countries, especially for the gene IMP-1. Detection and molecular characterization of MBL-producing P. aeruginosa strains are very important for infection control purposes. Currently, this study is still continued for detection of another MBL genes.
POLA MIKROBA PASIEN YANG DIRAWAT DI INTENSIVE CARE UNIT (ICU) SERTA KEPEKAANNYA TERHADAP ANTIBIOTIK DI RSUP SANGLAH DENPASAR BALI AGUSTUS - OKTOBER 2013 Hamdiyati, Rachmy; Pinatih, Komang Januartha Putra; Fatmawati, Ni Nengah Dwi
E-Jurnal Medika Udayana Vol 5 No 4 (2016): E-jurnal medika udayana
Publisher : Universitas Udayana

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Abstract

MICROBES AND THEIR SUSCEPTIBILITY PATTERN TO ANTIBIOTICS IN INTENSIVE CARE UNIT (ICU) SANGLAH HOSPITAL DENPASAR BALI AT AUGUST 2013 UNTIL OCTOBER 2013 Background: Hospital is a place where people seek medical attention, but also a reservoir for some microorganism. Expecially in ICU, because ICU often soiled by microorganism and also the patient is in immunocompromised state, invasife monitoring and treatment, and contact with health workers, can induced nosocomial infection. High usage of antibiotic also induced resistance in those microorganism. Microorganism pattern and the sensisitivity is essential in each hospital to control the usage of antibiotic, and give more accurate treatment. Method: This research use cross-sectional method. The samples are clinical specimen which received by Department of Clinical Microbiology RSUP Sanglah and. Susceptibility test conducted to 50 sample which fulfill inclusion criteria. Result and conclusion: Most frequent microorganism in those sample is Pseudomonas aeruginosa (18%), Acinetobacter baumanii (18%), Staphylococcus koagulase negatif (12%), Candida spp. (10%), and Staphylococcus aureus (8%). Gram positive bacteria resist to tetracycline dan erythromycin. Gram negatif resist to cefotaxime, amikacin, cefuroxime, cephalothin and chloramphenicol. Suggestion: This kind of research can be done continuously with better sample and method.
Detection of genes encoding ompW and ctxA of Vibrio cholerae isolated from shrimp and shellfish at Kedonganan fish market, Bali-Indonesia Kusuma Praja, Rian Arinta; Sukrama, I Dewa Made; Fatmawati, Ni Nengah Dwi
Oceana Biomedicina Journal Vol 2, No 1 (2019): Oceana Biomedicina Journal
Publisher : Universitas Hang Tuah

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (251.1 KB) | DOI: 10.30649/obj.v2i1.23

Abstract

Contamination of pathogenic bacteria in food can lead to the emergence of foodborne disease. One of foodborne disease which often occurs in some developing countries such as Africa, Southeast Asia, and Latin America is cholera which is caused by Vibrio cholerae. The disease is transmitted through beverages and food, especially contaminated seafood. V. cholerae has several virulence factors including the outer membrane protein W (ompW) and cholerae toxin (ctx).The ompW acts as a protective barrier and can also be used as a marker specific species of V. cholerae and cholerae toxin is an enterotoxin responsible for the incidence of diarrhea in a cholera outbreak produced by pathogenic V. cholerae. This study was an observational study to determine the level of contamination of V. cholerae by detecting the outer membrane protein W (ompW) and cholerae toxin subunit A (ctxA) gene of V. cholerae in shrimp and shellfish sold at Kedonganan fish market. Samples were taken using total sampling technique and obtained 24 samples consisting of 14 shrimp samples and 10 shellfish samples. Samples were examined using culture methods and biochemical tests, and then further tested using Duplex Polymerase Chain Reaction (dPCR) to detect ompW and ctxA gene. The dPCR assay results showed 8 out of 14 (57.1%) samples from shrimp and 1 out of 10 (10%) samples from the shellfish positive carried ompW gene, and found no positive samples carrying the ctxA gene in samples derived from shrimp and shellfish. Chi square test analysis results indicated contamination of V. cholerae in shrimp was higher than shellfish based on ompW gene (p<0.05). It can be concluded that the shrimp and shellfish at Kedonganan fish market are contaminated by V. cholerae. Further research is needed to detect the virulence factors besides ompW and ctxA of V. cholerae in seafood. Keywords: Foodborne disease, Vibrio cholerae, ompW gene, ctxA gene, and Duplex Polymerase Chain Reaction (dPCR).
KARAKTERISTIK FENOTIF ISOLAT KLINIK ESCHERICHIA COLI O157:H7 PADA MEDIA SORBITOL MAC CONKEY AGAR (SMAC) Hidayati, Wahyu; Temaja, I Gede Rai Maya; Fatmawati, Ni Nengah Dwi
Journal of Agricultural Science and Biotechnology Vol 7 No 1 (2018)
Publisher : Journal of Agricultural Science and Biotechnology

