Current Issues of Pharmacy and Medical Sciences

Molecular characterization of ESBL gene in Citrobacter spp. and antibacterial activity of omega-3 against resistant isolates

Curr Issues Pharm Med Sci., Vol. 30, No. 3, 156-161

Mayyada F. Darweesh

Biology Department, Faculty of Science, Kufa University, Najaf, Iraq



The study aimed to investigate the prevalence and resistance pattern of different Citrobacter spp., phenotypically and genotypically, to β-lactam antibiotics, then to evaluate the antibacterial activity of omega-3 extracted from flaxseed against isolates that harboring resistance genes. Herein, 19 Citrobacter isolates were isolated from 100 stool and urine samples taken from patients attended to AL-Sadar Hospital during June-December 2016. Clinical samples were then cultured on specific media, thereafter, isolates were identified depending on morphological, biochemical characteristics and VITK-2. The results showed that the Citrobacter spp. comprise 19/78 (24%) of positive bacterial growth on macConky agar, from which 14.1% were C. freundii, C. koseri represented 6.1% and C. farmeri were 3.8% of the total. The results of antibiotic susceptibility showed that all Citrobacter 100% isolates were resistant to ampicillin and cefoxitin, but were sensitive to imipinim. Moreover, the isolates initally showed different degrees of resistant to β-lactam antibiotics. Furthermore, by confirmatory test, the results observed that 17/19 (89.4%) of the isolated were extended-spectrum β-lactamase (ESBL – producers). Finally, using the PCR technique to detect blaGenes, the results revealed that 14/17 (82.3) of potential ESBL producing Citrobacter harbored one or more ESBL genes. These included 10 isolates of C. freundii and 4 isolates of C. koseri. In related work, extracts of essential fatty acid semicarbazide – omega3 (EFASC) from Linum usitatissium (Flaxseed) were tested to evaluate their activity against resistant isolates. The results demonstrate the broad spectrum antibacterial property of EFASC compounds against resistant bacteria. In conclusion, this study found increase prevalence of multi-drug resistance MDR Citrobacter spp. as causative agents in clinical cases. Considering the antibacterial activity of EFASC that displayed high activity against resistant pathogens, deservedly, attention must be paid to developing their use as alternative antibiotics.

Full text


Citrobacter spp., ESBL-genes, omega-3 antibacterial activity.


