Detection of Class 1 Integron Encoding Gene in Multidrug Resistance (MDR) Citrobacter freundii Isolated from Healthy Broiler Chicken

This study was aimed to find out that broiler chicken farms have problems with antibiotic resistance Citrobacter freundii and determined the prevalence and class 1 integron encoding gene. Multidrug resistance Citrobacter freundii was collected from broiler chicken among one hundred and sixty cloacal swab samples from 32 farms in Blitar for 3 months. The method of bacterial inoculation used MacConkey agar and biochemical test was conducted by IMViC and TSIA test. Citrobacter freundii for antibiotic sensitivity pattern was tested by disk diffusion, and the multidrug resistance encoding gene was tested by PCR. This study exposed 160 samples, and 13.75% (22/160) samples were positive of Citrobacter freundii . The antibiotic sensitivity pattern showed high resistances against ampicillin and erythromycin (77.27%), tetracycline (59.09%), trimetropim-sulfamethoxazole (50%), and streptomycin (22.72%). Isolates that were detected as multidrug resistance were continued with PCR testing to prove the existence of a class 1 integron encoding gene. Multidrug resistance Citrobacter freundii isolated from broiler chicken farms in Blitar were 81.82% (18/22), and were indicated that five were positive Class 1 Integron encoding gene. The results of this study showed that the prevalence and distribution of multidrug resistance Citrobacter freundii were high, so it can cause the spread of antimicrobial resistance to public health. Class 1 integron encoding gene was found 22.72% from multidrug resistance Citrobacter freundii by PCR. It was concluded that broiler chicken farms need assessment management to reduce and avoid multidrug resistance bacteria in animals and human. Therefore, the use of appropriate antibiotics is a good step to reduce the incidence of MDR in poultry. This study was designed to evaluate the class 1 integron in broiler poultry farms and assessment of the multidrug resistance encoding gene amongst Citrobacter . This study was designed to determine the pos sibility emerge by from in


INTRODUCTION
Antibiotic is commonly used to prevent bacteria infection in poultry (Mehdi et al., 2018). Poultry management applied more than one antibiotic to control bacteria (Roth et al., 2019). Multi antibiotics uses for treatment caused resistance to bacteria and became a global public health problem (Prestinaci et al., 2015). Antibiotics have been used for the treatment and prevention of disease in animals. Food of animal origin contains large amounts of antibiotic residues due to high antibiotic treatment, and this residue can be transmitted from animal products to humans (Landers et al., 2012). Antibiotic resistance was found and detected at the Class 1 integrons encoding gene in poultry (Asgharpour et al., 2018).
Integrons are gene to express encoding for antibiotic resistance (Kheiri et al, 2016). Integrons are discovered on the transposons and contain inserted gene cassettes. The characterization of integrons is to harbor gene cassettes coding for resistance to antimicrobial (Domingues et al., 2012;Permatasari et al., 2020). Class 1 integrons are frequently reported with high prevalence in Gram 1 integrons are found to be pathogen and made a worldwide significant bacterial infection in livestock (Karimi et al., 2020;Hidayatullah et al., 2020). Enterobacteriaceae is frequently found in poultry (Projahn et al., 2018), especially Citrobacter freundii. Citrobacter freundii is Gram negative, coccobacilli, motile using flagella and facultative anaerobic bacteria and commonly found in food, soil, water, and the intestines of animals and humans . Citrobacter freundii existed in gastrointestinal and distributed food borne diseases of animals (Bai et al., 2012;Prota et al., 2015). Antibiotics inhibited the growth of bacteria, and treatment of multiple drugs caused resistance in bacteria infection. Multidrug resistance has been the global public problem Ansharieta et al., 2020). Therefore, a detection method is needed at the molecular level to prove the spread of multidrug resistance (MDR) in poultry farms. The use of the Class 1 integron gene coding for the detection of MDR in C. fruendii on poultry is a new step in the right direction.
This study was designed to evaluate the class 1 integron in broiler poultry farms and assessment of the multidrug resistance encoding gene amongst Citrobacter freundii. This study was designed to determine the possibility of emerge multidrug resistance caused by class 1 integron from bacteria in poultry farms.

Ethical Clearance
Cloacal swabs were used in this study. Hence ethical clearance was not necessary. Cloacal swabs were collected from broiler chicken farms in Blitar, East Java province, Indonesia.

Sample Isolation and Identification
This cross-sectional study was conducted between June and August 2019. One hundred and sixty cloacal swab samples were collected from broiler chicken farms in Blitar, Indonesia. The Sampling of the farm was based on less maintenance, sanitation, cleanliness, and hygiene management . The cloacal swab samples were cultured and incubated in MacConkey agar plates (Oxoid, Cheshire, UK) at 37°C for 24 h (Al Humam, 2016). The positive of Citrobacter freundii were indicated by lactose fermenting in MacConkey agar and colonies of bacteria obtain acquire pure subcultures (Liu et al., 2017). Colonies of Citrobacter freundii were confirmed by biochemistry test. Indol test, Simmon Citrate Agar and TSIA were biochemistry tests to identify genus and species of Citrobacter freundii (Janda et al., 1994;Nayar et al., 2014).

