Iraqi J Pharm Sci, Vol.32( 1 ) 2023 Salmonella typhi and O-antigen encoded genes DOI: https://doi.org/10.31351/vol32iss1pp156-159 156 Isolation and Identification of Salmonella typhi from Clinical Samples with Molecular Detection of O-antigen Encoded Genes Huda J. *Mohemmad*,1, Semma H.Shalal*and Hadeel A.Ghajiri** *Department of Pharmaceutical Sciences, College of Pharmacy, Thi-Qar University, Iraq. **National University of Science and Technology, Thi-Qar, Iraq. Abstract Treatment of S. typhi is difficult as compared to treatment of acute infection. Antibiotic susceptibility test carried against S. typhi by using 6 kinds of antibiotics from different classes, their results showed that all isolates were high resistance to Ampicillin (99%), Gentamicin (98%), Amikacin (79%) and less resistances to Trimethoprim (55%), Imipenem (60%) and Ceftriaxone (66%).The present study focused on the molecular detection of Wzx flippase and Wzy polymerase genes in some Salmonella typhi isolates, Samples were collected from typhoid patients by classical lab work. Antibiotics susceptibility was investigated using the disc diffusion method. The DNA and molecular Wzx flippase, Wzy polymerase were implemented using specific primers. The results showed that there was 33.33% had Wzy gene. The Wzx gene did not observe in any Salmonella isolates. The present study concluded that there was an importance of the genetic diversity of O-antigen encoded genes included Wzx and Wzy, which may be effective in typhoid diagnosis and treatment. Keywords: Molecular Identification, Wzx flippase, Wzy polymerase genes salmonella typhi Oمن العينات السريرية مع الكشف الجزيئي للجينات المشفرة للمستضد S. Typhi عزل وتشخيص ** غجيريهديل علي و *سيماء حسن شالل ،1، *هدى جاسم محمد العراق قار، ذي ، قارجامعة ذي كلية الصيدلة، فرع العلوم الصيدالنية، * ** كلية طب االسنان، الجامعة الوطنية للعلوم والتكنولوجيا ، ذي قار، العراق . الخالصة 6باستخدام S.typhiتم تحديد المضادات الحيوية ضد . أن العالج بالمضادات الحيوية من التيفوئيد صعب مقارنة بعالج العدوى الحادة لألمبيسيلين مقاومة عالية ، وأظهرت نتائجها أن جميع العزالت كانت ذات مختلفة الجنتاميسين ( ٪99)أنواع من المضادات الحيوية من فئات ، (. ٪66)ياكسون ، سيفتر( ٪60)، إيميبينيم Trimethoprim (55 ٪ )وأقل مقاومة( ٪79)، أميكاسين ( 98٪) في بعض عزالت السالمونيال التيفية ، وتم Wzy polymeraseو Wzx flippaseركزت الدراسة الحالية أيضا على الكشف الجزيئي لجينات . تم فحص حساسية المضادات الحيوية باستخدام طريقة االنتشار القرصي. بري التقليديت جمع العينات من مرضى التيفوئيد عن طريق العمل المخ لم . Wzy٪ لديهم جين 33.33أن وأظهرت النتائج. باستخدام بادئات محددة Wzy polymeraseالجزيئي ، Wzx flippaseتمت دراسة وجود المتضمنة Oاستنتجت الدراسة الحالية أن هناك تنوًعا جينيًا مهًما في الجينات المشفرة لمستضد . في أي من عزالت السالمونيال Wzxيالحظ جين Wzx وWzy والتي قد يكون لها تأثير في تشخيص التيفوئيد وأنواع العالج. بكتريا التيفوئيد , Wzx , Wzyلكلمات المفتاحية : التشخيص الجزيئي , جينات ا Introduction Salmonella typhi is an enterobacteriaceae member responsible for typhoid fever, its Gram negative and serological positive to lipopolysaccharide antigens O9 and O12 in addition to polysaccharide capsular antigen Vi (1,2). The diversity of antigen –O in bacterial strains regarding to the genetic polymorphisms in the rfp cluster that coded the enzymes involved in the synthesis of construction of O-antigen (3), these genes included the enzymes that contributed in sugar synthesis, transferase for construction sugar and O subunit, and genes encoded proteins involved in construction O antigen via assembly subunits, these genes included wzx and wzy that encoded to O-antigen transporter or flippase and O-antigen polymerase respectively (4,5,6). The Wzx proteins in Salmonella enterica the O-antigen transport mechanism is dependent on the Wzx proteins (8(. The O-antigen variability which basis on the shigella serotypes schemes, there are 12 O-antigen clusters includes (2, 10, 12, 23, 25, 26, 32- 34, 66, 75 and 76) these clusters located between rep and aqpZ genes on chromosomes (4,9). Bacteria: The Salmonella typhi isolates were collected from patients suffering from typhoid fever who attended to the hospital centers. After clinical and serological diagnostic tests, collected samples using conventional methods by using blood agar, XLD agar and SSA agar The antibiotic susceptibility detected using disc diffusion method (Ampicillin, Gentamicin, Amikacin, Trimethoprim, Imipenem and Ceftriaxone). The multi-resistance isolates were used for molecular detection of Wzx flippase and Wzy polymerase genes. 