Evaluation of Citrobacter freundii as a Heat Labile (LT) Enterotoxin Producer

Dhia Shanan Al-Hissnawy,Salman Azez Al-Jibouri,Azhar Ammran AL-Thahab†
Authors Emails are requested on demand or by logging in
Keywords : Rabbit ligated ileal loop assay (RIL,Citrobacter freundii,Heat labile
Medical Journal of Babylon  9:1 , 2014 doi:1812-156X-9-1
Published :2012


This study included the investigation of the ability of C. freundii to produce Heat labile enterotoxin by both genotype and phenotype. Only 11 isolates were isolated from 422 clinical sample (282 stool and 140 urine samples), 8 isolates from stool samples and 3 from urine samples. All isolates identified by biochemical tests and confirmed with API 20 E. Molecular detection for gene responsible for heat- labile (lt, ltI-h and ltA) enterotoxin genes was achieved by PCR technique. The result showed that ltA was the heat-labile enterotoxin gene in 5 isolates (45.45%). Rabbit ligated ileal loop assay RIL, was applied to the 11 Isolates, only 4 of the bacterial isolates gave a positive results.


cfreundii is usually considered a commensal species of the human gut, although some isolates have acquired specific virulence traits that enable them to cause diarrhea. Therefore, virulence factors homologous, and some even identical, to those described in E. coli pathotypes were detected in C. freundii strains isolated from sporadic cases of infantile diarrhea[10, 12]. Locally, C. freundii isolated from different samples include stool, rectal swabs, urine, blood, sputum, cerebrospinal fluid, wounds, ear, nasal and throat swabs in Baghdad and Hilla cities [2, 3] The LT gene was cloned into E. coli and two proteins of molecular C Evaluation of Citrobacter freundii as a Heat Labile (LT) Enterotoxin Producer Dhia Shanan Al-Hissnawy Salman Azez Al-Jibouri* Azhar Ammran AL-Thahab† College of Dentistry, Kufa University, Najaf, Iraq. *College of Medicine , Kufa University, Najaf, Iraq. †College of Science, University of Babylon, Hilla, Iraq. M J B Medical Journal of Babylon-Vol. 9- No. 1 -2012 ???? ???? ??????- ?????? ??????- ????? ?????- 1021 Dhia Shanan Al-Hissnawy, Salman Azez Al-Jibouri and Azhar Ammran AL-Thahab 2 weights 11,500 (B subunit) and 25,500 (A subunits) were produced [5]. The LT A subunit structureal gene (eltA) was sequenced and the amino acid sequence deduced. The computed molecular weight of LT A is 29,673Da, The A subunit genes of CT and LT (LT-I) are 78.6% homologous, and the B subunit genes are 78% homologous[16]. The gene of LT-IIa was studied. It is organized in a transcriptional unit similar to those of CT and LT-I. The A subunit gene of LT-IIa was found to be 57% homologous with the A subunit gene of LTh-I and 55% homologous with the A gene of CT. Most of the homology derived from the region of the A gene which encodes the A1 fragment. The B gene of LTIIa was not homologous with the B gene of LTh-I or CT [13].

Materials and methods

Isolation and identification of C. freundii: During the period from July 2010 to October 2010, a total of 422 clinical samples (282 stool and 140urine) were taken from patients with suspected diarrhea and urinary tract infections, 50 healthy individuals (control),samples collected from three hospitals in Najaf (Al-Sadr Teaching, Al-Hakeem, and Al-Zahra Maternity and Children). Identification of Bacterial Isolates were identified to the level of species using the traditional morphological and biochemical tests [11]. All isolates were confirmed identification with API 20 E system. The bacterial isolates were preserved on nutrient agar slant at 4?C. The isolates were maintained monthly during the study by culturing on new culture media. For long preservation, nutrient broth supplemented with 15% glycerol was used and the isolates were maintained frozen (-20?C) for long term for several months [4]
Polymerase chain reaction assays were carried out in a 25 ?l reaction volume, and the PCR amplification conditions performed with a thermal cycler were specific to each single primer set depending on their reference procedure, as follows:.
Polymerase chain reaction: DNA was extracted by Salting out method [14]. DNA (extracted from bacteria cells) was used as a template in specific PCRs for the detection of enterotoxin genes. A pair of primers for each gene listed in table (1) was used for the amplification of a fragment that covers the entire gene. A single reaction mixture contained 5 ?l DNA extract, 12.5 ?l Master mix(Master mix 2X Promega : Go Tag DNA polymerase is supplied in 2x Green Taq Reaction buffer pH 8.5, 400?m dATP, 400?m dGTP, 400?m dCTP, 400?m dTTP, and 3mM MgCl2), 2?l of 10 pmol/?l of upstream primers specific and, 2?l of 10 pmol/?l of downstream primers specific the volume then completed to 25 ?l by nuclease-free water. All the additions were done in laminar flow on


