Molecular Detection of Flu, Csga Genes of E. coli in Apparently Healthy Student in AL-Muthannaa City
Yahye Mahdi Radi AL- Abady and Dr. Ashwak Bassim Jassim AL-Hashimy Institute for Genetic Engineering and Biotechnologies, University of Baghdad, Iraq Abstract:
Four hundred midstream urine sample (MSU) were collected from students apparently healthy in ten primary schools in Al-Muthannaa city from the beginning of October 2019 to the end of February 2020. The collected urine were culture on different media (MacConkey agar, Eosin Methylene Blue), and tested biochemically in order to find out the profile of E.coli.
polymerase chain reaction technique has been used to detect some of virulence genes Flu, and CsgA which encoding by E.coli isolates. The results demonstrated that out of total 40 isolates (20 urine + 20 stool) there were 25 isolate (62.5%) was positive for Flu gene, The prevalence of Flu gene in commensal was significantly lower (50%) than that in recurrent higher UPEC (75%). 31 isolate (77.5 %) were positive for CsgA gene, The result found a high prevalence (80%) of CsgA in UPEC isolates, while CsgA gene was detected in (75%) of the intestinal commensal isolates . In this study, describe the profile of E.coli bacteria isolated from healthy Iraqi children with no symptoms, for UTI or diarrhea by the identification of virulence genes.
Keywords: Molecular detection, Flu gene, CsgA gene, E. coli Introduction:
Commensal bacteria are an important reservoir of antibiotic resistance genes to pathogenic strains .there was carriage of multiple resistance commensal E.coli irrespective of exclusive mode of feeding ,or previous history of use of antibiotic or other parenteral drug .carriage of multiply resistant commensal E.coli was present even in infants with no history of use of any drug , there are various environmental and social reasons,(Oluyege et al ., 2015).
Young children tend to be the most exposed to antibiotics and several studies have found that the younger children have the highest risk of carrying resistance commensal bacteria (Duerink et al ., 2007).The nonpathogenic strains of E.coli referred to as commensal strains are harmless and are useful , not only digesting and breaking down food but also in protecting against harmful organisms which may be introduce into the gastrointestinal tract through food and water, (Chinen and Rudensky AY.,2012). However ,such commensal can have access to other part of the body such as urinary tract , blood or wounds and causes opportunistic infection particularly in immune-compromised individual , E.coli have been shown to develop resistance in response to antibiotic use and to be particularly capable of exchanging antibiotic resistance genes with Pathogenic bacteria, ( Karami et al .,2007)
Materials and Methods:
A total of 400 urine samples were collected from school age children (6-12) years apparently healthy during the period from October 2019 to the end of February 2020 .
The urine of these children were identified by methods of routine tests, colony depending on primary diagnosis , further stool samples were collected from positive E.coli urine culture children and cultured for isolation of E.coli further detection was done by PCR using TrpA gene . Molecular Detection:
DNA was extracted from activated pure culture of E.coli bacteria by using DNA genomic bacteria kit (promega) .detection of DNA band using agarose gel electrophoresis (1%) .Then Conventional PCR were used to detect the important virulance genes ( Flu , CsgA ) of E.coli bacteria using specific primers shown in table (1) .
Table 1- Sequence of primers of virulence gene.
Gene Primer sequence 5´---3´ Amplicon size Refrence
TrpA 5´-GCTACGAATCTCTGTTTGCC_3´ 784 (Plantamura
et al., 2019) 5´-GCTTTCATCGGTTGTACAAA-3´
CsgA For 5´-GATCTGACCCAACGTGGCTTCG-3´ 178 ( Silva et al.,2014) Rev 5´-GATGAGCGGTCGCGTTGTTACC-3´
Flu For 5´-CGGCGGGCAATGGGTACA-3´ 384 (Restieri et al.,2007) Rev 5-CAGCTCTCACAATCTGGCGAC-3´
PCR protocol :-
The PCR amplification mixture which used for detection of each gene include FIREPOL®
master mix12.5 , 4 µl of DNA template, 1 µl of each forward and reversed primer and 6.5 µl of nuclease free water to complete the amplification mixture to 25 µl table (2).
