Phylogenetic relationship of Citrobacter spp. and Other Enterobacteriaceae Isolated from Stool Samples of Healthy and Patients with Colorectal Cancer in Al-Najaf Al-Ashraf/Iraq

Prepared by the researche : Sahar Mohammad Jawad  ,  Leena Adeeb Mehdi – *Kufa University, Faculty of Medicine – Kufa University, Faculty of Pharmacy

DAC Democratic Arabic Center GmbH

International Journal of Environmental and Biological Sciences : First issue – January 2026

A Periodical International Journal published by the “Democratic Arab Center” Germany – Berlin

:To download the pdf version of the research papers, please visit the following link

https://democraticac.de/wp-content/uploads/2026/02/International-Journal-of-Environmental-and-Biological-Sciences-First-issue-%E2%80%93-January-2026.pdf

Abstract

    The current study aimed to detect phylogenetic relationships among enterobacteriaceae isolates. A total of 110 stool samples were collected from healthy and patients with coleractal cancer during the period 2016-2017. The results of PCR technique for amplification of tuf showed that 55 isolate (55%) were belong to Enterobacteriaceae (suspected Citrobacter) by appearance amplicon with molecular weight 871 bp while 45 isolate (45%) were belong to E.coli by appearance of amplicon with molecular wight 884 bp. Also, the results of electrophoresis of atpD1 and atpD2 showed that 36 isolates (56%) and 24 (42.8%) isolates were belong to E.coli by appearance of amplicon with molecular weight 884 bp while 32 isolates (57.1%) were belong to other Enterobacteriaceae (suspected Citrobacter) by appearance amplicon with molecular weight 871 bp. DNA sequencing of amplicon resulted from amplification of tuf, atpD1 and atpD2 was carried out to confirmed identification of Citrobacter spp as well as to evaluate the phylogenetic  relationship among bacterial isolates. Thirty isolates were selected for DNA sequencing depending one presence and absence of tuf, atpD1 and atpD2. The results of DNA sequencing and alignment with other enterobacterial sequences available in public database using MEGA 7 and BLAST showed that 4, 2 and 1 isolates gave strong alignment with E. coli, Klebseilla and Citrobacter.

Introduction:

Citrobacter is a Gram-negative, aerobic or facultative and rod-shaped bacterium which is considered a commensal species of the intestinal tract of humans and other animals (8). They were considered the intestinal inhabitants of human and animals, andhumansonly existed in sewage, water and soil and there are 12 recognized species within the genus Citrobacter, of which three were pathogenic bacteria in human. C. ferundii and C. koseri had caused meningitis in immunocompetent patients (6,12).

   Elongation factors (EF-Tu) are a set of proteins that are used in protein synthesis in the process of cell cycle and elongation in some cells. In the ribosome, they facilitate translational elongation, from the formation of the first peptide bond to the formation of the last one (7). It is encoded by tuf genes, carries aminoacyl- tRNA to the programmed ribosome during protein synthesis. Most enterobacterial genomes possess two copies of the tuf gene, tufA and tufB, in distinct operons, designated str and tRNA tufB, respectively (15). fusA gene, which encodes elongation factor G, is located upstream of tufA in the str operon of gammaproteobacteria (14). Four tRNA structural genes are located upstream of tufB in the tRNA-tufB operon, and the secE and nusG genes are downstream of tufB in most Enterobacteriaceae. tufA and tufB genes appear to evolve in concert through gene conversion events that maintain the sequence homology (10). A previous analysis of duplicated bacterial tuf genes revealed identical or very similar nucleic acid sequences that differ by less than 1.4%. (16). The two copies of the tuf gene (tufA and tufB) found in Enterobacteriaceae share high levels of identity (99 %) in Salmonella typhimurium and in Escherichia coli. A recombination phenomenon could explain the sequence homogenization between the two copies. (14, 17). Phylogenies based on protein sequences from elongation factor Tu and the F-ATPase b-subunit have shown good agreement with each other and with the rRNA gene sequence data.

        FATPase is present on the plasma membranes of eubacteria. It works mainly in ATP synthesis, and the b-subunit contains the catalytic site of the enzyme. The Elongation factors Tu and FATPase have been highly conserved throughout evolution and show functional constancy. Phylogenies based on protein sequences from elongation factor Tu and the F-ATPase b-subunit has shown good agreement with each other and with the rRNA gene sequence data (18).

In order to evaluate the discriminate role of tuf and atpD in identification of enterobacteriaceae member, this study aimed to investigate the phylogenetic relationships of Citrobacter spp with other enterobacteriaceae member isolated from patient with colorectal patients and healthy.

