DETEKSI GEN PENYANDI RESISTENSI ANTIBIOTIK PADA ...

DETEKSI GEN PENYANDI RESISTENSI ANTIBIOTIK PADA Escherichia coli YANG DIISOLASI DARI KUCING DAN

LINGKUNGAN KLINIK HEWAN DI KOTA BOGOR

Yamin Yaddi

(B253180051)Prof. Dr .drh. Fachriyan Hasmi Pasaribu

Dr. drh. Safika, M.Kes

Komisi Pembimbing:

PRASEMINAR

PROGRAM STUDI MIKROBIOLOGI MEDIKSEKOLAH PASCASARJANA

IPB UNIVERSITY 2020

intestinal

intraintestinal

ekstraintestinal

Patogen

Peningkatan populasi

Berada di luar habitatnya

LATAR BELAKANGMakanan

Lingkungan

Flora normal

IntraIntestinal**EkstraIntestinal*

UTI

SEPEC

PYOMETRA

PROSTITIS

Patogen

EPEC

ETEC

EHEC

EAEC

EIEC

LATAR BELAKANG

VAGINITIS

**(Mitchell dan Gu 2010)

AntibiotikInfeksi primer/ sekunder

Seleksi bakteri

resisten(Nam et al. 2010; Rini et al. 2017)

InangPeningkatan populasi bakteriresisten (Bryan et al. 2004)

LingkunganCemaran bakteri resisten dilingkungan

(Ishii dan Sadowsky 2008)

*(Gyles et al. 2010)

Penggunaan antibiotik yang irasional (Papic 2013; Bourely

et al. 2020)

Tujuan Penelitian

LATAR BELAKANG

1. Mengisolasi Escherichia coli dari kucing dan lingkungan dari klinikhewan di Kota Bogor

2. Mengukur tingkat resistensi Escherichia coli terhadap antibiotikgolongan tetrasiklin (OTC, TE, DO), kuinolon (CIP, ENR) dan β-laktam(AMP, AML)

3. Mendeteksi gen penyandi resistensi antibiotik isolat Escherichia coliyang menunjukan resisten dan intermediate pada uji resistensi

METODE PENELITIAN

September 2019 – September 2020

➢ Isolasi, identifikasi dan uji resistensi (Lab Bakteriologi)

➢ Deteksi gen penyandi resistensi (Lab terpadu, Divisi Mikrobiologi Medik, FKH-IPB)

84 sampel64 sampel usap

rektal

20 sampel usap lingkungan

10 sampel usap lantai ruang pemeriksaan

10 sampel usap saluran pembuangan(10 klinik hewan)

TSA

Biokimia

Koloni SpesifikHijau metalik

EMBA

TSIA

FermentasiKarbohidrat

Urea

Simmons Citrate Agar

MR-VP broth

Sulfida Indol Motility

I M

V i

C

Isolasi dan Identifikasi

Gram negatif

Merah/ batang

Ektraksi DNAPrestoTM Mini gDNA bacteria kit (Geneaid)

Gen target (gen uspA, 884 bp*)

(F) 5′-CCGATACGCTGCCAATCAGT-3′

(R) 5′-ACGCAGACCGTAGGCCAGAT-3′Vol total reaksi 25 μl :

(12 μl MytaqTM HS Red Mix 3 μl template DNA

2 μl primer forward2 μl primer reverse 6 μl ddH2O

Master Mix PCR

1

Predenaturasi 95 °C, 3 Menit

Amplifikasi DNA 30 siklus :

Denaturasi 95 °C, 30 detik

Annealing 58 °C, 15 detik

Ekstensi 72 °C, 1 menit

Ekstensi akhir 72 °C, 5 menit

Thermocycler2

Gel elektroforesis 1% agarosaTEA buffer 1 (×)

Pewarnaan 1 μl FloroSafe DNA StainLeader DNA 100 bp

Visualisasi

3

*Chen dan Griffiths 1998

METODE PENELITIAN

Makroskopis, Mikroskopis, Biokimia

Molekuler

PCR

Uji Resistensi Antibiotik

Metode Disk Diffusion Kirby-bauer

Isolat (+) E.coli

Mcfarland 0,5

(1.5 x 108 CFU/ml)

1 ml suspensi

Pada Muller-Hilton

Disk diffusion Antibiotik MHA

Pembacaan Hasil

TSANaCl Fisiologis

* CLSI 2018

Standar diameter zona hambat*

Deteksi Gen Penyandi

Resistensi

Sumber: aRandal et al. (2004), bRobicsek et al. (2006), cColomb et al. (2003), dVan et al. (2008), e Lyimo et al. 2016.