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Abstract

Phenotypic Characteristic of Escherichia coli O157: H7 Clinical Isolates on MacConkey Agar with Sorbitol (SMAC)Escherichia coli is a Gram-negative, rod-shaped bacterium, and commonly has flagella. Most of E. coli strains are normal flora in digestive tract of human, but some serotypes are pathogen for human and animal. One of pathogenic E. coli strain that causes a severe infection in humans (hemorrhagic colitis and hemolytic uremic syndrome) is known as Enterohemorrhagic Escherichia coli (EHEC) with cattle as their natural reservoir. E. coli O157:H7 is the most important and pathogenic serotype of EHEC which responsible for outbreak of Hemorrhagic Colitis and Hemolytic Uremia Syndrome. Accurate and cheap method detection of E. coli O157:H7 is needed to help early detection diagnosis and therapy. Therefore, the aim of this study is investigate phenotypic characteristics of E. coli O157:H7 isolated from clinical specimens using MacConkey Agar with Sorbitol (SMAC) media. Three E. coli clinical isolates from Sanglah General Hospital showed colorless non-sorbitol fermenting colony in SMAC media as phenotypic characteristic of E. coli O17:H7, therefore SMAC may beused on are of confirmation methods for E. coli O157:H7 detection from clinical isolats.
DETEKSI MOLEKULER GEN AMPC PADA ISOLAT KLINIS KLEBSIELLA PNEUMONIAE PENGHASIL ESBL DI RSUP SANGLAH DENPASAR Priyaka, I Gusti Ngurah Krishna; Tarini, Ni Made Adi; Fatmawati, Ni Nengah Dwi
E-Jurnal Medika Udayana Vol 8 No 11 (2019): Vol 8 No 11 (2019): E-Jurnal Medika Udayana
Publisher : Universitas Udayana

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (372.725 KB) | DOI: 10.24843/MU.2019.V8.i11.P9

Abstract

Sifat resistensi yang dimiliki oleh bakteri merupakan salah satu masalah pada kasus -  kasus Healthcare Associated Infections (HAIs). Klebsiella pneumoniae dapat bersifat resistenterhadap antibiotik jenis cephalosporin akibat diproduksinya enzim AmpC yang dikode oleh genampC. Penelitian ini bertujuan untuk mengetahui prevalensi gen ampC dari isolat klinis K.pneumoniae penghasil ESBL di RSUP Sanglah tahun 2013. Penelitian ini menggunakan metodedeskriptif cross-sectional. K. pneumoniae diisolasi dari spesimen klinis di Instalasi MikrobiologiRumah Sakit Umum Pusat (RSUP) Sanglah selama tahun 2013. Uji fenotif DDST dilakukan padasampel untuk mendeteksi enzim ESBL. Selanjutnya uji genotif PCR dan sequencing dilakukanuntuk mendeteksi gen ampC. Dalam penelitian ini ditemukan hasil PCR 4 sampel positif denganpanjang pita yang tidak sesuai. Salah satu sampel lalu di-sequencing untuk mengidentifikasi genyang terdeteksi tersebut. Uji sequencing menemukan gen yang terdeteksi adalah fragmen genyang mengkode enzim alpha?amylase pada K. pneumoniae. Penelitian ini menunjukan genampC pada K. pneumoniae belum berhasil teramplifikasi melalui uji genotif PCR. Empat sampelterdeteksi memiliki fragmen gen yang mengkode enzim alpha-amylase dari hasil sequencing dananalisis. Perlu dilakukan penelitian lanjutan dengan menggunakan primer dengan target genpengkode AmpC lainnya. Kata Kunci: Klebsiella pneumoniae, HAIs, AmpC, ESBL, PCR
Positivity of ExoU Gene of Type III Secretion System and Fluoroquinolone Resistance of Psedomonas aeruginosa from Sputum of Nosocomial Pneumonia Patients in Sanglah Hospital, Bali Saputra, I Wayan Agus Gede Manik; Mertaniasih, Ni Made; Fatmawati, Ni Nengah Dwi
Folia Medica Indonesiana Vol 54, No 2 (2018): June
Publisher : Faculty of Medicine, Universitas Airlangga

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (209.634 KB) | DOI: 10.20473/fmi.v54i2.8863

