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.

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