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

  • A. M. Witaningrum Department of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga
  • F. J. Wibisono Department of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Wijaya Kusuma
  • D. A. Permatasari Department of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga
  • M. H. Effendi Department of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga
Keywords: broiler chicken, Citrobacter freundii, Class 1 integron encoding gene, multidrug resistance, public health


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.


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Akrami, F., M. Rajabnia, & A. Pournajaf. 2019. Resistance integrons; A mini review. Caspian J. Intern. Med. 10:370-376.

Aminharati, F., M. H. Ehrampoush, S. M. Dallal, S. M., M. Yaseri, D. A. Tafti, & Z. Rajabi. 2019. Citrobacter freundii Foodborne disease outbreak related to environmental conditions in Yazd Province, Iran. Iran J. Public Health. 48:1099-1105.

Ansharieta, R., M. H. Effendi, & H. Plumeriastuti. 2020. Detection of multidrug-resistant (MDR) Escherichia coli isolated from raw milk in East Java Province, Indonesia. Indian J. Forensic Med. Toxicol. 14:4303-4307

Asgharpour, F., S. Mahmoud, A. Marashi, & Z. Moulana. 2018. Molecular detection of class 1, 2 and 3 integrons and some antimicrobial resistance genes in Salmonella infantis isolates. Iran J. Microbiol. 10:104-110.

Bai, L., S. Xia, R. Lan, L. Liu, C. Ye, Y. Wang, D. Jin, Z. Cui, H. Jing, Y. Xiong, X. Bai, H. Sun, J. Zhang, L. Wang, & J. Xu. 2012. Isolation and characterization of cytotoxic, aggregative Citrobacter freundii. PLoS ONE 7:e33054.

Clinical and Laboratory Standarts Institute. 2017. Performances Standards for Antimicrobial Susceptibility Testing. 27th ed. CLSI supplement M100. Clinical and Laboratory Standards Institute, Wayne.

Cury, J., T. Jové, , M. Touchon, B. Néron, & E. P. Rocha. 2016. Identification and analysis of integrons and cassette arrays in bacterial genomes. Nucleic Acids Res. 44:4539-4550.

Dehkordi, M. K., M. Halaji, & S. Nouri. 2020. Prevalence of class 1 integron in Escherichia coli isolated from animal sources in Iran: a systematic review and meta-analysis. Trop. Med. Health. 48:1-7.

Deng, Y., X. Bao, L. Ji, L. Chen, J. Liu, J. Miao, D. Chen, H. Bian, Y. Li, & G. Yu. 2015. Resistance integrons: class 1, 2 and 3 integrons. Ann. Clin. Microbiol. Antimicrob. 14:1-11.

Domingues, S., G. J. da Silva, & K. M. Nielsen. 2012. Integrons: Vehicles and pathways for horizontal dissemination in bacteria. Mobile genetic elements 2:211-223.

Effendi, M. H., I.G. Bintari, E. B. Aksono, & I. P. Hermawan. 2018. Detection of blaTEM gene of Klebsiella pneumoniae isolated from swab of food-producing animals in East Java. Trop. Anim. Sci. J. 41:174-178.

Effendi M.H., N. Harijani, S. M. Yanestria & P. Hastutiek. 2018. Identification of shiga toxin-producing Escherichia coli in raw milk samples from dairy cows in Surabaya, Indonesia. Philipp. J. Ved. Med. 55:109-114.

Effendi, M.H., N. Harijani, Budiarto, N. P. Triningtya, W. Tyasningsih, & H. Plumeriastuti. 2019. Prevalence of pathogenic Escherichia coli Isolated from Subclinical Mastitis in East Java Province, Indonesia. Indian Vet. J. 96:22-25.

Falagas, M. E., P. K. Koletsi, & I. A. Bliziotis. 2006. The diversity of definitions of multidrug-resistant (MDR) and pandrug-resistant (PDR) Acinetobacter baumannii and Pseudomonas aeruginosa. J. Med. Microbiol. 55:1619-1629.

Gould, I. M. 2008. The epidemiology of antibiotic resistance. Int. J. Antimicrob. Agents. 32 (suppl 1): 2-9.

Hidayatullah, A. R., M. H. Effendi, H. Plumeriastuti, F. M. Wibisono, E.B. Hartadi, & E. D. Sofiana. 2020. A Review of the opportunistic pathogen Citrobacter freundii in piglets post weaning : Public Health Importance. Sys. Rev. Pharm. 11:767-773.

