MicroBio Pharmaceuticals and Pharmacology | Online ISSN 2209-2161
RESEARCH ARTICLE   (Open Access)

Multi-Antibiotic Resistant Citrobacter freundii in Eggs: A Silent Public Health Threat

Jinia Afroz1, Md. Al Masud1, Esrat Jahan1, Ajoy Chowdhury1, Md. Fakruddin2, Md. Asaduzzaman Shishir1,3*

+ Author Affiliations

Microbial Bioactives 6 (1) 1-10 https://doi.org/10.25163/microbbioacts.61910A

Submitted: 10 October 2023 Revised: 09 November 2023  Published: 18 November 2023 


Abstract

Background: Improper handling of poultry, particularly in developing countries like Bangladesh, can lead to foodborne illnesses. Citrobacter freundii, a Gram-negative bacteria, is a silent emerging threat causing diseases and spreading antibiotic resistance. This study aimed to identify the multidrug resistant (MDR) C. freundii from poultry egg samples through biochemical and molecular characterization. Materials and Methods: A total of 50 poultry egg samples (both outer and inner) were collected from different farms located in Dhaka, Bangladesh within six months. Isolation of pathogens with selective bacteriological media such as McConkey agar, Xylose Lysine Deoxycholate agar, Salmonella-Shigella agar, and specific biochemical tests were performed to identify the Citrobacter isolates. The antibiotic sensitivity pattern of the isolates were determined by the disk diffusion method. Multidrug-resistant isolates were further identified by 16S rRNA gene sequencing and BLASTn analysis. Results: Out of 50 samples, 60% (30) were found positive for Gram negative Enterobacteriaceae. Among them, 50% of the samples were found to be contaminated with Salmonella enterica (15), 20% with E. coli (6), 6.67% with Pseudomonas spp. (2) and 23.33% with Citrobacter spp. They were highly resistant to erythromycin (83%), cefixime (82%), and amoxicillin (79%) antibiotics. On the other hand, they were sensitive against imipenem (93%) and intermediate for azithromycin (46%). Among all isolates, the most potent multidrug-resistant isolate was identified based on its 16S rRNA gene sequence, with one having the highest similarity (100%) to Citrobacter freundii strain A3.Conclusion: Poultry eggs can be a source of transmission of multidrug-resistant Citrobacter freundii from animal to human. It could be a serious health concern for developing countries since very little effort has been put into this issue.

Keywords: Citrobacter freundii, human health, poultry eggs, multidrug resistance.

References


Akbar A, Anal AK. Food safety concerns and foodborne pathogens, Salmonella, Escherichia coli and Campylobacter. FUUAST J Biol 2011; (1): 5-17.    

Bai, L., Xia, S., Lan, R., Liu, L., Ye, C., Wang, Y., et al. (2012). Isolation and characterization of cytotoxic, aggregative Citrobacter freundii. PLoS ONE 7:e33054. doi: 10.1371/journal.pone.0033054 https://doi.org/10.1371/journal.pone.0033054; PMid:22470435 PMCid:PMC3310003      

Balagué C, Véscovi EG. Activation of multiple antibiotic resistance in uropathogenic Escherichia coli strains by aryloxoalcanoic acid compounds. Antimicrob Agents Chemother 2001; 45:1815-22. https://doi.org/10.1128/AAC.45.6.1815-1822.2001 PMid: 11353631 PMCid: PMC90551    

Chousalkar KK, Flynn P, Sutherland M, Roberts JR, Cheetham BF. Recovery of Salmonella and Escherichia coli from commercial egg shells and effect of translucency on bacterial penetration in eggs. Int J Food Microbiol. 2010 Aug 15; 142(1-2):207-13. Epub 2010 Jul 3. PMID: 20663580. https://doi.org/10.1016/j.ijfoodmicro.2010.06.029 PMid: 20663580 

Cockerill FR, Wikler M, Bush K, Dudley M, Eliopoulos G, Hardy D, et al. Performance standards for antimicrobial susceptibility testing: twenty-second informational supplement. Clinical and laboratory standards institute: Pennysylvania, USA, 2012.        

