MULTIDRUG-RESISTANT Salmonella sp . ISOLATED FROM SEVERAL CHICKEN FARMS IN WEST JAVA, INDONESIA

This study was aimed at isolating and identifying Salmonella sp . and then conducting an antibiotics susceptibility test in order to detect resistant genes. One hundred and five chicken cloaca swab samples were used in this study. 30 samples were taken from a layer farm in Bogor, 45 from a broiler farm in Sukabumi and 30 from a broiler farm in Cianjur. In order to isolate and identify the bacteria, a tetrathionate broth was used, which was then cultured in a Salmonella-Shigella agar, and finally a Gram stain and biochemical test was conducted. To confirm the presence of Salmonella sp. , a pair of primers were used for the polymerase chain reaction (PCR) method to determine the presence of the i nvA gene . . An antibiotics susceptibility test was used with the Kirby-Bauer disk diffusion method. Nine antibiotics were used in this study. Each primer pair was used for the detection of tetA , blaTEM , aac(3)-IV , gyrA and ermB genes, and for genes encoding antibiotic resistance a PCR test was used. Eight (7.6%) Salmonella sp. were isolated in this study. All isolates showed positive results with PCR confirmation. The results of the antibiotics susceptibility test showed that Salmonella sp . isolates were resistant to tetracycline (75%), oxytetracycline (75%), amphicillin (75%), gentamicin (12.5%), nalidixic acid (100%), ciprofloxacin (12.5%), enrofloxacin (0%), erythromycin (100%), and chloramphenicol (0%). The distribution of antibiotic resistance genes in Salmonella sp. were tetA (33.3%), blaTEM (100%), aac(3)-IV (0%), gyrA (100%) and ermB (0%) positive. In conclusion, Salmonella sp. was isolated. All isolates showed positive results in the PCR confirmation. Salmonella sp. isolates were resistant to tetracycline, oxytetracycline, amphicillin, gentamicin, nalidixic acid, ciprofloxacin, and erythromycin. Only the tetA , blaTEM, and gyrA genes were detected in Salmonella sp. isolates.


INTRODUCTION
Antibiotics have saved humans against disease infections for almost seven decades. Animals that are used as food sources are also given antibiotics in an effort to fight harmful bacteria or to increase production. The use of antibiotics for increased production is considered to be a very controversial practice. The use of low-dose antibiotics has a positive effect according to farmers, namely by increasing 4-5% of the average growth of livestock. However, the use of antibiotics also carries with it a negative impact which is an increase in the incidences of bacterial resistance (Diarra et al. 2007).
The use of antibiotics on farms plays a major role in the development of commensal and pathogenic bacterial resistance to antibiotics. Risk to humans can also occur due to the developed resistant bacteria (WHO 2010). Deaths due to bacterial infection are estimated to increase every year from 700.000 to 10.000.000 by 2050. Not only do these resistant bacteria pose a threat to human health, but also the worldwide economic cost of fighting these bacteria is estimated to reach US$ 100 trillion (O'Neill 2014). The occurrence of resistant bacteria complicates the treatment process of bacterial infections due to the antibiotics being ineffective at treating the illness. Antibiotic resistant bacterial infections in humans increases material losses in the form of life quality loss, increased death, and reduced health improvement programs (WHO 2010).
Several studies on bacterial resistance to antibiotics have been carried out in Indonesia. Salmonella species have been found to be resistant to nalidixic acid (44.4%) and ampicillin sulbactam (11.1%) (Aprilian et al. 2015). According to Loisa et al. (2016), Salmonella sp. has been found to be resistant to erythromycin (66.7%), streptomycin (33.3%) and chloramphenicol (33.3%). Salmonella sp. is still relatively susceptible to enrofloxacin, tetracycline, and gentamicin.
The emergence of bacterial resistance to antibiotics is a major concern to the world. Programs to monitor such resistance are mostly carried out in various countries to protect human and animal health (Li et al. 2012). Concerns about bacterial and multidrugresistance is also being studied in Salmonella. Increased resistance in Salmonella not only poses a threat to animal life but also to human life as well because Salmonella is a zoonotic agent (Nahar and Ridwan 2018).
One characteristic of bacteria that makes them deadly is their natural resistantability to protect themselves from harmful agents. These resistant properties can be obtained from mutations, gene transfer by conjugation or transformation, transposon, integron and bacteriophage (Cogliani et al. 2011). At present the use of molecular detection, specifically polymerase chain reaction (PCR), is widely used to detect resistant genes.
This study was aimed at isolating and identifying Salmonella sp. and then conducting an antibiotics susceptibility test in order to detect resistant genes. The isolates used in this study were taken from Chicken Farms in West Java, Indonesia.