Humam, N. A. 2016. Special biochemical profiles of Escherichia coli strains isolated from humans and camels by the VITEK 2 automated system in Al-Ahsa, Saudi Arabia. Afr. J. Microbiol. Res.10: 783-790.

Janda, J. M., S. L. Abbott, W. K. W. Cheung, & D. F Hanson. 1994. Biochemical identification of Citrobacteria in the clinical laboratory. J. Clin. Microbiol. 32: 1850-1854.

Kallen, A. J., A. I. Hidron, J. Patel, & A. Srinivasan. 2010. Multidrug resistance among gram-negative pathogens that caused healthcare-associated infections reported to the National Healthcare Safety Network, 2006-2008. Infect. Control Hosp. Epidemiol. 31:528-531.

Kheiri, R. & L. Akhtari. 2016. Antimicrobial resistance and integron gene cassette arrays in commensal Escherichia coli from human and animal sources in IRI. Gut pathog. 8:1-10.

Krauland, M. G., J. W. Marsh, D. L. Paterson, & L. H. Harrison. 2009. Integron-mediated multidrug resistance in a global collection of nontyphoidal Salmonella enterica isolates. Emerg. Infect. Dis. 15:388-396.

Kristianingtyas, L., M. H. Effendi, W. Tyasningsih, & F. Kurniawan. 2020. Genetic identification of blactx-M gene and blatem gene on Extended Spectrum Beta Lactamase (ESBL) producing Escherichia coli from dogs. Indian Vet. J. 97:17-21

Kwoji, I. D., J. A. Musa, N. Daniel, D. L. Mohzo, A. A. Bitrus, A. A. Ojo, & K. U. Ezema. 2019. Extended-spectrum beta-lactamase-producing Escherichia coli in chickens from small-scale (backyard) poultry farms in Maiduguri, Nigeria. Int. J. One Health. 5:26-30.

Landers, T. F., B. Cohen, T. E. Wittum, & E. L. Larson. 2012. A review of antibiotic use in food animals: perspective, policy, and potential. Public Health Rep. 127:4-22.

Liu, L., D. Chen, L. Liu, R. Lan, S. Hao, W. Jin, H. Sun, Y. Wang, Y. Liang, & J. Xu. 2018. Genetic diversity, multidrug resistance, and virulence of Citrobacter freundii from diarrheal patients and healthy individuals. Front. Cell. Infect. Microbiol. 8:1-10

Liu, L. H., N. Y. Wang, A. Y. J. Wu, C. C. Lin, C. M. Lee, & C. P. Liu. 2018. Citrobacter freundii bacteremia: Risk factors of mortality and prevalence of resistance genes. J. Microbiol. Immunol. Infect. 51:565-572.

Liu, L., R. Lan, L. Liu, Y. Wang, Y. Zhang, Y. Wang, & J. Xu. 2017. Antimicrobial resistance and cytotoxicity of Citrobacter spp. in Maanshan Anhui Province, China. Front. Microbiol. 8:1357.

Magiorakos, A.P., A. Srinivasan, R. B. Carey, Y. Carmeli, M. E. Falagas, C. G. Giske, S. Harbarth, J. F. Hindler, G. Kahlmeter, B. O. Liljequist, D. L. Paterson, L. B. Rice, J. Stelling, M. J. Struelens, A. Vatopoulos, J. T. Weber, & D. L. Monnet. 2012. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect. 18:268-281.

Mehdi, Y., M. P. L. Montminy, M. L. Gaucher, Y. Chorfi, G. Suresh, T. Rouissi, S. K. Brar, C. Côté, A. A. Ramirez, & S. Godbout. 2018. Use of antibiotics in broiler production: Global impacts and alternatives. Anim. Nutr. 4:170-178.

Mostafa, M., , S. D. Siadat, F. Shahcheraghi, F. Vaziri, A. J. Nejad, J. V. Yousefi, B. Rajaei, E. H. Mood, N. E. Zadeh, A. Moshiri, S. A. S. Siamdoust, & M. Rahbar. 2015. Variability in gene cassette patterns of class 1 and 2 integrons associated with multi drug resistance patterns in Staphylococcus aureus clinical isolates in Tehran-Iran. BMC microbial. 15:152.

Nagachinta, S. & J. Chen. 2009. Integron-mediated antibiotic resistance in Shiga Toxin-producing Escherichia coli. J. Food Prot. 72:21-27.

Nassar, M. S. M, W. A. Hazzah, & W. M. K. Bakr. 2019. Evaluation of antibiotic susceptibility test results: how guilty a laboratory could be?. J. Egypt Public Health Assoc. 94:4.