1Corresponding author E-mail: huda.jassim@utq.edu.iq Received: 9/1 /2022 Accepted: 10/5 /2022 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol32iss1pp156-159 Iraqi J Pharm Sci, Vol.32( 1 ) 2023 Salmonella typhi and O-antigen encoded genes 157 PCR experiment Bacterial DNA was extracted from all strains using the appropriate kit. The molecular detection of Wzx flippase and Wzy polymerase genes were implemented using the following primers (5). wzx (F): CCG GGT TTC GAT TTG TGA AGG TTG, (R): CAC AAC AGC CAC TAC TAG GCA GAA wzy (F): GAA ATT ATG CCA TCT TGG CGA GCG (R): CAT GTG AAG CCT GAA GGC AAA CTC The following conditions were used for PCR. 5 min 94˚C, 35 cycles consist of 30 s 94˚C, 30 s 59˚C, 30 s 72˚C, and 10 min at 72˚C for both primers in mono-plex reactions (10). PCR products were visualized in 1% agarose, TBE 0.5 X for 1 hour, 70 V and 20 mA under UV trans-illuminator. Results Bacterial Isolation: strains of S. typhi were recovered from blood samples of outpatients confirmed by conventional method plus Vitek identification (Table1) Table 1. Bacterial isolation during the study Sample No. Bacteria Widal test Conventional and Vitak 1 Salmonella typhi + + 2 Salmonella typhi + + 3 Salmonella typhi + + 4 Salmonella typhi + + 5 Salmonella typhi + + 6 Salmonella typhi + + 7 Salmonella typhi + + 8 Salmonella typhi + + 9 Salmonella typhi + + Antibiotic susceptibility test The results of the present study show that, there were 9 isolates were multi-resistance to antibiotics in different manner, Figure1. Figure 1. Multi drug resistances Salmonella typhi isolates (n=9) Molecular detection of Wzx and Wzy genes Genomic DNA was extracted from bacterial cells using a DNA extract kit. Figure2. Figure 2. Gel electrophoresis 2% of DNA extracted from Salmonella typhi isolates Polymerase chain reaction were applied for detection of Wzx and Wzy genes The molecular detection of Wzx and Wzy were showed that some isolates of S. typhi had wzy gene while no isolates detected that have wzx gene. Figure 3. Figure 3. PCR product of Salmonella typhi isolates carrying wzy gene with molecular weight of 451bp visualized on 1% agarose (conditions). Percentage of Wzx gene was 33.33% from all samples while the percentage of presence of Wzy was 0%. Table 2 and Figure 4. Iraqi J Pharm Sci, Vol.32( 1 ) 2023 Salmonella typhi and O-antigen encoded genes 158 Table 2. Presence of Wzy and Wzx gene in different isolates of Salmonella typhi. Sample No. Bacteria Wzy gene Wzx gene 1 Salmonella typhi + - 2 Salmonella typhi - - 3 Salmonella typhi - - 4 Salmonella typhi + - 5 Salmonella typhi - - 6 Salmonella typhi + - 7 Salmonella typhi - - 8 Salmonella typhi - - 9 Salmonella typhi - - Figure 4. The percentage of Wzy and Wzx genes in Salmonella typhi. Discussion In the present research, the resistance to common antibiotics used in the treatment of S. typhi was tested and appeared that all the persisting isolates of S. typhi obtained from patients who suffered typhoid fever showed high resistance to gentamycin, ampicillin amikacin, and ceftriaxone; these results agree with Andualem et al. and Al- aarajy et al. (11, 12) they were found that all isolates were resistant to four or more classes of antibiotics as antimicrobials. The present study implemented to identifyWzx and Wzy genes in Salmonella typhi isolates that have an important role in typhoid diagnosis via O-antigen, although of the low sensitivity and specificity of Widal test in typhoid diagnosis, it is still used in some labs (11), on the other hands the false negative results (13, 14) may be associated with genetic diversity of O-antigen genes which formed diversity in O-antigen proteins. The evidence found that according to The Kauffmann-White scheme there are 46 somatic O antigens of Salmonella, thus the diversity of O-antigens encoded genes can be used in the PCR development test to molecular serotypes identification. The negative results in the present study of Wzx gene and in Wxy (66.66%) maybe because of genetic diversity in target loci such as duplication, formation of the pseudogene, deletion, and insertion of the bacteriophage elements that occur ubiquitously through serogroups (15). The present finding needs more investigation to detect the genetic diversity of O-antigen in S. typhi which may have an important role in serotypes identification