Prevalence of Salmonella Spp . Bacteria Antibiotic Resistency Indigestion Tract in the Broiler Farms of Subang District

This study is aimed to estimate the prevalence of resistant Salmonella spp., determine the spread of bacterial resistance and investigate the serotypes of bacteria in the chicken’s digestion tract in the broiler farms in Subang District. As many as 74 farms were chosen, five poled caeca samples were taken from each farm and tested for isolation and identification of Salmonella spp. Salmonella isolates obtained were tested antimicrobial susceptibility against 8 antibiotics using the agar dilution method. The antibiotics were gentamycin, tetracycline, ciprofloxacin, nalidixic acid, ampicillin, chloramphenicol, trimethoprim, and sulfamethoxazole. The result showed that 8 out of 74 samples were positive for Salmonella. The prevalence of Salmonella spp. in the digestion tract was 10.8%; 95% confidence interval 3.7%-17.9%. Based on the serological test eight serotypes obtained were Salmonella enteritidis, Salmonella oslo, Salmonella narashino, Salmonella nakuru, and Salmonella nordufer. The result of antibiotic resistance test showed that from 8 Salmonella isolates obtained, 12.5% were found to be sensitive, 75% isolates were resistant to one or two antibiotics, and the remaining 12.5% isolates were resistant to more than two antibiotics; 95% confidence interval (0%-35.4%). The prevalence of resistant Salmonella spp. bacteria in chicken digestion tract in broiler farms in Subang District was high, so integrated control program to reduce antimicrobial resistance problem in broiler farm are greatly needed.


Introduction
Multidrug resistance (MDR) of foodborne pathogens such as Salmonella spp is a major concern for public health nowadays.imprudent use of antibiotics in broiler chicken farms increases the incidence of Salmonella spp.bacteria resistance.The spread of resistant bacteria can occur through direct or indirect contact, through food, water, and animal feces applied to agricultural land.Salmonella spp.resistant can spread to the environment through broiler feces and can transmit resistant genes to other bacteria, in line with Marshal and Levy (2011) which states that resistance can spread by horizontal transfer of genetic elements through conjugation, transduction and translation mechanism.
Resistant Salmonella spp.can enter the food chain through the animal origin food carrying resistant bacteria and contamination due to unhygienic animal origin food handling.
Contaminated agricultural products due to the use of manure containing resistant Salmonella spp.can increase the risk of foodborne diseases caused by resistant bacteria (Hanning et al., 2009).
The illness caused by Salmonella species was estimated 93,800,000 cases and resulting 155,000 human deaths globally in 2015 and estimated 85.6% of these cases are foodborne diseases (Eguale et al., 2015).Subang district has about 7,959,370 broiler chickens population in 2016 (BPS 2018).Broiler chicken from farms in Subang district distributed to surrounding West Java and DKI Jakarta area.The incidence of foodborne diseases due to resistant bacteria is very important to control.Research needs to be done to estimate the prevalence of resistant Salmonella spp. to antibiotics used both in human medicine and broiler farm in Subang District.