Tetrasiklin

Kuinolon

β-laktam

HASIL DAN PEMBAHASAN

Isolasi dan Identifikasi

*Ijong dan Dien 2011

EMBA

Biokimia

Asal kucing

Asallingkungan

87,50 % (56) E. coli

55% (11/20) pertumbuhan koloni35% (7/20) koloni dugaan E. coli

(20% (4) UP & 15% (3) UL)

Morfologi 100% (71) Gram negatif

Asal kucing

Asal lingkungan 35% (7) E. coli

100% (64) diduga E. coli

Konfirmasi E.coli dengan PCR

23 isolat usap rektalA

B

Kelangsungan hidup E. coli selama masapertumbuhan, membantu adhesi danmotilitas (Mishra et al.2017) yang diinduksiakibat tekanan dari lingkungan (Nacin et al.2005).

Produksi protein sitoplasmik saat hambatanpertumbuhan. Transkripsi dari gen uspAdihentikan bila kebutuhan nutrien telahterpenuhi

(Nystrom dan Neidhardt 1992 )7 isolat usap lingkungan

Gen uspA

30 isolat positif gen uspA 884 bpbp

bp

bpbp

bp

bp

bp

bp

Uji Resistensi

Permeabilitas dinding sel

(Giguere et al. 2013)

Sintesis protein

(Markley danWencewicz 2018)

Sintesis asam nuleat

(Bradley dan Jackson 2006)

Mekanisme Resistensi

• Biokimia: enzimatik (inaktivasi antibiotik), efflux pumps, dan merubah/

modifikasi target

• Genetik (mutasi dan transfer gen horizontal )Guilfole 2007

AntibiotikHasil uji resistensi

R I S (%)

β-laktamAmpisilin 22a/ 7b - a/ -b 1a/ -b 95,65a/ 100b

Amoksisilin 20a/ 7b 2a/ -b 1a/ -b 86,96a/ 100b

KuinolonCiproflokasin 11a/ 2b 3a/ 2b 9a/ 3b 47,83a/ 28,57b

Enrofloksasin 16a/ 3b 4a/ 3b 3a/ 2b 69,57a/ 42,86b

Tetrasiklin

Tetrasiklin 18a/ 7b 3a/ -b 2a/ -b 78,26a/ 100b

Oksitetrasiklin 21a/ 7b - a/ -b 2a/ -b 91,30a/ 100b

Doksisiklin 11a/ 4b 9a/ 3b 3a/ -b 47,83a/ 57,14b

Keterangan: R (resistant), I (Intermediate), S (Susceptible).aisolat asal kucing (n=23), bisolat asal lingkungan (n=7).

Jumlah

antibiotik

Jumlah Isolat

Resisten

Persentase (%)

1 -a/ -b 0a/ 0b

2 1a/ 1b 4,37a/ 14,29b

3 3a/ -b 13,04a/ 0b

4 5a/ 1b 21,73a/ 14,29b

5 4a/ 3b 17,39a/ 42,85b

6 5a/ 1b 21,73a/ 14,29b

7 5a/ 1b 21,73a/ 14,29b

Uji Resistensi

Multiresisten

• Akumulasi gen yang menyandi sifat resisten terhadap satu antibiotik dalam plasmid

• Peningkatan ekspresi gen penyandi pompa efflux(Nikaido 2009)

Perpindahan gen resistenBakteri Gram negatif (Aleksun dan Levy 2015)

Melalui- transduksi (bakteriofag) - konjugasi (plasmid dan transposon) - transformasi (mutasi DNA)

(Levy dan Marshall 2004)

Keterangan: R (resistant), I (Intermediate), S (Susceptible).aisolat asal kucing (n=23), bisolat asal lingkungan (n=7).

95,65% (22/23) isolat asal kucing

85,71% (6/7) isolat asal lingkungan

Deteksi Gen PenyandiResistensi

Isolat asal kucing & lingkungan

Ia

IIb

Protein Tet mediator mekanisme efflux, proteksi protein dan inaktivasi enzim (Bryan et al. 2004)Protein qnr melindungi DNA gyrase dan topoisomerase IV (Rezazadeh et al. 2016), berperan dalam inaktivasi

antibiotik dan penurunan konsentrasi intraseluler (efflux pumps) (Baguy et al. 2014)Beta-Lactamase (bla) Enzim Beta-lactamase menonaktifkan cincin karbon/nitrogen (Paterson dan Bonomo 2005).

