Whole Genome Sequencing Isolat Salmonella Resistan Antibiotik Asal Karkas Ayam Kampung dan Broiler

Abstract

Daging ayam yang merupakan komoditi dengan tingkat konsumsi tertinggi di Indonesia namun mengandung potensi bahaya kontaminasi Salmonella penyebab Salmonelosis yang berkaitan erat dengan higienitas penanganan dan pengolahan karkas ayam. Konsumsi daging ayam di Indonesia cenderung terus meningkat akan tetapi beberapa tahun terakhir kecenderungan resistansi terhadap beberapa jenis antibiotik (multiresistan) pada Salmonella spp. yang diisolasi dari bahan pangan semakin meningkat. Hal ini diduga akibat penggunaan antibiotik yang tidak tepat dan berulang sehingga berdampak pada pengobatan infeksi yang semakin sulit. Saat ini, data yang berkaitan dengan tingkat resistansi antibiotik di Indonesia masih minim, terutama data terhadap isolat lokal. Keberadaan informasi mengenai tingkat resistansi antibiotik ini dapat menjadi dasar penting dalam penentuan kebijakan. Penelitian bertujuan mengidentifikasi profil resistansi antibiotik isolat Salmonella spp. asal karkas ayam kampung dan asal karkas ayam broiler secara fenotipik, menganalisis sekuensing genom total isolat Salmonella spp. asal karkas ayam yang memiliki karakteristik resistansi tertentu dan mengidentifikasi hasil sekuensing genom total isolat Salmonella spp. terhadap profil genotipe penyandi resistansi antimikroba, faktor virulensi, gen plasmid terkait serta identifikasi Salmonella pathogenicity island. Penelitian dilakukan dengan menguji sifat resistansi antibiotik 25 isolat Salmonella spp. menggunakan metode KirbyBauer Disc Diffusion Test terhadap 8 jenis antibiotik. Sebanyak 3 isolat dengan karakter paling multiresistan kemudian diisolasi DNAnya dan disekuensing genom total menggunakan GridION (Teknologi Oxford Nanopore). Assembly genom total isolat hingga analisis genom total dilakukan pada platform Galaxy. Sebanyak 52% isolat Salmonella spp. asal karkas ayam (2/8 asal ayam kampung dan 11/17 asal karkas ayam broiler) memiliki sifat resistansi terhadap minimal 1 jenis antibiotik uji. Isolat BCID6.4 asal karkas ayam broiler memiliki sifat resistansi terhadap antibiotik uji tertinggi dibandingkan semua isolat uji dengan pola resistansi TE-OT-AMP-NA-CIP-E. Sekuensing genom total dilakukan pada isolat BCID6.2, BCID6.4 dan KCID6.5 menghasilkan genom lengkap dengan ukuran sekitar 4,80Mbp. Sebanyak 33 gen penyandi resistansi antibiotik, 104 faktor virulensi dan 8 jenis Salmonella Pathogenicity Island ditemukan pada genom total ketiga isolat uji. Plasmid kecil ditemukan pada sekuen genom isolat BCID6.4 tetapi tidak ditemukan gen penyandi resistansi antimikroba dan faktor virulensi. Hasil serotyping secara in silico menunjukkan ketiga isolat merupakan Salmonella enterica serovar Kentucky.

Chicken meat is a commodity with the highest consumption level in Indonesia but contains a potential danger of Salmonella contamination which causes salmonellosis that might be closely related to the hygiene of handling and processing of the chicken carcasses. Consumption of chicken meat in Indonesia tends to increase every year, but in recent years there has been an increase in resistant Salmonella spp. isolated from food that also tends to be resistant to several types of antibiotics (multi-resistant). This is thought to be due to the inappropriate and repeated use of antibiotics thus could increase the difficulty of the infection treatment. Currently, data relating to the level of antibiotic resistance in Indonesia's predominantly local-based data is minimal. Whereas, information on the level of antibiotic resistance could be an important basis in determining related policy. The study aimed to determine the antibiotic resistance profiles of Salmonella spp. isolates from Kampung chicken carcasses and broiler carcasses phenotypically, analyze the total genome sequencing of Salmonella spp. isolates. from chicken carcasses that have certain resistance characteristics and identify the whole genome sequencing results of Salmonella spp. isolates genotype profile encoding antimicrobial resistance, virulence factors, related plasmid genes, and identification of Salmonella pathogenicity island. Antibiotic resistance properties were tested on 25 isolates of Salmonella spp. using the Kirby-Bauer Disc Diffusion Test method against 8 types of antibiotics. DNA from 3 isolates with the most multiresistant profiles were then isolated and the whole genome was sequenced using GridION (Oxford Nanopore Technology). The total genome assembly to total genome analysis was carried out on the Galaxy platform As many as 48% Salmonella spp. isolated from chicken carcass (2/8 from Kampung chicken and 10/17 from broiler) had resistance to at least 1 type of test antibiotic. Isolate BCID6.4 from broiler carcass had the highest resistance to the test antibiotics compared to all test isolates with the TE-OT-AMP-NA-CIP-E resistance pattern. Total genome sequencing was performed on isolates BCID6.2, BCID6.4 and KCID6.5 resulting in complete genomes with the size of approximately 4,80Mbp. A total of 33 genes encoding antibiotic resistance, 104 virulence factors and 8 types of Salmonella Pathogenicity Island were found in the total genome of the three isolates. One small plasmid was found in the genomic sequence of isolate BCID6.4 but no genes encoding antimicrobial resistance and virulence factors were found. The results of in silico serotyping showed that all three isolates were Salmonella enterica serovar Kentucky.