Detection of Class 1 Integron Encoding Gene
Citrobacter freundii was revealed multidrug resistance using PCR method and analysis subtypes Class I Integron encoding gene identification. DNA isolation was done with the QIAamp® DNA mini kit (QIAGEN, Germany). The primers encoding the gene Class 1 Integron were hep 58 (TCATGGCTTGTTATGACTGT) and hep 59 (GTAGGGCTTATTATGCACGC). We used GoTaq Green mastermix (Promega, USA) and PCR condition with denaturation temperatures for 2 minutes at 94ᵒC; extended denaturation at 94ᵒC for 30 seconds; annealing at 55ᵒC for 45 seconds; extension at 72ᵒC for 45 seconds; final extension at 72ᵒC for 7 minutes, this reaction is carried out for 30 cycles. PCR results were confirmed by electrophoresis using 2% agarose gel (Invitrogen, USA), the amplicon using primer hep 58 and hep 59 was 200 bp (Singh et al., 2017).

RESULTS
Cloacal swab samples were collected from 160 chickens in broiler poultry farms in Blitar and the appearance of multidrug resistance (MDR) Citrobacter freundii was detected. The sample was obtained 13.75% (22/160) Citrobacter freundii and 81.82% (18/22) multidrug resistance cases in broiler poultry farms.
The amplification Class 1 Integron of Citrobacter freundii from 22 cloacal swab samples of broiler poultry was positive for 200 bp using the primer hep 58 and hep 59 (Nagachinta et al., 2009). The results indicated that five were positive Class 1 Integron encoding genes ( Table 2).
The results showed that several resistance antibiotics of Citrobacter freundii are related to the appearance of the Class 1 Integron. Figure 1 shows Citrobacter freundii are resistance to several antibiotics. Figure 2 shows that multidrug resistance to antibiotics. The multidrug resistance Citrobacter freundii by antibiotic susceptibility testing was confirmed 18/22 (81.82%), and Class 1 Integron encoding gene positive by PCR testing was showed 5/22 (22.72%) (Figure 3).
Organisms that are resistant to three or more classes of antimicrobials are referred to as multidrug resistant (MDR) (Magiorakos et al., 2012;. One method frequently used by various researchers to characterize organisms as MDR is based on the results of in vitro antimicrobial susceptibility testing (Kallen et al., 2010). The most commonly used definition is for Gram-negative bacteria that are resistant to three or more classes of antimicrobials (Gould, 2008, Kristianingtyas et al., 2020. The variability of this definition is provided in the comprehensive MDR review (Falagas et al., 2006), which is used by some researchers as a reference that a large number of studies do not propose a specific definition for MDR.
There were 18 MDR of Citrobacter freundii isolates in this study, shown in Table 2, although 5 isolates contain Class 1 integrons gene of Citrobacter freundii in this study. Other isolates are shown negative of Class 1 integrons gene because globally pathogens of Class 1 integrons discovered on the chromosomes of environmental bacteria. Four classes of integron have been described, each of which codes for a distinct but related integrase enzyme. Class 1 integrons are the most widely studied (Rosser & Young, 1999). Class 1 integrons are bounding in conjugative plasmids, transposons conjugative plasmids, and spreading by lateral gene transfer. Therefore, Class 1 integrons have overspread to almost all species of Gramnegative pathogens. Class 1 integron is a major competence in the global spread of multidrug resistance and important to recover management of the farm to decrease the prevalence of antibiotic resistance and improving the balance of healthy life.
Consumption of antibiotics increased antibiotic resistance containing important MDR organisms in poultry. These MDR organisms can be transmitted to humans through direct contact or consumption, and C. freundii causes economic losses and levels of food contamination. Therefore antibiotic-resistant genes have the potential to spread to other populations. The abundant use of antibiotics in poultry farming has been linked to treatment failure and the development of antibiotic resistance itself.
Integrons are genes encoding multidrug resistance and reduction of treatment for bacterial infections (Mostafa et al., 2015). The classification of integrons is based on differences in the gene structure of integrases (Cury et al., 2015). The spread of Class 1 integrons have been found commonly in Gram-negative bacteria and showed the existence of Class 1 integrons of Citrobacter freundii in this study. The appearance result of amplification class 1 integron from positive isolates were 22.72%. The amplification using primer hep 58 and hep 59 for 200bp represent class 1 integron of Citrobacter freundii. Multidrug resistance was reported to be spread by Class 1 integron genes in Gram-negative bacteria.
The multidrug resistance nature of this isolate can be explained by the fact that it is mediated by plasmids carrying multiresistant genes and by transposons, and by integrons that are easily transferred to other bacteria, not necessarily of the same species (Widodo et al., 2020). Bacteria with various resistance to antibiotics are widespread in animals and the environment (Kwoji et al., 2019, Riwu et al., 2020. Recent surveys from China (Gao et al., 2015), Thailand (Runcharoen et al., 2017), and Indonesia , have illustrated an alarming trend regarding resistance among broad-spectrum beta lactamase-producing organisms (ESBLs), which are also multidrug-resistant isolated from animals and the environment .
Farm animals are frequently found to be infected by pathogenic bacteria and the infection can be transfered from animals to humans, especially Citrobacter freundii (Zhou et al., 2019;Liu et al., 2017). The treatment to pathogenic bacteria used large amounts of antibiotics to reduce attack infection and caused resistance to multiple drugs. Multidrug resistance bacteria are most important to observation concern because influence public health problem.

CONCLUSION
Citrobacter freundii was discovered in broiler chicken. This study showed that multidrug resistance Citrobacter freundii was confirmed 18/22 (81.82%), and Class 1 Integron encoding gene positive by PCR testing was 5/22 (22.72%). The result revealed multidrug resistance Gram negative bacteria emerge in poultry farms and need evaluation management plans to prevent the transfer of multidrug resistance bacteria from poultry to humans and the environment.

CONFLICT OF INTEREST
The authors declare that there is no conflict of interest with any financial, personal, or other relationships with other people or organization related to the material discussed in the manuscript.