Sample
Isolation and identification of bacteria was obtained from 105 poultry cloaca swab samples. Samples were collected randomly from three districts (Bogor, Sukabumi, and Cianjur) in the West Java region. Thirty samples were taken from a layer poultry farm in Bogor, 45 samples were taken from a broiler poultry farm Sukabumi and 30 samples were taken from a broiler poultry farm in Cianjur. Samples were put into a buffered peptone water media and stored in an icebox then transported to the Medical Microbiology Laboratory, Department of Animal Disease and Veterinary Public Health, Faculty of Veterinary Medicine, IPB University, Indonesia.

Microbiology Analysis
The samples were inoculated in 10 mL of tetrathionate broth media aseptically and incubated at 37° C for 48 hours (Yue et al. 2014). Bacteria grown in the tetrathinate broth media were cultured on a Salmonella-Shigella agar media and then incubated at 37° C for 24 hours. Colonies referring to Salmonella were cultured on a tryptic soy agar and then incubated at 37° C for 24 hours. The test to distinguish Enterobactericeae groups from other Gram negative bacil bacteria, such as Pseudomonas, can be assessed with the use of a triple sugar iron agar. The biochemical test carried out in this study was urease and indole, methyl red, Voges Proskauer and citrate (IMViC). Bacterial incubation on the urea media was carried out at 37° C for 18-24 hours. Testing of indole, methyl red, Proskauer and citrate was conducted at 37° C for 48 hours. The 0.5 mL Erlich reagent was added to the culture in the media, and then the tube was shaken and left to sit for a while. A red indole ring showed positive results. One drop of methyl red was dropped on the culture in the media. Positive results were indicated by a change in color to red. A Voges Proskauer test was carried out by dripping 2 drops of αnaphthol 5% followed by KOH 40%. Positive results were indicated by a change in color to red (Markey et al. 2013).

Extraction of DNA Salmonella Isolates
The bacteria that had grown on the tryptic soy agar were then inoculated on a tryptic soy broth media and incubated at 37° C for 24 hours. Extraction of the bacterial DNA used the Presto TM Mini gDNA bacteria kit (Geneaid Biotech, Taiwan) according to manufacturing procedures. The extraction results were then amplified using the PCR method using a specific primer Salmonella sp. and gene-specific primers encoding antibiotic resistance ( Table 1).

Confirmation of Salmonella sp. Isolates by PCR Method
The specific primer of Salmonella sp. used was the nucleotide base sequence forward 5´-TCA TCG CAC CGT CAA AGG AAC C -3´and reverse 5´ -GTG AAA TTA TCG CCA CGT TCG GGC AA-3´ in the invA gene with 284 bp amplification product (Li et al. 2012). The total volume of the PCR reaction used for amplification of the invA gene was 25 μL consisting of 12 μl MyTaq TM Red Mix (Bioline, Singapore), 2 μL primary (forward), 2 μL primer (reverse), 4 μL DNA templates and ddH 2 O added to 25 μL. Amplification using T100 TM Thermal Cycler (Bio-Rad, Singapore) with 30 cycles consisting of 5 minutes initial denaturation at 95° C, 30 seconds denaturation at  95° C, 1 minute annealing at 58° C, 5 minutes extension at 72° C and final extension 10 minutes at 4° C. Visualization of PCR results was carried out by electrophoresis on 1% agarose gel, Tris-Acetate-EDTA (TAE) buffer (1x), FloroSafe DNA staining, 1 mL (1 st BASE, Singapore). The DNA ladder used was 100 bp as a standard size.