  1. Janda, J.M. et al. Biochemical identification of citrobacteria in the clinical laboratory. J. Clini Microbi. 32(8): 1850-1854, 1994.Google Scholar
  2. Nada, T. et al.: Small outbreak of third generation cephem-resistant Citrobacter freundii infection on a surgical ward. JPN. J. Infectious Diseases. 57:181-2,2004.Google Scholar
  3. Shih, C.C. et al.: Bacteremia due to Citrobacter species: significance of primary intraabdominal infection. Clinical Infectious Diseases. 23(3): 543-549,1991.CrossrefGoogle Scholar
  4. Al-Hasnawi, A. A.: Comparison of biochemical tests, Api system, Vitek 2 system and PCR of the enteropathogenic bacteria isolated from children with persistent diarrhea. And the occurrence of virulence factors and antibiotic resistance in the isolates. Master Thesis. Faculty of Science, University of Kufa 2014.Google Scholar
  5. Al-Hissnawy, D.; AL-Thahab, A.A.; Al-Jubori,S.A.: Evaluation of Citrobacter freundii isolated in Najaf governorate as an enterotoxin producer. Medical J. Babylon. 9(1): 1-5, 2012.Google Scholar
  6. Tuwaij, N.S.: Molecular Study of Quinolone Resistance in Klebsiella pneumoniae and Citrobacter freundii Isolates. Al-Kufa University J. for Biology 8(3): 300-312, 2016.Google Scholar
  7. Harvey, R.A. and Champe, D.C.: Lippincott’s Illustrated Reviews: Pharmacology 5th ed., Lippincott Williams and Wilkins, USA. 382-385, 2012.Google Scholar
  8. Fuad, al.: In Vitro Antibacterial Activity of Common Antibiotics and Herb Extracts to Clinical Isolates of Escherichia coli Collected from UTI Patient. Int. J. of Rese in Pharmacel and Biomedi Sciences. 3(2): 987-992, 2012.Google Scholar
  9. Wang, H. et al.: Comparison of phytochemical profiles and health benefits in fiber and oil flaxseeds (Linum usitatissimum L.). Food Chem. Jan 1;214: 227-33, 2017.CrossrefGoogle Scholar
  10. Desbois,A.P. and Lawlor K.C.: Antibacterial Activity of Long-Chain Polyunsaturated Fatty Acids against Propionibacterium acnes and Staphylococcus aureus. Mar. Drugs 11: 4544-4557, 2013.Google Scholar
  11. Shin,S.Y., Bajpai, H.R. and Kang,S.C.: Antibacterial activity of ecosapantaenoic acid (EPA) against foodborne and food spoilage microorganism.LWT-Food Sci. Tech.,40: 1515-1519, 2007.Google Scholar
  12. Singhal, M. and Paul, A.(2011): Antibacterial evaluation of synthesized methyl semicarbazone derivatives. IJPSR. 2(10): 2602-2604.Google Scholar
  13. MacFaddin, J.E.: Individual Biochemical Tests For Identification of Medical Bacteria. 3th ed. Lippincott Williams Wilkins, London: 57-424, 2000.Google Scholar
  14. CLSI. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Fourth Informational Supplement M02-A11, M 0-A11, and M11-A8. Wayne, PA, USA.2014.Google Scholar
  15. Batchoun, R.G.; Swedan, S.F. and Shurman, A.M.: Extended spectrum β-lactamases among Gram-negative bacterial isolates from clinical specimens in three major hospitals in Northern Jordan. J. Micro. Res. Article, 2009,ID 513874.Google Scholar
  16. Hassan, al.: Detection of extended spectrum beta-lactamasesproducing isolates and effect of AmpC overlapping. Infect. Dev., 7(8): 618-629, 2013.Google Scholar
  17. Colom,K et al.: Simple and reliable multiplex PCR assay for detection of blaTEM, blaSHV and blaOXA-1 genes in Enterobacteriaceae. FEMS Microbiology Letters, 223: 147-151, 2013.Google Scholar
  18. Svard, L. (2007): Evaluation of phenotypic and genotypic extended spectrum beta-lactamase detection method. M.Sc. Thesis. School of Biological Sciences, Dublin Institute of Technology, Uppsala University, Germany.Google Scholar
  19. AL-Ramahi, A.A.; Darweesh, M. A., Ahmad A. M.: The Antibacterial of Essential Fatty Acid Semicarbazide Extracted from Flaxseed Oil Against Some Nosocomial Infection Bacteria in Iraq. IJCPR; 8(1) January-February: 31-39, 2017.Google Scholar
  20. Harborne, J. B. (1984): Phytochemical Methods.; A Guide to Modern Techniques of Plant Analysis, 2nd ed. Chapman and Hall, London.Google Scholar
  21. Borhade, S.: Synthesis, Characterisation and Antimicrobial Activity of Essential Fatty Acid of Semicarbazide. Int.J. of Chem.Scie.and Applic. 5(2): 46-55, 2014.Google Scholar