and development of knew molecular tests for typhoid fever diagnosis. The detection of these genes in S. typhi was poor in the last decades, Yara et al (13) in a study dealing with the pangenome of Salmonella O-Antigens, they found high metabolic and genetic differences within and across O-antigen groups in Salmonella strains. In Iraq, there were high multi-resistance bacterial isolates in the last decade which contributed to the health problems of diseases infection and this need to focused efforts for developing a new method for diagnosis and treatments based on the new finding like O-antigen encoded genes. Conclusion The analysis of the drug susceptibilities of the isolates was observed that all isolates were multidrug-resistant (MDR). Besides, Salmonella typhi is shown to persist chronically within the patients and molecular methods are more rapidly than the conventional method. The present study concluded that there was an importance of the genetic diversity of O-antigen encoded genes including Wzx and Wzy which may be effective in typhoid diagnosis and treatment types. References 1. Falcao, D. P., Trabulsi, L. R., Hickman, F. W., & Farmer 3rd, J. J. Unusual Enterobacteriaceae: lactose-positive Salmonella typhimurium which is endemic in Sao Paulo, Brazil. Journal of Clinical Microbiology.1975;2(4):349-353. 2. Ashurst JV, Truong J, Woodbury B. Salmonella typhi.In: StatPearls. Treasure Island (FL): StatPearls Publishing; Jan2020. 3. Tarr PI, Schoening LM, Yea YL, Ward TR, Jelacic S, et al. Acquisition of therfb-gndcluster inevolution ofEscherichia coliO55 and O157. J Bacteriol . 2000; 182: 6183 – 6191. 4. Samuel G, Reeves P Biosynthesis of O-antigens: genes and pathways involved in nucleotidesugar precursor synthesis and O-antigen assembly. Carbohydr Res .2003; 338: 2503–2519. 5. Patel, K. B., Toh, E., Fernandez, X. B., Hanuszkiewicz, A., Hardy, G. G., Brun, Y. V., Bernards, M. A., & Valvano, M. A. Functional characterization of UDP- glucose:undecaprenyl- phosphate glucose-1-phosphate transferases of Escherichia coli and Caulobacter crescentus. Journal of bacteriology.2012;194(10):2646– 2657. 6. Vinés, E. D., Marolda, C. L., Balachandran, A., & Valvano, M. A.. Defective O-antigen Iraqi J Pharm Sci, Vol.32( 1 ) 2023 Salmonella typhi and O-antigen encoded genes 159 polymerization in tolA and pal mutants of Escherichia coli in response to extracytoplasmic stress. Journal of Bacteriology.2005;187(10): 3359–3368. 7. Liu, D., R. A. Cole, and P. R. Reeves. An O- antigen processing function for Wzx (RfbX): a promising candidate for O-unit flippase. J. Bacteriol. . 1996 ; 178:2102-2107. 8. Fitzgerald, C., Sherwood, R., Gheesling, L. L., Brenner, F. W., & Fields, P. I. Molecular analysis of the rfb O antigen gene cluster of Salmonella enterica serogroup O:6,14 and development of a serogroup-specific PCR assay. Applied and Environmental Microbiology. 2003; 69 (10) , 6099–6105. 9. Liu B, Knirel YA, Feng L, Perepelov AV, Senchenkova SN, Reeves PR, Wang LStructural diversity in Salmonella O antigens andits genetic basis. FEMS Microbiol. Rev. 2014;38: 56-89. 10. Al-Terehi M, Khazaal S, Muhammed H, Behjet R. Molecular Detection of wzx1 and wzy Genes in Multi Drugs Resistance E. coli Isolates. International Journal of Pharmaceutical Quality Assurance 2018; 9(3); 304-307 11. Andualem, G., Abebe, T., Kebede, N., Gebre- Selassie, S., Mihret, A., & Alemayehu, H. A comparative study of Widal test with blood culture in the diagnosis of typhoid fever in febrile patients. BMC research notes.2014; 7: 653. 12. Al-aarajy, A. Nabeel. Assessment of Silver Nanoparticles as Anti- Salmonella Agent: Phenotypic, Genotypic and Histological Study .Thesis Degree of Doctorate of Philosophy of College of Science- University of Anbar2020. 13. AlObaidi Ahmed Flyyih; Mohammed N. T.; Abdulrazzaq Mohammed and Basim Basima.Molecular detection of Salmonella typhi isolated from diarrheal patients in Al- Najaf governorate .Annals of Tropical Medicine & Public Health . 2019; 22(9). 14. Olopoenia LA, King AL. Widal agglutination test - 100 years later: still plagued by controversy. Postgrad Med J. 2000;76:80–84. 15. Yara Seif, Jonathan M. Monk, Henrique Machado, Erol Kavvas, Bernhard O. Palsson Systems Biology and Pangenome of Salmonella O-Antigens, Ecological and Evolutionary Science .2019; 10 (4) : e01247-19. This work is licensed under a Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/