Isolation and Identification of Salmonella typhi from Clinical Samples with Molecular Detection of O-antigen Encoded Genes

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


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.

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) (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)

Antibiotic susceptibility test
The results of the present study show that, there were 9 isolates were multi-resistance to antibiotics in different manner, Figure1.

Molecular detection of Wzx and Wzy genes
Genomic DNA was extracted from bacterial cells using a DNA extract kit. Figure2.

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. Percentage of Wzx gene was 33.33% from all samples while the percentage of presence of Wzy was 0%. Table 2 and Figure 4.

Sample
No.

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 Alaarajy 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.