Molecular Detection of heat labile enterotoxin of C. freundii isolates: DNA was extracted from all isolates in this study and used as a template for PCR assays to heat-labile enterotoxin genes (lt, lt1-h and ltA) .PCR results showed that C. freundii retain ltA as a heat-labile enterotoxin gene in 5 isolates(C1,2,3,6 and 10) with percent 45.45%(Fig. 1). Other heat-labile enterotoxin genes showed negative result at least in thermocycles listed in table (2). ? Lanes (C1, 2, 3, 6 and 10) show positive results with ltA gene. ? Lanes (C4,5,7,8,9 and 11) show negative results with ltA gene ? Lane (L), DNA molecular size marker (100-bp ladder) Heat-labile enterotoxin bioassay: The Rabbit ligated ileal loop assay RIL (Fig. 2) was applied to the isolates of C. freundii isolated in this study. The results showed that only 4 of the bacterial isolates gave a positive results (C1: 0.74, C2: 0.86 C3: 0.76 and C10: 0.76) ml/cm with percent 36.36%, while other bacterial isolates gave a negative result compared with cholera toxin as a positive control (1.1 ml/cm) and (TSB+ 6% yeast extract) as a negative control (0.15 ml/cm) as shown in table (3).


Molecular Detection for heat-labile enterotoxin of C. freundii isolates: From figure (1), C. freundii showed positive result for ltA (696bp) as genes responsible for heat-labile enterotoxin by PCR. C. freundii known to be enterotoxin producer. identification and characterization a gene encoding a homologue of the B subunits of cholera toxin (CTB) and heat-labile enterotoxin (LTB) of E. coli from a clinical isolate of C. freundii that was found to produce a factor in the culture supernatant that cross-reacted with antibodies to CTB and LTB when assayed by enzyme-linked immunosorbent assay (ELISA) [10]. The gene encoding the ELISA-positive factor, cfxB, consisted of 375 nucleotides and was located downstream of an 852-nucleotide open reading frame, cfxA, with a 56-nucleotide intergenic space. The cfxB gene was predicted to encode a 125-amino-acid polypeptide. Strains of C. freundii isolated from environmental water were identified as heat-labile enterotoxin (LT) producing strains by immunological methods and polymerase chain amplification. A 322 bp amplified fragment was obtained [9]. Enterotoxin bioassay showed in table (3) recorded a marked fluid accumulation in only 4 of the bacterial isolates gave a positive results (C1-3 and C10) ml/cm with percent 36.36% in rabbit ligated ileal loop assay as indicator of heat-labile enterotoxin activity. The ability all strains were capable of producing heat-labile enterotoxin with (100%) by delayed permeability test of rabbit skin and mice paw oedema test taking into account the difference in the toxicity degree among producing strains of this toxin[1]. Strain isolated from urine and stool showed a negative result with RIL[7]. The enterotoxin bioassay has the advantage over cell-culture systems which detect only cytotoxicity [17]




1. Al-Hashimi, E.S.N.H. (2002). Pathogenicity of Citrobacter freundii Bacterium and Toxins Isolated from Some Diarrheic Cases in Infant at Mosul City. MSc thesis. college of science. Mousl University, Iraq.

2. AL-Khafagee, N. S. K. (2010). Bacteriological and Immunological Study of Citrobacter freundii Bacteria in Rabbit. M.Sc thesis. College of science. Babylon University, iraq.

3. Al-Muslemawi , T. A. J.(2007). Study of Some Biochemical, Biological And Pathological Properties Of Lipopolysaccharide Extracted From Citrobacter freundii. Ph.D. Thesis. Baghdad University.

4. Collee, J.G., Fraser, A.G., Marion, B.P., and Simmon, A.C.(1996). Mackie and McCarteny. Practical Medical Microbiology. 4th ed. Livingstone Inc., USA.

5. Dallas,W.S., Gill,D.M., Falkow,S. (1979). Cistrons encoding Escherichia coli heat-labile toxin. Journal of Bacteriology. 139, 850-858.