  22. Egharevba, H. O. et al.: Phytochemical analysis and antimicrobial activity of Punica granatum L. (fruit and leaves). New York Scie. J. 3 (12): 91-98, 2010.Google Scholar
  23. AL-Muslemawi, TH. A. (2007): Study of some biochemical, biological and pathological properties of lipopolysaccharide extracted from Citrobacter freundii. Ph.D.Thesis.Baghdad univ.Google Scholar
  24. Salih, M.K., Alrabadi, N.I., Thalij, K.M. and Hussien, A.S.: Isolation of Pathogenic Gram-Negative Bacteria from Urinary Tract Infected Patients. Open Journal of Medical Microbiology, 6, 59-65, 2016.Google Scholar
  25. Stewart, Z. E. ; Shaker M.; Baxter, J. D.: Inflammation and Infection Urinary Tract Infection Caused by Citrobacter koseri in a Patient With Spina Bifida, an Ileal Conduit and Renal Caluli Progressing to Peri-nephric Abscess and Empyema. Urology Case Reports 11: 22-24, 2017.Google Scholar
  26. Warren, J. et al.: Out break of nosocomial infection due to extended - spectrum beta- lactamase producing strain of enteric group 137, a new member of the family Enterobacteriaceae closely related to citrobacter farmer and C. amalonatica. J. Clin. Microbio. 38(11): 3946-52, 2000.Google Scholar
  27. Hassan, M.M. et al.: Molecular characterization of antibiotic resistance genes in pathogenic bacteria isolated from Patients in Taif Hospitals, KSA. AJPCT. 2(8): 939-951, 2014.Google Scholar
  28. AbdAlhussen, L.S. and Darweesh, M.F.: Prevelance and antibiotic susceptibility patterns of Pantoea spp. isolated form clinical and environmental sources in Iraq. I.J.of ChemTech Research, 9 (08): 430-437, 2016.Google Scholar
  29. Metri, B.C.; Jyothi, V.X. and Peerapur B. V.: Antibiotic resistance in Citrobacter spp. isolated from urinary tract infection. Urol Ann. Oct-Dec; 5(4): 312-315,2013.Google Scholar
  30. Sami, al.: Citrobacter as a uropathogen, its prevalence and antibiotics susceptibility pattern. 4 (1): 23-26,2017. Google Scholar
  31. Bush, K.; Jacoby, G. and Medeiros A.: A functional classification scheme for β-lactamases and its correlation with molecular structure. Anti. Agents Chemo.,39: 1211-1233, 1995.Google Scholar
  32. Erlandsson, M. (2007). Surveillance of antibiotic consumption and antibiotic resistance in Swedish intensive care units. Linkoping University Medical Dissertations No. 1019 Sweden.Google Scholar
  33. Al-Muhannak, F. H. N. (2010): Spread of Some Extended Spectrum Beta-Lactamases in Clinical Isolates of Gram Negative Bacilli in Najaf. M.Sc. Thesis. College of Medicine, University of Kufa.Google Scholar
  34. Shahid, M.: Citrobacter spp. Simultaneously harboring bla- CTX-M, blaTEM, blaSHV, bla-ampC, and insertion sequences IS26 and orf513: an evolutionary phenomenon of recent concern for antibiotic resistance. J Clin. Microbi. 48(5): 1833-1838,2010.Google Scholar
  35. Perilli, M. et al.: Novel TEM-type extended-spectrum beta-lactamase, TEM-134, in a Citrobacter koseri clinical isolate. Antimicrob. Agents Chemother. 49: 1564-1566, 2005.Google Scholar
  36. Huang, Z.M. et al.: Study on molecular epidemiology of SHV type beta-lactamase encoding genes of multiple-drug - resistant Acinetobacter baumannii. Zhonghua Liu. Xing Bing Xue Za Zhi.,25: 425-427,2004.Google Scholar
  37. Pandya, P., Harisha, C.R., Shandla,V.J. and Chandola, H. M.: Pharmoacogostical and Photochemical evaluation Atasi (Linum ustatissimmum L.). Indian Journal of Tradition Knowledge. 12(4): 688-692, 2013. Google Scholar
  38. Seidel, V., Taylor, P.W.: In vitro activity of extracts and constituents of Pelagonium against rapidly growing mycobacteria. Int. J. Antimicrob. Agents,23: 613-619, 2004.CrossrefGoogle Scholar
  39. Mogensen, T.H.: Pathogen recognition and inflammatory Signaling in innate immune. Defenses. Clin.Micro. Rev. 22(2): 240-273,2009. Google Scholar
  40. Ibarguren, M.; Lopez, D. and Escriba, P.: The effect of natural and synthetic fatty acids on membrane structure, microdomain organization, cellular functions and human health. Bioph. Acta. 1838: 1518-1528, 2014.Google Scholar


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