Antibiotics Susceptibility Test
The Salmonella sp. isolates were assessed against varying antibiotics, namely amphicillin (AMP) 10 μg, tetracycline (TE) 30 μg, oxytetracycline (OT) 30 μg, gentamicin (CN) 10 μg, chloramphenicol (C) 15 μg, nalidixic acid (NA) 30 μg, ciprofloxacin (CIP) 5 μg, enrofloxacin (ENR) 5 μg and erythromycin (E) 30 μg. This antibiotic resistance test used the Kirby-Bauer diffusion disk method on Mueller-Hinton agar media. The bacterial suspension of the tryptic soy agar was diluted with a sterile physiological NaCl until it matched the McFarland standard of 0.5 or equivalent to 1.5 × 10 8 CFU/mL. The suspension was cultured using sterile cotton buds on Mueller-Hinton agar, and then set to dry for around 10-15 minutes. It was then placed on antibiotic discs on a Mueller-Hinton agar using sterile tweezers. The maximal antibiotic discs used in one 100 mm diameter Petri dish were six pieces. Tests were carried out in triple replications, and incubation was carried out at 35° C for 16-18 hours (CLSI 2018).

Detection of Genes Encoding Antibiotic Resistance
Positive confirmation of Salmonella sp. from PCR was detected as well as its antibiotic resistance encoded gene. Detection of resistance encoded genes was only carried out on isolates belonging to the group resistant to the antibiotics tested. The bacteria were extracted using a Presto TM Mini gDNA bacteria kit (Geneaid, Taiwan) in the previous stage. A PCR reaction was used to detect antibiotic resistant gene coding using MyTaq TM HS Red Mix (Bioline, Singapore). The total volume of PCR reactions of 25 µL consisted 4 μL templates, 2 μL reverse primers (20 μM), 2 μL forward primers (20 μM), 12 μL MyTaq TM Red Mix, 2x and ddH 2 O which were added to 25 μL. Primary targets for detection of the bacterial resistant genes against antibiotics are presented in Table 1. Amplification using Thermal Cycler T100 TM (Bio-Rad, Singapore) with 30 cycles consisting of a 5 minute initial denaturation at 95° C, 30 second denaturation at 95° C, 1 minute annealing (temperature in Table 1), 5 minute extension at 72° C and final 10 minute extension at 4° C. Observation of the results is carried out by electrophoresis on 1% agarose gel, Tris-Acetate-EDTA (TAE) buffer (1x) and FloroSafe DNA Staining, 1 mL (1 st BASE, Singapore). The DNA ladder used was 100 bp as a standard size.