Nayar, R., I. Shukla, & A. Sultan. 2014. Epidemiology, prevalence and identification of Citrobacter species in clinical specimens in a tertiary care hospital in India. International Journal of Scientific and Research Publications 4:4.

Permatasari, D. A., A. M. Witaningrum, F. J. Wibisono, & M. H. Effendi. 2020. Detection and prevalence of Multidrug Resistant Klebsiella pneumoniae strains isolated from poultry farms in Blitar, Indonesia. Biodiversitas 21:4642-4647.

Pormohammad, A., R. Pouriran, H. Azimi, & M. Goudarzi. 2019. Prevalence of integron classes in Gram-negative clinical isolated bacteria in Iran: A systematic review and meta-analysis. Iran J. Basic Med. Sci. 22:118-127.

Prestinaci, F., P. Pezzotti, & A. Pantosti. 2015. Antimicrobial resistance: A global multifaceted phenomenon. Pathog. Glob. Health. 109:309-318.

Projahn, M., E. Pacholewicz, E. Becker, G. C. Carreira, N. Bandick, & A. Kaesbohrer. 2018. Reviewing interventions against Enterobacteriaceae in Broiler processing: Using old techniques for meeting the new challenges of ESBL E. coli?. Biomed Res. Int. 2018:7309346.

Prota, M. A., A. P. Sandoval, M. G. Clemente, R. Fernández, & P. Casan. 2015. Community-acquired pneumonia and empyema caused by Citrobacter koseri in an immunocompetent patient. Case Rep. Pulmonol. 2015:1-6.

Riwu, K. H. P., M. H. Effendi, & F. A. Rantam. 2020. A review of extended Spectrum β-Lactamase (ESBL) producing Klebsiella pneumoniae and Multidrug Resistant (MDR) on companion animals. Sys. Rev. Pharm. 11:270-277.

Rosser, S. J. & Young, H. K. 1999. Identification and characterization of class 1 integrons in bacteria from an aquatic environment. J. Antimicrob. Chemother. 44:11-18.

Roth, N., A. Käsbohrer, S. Mayrhofer, U. Zitz, C. Hofacre, & K. J. Domig. 2019. The application of antibiotics in broiler production and the resulting antibiotic resistance in Escherichia coli: A global overview. Poult. Sci. 98:1791-1804.

Runcharoen, C., K. E. Raven, S. Reuter, T. Kallonen, S. Paksanont, & J. Thammachote. 2017. Whole-genome sequencing of ESBL producing Escherichia coli isolated from patients, farm waste and canals in Thailand. Gen. Med. 9:81-93.

Singh, T., S. Das, V. G. Ramachandran, S. Wani, D. Shah, K. A. Maroof, & A. Sharma. 2017. Distribution of integrons and phylogenetic groups among enteropathogenic Escherichia coli isolates from children <5 years of age in Delhi, India. Front. Microbiol. 8;561.

Wibisono, F. J., B. Sumiarto, T. Untari, M. H. Effendi, D. A. Permatasari, & A. M. Witaningrum. 2020. The presence of Extended Spectrum Beta-Lactamase (ESBL) producing Escherichia coli on layer chicken farms in Blitar area, Indonesia. Biodiversitas 21: 2667-2671.

Wibisono, F.J., B. Sumiarto, , T. Untari, , M.H. Effendi, , D.A. Permatasari, & A.M. Witaningrum. 2020. Short Communication: Pattern of antibiotic resistance on extended-spectrum beta-lactamases genes producing Escherichia coli on laying hens in Blitar, Indonesia. Biodiversitas 21: 4631-4635.

Widodo, A., M. H. Effendi, & A. R. Khairullah. 2020. Extended-spectrum beta-lactamase (ESBL)-producing Eschericia coli from livestock. Sys. Rev. Phar. 11: 382-392.

Zhou, W., Q. Chen, C. Qian, K. Shen, X. Zhu, D. Zhou, W. Lu, Z. Sun, H. Liu, K. Li, T. Xu, Q. Bao, & J. Lu. 2019. In Vitro susceptibility and florfenicol resistance in Citrobacter isolates and whole-genome analysis of multidrug-resistant Citrobacter freundii. Int. J. Genomics. 2019:1-15.

How to Cite
Witaningrum, A. M., Wibisono, F. J., Permatasari, D. A., & Effendi, M. H. (2021). Detection of Class 1 Integron Encoding Gene in Multidrug Resistance (MDR) Citrobacter freundii Isolated from Healthy Broiler Chicken. Tropical Animal Science Journal, 44(3), 363-368.