 Methods:

Samples collection

A total of 110 stool samples has been collected from healthy people (100 samples) and patients with colorectal cancer (10 samples) during the period from December 2016 to March 2017. All samples were collected in appropriate technique to avoid any possible contamination and cultured on MacConkey agar, Blood Agar Base and SS agar for primary isolation of enterobacteriaceae.

Identification of bacterial isolates

              All bacterial isolates were identified according to conventional methods using microscopic exam, culturing characteristics and biochemical test (MacFaddin, 2000). Furthermore, all bacterial isolates were re cultured on GHROM agar (Chromagar, France) to confirmed identification of enterobacteriaceae.

Molecular technique

PCR technique was used to confirm identification of bacterial isolates and to detect the phylogenetic relationship between isolates using elongation factors Tu gene (tuf) and F-ATPase β-sub unite genes (atpD1 and atpD2).

Extraction of Genomic DNA:

  Extraction of bacterial DNA using Boiling method has been followed as described previously (14). Briefly bacterial cell suspension boiled for 5 minutes then incubated in water bath at boiling temperature for 5 minutes, then, ice bath incubation for another 5 minutes. The lyses mixture was centrifuge at 15000 rpm/min then, the DNA was precipitated by mixing with isopropanol for 24 hrs and centrifuged again at 10000 rpm/min.

The DNA precipitate was conserved in TE solution and DNA concentration was measure by DNA-RNA spectrophotometer (Bio-Droop, England).

Amplification Reaction

The oligo-synthesis nucleotide sequences (iNtRON, Korea) that used in PCR technique were mention in table 1. PCR mixture was prepared to the final volume of 20µl by adding 3µl of the forward and revers primers and 6µl of DNA template to the reaction mixture of PCR (Maxime PCR PreMix Kit, iNtRON, Korea), then the volume was completed to 20μl of Nuclease-free water.  The thermo-cyclic conditions of each gene were set by thermocycler (Biometra, Germany) as shown in table 2 using gradient PCR. The amplification products were electrophoresing on 1% agarose gel stained with ethidium bromide at 70 V volt for 50 min. Then, the results were record using a gel documentation system (Biometra, Germany).

Table (1) The oligo-synthesis nucleotide sequences.            

Table (2) The Amplification Condition

DNA Sequencing

A partial sequencing of tuf, atpD1 and atpD2 has been carried out. The DNA sequences alignment was done using data available in NCBI depending on BLAST, also, MEGA 7 was used to analyze the phylogenetic relationships between bacterial isolates.

Results and Discussion:

The results of primary isolation and identification of Citrobacter spp. as well as other Enterobacteriaceae members on MacConkey agar, blood agar base and SS agar showed a wide distribution of Enterobacteriaceae members among all collected samples in which all 110 (100%) sample gave +ve culture.

       CHROM Agar and different selective culture media were used to confirm the primary isolation of Citrobacter and other species. The results showed a variable percentage of isolation of Citrobacter and other enterobacterial species in which a high percentage was detected to E. coli (Table 3)

Table (3) The percentage of bacterial isolates in clinical samples

Sixty bacterial isolates (10 isolates from colorectal cancer and 50 isolates from healthy) from CHROM agar were undergoes PCR technique to amplified tuf, atpD1 and atpD2 to confirmed identification of bacterial isolates. The results of agarose gel electrophoresis of amplicon resulted from amplification of tuf gene showed that 26 isolates (43%) belonged to E. coli while 40 isolates (66%) were belonged to other enterobact-eriaceae. Also, agarose gel electrophoresis of amplicon resulted from amplification of atpD1and atpD2 showed that 4 (6.6%) isolates and 9 (15%) isolates were belonged to E. coli respectively while 11 (18.3%) isolates and 18 (30%) isolates were belonged to other enterobacteriaceae respectively (Table4).

   Table (4) Comparison between conventional and molecular technique for identification of bacterial isolates.

The results of DNA sequencing of tuf, atpD1 and atpD2 amplicon showed a successful result for all amplicons and the results of DNA aligment with other nucleotide sequences available in NCBI using BLAST showed that a high percentage of identity of local DNA sequences of isolates to a DNA sequence available in NCBI data base as mention in table5.