Protein qnr

Deteksi Gen PenyandiResistensi

Isolat asal lingkungan

▪ Sumber air (Sarker et al. 2019) ▪ lingkungan peternakan sapi (Sobur et al. 2019) ▪ lingkungan klinik hewan (Tuerena et al. 2016)

Deteksi Gen PenyandiResistensi

Amplifikasi Gen ESBL

Isolat asal kucing(A)

8,70% gen ESBL

- gen blaCTXM (866 bp), gen blaSHV (768) gen blaTEM (516) 2 isolat

- gen blaCTXM (866 bp), gen blaTEM (768) 1 isolat

- gen blaSHV (768) gen blaTEM (516) 1 isolat

Isolat asal lingkungan (B)

14,29% gen ESBL

- gen blaCTXM (866 bp) dan gen blaTEM (516) 1 isolat

- gen blaSHV (768) negatif

A

BTransfer gen horizontal dalam satu spesies maupunberbeda (Manoharan et al. 2011).

KESIMPULAN DAN SARAN

Kesimpulan

1. Escherichia coli dapat diisolasi dari kucing dan lingkungan klinik

hewan di Kota Bogor

2. Isolat Escherichia coli dari kucing dan lingkungan menunjukan

resisten terhadap antibiotik golongan β-laktam, kuinolon dan

tetrasiklin dengan variasi yang berbeda pada setiap golongan dan

klinik (terdapat isolat yang multiresisten)

3. Gen blaTEM, blaCTXM, tetA dan qnrS terdeteksi pada isolat asal

kucing dan lingkungan, sedangkan gen blaSHV hanya terdeteksi

pada isolat asal kuncing

Saran

KESIMPULAN DAN SARAN

Perlu adanya regulasi yang mengatur tentang standar penggunaan

antibiotik dalam penanganan kasus infeksius maupun non infeksius

pada klinik hewan.

Diperlukan penelitian lanjutan untuk mengukur tingkat resistensi

Escherichia coli isolat hewan kesayangan lainnya (anjing) terhadap

antibiotik yang sering digunakan di klinik hewan serta mengukur

tingkat homologi gen penyandi resistensi antara hewan, lingkungan,

karyawan serta pemilik hewan.

DAFTAR PUSTAKA

Adelowo O, Fagade O. 2009. The tetracycline resistance gene tet39 is present in bothGram‐negative and Gram‐positive bacteria from a polluted river, Southwestern Nigeria.Lett Appl Microbiol: SFAM. 48:167–72.

Baguy OM, Nathalie GK, David CN, Daniel SN, Julien CK, Rose KN, Djeneba OG, Valerie G,Bertin TK, Mireille D. 2014. Firs Report of qnr Genes in Multidrugs Resistant (ESBL)Enterobacteria Isolated from Different Ecosystem in Abidjan, Ivory Coast.AASCIT.1(4):170–175.

Bourley C, Coeefic T, Caillon J, Thibaut S, Cazeau G, Jouy E, Jarrige N, Chauvin C, Madec JY,Haenni M, Leblond A, Gay E. 2020. Trends in Antimicrobial Resistance amongEscherichia coli from Defined Infections in Humans and Animals. JAC.75(6):1525–1529.

Bradley JS, Jackson MA. 2006. The use of systemic fluoroquinolones. J Pediatr. 118:1287–1292.Bryan A, Shapir N, Sadowsky MJ. 2004. Frequency and Distribution of Tetracycline Resistance

Genes in Genetically Diverse, Nonselected, and Nonclinical Escherichia coli StrainsIsolated from Diverse Human and Animal Sources. AEM. 70(4):2503–2507.

[CLSI] Clinical and Laboratory Standards Institute. 2018. Performance Standards forAntimicrobial Susceptibility Testing. 28th Edition. Wayne (US): Clinical andLaboratory Standards Institute.

Colom KL, Perez J, Alonso R, Fernandez AA, Larino R, Cisterna R. 2003. Simple and ReliableMuliplex PCR Assay of Detection of blaTEM, bla (SHV) and blaOXA-1 GenesEnterobacteriacea. FEMS Mikrobiol Letters. 223(2):147–151.

Correia S, Poeta P, Hebraud M, Capelo JL, Igrejas G. 2017. Mechanisms of Quinolone Actionand Resistance. JMM. 66:551–559.