6. Evans, D.G.; Evans, D.J. and Pierce N.F.(1973). Differences in the Response of Rabbit Small Intestine to
Heat-Labile and Heat-Stable Enterotoxins of Escherichia coli. Infection and Immunity. 7, 873-880.

7. Guarino A., Capano G., Malamisura B., Alessio M., Guandalini S. and Rubino A. (1987). Production of Eschericia coli STa-Like heat-stable enterotoxin by Citrobacter freundii isolated from humans. J. Clin. Microbiol., 25, 110-114.

8. Itoh, F.; Ogino, T.; Itoh, K.; and Watanabe, H. (1992). Diffrentiation and detection of pathogeneic determinants among diarrheagenic Escherichia coli by polymerase chain reaction using mixed primers.Jpn. J. Clin. Med., 50, 343-347.

9. Jolivet-Gougeon, A.; Tamanai-Shacoori, T.; Sauvager, F. and Cormier. M. (1997). Production of Escherichia coli group I-like heat-labile enterotoxin by Enterobacteriaceae isolated from environmental water. Microbios. 90,209–218.

10. Karasawa, T.; Ito, H.; Tsukamoto, T.; Yamasaki, S.; Kurazono, H.; and Faruque, S.M. (2002). Cloning and characterization of genes encoding homologues of the B subunit of cholera toxin and the Escherichia coli heat-labile enterotoxin from clinical isolates of Citrobacter freundii and E. coli. Infect Immun. 70, 7153-7155.

11. MacFaddin, J. F. (2000). Biochemical tests for identification of medical bacteria (3rd ed.), Lippincott Williams and Wilkins, USA.

12. Pereira, A. L.; Silva, T. N.; Gomes, A. C.; Araujo, A. C.; Giugliano, L. G. (2010). Diarrhea-associated biofilm formed by enteroaggregative Escherichia coli and aggregative Citrobacter freundii: a consortium mediated by putative F pili. BMC Microbiology 10:57 pp. 1-18.

13. Pickett,C.L., Weinstein,D.L., Holmes,R.K. (1987). Genetics of type IIa heat-labile enterotoxin of Escherichia coli: operon fusions,
Medical Journal of Babylon-Vol. 9- No. 1 -2012 ???? ???? ??????- ?????? ??????- ????? ?????- 1021
Dhia Shanan Al-Hissnawy, Salman Azez Al-Jibouri and Azhar Ammran AL-Thahab 7
nucleotide sequence, and hybridization studies. Journal of Bacteriology 169, 5180-5187.

14. Pospiech, T. and Neumann, J. (1995). In Genomic DNA isolation T. Kiesereds. John Innes Center. Norwich NR4 7UH.U.K.

15. Schultz, C, Pool GJ, van Ketel R, de Wever B, Speelman P, Dankert J (1994). Detection of enterotoxigenic Escherichia coli in stool samples by using nonradioactively labeled oligonucleotide DNA probes and PCR. J Clin Microbiol 32, 2393-2397.

16. Spicer, E.K., Noble, J.A. (1982). Escherichia coli heat-labile enterotoxin. Journal of Biological Chemistry 257, 5721-5782.

17. Trower, C. J.; Abo, S.; majeed, K. and voni Itzsteyn, M. (2000). Production of an Enterotoxin by a gastro-entertis associated Aeromonas strain. J Med. Microbiol. 49,121-126.

18. Yamamoto, T. and T. Yokota. (1983). Sequence of heat-labile enterotoxin of Eschericlhia coli pathogenic for humans. J. Bacteriol. 155,728-733.

19. Zhang, W.; Zhang, C.; Francis, D.H.; Fang, Y.; Knudsen, D.; Nataro, J.P. and Robertson, D.C. (2010). Genetic Fusions of Heat-Labile (LT) and Heat-Stable (ST) Toxoids of Porcine Enterotoxigenic Escherichia coli Elicit Neutralizing Anti-LT and Anti-STa antibodies. Infection and Immunity.78, 316–325.

The complete article is available as a PDF File that is freely accessible. The fully formatted HTML version can be viewed as HTML Page.

Medical Journal of Babylon

volume 9 : 1

Share |

Viewing Options

Download Abstract File

Related literature

Cited By
Google Blog Search
Other Articles by authors

Related articles/pages

On Google
On Google Scholar
On UOBabylon Rep

User Interaction

2870  Users accessed this article in 1 year past
Last Access was at
17/01/2017 01:12:07