Collignon AM, Castillo-Dali G, Gomez E, Guilbert T, Lesieur J, Nicoletti A, Acuna-Mendoza S, Letourneur D, Chaussain C, Rochefort GY, Poliard A (2019) Mouse Wnt1-CRE-RosaTomato dental pulp stem cells directly contribute to the calvarial bone regeneration process. Stem Cells 37: 701-711. https://doi.org/10.1002/stem.2973 PMid:30674073        

De Reu K., Messens W., Herman L. (2008). Bacterial contamination of table eggs and the influence of housing systems. World's Poultry Science Journal, 64: 6-19. https://doi.org/10.1017/S0043933907001687 

Fair, R.J. and Tor, Y. (2014) Antibiotics and Bacterial Resistance in the 21st Century. Perspectives in Medicinal Chemistry, 6, PMC-S14459. https://doi.org/10.4137/PMC.S14459  PMid: 25232278 PMCid: PMC4159373     

Fakruddin M, Chowdhury A, Hossain MN, Ahmed MM. Characterization of aflatoxin producing Aspergillus flavus from food and feed samples. Springerplus. 2015 Apr 1;4:159. https://doi.org/10.1186/s40064-015-0947-1 PMid:25883886 PMCid:PMC4394062      

Fakruddin, M., M.M. Rahaman, M.M. Ahmed, & M. Hoque. 2014. Antimicrobial resistance and virulence factors of Enterobacteriaceae isolated from food samples of Bangladesh. Intl. J. Microbiol. Immunol. Res. 3(1): 12-18.

Fardows J, Shamsuzzaman SM. Detection of potential pathogenic aerobic bacteria from egg shell and egg contents of hen collected from poultry. Bangladesh Med Res Counc Bull. 2015 Aug; 41(2):67-72. https://doi.org/10.3329/bmrcb.v41i2.29983 PMid:29624284   

Gantois I, Ducatelle R, Pasmans F, Haesebrouck F, Gast R, Humphrey TJ, Van Immerseel F. Mechanisms of egg contamination by Salmonella Enteritidis. FEMS Microbiol Rev. 2009 Jul; 33(4):718-38. doi: 10.1111/j.1574-6976.2008.00161.x. Epub 2009 Jan 21. PMID: 19207743. https://doi.org/10.1111/j.1574-6976.2008.00161.x; PMid: 19207743 

Hashim & Alkhafaki, 2018. Isolation and identification of Citrobacter freundii from chicken meat samples using cultural and molecular techniques Mohammed Hayder Hashim*1, Marwa Hameed AlKhafaji2. Iraqi Journal of Science, 2018, Vol. 59, No.3A, pp: 1216-1224 https://doi.org/10.24996/ijs.2018.59.3A.9

Igbinosa EO, Rathje J, Habermann D, Brinks E, Cho GS, Franz CMAP. Draft Genome Sequence of Multidrug-Resistant Strain Citrobacter portucalensis MBTC-1222, Isolated from Uziza (Piper guineense) Leaves in Nigeria. Genome Announc. 2018 Mar 1;6(9):e00123-18. doi:10.1128/genomeA.00123-18  PMID: 29496838; PMCID: PMC5834338. https://doi.org/10.1128/genomeA.00123-18 PMid: 29496838 PMCid: PMC5834338     

Jahantigh, M. Antimicrobial resistance to Citrobacter spp. and Salmonella spp. isolated from goose eggs. Comp Clin Pathol 22, 1-4 (2013). https://doi.org/10.1007/s00580-011-1360-y  

Johnson JL, Adkins D, Chauvin S. A Review of the Quality Indicators of Rigor in Qualitative Research. Am J Pharm Educ. 2020 Jan; 84(1):7120. https://doi.org/10.5688/ajpe7120 PMid:32292186 PMCid:PMC7055404   

Kanamori H, Yano H, Hirakata Y, Endo S, Arai K, Ogawa M, Shimojima M, Aoyagi T, Hatta M, Yamada M, Nishimaki K, Kitagawa M, Kunishima H, Kaku M. High prevalence of extended-spectrum β-lactamases and qnr determinants in Citrobacter species from Japan: dissemination of CTX-M-2. J Antimicrob Chemother. 2011 Oct;66(10):2255-62. Epub 2011 Jul 6. PMID: 21733965. https://doi.org/10.1093/jac/dkr283 PMid: 21733965      

Khan S, McWhorter AR, Moyle TS, Chousalkar KK. Refrigeration of eggs influences the virulence of Salmonella Typhimurium. Sci Rep. 2021 Sep 9; 11(1):18026. https://doi.org/10.1038/s41598-021-97135-4 PMid:34504138 PMCid:PMC8429434     

Kowalczyk, J.; Czokajlo, I.; Ganko, M.; Smialek, M.; Koncicki, A. Identification and Antimicrobial Resistance in Klebsiella spp. Isolates from Turkeys in Poland between 2019 and 2022. Animals 2022, 12, 3157. https://doi.org/10.3390/ani12223157 PMid: 36428385 PMCid: PMC9686990