Giguère S, Prescott JF, Dowling PM. 2013. Antimicrobial Therapy in Veterinary Medicine, 5thed. Wiley Blackwell, Ames, Iowa (US).

Gyles CL, Prescott JF, Songer JG, Thoen CO. 2010. Pathogenesis of Bacterial Infection inAnimals. Ames (US). Blackwell Pub.

Ijong FG, Dien HA. 2011. Karakteristik bakteri pereduksi merkuri (Escherichia Coli) diisolasidari perairan pantai teluk manado. Perikanan dan Kelautan Tropis. Vol 7(3): 103-108.

Ishii S, Sadowsky MJ. 2008. Escherichia coli in the Environment: Implication for quality andhuman health. JSME. 23(2):101–108. doi.org/10.1264/jsme2.23.101.

Leboffe MJ, Burton EP. 2011. A Photographic Atlas for the Microbiology Laboratory. 4th Ed.United States of America (US): Morton Publishing.

Levy SB, Marshall B. 2004. Antibacterial Resistance Worldwide: Causes, Challenges and Esponses. Nat Med.10(12):122–129.

Lyimo B, Buza J, Subbiah M, Smith W, Call DR. 2016. Comparison of antibiotic resistant Escherichia coliobtained from drinking water sources in northern Tanzania: a cross-sectional study. BMCMicrobiol. 16(254):1–10.

Manoharan A, Premalatha K, Chatterjee S, Mathai D. 2011. Correlation of TEM, SHV, CTX-M Estended-spectrum Betalactamases Among Enterobacteriaceae with their in vitro Antimicrobial Susceptibility.IJMM. 29(2):161–164.

Markley JL, Wencewicz TA. 2018. Tetracycline-inactivating Enzymes. Front Microbiol. 9(1058):1–22.Mitchell R, Gu JD. 2010. Enviromental Microbiology 2th Ed. New Jersey (US): Wiley-Blackwell.Nguyen F, Starosta AL, Arenz S, Sohmen D, Donhofer A, Wilson DN. 2014. Tetracycline Antibiotics and

Resistance Mechanisms. Biol Chem. 395(5):559–575.Nikaido H. 2009. Multidrug resistance in bacteria. Annu Rev Biochem. 78(22):119–146.Nystrom T, Neidhardt FC. 1992. Cloning, Mapping and Nucleotide Sequencing of a Gene Encoding a

Universal Stress Protein in Escherichia coli. Mol Microbiol. 6(21):3187–3198.Pao SS, Paulsen IT, Saier MH. Major facilitator superfamily. 1998. Microbiol Mol Bio. 62(1):1–34.Price LB, Graham JP, Lackey LG, Roess A, Vailes R, Silbergeld E.2007. Elevated risk of carrying

gentamicin-resistance Escherichia coli among U.S. poulytry workers. EHP. 115(12):1738–1742.Randal LP, Cooles SW, Osborn MK, Piddock LJV, Woodward MJ. 2004. Antibotic Resistance Gene,

Integrons and Multiple Antibiotic Resistance in thirty-five Serotypes of Salmonella enteriticaIsolated fromhumans and animals in the. (UK). Antimicrobial Chemoteraphy. 53:208–216.

Rezazzadeh M, Baghchesaraei H, Peymani A. 2016. Plasmid-Mediated Quinolone-Resistance (qnr) Genes inClinical Isolated of Escherichia coli Collected from Several Hospitasl of Qazvin and ZanjanProvinces, Iran. J PHRP. 7(5):307–312.

Rini SH, Selma S, Max JH. 2017. Resistensi antimikroba dan Penerapan Kebijakan Pengendalian di RumahSakit di Indonesia. MPK. 1(2): 131–140.

Riviere JE, Papich MG. 2009. Veterinary pharmacology and therapeutics. 9th ed. New Jersey, WileyBlackwell (US).

Robicsek A, Strahilevitz J, Sahm DF, Jacoby GA, Hooper DC. 2006. qnr Prevalence in Ceftazidime-ResistantEnterobacteriaceae Isolates from the United States. AAC. 50(8):2872–2874.

Ryan KJ, Ray CG. 2014. Antibacterial Agents and Resistance in Sherris Medical Microbiology. 6th Edition.Mc Graw Hill. New York (US).

Van TTH, Chin J, Chapman T, Tran LT, Coles PJ. 2008. Safety of Raw Meat and Shellfish in Vietnam: anAnalysis of Escherichia coli Isolation for antibiotic Resistance and Virulance Genes. ICFMH.124(3):217–223.