Table (2):- The PCR mixture components for each gene .
Component Volume (µl) Reference
Forward primer 1 µl (Plantamura et al., 2019
)
Reverse primer 1 µl
DNA templet 4µl
Master mix 12.5µl
Deionize water 6.5µl
Final volume 25µl
After preparing the reaction volume in PCR tube the Mixture was spin down and then PCR tube placed in the PCR thermo cycler and the amplification reaction and condition for each gene was started according to the program described in table (3).
Table( 3):- PCR thermo cycler program for DNA amplification and condition of E. coli genes
.
Stage temperature Time Number of cycle
Initial denaturation 95cº 5min 1
Denaturation 95cº 30 sec
Annealing xº 30sec 30
Extension 72cº 60sec
Final extension 72cº 5min 1
( Xº ), annealing temperature for each primer of virulence gene as fellow; TrpA , Flu , and CsgA as 58c ◦55cº, 60cº respectively.
Results and discussion:-
A total of 400 urine sample were collected from school age children (6-12) from October 2019 to the end of February 2020 from both gender. All colonies were diagnosed in the laboratory as E.coli bacteria due to characteristic lactose fermenting colonies , daunt shaped and dark pink in color due to reduce pH below 6.8 Figure (1, A),(1,B).
Figure (1 ):- A-Colonies of E.coli on MacConkey shape , B- colonies of E.coli on EMB agar . While the colonies on EMB agar plates appeared metallic shine, which is characteristic for E.coli bacteria from other bacteria strains belong to the intestinal family to contain this media eosin and methylene blue dye Figure (1,A,B).
It was found that 64 (80%),(32) sample urine and (32) sample stool of the total samples were positive for culture for E.coli were as 16 (20%) negative ,but positive for other type bacteria .the
extraction of chromosomal DNA was done for(50/64)isolates that diagnosed by vitek 2 system.
The results of gel electrophoresis showed presence of DNA band in the same level in 50 isolate only Figure (2).then the concentration and purity of extraction DNA was measured by Quant flour.
Figure(2): Gel Electrophoresis of genomic DNA extracted from E.coli isolates in 1%
agarose gel at 75 Volt/Cm for 30 min then visualized under U.V .after staining with Ethid bromide.
Further molecular diagnosis of E.coli bacteria by using TrpA gene specific primer and the results were detected by electrophoresis 2% agarose and exposed to U.V light in which the results indicates that only 40 isolates give the same results for the biochemical diagnosis Figure (3).
Figure (3): Gel Electrophoresis of PCR amplified products of E.coli TrpA gene Lane L:
DNA molecular weight Marker (1500pb), Lan ( 40 ) positive band at (784 pb) for TrpA gene, Lan (10) negative control .
The result revealed that clear bands appeared for Flu gene in 25 (62.5%), the prevalence of Flu gene in commensal was significantly lower 10(50%) than in UPEC 15 (75%). This gene representive member of the auto transporter family which have functions ranged from cell adhesion to the secreted of toxin Ag43 figure (4).
Figure (4):Gel electrophoresis of amplified Flu gene (384bp) from E.coli using conventional PCR. Agarose 1%, 70 V/cm for 80 min, stained with ethidium bromide dye and visualized on a UV trans illuminator. Lane (M): 100 pb DNA ladder. Lane (1-15 ): Amplicon of Flu gene.
Furthermore the investigation showed the presence of CsgA gene in 31 isolates (77.5%) in a total as shown in Figure (5).