Table (5) The DNA sequences alignment of tuf, atpD1 and atpD2

Furthermore, Phylogenetic tree between bacterial isolates using tuf , atpD1 and atpD2 was carried out using MEGA 7 program and depending on construct/ test neighbor – joining Tree. The results of phylogenetic relatedness showed variable consideration which could be explained by the following:

• The sequencing atpD1 separated five bacterial isolates included in this study in to tow group the first one included SAHAR 5 and 4 which is closely related to each other and also related with SAHAR 2 while the other group include SAHAR 3 and SAHAR 1. Both groups were closely related to Citrobacter from AX109531.1(Figure 1-A).
• In state of atpD2, variable results were obtained in which Citrobacter for cure closely related with SAHAR 10 while SAHAR 7 and 11 gave high similarity to each one. Whereas other isolates gave variable relatedness with both groups (Figure 1-B).
• Five groups were obtained when phylogenetic tree was depended on atpD1– atpD2, each group consist of 2 bacterial isolates that showed closely related to each other except one group that involved SAHAR 3 and 9 which appear the same isolates and gave relatedness with SAHAR 4 (Figure 2-A) .
• The results of sequencing of tuf segregate the bacterial isolates in to 3 group consist of 2 isolates in which were closely related together and SAHAR 18 and 19 appear as the same isolates. The relatedness between these isolates was more than that occur in atpD (Figure 2-B).
• The results of phylogenetic tree depending on atpD–tuf sequences showed that the isolates were segregated into four groups with low distances between each group (Figure 3).

Figure (1)   phylogenetic trees based on sequence data from (A) atpD1 (B) atpD2. The phylogenetic analysis was performed with the neighbor-joining test. Values on each branch indicate the display branch length. The percentage of identity between each branch was 100%. AX109531.1refer to Citobacter ferundi.

Figure (2) Phylogenetic trees based on sequence data from: (A) atpD1- atpD2 and (B) tuf. The phylogenetic analysis was performed with the neighbor-joining test. Values on each branch indicate the display branch length. The percentage of identity between each branch was 100%. AX109531.1refer to Citobacter ferundi.

Figure (3) Phylogenetic trees based on sequence data from atpD- tuf. The phylogenetic analysis was performed with the neighbor-joining test. Values on each branch indicate the display branch length. The percentage of identity between each branch was 100%. AX109531.1refer to Citobacter ferundi.

 Most Enterobacteriaceae are opportunistic pathogens, especially Escherichia coli, Klebsiella spp., Serratia spp., Enterobacter spp. and Salmonella, and are associated with significant morbidity and mortality (2,5). These bacteria can cause many different kinds of infections in wounds (sepsis), brain (meningitis) and diarrhea (4). (9) showed that   42.3% of isolates belonged to Citrobacter isolated from animals and other studies have been confirmed that Citrobacter, associated with bacteremia most commonly occurred in patients with malignancies (48.9%) or hepatobiliary stones (22.2%) or Intra-abdominal tumors (59.1%) (3, 21). (16) reported that the percentage of Citrobacter isolation from intraabdominal infection was 61.1% while from urinary tract was 8.3% and intravascular catheter was 5.6%, whereas soft tissue was 2.8%, in comparison with (19) pointed out the percentage of isolation of Citrobacter from stool sample of children with diarrhea was 14% . Bacteremia, which commonly originated from sites of infection with Citrobacter such as the abdominal cavity, urinary tract and lung, consist of 51.1%, 20%, 11.1% respectively (11,17, 20).

A single or dinucleotide polymorphism made tuf, atpD1 and atpD2 more sufficiently in identification of bacterial isolates to species level.  tuf and atpD tree exhibit very short genetic distances between taxa belonging to same genetic species including species segregated on the basis of clinical considerations (2 (.The variation in the results of phylogenetic trees referred to the presence of SNP in atpD and tuf  that lead to identification of bacterial isolates to sub species levels. One to three of SNP have been found in tuf and atpD. This SNP may result from hybridization between closely related species. Many studies referred to a closer relationship between Salmonella and C. freundii (10,19). (7) reported that tuf and atpD distances provide higher discriminating power at the species level.

Conclusions:

Elongation factor Tu and FATPase have been highly conserved throughout evolution and show functional constancy. Phylogenies based on protein sequences from elongation factor Tu and the F-ATPase b-subunit have shown good agreement with each other and with the rRNA gene sequence data. (19)

Reference:

1. Abdul karim F and Hughes D .1996. Homologous recombination between the tuf genes of Salmonella typhimurium. J Mol Biol. 260 (4): 506-22.
2. Adamson V; Mttt P; Pisarev H; Metsvaht T; Telling K; Naaber P and Maimets M. 2012. Prolonged outbreak of Serratia marcescens in Tartu University Hospital: a case–control study. Bio Med Cent Infec Dise. 12 (1): 281-286.
3. Adékambi T; Berger P; Raoult D; Drancourt M. 2006. rpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacterium bolletii nov., Mycobacterium phocaicum sp. nov. and Mycobacterium aubagnense sp. nov. Int J Syst Evol Microbiol. 56 (1):133-143.
4. Bister B; Bischoff D; Nicholson G J; Valdebenito M; Schneider K; Winkelmann G; Hantke K and Süssmuth RD.2004.The structure of salmochelins: C-glucosylated enterobactins of Salmonella enterica. Biometals Biolog Chemis 17 (4):471-481.
5. Budic M; Rijavec M; Petkovšek, Ž and Žgur-Bertok D. 2011. Escherichia coli Bacteriocins: Antimicrobial Efficacy and Prevalence among Isolates from Patients with Bacteraemia. Plos one. 6 (12): 1-11.
6. Clermont D; Motreff L; Passet V. 2015. Multilocus sequence analysis of the genus Citrobacter and description of Citrobacter pasteurii Syst Evol Microbiol.65 (5):1486-1490.
7. Drancourt M; Bollet C; Carta A; and Rousselier P. Phylogenetic analyses of Klebsiella species delineate Klebsiella and Raoultella gen. Syst Evol Microbiol. 51(3): 925–932.
8. Duman U; Saticioglu I B and Buyukekiz A G. 2017. Molecular characterization and antimicrobial resistance profile of atypical Citrobacter gillenii and Citrobacter isolated from diseased rainbow trout Glob Antimicrob Resist.10(5):136-42.
9. Hammerum A M. 2016.Use of WGS data for investigation of a long-term NDM-1-producing Citrobacter freundii outbreak and secondary in vivo spread of blaNDM-1 to Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca. J Antimicrob Chemother 71(11): 3117–3124.
10. Hazen T H; Sahl J W and Fraser C M .2013. Refining the pathovar parading via phylogenomics of the attaching and effacing Escherichia coli. Proc Natl Acad Sci USA.110 (3) :12810-12815.
11. Hoban D. J. In vitro susceptibility and distribution of beta-lactamases in Enterobacteriaceae causing intra-abdominal infections in North America 2010–2011.  Microbiol Infect Dis. 79 (3):367–372.
12. Honglian C; Yongjie W; Jing Z; Yunsheng C and Minglin W. 2018. Isolation and identification of Citrobacter from the intestine of Procambarus clarkii. J Fish Res. 2 (1): 1-6.
13. Klingenberg C; Sundsfjord A; Rønnestad A; Mikalsen J; Gaustad P and Flaegstad T. 2004. Phenotypic and genotypic aminoglycoside resistance in blood culture isolates of coagulase-negative staphylococci from a single neonatal intensive care unit, 1989– 2000. J. Antimicrob.Chemother. 54 (5): 889–896.
14. Kondrashov F; Gurbich A; and Vlasov K. 2007. Selection for functional uniformity of tuf duplicates in gamma-proteobacteria. Trends Genet. 23 (30) :215–218.
15. Lathe W and Bork P. 2001. Evolution of tuf genes: ancient duplication, differential loss and gene conversion. FEBS Lett. 502 (3):113–116.
16. Li-Hsiang L; Wang N; Alice u; Chih L; Chang-Pan L. 2018. Citrobacter freundii bacteremia: Risk factors of mortality and prevalence of resistance genes. J Microbiol Immunol Infect.51 (4):565-572.
17. Liyun L; Liqin L; Yonglu A; Yushi Z; Yiting W; and Jianguo X . 2017. Antimicrobial Resistance and Cytotoxicity of Citrobacter in Maanshan Anhui Province, China. Front Microbiol; 8 (5): 1357.
18. Ludwig W; Weizenegger M; Betzl D; Leidel E; Lenz T; Ludvigsen, A; Mollenhoff D; Wenzig P; and Schleifer K H. 1990. Complete nucleotide sequences of seven eubacterial genes coding for the elongation factor Tu: functional, structural and phylogenetic evaluations. Arch Microbiol. 153 (3): 241–247.
19. Paradis S; Boissinot M; Paquette N; Martel E A; Boudreau D K; Picard F J; Roy P H and Bergeron M G. 2013. Phylogeny of the Enterobacteriaceae based on genes encg elongation factor TU and F-ATPase -subunit. Int J Syst Evol Microbiodinol. 55(11): 2013– 2025.
20. Parte A C. 2018. LPSN – List of Prokaryotic names with Standing in Nomenclature (bacterio.net), 20 years on. International Journal of Systematic and Evolutionary Microbiology. 68(6): 1825-1829;
21. Tortoli E; Tutunji M F; Husam M I; Khabbas M H and Tutunji LF. 2009. Simultaneous determination of bisoprolol and hydrochlorothiazide in human plasma by HPLC coupled with tandem mass spectrometry. Jou of Chromat. 877(4): 1689–1697.