RESULTS AND DISCUSSION
The use of unregulated antibiotics in chicken maintenance systems causes many incidences of resistant bacteria, one of which is Salmonella sp. These bacteria are enteric bacteria that can cause foodborne diseases (Newell et al. 2010). In this study, 7.6% (8 of 105 samples) of Salmonella sp. isolates were grown on a media so that the Salmonella-Shigella agar corresponded to a transparent colony with a black center in the middle. According to Leboffe and Burton (2011), these black dots are precipitates formed from the reaction of ferric citrate in the medium and hydrogen sulfide produced from the reduction of sulfur by the bacteria. Microscopically, Salmonella sp. cells were stem-shaped and had a pink color, thus it belongs to the category of Gram-negative bacteria. In the triple sugar iron, isolates presented a red (base) in the slant and yellow (acid) in the butt part of the media and produced hydrogen sulfide. Acidic conditions describe the ability of bacteria to ferment lactose and /or sucrose and glucose. Bacteria that cannot ferment sucrose or lactose under limited glucose conditions will use pepton in the media to produce energy. Pepton metabolites are alkaline and cause red slants (Markey et al. 2013). Urea tests showed negative results. Salmonella sp. do not use the urease enzyme to hydrolyze urea to ammonia and carbon dioxide (Leboffe and Burton 2011).
IMViC test results were negative indole, positive methyl red, negative Voges Proskauer and citrate. Corresponding to the indole test, Salmonella sp. did not use the enzyme tryptophanase to produce indole. All Salmonella sp. isolates were motile. Based on the literature, Leboffe and Burton (2011), Salmonella sp. isolates were able to produce mixed acids known from the positive methyl red test results. The Voges Proskauer test shows that Salmonella sp. was unable to produce acetoin from glucose. Salmonella sp. can use citrate as the only source of carbon. Citrate (citric acid) is produced as acetyl coenzyme A (from pyruvate oxidation or β-oxidation fatty acids) which reacts with oxaloacetate when entering the Krebs cycle. The 8 isolates detected as Salmonella sp. were code C7.2 and G7.5 (from Bogor); A.56 and A.72 (from Sukabumi); and K4.13, K4.16, K4.18, K4.26 (from Cianjur).
The invA gene in 8 Salmonella sp. isolates was successfully amplified by the PCR method. Visualization of the invA gene PCR products showed an amplicon length of 284 bp (Figure 1). The invA gene works to encode membrane proteins in bacteria that are responsible for invading the host intestine cells (Sharma and Khasmiri 2016). The invA gene in Salmonella is located in Salmonella Pathogenicity Island-1 (SPI-1) which has a very important role in invading the epithelial cells of the host. This gene is very specific in most Salmonella serotypes (Li et al. 2012).
The results of the antibiotic susceptibility test were shown in Table 2. Most of Salmonella sp. isolates were resistant against several antibiotics. Only 3 isoates showed intermediate results. All isolates were susceptible to enrofloxacin. Based on the bacterial resistance test for antibiotics, all isolates showed multidrug-resistant (MDR) properties. Multidrug-resistant properties are bacterial that are resistant to three or more antibiotic groups (Magiorakos et al. 2012). Multidrug resistance in bacteria is caused by two mechanisms: first, the accumulation of several genes, each of which encodes the resistance to one type of antibiotic in a single cell. This accumulation occurs on plasmid R (resistance).
The second mechanism is due to the increase in gene expression that encodes the efflux pump for several antibiotics (Nikaido 2009).
The tetracycline group has been marketed under various trade names. Adesiji et al. (2014) investigated Salmonella sp. gathered from human, poultry and seafood samples found that resistance to tertracyclin was 66.7%. In this study, 6 (75%) of the isolates were resistant to tetracycline and oxytetracycline. The resistance of Gram-negative bacteria, one of which being Salmonella sp., to the tetracycline group occurs mostly due to the efflux pumps. The genes for efflux pumps are related to mobile elements, one of which is tetA (Poole 2005). As many as 2 (33.3%) of the isolates are positive for the tetA gene, meaning efflux pump activity secretes tetracyclines from cells using protons as a successful energy source. According to Nguyen et al. (2014) tetracycline will exit to the bacterial surface periplasmic space at the time of proton exchange (H + ) during active transport so that it cannot bind to the target ribosome subunit of 30s bacteria. This study also found 2 isolates which were negative to tetA. It is possible that other tet genes are involved in tetracycline group resistance in these isolates.
In general, resistance to β-lactam groups is caused by β-lactamase enzymes (Munita and Caesar 2016). Resistance to β-lactam class antibiotics in Gramnegative bacteria occurs mostly due to extended spectrum β-lactamase (ESBLs) (Rawat and Deepthi 2010). Extended spectrum β-lactamase encoded by the blaTEM gene will hydrolyze the β-lactam ring in the periplasmic space. Breaking bonds causes antibiotics not to function so there is no antibiotic reaction with penicillin-binding proteins (PBPs) (Munita and Caesar 2016). The blaTEM gene was detected in all Salmonella sp. isolates which is resistant to ampicillin.
The gyrA gene was detected in 8 (100%) isolates, which is resistant to quinolone and fluoroquinolone. Amino acid substitution that occurs in quinolon resistance determining region (QRDR) of the gyrA gene can cause resistance (Munita and Caesar 2016). Ogbolu et al. (2012) reported that mutation of the gyrA protein at codon position 83 (Serin → Leusin) in Gramnegative bacteria causes resistance to nalidixic acid and ciprofloxacin. Gram-negative-resistant enrofloxacin had amino acid substitution at codon position 83 (Serin → Leusin) and 87 (Aspartat → Glisin) (Jurado et al.   (3)-IV genes were not detected in the 8 Salmonella sp. isolates. According to Munita and Caesar (2016) more than 30 different ermB genes have been found in nature. Erythromicin-resistance is caused by the impermeable characteristics of Gramnegative bacteria against macrolide (Cesur and Demiroz 2013). Sandra et al. (2018) revealed that there are several genes encoding the aminoglycoside N-8-acetyltrasferase enzyme besides aac(3)-IV which can cause bacterial resistance to gentamicin is aac (6)

CONCLUSION
In conclusion, Salmonella sp. was isolated. All isolates showed positive results in the PCR confirmation. Salmonella sp. isolates were resistant to tetracycline, oxytetracycline, amphicillin, gentamicin, nalidixic acid, ciprofloxacin and erythromycin. Only the tetA, blaTEM and gyrA genes were detected in Salmonella sp. isolates.