Figure (5) :- Gel Electrophoresis of amplified CsgA gene (178 pb) from E.coli Agarose 2% , 70 v/cm for 80min , stained with ethidium bromide dye and visualized on UV. trans illuminator . Lan (M) : 100pb DNA ladder.Lan (1-5) : Amplicon of CsgA genes .
We found a high prevalence (16/31) of CsgA in UPEC isolates , while CsgA was detected in (15/31) of intestinal E.coli .
The result presented her go a long nicely with the results recorded by Hung et al .,(2014) who found that CsgA gene presents in 16 (73%) from 22 E.coli isolates from children with UTI in India.
Curli fimbria is a fibrous surface protein that is important for biofilm development by E.coli and its presence is associated with sever human infection fimbria interacts specifically with host matrix proteins such as fibronectin, laminin and plasminogen to initiate a adherence to and
colonization of the host Nhu et al (2018).
References
1. Chinen T ; and Rudensky A.Y.(2012). The effects of commensal microbiota on immune cell subsets and inflammatory response, Immunol Rev. 2012 Jan;245(1):45-55. doi:
10.1111/j.1600-065X.2011.01083.x.
2. Duerink D.O ; Lestari E.S ; Hadi U ; Nagelkerke N.J ; Severin J.A ; and Verbrugh H.A, et al(2007). Study Group Antimicrobial resistance in Indonesia: Prevalence and Prevention (AMRIN): Determinants of carriage of resistant Escherichia coli in the Indonesian population inside and outside hospitals. J. Ant micro Chemotherapy, 2007 (60): 377-384.
3. Hung C ; Marschall J ; Burnham C.A.D ; Byun A.S ; Henderson J.P.(2014). The Bacterial Amyloid Curli Is Associated with Urinary Source Bloodstream Infection . PLOS one | Volume 9 | Issue 1 | e86009 .
4. Karami N ; Martner A ; Enne V.I ; Swerkesson S ; Adlerberth I; and Wold A.E.(2007).
Transfer of an Ampicillin resistance gene between two Escherichia coli strains in the bowel microbiota of an infant treated with antibiotics. Journal on Antimicrobial Chemotherapy. 2007. 60 (5): 1142-5.
5. Nhu N.T.K ; Phan M.D ; Peters K.M ; Lo A.W ; Forde B.M;and Chong T.M. ; et al .(2018). Discovery of New Genes Involved in Curli Production by a Uropathogenic Escherichia coli Strain from the Highly Virulent O45:K1:H7 Lineage. mBio Journal, Volume 9 Issue 4 e01462-18.
6. Oluyege A.O ; Ojo-Bola O ; and Oludada O.E.(2015). Carriage of Antibiotic Resistant Commensal E. coli in Infants below 5 Months in A do-Ekiti, Int.J.Curr.Microbiol.App.Sci (2015) 4(5): 1096-1102.
7. Plantamura J ; Bousquet A ; Védy S. S ; Larréché S ; Bigaillon C ; and Delacour H ; et al . (2019) . Molecular epidemiological of extended-spectrum β-lactamase producing Escherichia coli isolated in Djibouti. J Infect Dev Ctries . 2019 Aug 31;13(8):753-758.
Doi:10.3855/jidc.11283. PMCID: PMC3890832 PMID: 24335802Microbiolog.
8. Restieri C ; Garriss G ; Institutet K ; Locas M.C ; and Dozois M.C (2007).
Autotransporter-Encoding Sequences Are Phylogenetically Distributed among Escherichia coli Clinical Isolates and Reference Strains, April 2007Applied and Environmental Microbiology 73(5):1553-62 DOI:10.1128/AEM.01542-06.
9. Silva V.O ; Soares L.O ; Júnior A.S ; Mantovani H.C ; Chang Y. ; and Moreira M.S.
(2014) . Biofilm Formation on Biotic and Abiotic Surfaces in the Presence ofAntimicrobials by Escherichia coli Isolates from Cases of BovineMastitis. Applied and Environmental Microbiology 80(19).
