THE DR FAMILY - AFIMBRIAL ADHESIN GENE IN UROPATHOGENIC ESCHERICHIA COLI ISOLATED FROM PATIENTS SUSPECTED WITH URINARY TRACT INFECTION.

DNA extraction done followed by PCR amplication were processed for detection of afa genes using specific primer and the specific base pair bands were noted using gel documentation. Results: The afa gene was found in 49 isolates (23.1%) of UPEC out of 212 isolates, which were distributed among male patients 19(38.8%) and 30 (61.2%) in female patients with more prevalence among less than 20 years of age group. Conclusion: afa, though a low frequency gene in UPEC showed high percentage in this study comparing the other geographical areas out of its low frequency. Conclusion:-Afa gene, a low frequency gene found in UPEC compared to other adhesin genes. Noting the rate of prevalence of afa among different geographical areas, this study has higher percentage of afimbrial adhesin which is noted for its significance.


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can only be detected by molecular method. The aim of the study is to determine the prevalence of afa gene in UPEC by PCR.

Materials and methods:-Bacterial isolates:
A total of 212 E. coli isolated from suspected UTI patients were identified by standard culture using Blood agar, Mac Conkey agar and Hichrome agar and the biochemical identification processed were Catalase, Oxidase, Indole, Methylred -Voges prauskauer, Citrate, Urease, Triple sugar iron agar, Mannitol motility medium, glucose fermentation. Institutional Ethical Committee (IEC) approval was obtained and the study was progressed [4].
Genotypic detection of papG and fimH gene by PCR: DNA extraction: DNA extraction was performed by using boiling lysis method. E. coli isolates were cultured in Luria Bertani broth at 37 o C for 18 hours. Bacteria were pelleted from 1.5 ml LB broth using centrifuge at 3000 rpm then suspended in 200 ml of sterile deionized water and kept in water bath at 100 o C for 10 min. After centrifuging, the supernatant was used as template DNA and stored at -20 o C [5].

PCR amplification:
PCR amplification of virulence genes were used to reveal the prevalence of afimbrial adhesin, afa gene using specific primers. The amplification of virulence genes was carried out in a Thermal Cycler (Eppendorf Master Cycler) after standardizing the PCR conditions: an initial denaturation at 94 ο C for 2 min, followed by 30 cycles of denaturation at 94 ο C for 1 min, annealing at 60 ο C for 30 s, and extension at 72 ο C for 1min 30 sec, with a final extension at 72 ο C for 5 min.\ PCR products were then loaded in 1% agar gel electrophoresis and amplified DNA fragments were detected by UV fluorescence transilluminator and the size of the amplicons was estimated by comparing with 100bp DNA ladder.

Results:-
All the bacterial isolates were identified using standard bacteriological methods. The afa gene was amplified using PCR with specific primers and the specified band of 672 bp on polyacrylamide gel compared with 100 bp ladder was determined to be the presence of afa gene (Figure 1). Afimbrial adhesin gene was found in 49 isolates (23.1%) of UPEC out of 212 isolates.  The total of 49 (23.1%) afa genes were distributed in the ratio of 19 (38.8%) and 30 (61.2%) among E. coli isolated from male and female patients respectively ( Table 2). afa is more prevalent in the age group less than 20 years, followed by 41 -60 years (28.6%) and least in the age group of > 80 years (2%), (Table 3).

Discussion:-
Afimbrial adhesin of the Dr Family have a specific renal tissue tropism involved in UTI. It has a potential property which favors the establishment of chronic and recurrence of UTI, suggested by experimental and clinical findings [7]. These adhesins strength of binding is potential as it is responsible for chronic interstitial nephritis and chronic pyelonephritis [8].
The Dr Family afimbrial adhesin possess Afa-I and Afa-III found in many UPEC strains binds to the receptor on the Dr Blood group antigens present on the decay accelerating factor (DAF), which prevents the lysis of the cell by complement action. Once after binding to the host cells it activates signal pathways and cascade reactions and induces the cytopathic effect through development long cellular extensions wrapping around the adherent bacteria, which is the characteristic of Dr Family group of adhesins [2].
This study found the prevalence of 49 (23.1%) of afa gene, which are distributed among 38.8% of male and 61.2% female patients where it is less in a study conducted by Mohajeri et al., 17 (8.2%) afa genes, 9.5% and 8% among male and female respectively. Among age groups, the higher percentage were noted in <20 years 30.6%, similar to the study Mohajeri et al., were the age group between 1-10 years and 11-20 years is higher comparing to other age groups.  Table 4 represents the prevalence and distribution of afa genes in different areas. Though the prevalence of afa gene is less comparing to other adhesin factors, this afa gene distribution is higher in this study while reviewing other studies. So, the distribution of afa gene differed in different geographical regions.