Kumar S., Stecher G., & Tamura K. (2016). MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular biology and evolution, 33(7), 1870-1874. https://doi.org/10.1093/molbev/msw054 PMid:27004904 PMCid:PMC8210823      

Liu L, Lan R, Liu L, Wang Y, Zhang Y, Wang Y, Xu J. Antimicrobial Resistance and Cytotoxicity of Citrobacter spp. in Maanshan Anhui Province, China. Front Microbiol. 2017 Jul 20;8:1357. https://doi.org/10.3389/fmicb.2017.01357 PMid:28775715 PMCid:PMC5518651   

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 https://doi.org/10.3389/fcimb.2018.00233  PMid:30050870 PMCid:PMC6052900      

Liu, L., Lan, R., Liu, L., Wang, Y., Zhang, Y., Wang, Y., et al. (2017a). Antimicrobial Resistance and cytotoxicity of Citrobacter spp. in Maanshan Anhui Province, China. Front. Microbiol. 8:1357. https://doi.org/10.3389/fmicb.2017.01357; PMid:28775715 PMCid:PMC5518651      

Machuve, D., Nwankwo, E., Mduma, N., & Mbelwa, J. (2022). Poultry disease diagnostics models using deep learning. Frontiers in Artificial Intelligence (Vol.  5). https://doi.org/10.3389/frai.2022.733345; https://doi.org/10.3389/frai.2022.1016695; PMid: 36117782 PMCid:PMC9477006

Md. Ekhlas Uddin, H. M. Faruquee, Md. Firoz Alam. (2014). Isolation and characterization of proteases enzyme from locally isolated Bacillus sp. American J. of Life Sciences. 2(6): 338-344. https://doi.org/10.11648/j.ajls.20140206.12

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. https://doi.org/10.1016/j.aninu.2018.03.002 PMid: 30140756 PMCid: PMC6103476     

Messens W., Grijspeerdt K., Herman L. (2005). Eggshell penetration by Salmonella: A review. World's Poult. Sci. J., 61: 71-85. https://doi.org/10.1079/WPS200443

Monzur Morshed Ahmed, Md. Siddique Hossain, Khandaker Rayhan Mahbub, Himel Nahreen Khaleque, Zakir Hossain, Md. Fakruddin, Abhijit Chowdhury, Md. Nur Hossain and Md. Zobaidul Alam, 2012. Performance Analysis of Multiplex-PCR based Detection of Salmonella sp. and Salmonella Typhimurium in Chicken Egg Samples. Singapore Journal of Scientific Research, 2: 25-32. https://doi.org/10.3923/sjsres.2012.25.32

Musgrove M.T., J.K. Northcutt, D.R. Jones, N.A. Cox, M.A. Harrison. Enterobacteriaceae and Related Organisms Isolated from Shell Eggs Collected During Commercial Processing. Poultry Science, Volume 87, Issue 6, 2008, Pages 1211-1218, https://doi.org/10.3382/ps.2007-00496 PMid:18493013

Nandi SP, Sultana M, Hossain MA. Prevalence and characterization of multidrug-resistant zoonotic Enterobacter spp. in poultry of Bangladesh. Foodborne Pathog Dis. 2013 May;10 (5):420-7. Epub 2013 Apr 6. PMID: 23560422. https://doi.org/10.1089/fpd.2012.1388

Papadopoulou C, Dimitriou D, Levidiotou S, Gessouli H, Panagiou A, Golegou S, Antoniades G. Bacterial strains isolated from eggs and their resistance to currently used antibiotics: is there a health hazard for consumers? Comp Immunol Microbiol Infect Dis. 1997 Jan; 20(1):35-40. https://doi.org/10.1016/S0147-9571(96)00024-0 PMid:9023039     

Park, Y. J., Park, S. Y., Oh, E. J., Park, J. J., Lee, K. Y., Woo, G. J., et al. (2005). Occurrence of extended-spectrum beta-lactamases among chromosomal AmpC-producing Enterobacter cloacae, Citrobacter freundii, and Serratia marcescens in Korea and investigation of screening criteria. Diagn. Microbiol. Infect. Dis. 51, 265-269. https://doi.org/10.1016/j.diagmicrobio.2004.11.009; PMid:15808318   

Plakkal N, et al. Citrobacter freundii brain abscess in a preterm infant: a case report and literature review. Pediatr Neonatol 2013; 54:137-140. https://doi.org/10.1016/j.pedneo.2012.10.004 PMid:23590960   