Materials and Methods
This research was conducted from March to September 2018.The Isolation and identification of bacteria was carried out at the Subang Disease Investigation Centre Laboratory (BVet Subang) and serological (confirmation) testing of Salmonella spp.conducted at the Bogor Research Center For Veterinary Science (BBLitvet).Antibiotic resistance testing was carried out at the Bogor Quality Testing and Animal Product Certification Centre (BPMSPH).
Sampling was taken in partnership and independent broiler farms in Subang District using stratified random sampling method.The farms were selected by population criteria of 3,000 to 50,000 and at least 21 days old chicken.The results of broiler farm profiling obtained 126 eligible farms.The sample size was calculated by assuming a 95% of confidence level and 8% precision and the proportion (p) 0.5.The formula used was n = 4pq/L 2 , after obtaining n1 above 10%, the calculation of sample size was adjusted with the formula = , and the sample size was 74 samples.
Seventy-four samples of chicken cecum were tested for the identification of Salmonella spp.The method consisted of several steps, that is: (1) the selective enrichment stage used Hajna Tetrathionate Broth (HTTB), 1-2 ml sample Transferred to HTTB broth and incubated for 20 hours at 42°C; (2) one loopful HTTB mixture isolated to Deoxycholate Hydrogen Sulfide Lactose (DHL) Agar and incubated overnight at 37°C to select bacteria that can use lactose and sucrose and produce H2S; (3) no pink and red colonies from DHL agar isolated into mac Conkey agar (MCA) and incubated overnight at 37°C; (4) Bacterial colonies that grown on MCA was inoculated to triple sugar iron agar (TSI); (5) then the colonies tested by biochemical testing using lysine decarboxylase (LD), sulphite indol motility (SIM) and Simmons' citrate agar (Kishima, 2014).Positive isolates were continued to be Serological tested carried out in Bogor Research Center ForVeterinary Science.
Salmonella spp.Isolated f r o m cecum samples were tested for antimicrobial susceptibility.The antimicrobial susceptibility was performed by disk dilution method following the Clinical and Laboratory Standards Institute protocol (CLSI, 2015).The antimicrobial susceptibility was tested against gentamicin (aminoglycoside class), tetracycline (tetracycline class), nalidixic acid, ciprofloxacin (fluoroquinolone class), ampicillin (penicillin class), chloramphenicol (chloramphenicol class), trimethoprim, and sulfamethoxazole (sulfonamide class).The method consisted of several steps, that is: (1) Inoculum and agar dilution preparation (2) Prepare a standardized inoculum for the agar dilution method by either growing Salmonella spp. to the turbidity of the 0.5 McFarland standard (3) Each standard of antibiotics solution were taken as much as 1 mL and poured to a petri dish, then 9 mL Mueller Hilton media (MHA) added into a petri dish.(4) the 0.5 McFarland suspension diluted 1:10 in sterile broth or saline to obtain a concentration of 107 CFU/mL.(5) then the solution containing Salmonella spp.inoculated using a multi inoculator into the antibioticcontaining MHA medium and then incubated at 37°C for 18-24 hours.(6) The level of bacterial susceptibility is determined by comparing the smallest concentration of antibiotics that can inhibit bacterial growth with a minimum standard of inhibitory concentration (MIC) for each antibiotic.
Data on Salmonella spp. the resistance obtained from this study was analyzed descriptively by using Microsoft Excel.Prevalence data is estimated using the confidence level of 95% and calculated by the estimator formula of proportions as follows: p ̂±  /2 √ (1−)  .
The diversity of Salmonella serotypes that infect different host is strongly related to the ability of Salmonella serotypes to survive and thrive in different host.The Ability to survive from Salmonella spp.influenced by some interconnected factors, including differences in host environments such as pH, temperature, and binding sites and other factors (Foley et al., 2013).
Salmonella oslo has been reported as a causative agent of food poisoning due to eating contaminated Persian cucumbers (Bottichio et al., 2016).Salmonella oslo has been found in seafood made from squid and oysters originating from the southwest coast of India (Deekshit et al., 2016) and also found in Harbor porpoises (Haase et al., 2015) and pigs (EFSA, 2015).Salmonella garba has found in lizards (Bauwens et al., 2006) and Shortnose crocodile (Reasoner, 1982).
Salmonella spp.has been known to have more than 2,500 serotypes spread in environment (W HO, 2018).This study found 5 Salmonella serotypes.Three serotypes could infect humans, the serotypes were Salmonella enteritidis, Salmonella oslo and Salmonella narashino, while Salmonella nakuru and Salmonella Nordfer have not been reported to cause infection in humans since 2006 (CDC, 2015).Salmonella isolates were tested to determine resistance to 8 types (6 class) of antibiotics using agar dilution method.The test results showed all isolates were sensitive to gentamicin, trimethoprim and chloramphenicol.The isolates were most resistant to ciprofloxacin, tetracycline and nalidixic acid.It also experienced resistance to sulfamethoxazole and ampicillin (Figure 1).
Results of the antimicrobial susceptibility test showed 1 2 .5 % o f Salmonella spp.Isolates were sensitive to all antibiotics tested.In general, resistance occurs in 2 classes of antibiotics.Multidrug resistance occurs in 12.5% Salmonella spp.Isolates (Figure 2).The resistance of Salmonella spp.known to be associated with the trend of subtherapeutic dose antibiotics in broiler farms (CDC, 2015).This study found resistance of Salmonella spp.occurs in farms that use 1 to 4 types of antibiotics in one cycle for disease prevention programme.According to Marshall and Levy (2011), antibiotic subtherapeutic uses are also clearly linked to the propagation of multidrug resistance (MDR), including resistance against drugs that were never used on the farm Imprudent use of antibiotics in animals husbandries can lead to antimicrobial resistance.The Increase of antimicrobial resistance leads to reduce the efficacy of antimicrobial, leading to increased morbidity, mortality, and health care expenditure.health care expenditure Increasing caused by additional treatment, longer hospitalizations, more complex diagnostic tests, higher professional service fees and other medical measures (CDC, 2015).According to Arisanti et al. (2018) in the period from 2000 to 2015, 61,119 foodborne deaseas were reported resulting 291 deaths (CFR 0.4%) in Indonesia, there were 1,176 outbreaks, most of it occurred in Central Java and W est Java.The most causative agent was pathogenic bacteria (74.9%), 4.6% of outbreak caused by Salmonella bacteria.
The most risk factors were bad h a n d l i n g d u r i n g food processing and improper storage.Resistant bacteria can be transmitted to humans through consumption of contaminated food from animals or through direct contact with infected animals and cross contamination.resistant Salmonella bacteria can lead to more seriously foodborne diseases.Res is ta nt p ath og en s ar e abl e to c au s e s eri o us di s ea s e a n d t hi s i s a m ajor p u blic h ea lt h p ro bl em .
Resistance can arise spontaneously through the process of mutation, besides that resistant genes can be inherited or can be obtained from other bacteria through cellular genetic elements transfers such as plasmids.Horizontal gene transfer (HGT) can occur between bacteria so that the discovery of resistance events in Salmonella spp. in the environment in chicken house and digestive tracts on farms in the Subang District will increase the risk of resistance in other bacteria.This will be an important problem for human health given a large number of pathogenic bacteria that are spread in the environment and can freely contact bacteria that have experiencing resistance (Marshal and Levy 2011).

Conclusions
This study has found the rates of Salmonella spp.antibiotic resistance was quite high (9.46%) in the digestive tract of broiler chickens in Subang district.Salmonella serotypes found in the digestive tract are mostly bacteria that can cause disease in humans.Salmonella spp.most found to have experienced resistance to 2 classes of antibiotics to MDR.Salmonella spp.Antibiotic-resistant has found in 8 farms in 7 subdistricts that spread in Subang district.

Table 1 .
Salmonella serotypes found in chicken digestion tract in broiler farms of Subang Regency