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Pseudomonas aeruginosa is one of the Gram-negative rods bacteria that frequently cause nosocomial pneumonia. One of the main virulent effector proteins on Type III secretion system (TTSS) of P. aeruginosa is Exoenzyme U ( ExoU). ExoU works as a phospholipase A2 activity and exhibits lung tissue injury effect in pneumonia. As an antibiotic that has activity against P. aeruginosa, fluoroquinolone resistance has increased as many as three fold since the last decade. Infections caused by P. aeruginosa that are fluoroquinolone resistant and positive for ExoU gene show worse clinical outcome. The aim of this study was to determine the positivity of ExoU gene TTSS and fluoroquinolone resistance of P. aeruginosa that isolated from sputum of nosocomial pneumonia patients in Sanglah Hospital, Bali. P. aeruginosa isolated from sputum of patient that diagnosed as nosocomial pneumonia, isolates had been identified phenotypically by Vitek2 Compact system (bioMérieux, Inc., Marcy-lEtoile - France), and then continued by genotypic detection by PCR. The susceptibility testing of P. aeruginosa isolates to Ciprofloxacin were conducted by Vitek2 Compact, whereas ExoU genes were detected by PCR. Fifty-three P. aeruginosa isolates were identified in this study, in which 35 isolates (66.1%) had ExoU gene and 22 isolates (41.5%) were resistant to Ciprofloxacin. Based on nosocomial pneumonia type, the highest proportion of isolates genotipically ExoU+ and phenotypically Ciprofloxacin were on VAP group accounted for 57.1% and 54.5%, respectively. Chi-square analysis showed significant correlation between Ciprofloxacin resistance and ExoU gene (p=0.001). As a conclusion, the positivity of ExoU+ isolates were more likely found in Ciprofloxacin resistant group.
UJI DAYA HAMBAT EKSTRAK ETIL ASETAT DAUN KAMBOJA (PLUMERIA RUBRA VAR. ACUTIFOLIA) TERHADAP PERTUMBUHAN BAKTERI METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS SECARA IN VITRO Rasmawati, Ni Luh Made; Fatmawati, Ni Nengah Dwi
E-Jurnal Medika Udayana Vol 9 No 4 (2020): Vol 9 No 04(2020): E-Jurnal Medika Udayana
Publisher : Universitas Udayana

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Abstract

ABSTRAKPenggunaan antibiotik yang kurang bijaksana dapat memicu terjadinya resistensi bakteri terhadapantibiotik. Salah satu bakteri yang kini mengalami resistensi terhadap berbagai macam antibiotikadalah Methicillin-Resistant Staphylococcus aureus (MRSA). Kandungan flavonoid dan alkaloidyang dilaporakan pada daun tanaman kamboja (Plumeria rubra var. acutifolia) memiliki aktivitasanti bakteri sehingga penelitian lebih mendalam terkait efikasi senyawa tersebut akan menarik untukdikaji. Tujuan penelitian ini adalah untuk mengetahui apakah ekstrak etil asetat daun kamboja(Plumeria rubra var. acutifolia) dapat menghambat pertumbuhan bakteri Methicillin-ResistantStaphylococcus aureus (MRSA) secara in vitro. Metode yang digunakan adalah metode difusi agar.Rerata diameter zona hambat kontrol negatif dengan etil asetat (K1)= 0 mm, konsentrasi 10% (P1)= 0 mm, 20% (P2) = 0 mm, 40% (P3)= 7,25 mm, dan 60% (P4) = 7,75 mm. Secara statistik ekstraketil asetat daun kampoja (Plumeria rubra var. acutifolia) konsentrasi 40% dan 60% mampumenghambat pertumbuhan bakteri MRSA jika dibandingkan dengan kontrol negatif. Perlunyadilakukan penelitian lebih lanjut untuk fraksinasi ekstrak sehingga dapat diketahui senyawa manakahyang paling berperan sebagai antibakteri. Kata Kunci: MRSA, daun kamboja, fitokimia
CAPSULAR GENOTYPING OF KLEBSIELLA PNEUMONIAE ISOLATED FROM CLINICAL SAMPLES AT SANGLAH GENERAL HOSPITAL IN 2013 Saskara, I Made Sutha; Fatmawati, Ni Nengah Dwi; Budayanti, Ni Nyoman Sri; Budayanti, Ni Nyoman Sri; Hidayati, Wahyu
Journal of Health Sciences and Medicine Vol 1 No 1 (2017): JHSM (Febuary 2017)
Publisher : Institute for Research and Community Services Udayana University

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (170.815 KB) | DOI: 10.24843/JHSM.2017.v01.i01.p06

Abstract

Klebsiella pneumoniae, one of hospital associated infections agents, become more prevalence in worldwide. It is an opportunistic pathogen that can cause wide spectrum of diseases such as pneumonia and sepsis. Capability of this bacterium in causing diseases is influenced by its virulence factors. Capsule (K) is considered as the major virulence factor responsible in avoiding first defense mechanism of host cells. Almost no study about molecular characterization of capsule is reported in Bali; therefore, this study is aimed to determine the capsular genotype of  K. pneumoniae isolated from clinical specimens at Sanglah General Hospital. Samples were collected between January until Desember 2013 in Sanglah General Hospital at Clinical Microbiology Laboratory. Total of 56 samples were examined taken from blood, urine, pus, sputum, and other sources. All  K. pneumoniae DNA were then subjected to PCR using specific primer pairs against K1, K2 and K5. The results showed that from 56  K. pneumoniae clinical isolates only 12 (21.4%) were PCR positive, and all (100%) of them were positive for K2 capsule gene. Ten (83.3) of them were from blood, 1 (8.3%) from sputum, and 1 (8.3%) from other specimens. Finding of K2 capsule gene in most K. pneumoniae clinical isolates at Clinical Microbiology Laboratory in Sanglah General Hospital might figure out the relationship of capsular type with severity of diseases. However, further study of capsule in Bali with higher isolates number will help in understanding of pathogenicity of K2 capsule in order to treat the infections itself.