Prestinaci, F., P. Pezzotti, & A. Pantosti. 2015. Antimicrobial resistance: A global multifaceted phenomenon. Pathog. Glob. Health. 109:309-318. https://doi.org/10.1179/2047773215Y.0000000030 PMid:26343252 PMCid:PMC4768623   

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. https://doi. org/10.1155/2018/7309346; https://doi.org/10.1155/2018/7309346; PMid: 30426012 PMCid: PMC6218796       

Rahman, M.S., Jang, D.H. and Yu, C.J. (2017) Poultry Industry of Bangladesh: Entering a New Phase. Korean Journal of Agricultural Science, 44, 272-282. https://doi.org/10.7744/kjoas.20170027 https://doi.org/10.7744/kjoas.20170027

Reygaert, W. C. (2018). An overview of the antimicrobial resistance mechanisms of bacteria. AIMS Microbiology, 4(3), 482-501. https://doi.org/10.3934/microbiol.2018.3.482 PMid:31294229 PMCid:PMC6604941      

S. Rodriguez-Mozaz, S. Chamorro, E. Marti, B. Huerta, M. Gros, A. Sànchez-Melsió, C.M. Borrego, D. Barceló, J.L. Balcázar. Occurrence of antibiotics and antibiotic resistance genes in hospital and urban wastewaters and their impact on the receiving river. Water Res., 69 (2015), pp. 234-242. https://doi.org/10.1016/j.watres.2014.11.021 PMid:25482914   

Sai, S., Mani, R., Ganesan, M. (2023). Isolation and Identification of Citrobacter Species. In: Thomas, J., Amaresan, N. (eds) Aquaculture Microbiology. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3032-7_4

Shao, Y., Xiong, Z., Li, X., Hu, L., Shen, J., Li, T., et al. (2011). Prevalence of plasmid-mediated quinolone resistance determinants in Citrobacter freundii isolates from Anhui province, PR China. J. Med. Microbiol. 60, 1801-1805. https://doi.org/10.1099/jmm.0.034082-0 PMid: 21816943         

Shrestha A, Bajracharya AM, Subedi H, Turha RS, Kafle S, Sharma S, Neupane S, Chaudhary DK. Multi-drug resistance and extended spectrum beta lactamase producing Gram negative bacteria from chicken meat in Bharatpur Metropolitan, Nepal. BMC Res Notes. 2017 Nov 7; 10(1):574. PMID: 29116010; PMCID: PMC5678746. https://doi.org/10.1186/s13104-017-2917-x PMid: 29116010 PMCid:PMC5678746      

Stepien-pysniak D. Occurrence of Gram-negative bacteria in hens' eggs depending on their source and storage conditions. Polish J Vet Sci 2010; 13 (3): 507- 13.

Sultana F, Kamrunnahar, Afroz H, Jahan A, Fakruddin M, Datta S. Multi-antibiotic resistant bacteria in frozen food (ready to cook food) of animal origin sold in Dhaka, Bangladesh. Asian Pac J Trop Biomed. 2014 May; 4(Suppl 1):S268-71. PMID: 25183094; PMCID: PMC4025334. https://doi.org/10.12980/APJTB.4.2014B85 PMid:25183094 PMCid:PMC4025334      

Treves D. S. (2010). Review of three DNA analysis applications for use in the microbiology or genetics classroom. Journal of Microbiology & Biology Education: JMBE, 11(2), 186. https://doi.org/10.1128/jmbe.v11i2.205 PMID: 23653728, PMCid:PMC3577175      

Velazquez-Meza ME, Galarde-López M, Carrillo-Quiróz B, Alpuche-Aranda CM. Antimicrobial resistance: One Health approach. Vet World. 2022 Mar;15(3):743-749. Epub 2022 Mar 28. https://doi.org/10.14202/vetworld.2022.743-749 PMid:35497962 PMCid:PMC9047147      

Weinstein, M.P. and Lewis, J.S. (2020) The Clinical and Laboratory Standards Institute Subcommittee on Antimicrobial Susceptibility Testing: Background, Organization, Functions, and Processes. Journal of Clinical Microbiology, 58, e01864-19. https://doi.org/10.1128/JCM.01864-19 PMid:31915289 PMCid:PMC7041576      

White DG, Piddock LJ, Maurer JJ, Zhao S, Ricci V, Thayer SG. Characterization of fluoroquinolone resistance among veterinary isolates of avian Escherichia coli. Antimicrob Agents Chemother 2000; 44:2897-9. https://doi.org/10.1128/AAC.44.10.2897-2899.2000 PMid: 10991884 PMCid:PMC90175

PDF
Abstract
Export Citation

View Dimensions


View Plumx


View Altmetric




Save
0
Citation
599
View

Share