THE CHARACTERIZATION OF MULTIDRUG RESISTANT TYPE 1 S-FIMBRIATED ESCHERICHIA  COLI FROM WOMEN WITH RECURRENT URINARY TRACT INFECTIONS (RUTIS) IN BANGLADESH 

Hasan1, M. K., Momtaz2, F., Foysal1,3*, M.J., Ali1, M.H., Islam1, K., Prodhan1, S.H. 
Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet 3114, 
Bangladesh;

Department of Microbiology, University of Chittagong, Chittagong 4331, Bangladesh;

School of  Molecular and Life Sciences, Curtin University, WA 6845, Australia. 
Correspondence: Md Javed Foysal*, School of Molecular and Life Sciences, Curtin University, WA6845, Australia & 
Assistant Professor, Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and 
Technology, Sylhet-3114, Bangladesh. Email: [email protected], [email protected] 
Corresponding author 
The characterization of multidrug resistant type 1 S-fimbriated Escherichia coli from women with recurrent urinary tract 
infections (RUTIs) in Bangladesh

 

 

INTRODUCTION 
Urinary tract infections (UTIs) are the systemic bacterial infections that are known to affect the urethra, urinary bladder, and kidneys. Females are mostly infected due to their anatomical arrangement- a shorter urethra, resulting in an easier travel by the bacteria. Previous studies have revealed that around 50–60% of women are likely to develop UTIs in their lifetime. The numerous causative agents, responsible for this disease; however, E. coli alone accounts for 80–85% of the global UTIs. Recurrent urinary tract infections (RUTIs) are the reinfections that are generally caused by the original bacterial isolates at a young age. Frequent sexual intercourse increases the chance of getting RUTIs. 

 

Approximately one third of women are found to be positive for RUTIs by the same bacteria. In some cases, RUTIs can be lethal when the bacteria persist for a long time in a position. Drug resistance in E. coli is one of the most common barriers for treating UTI patients worldwide. However, the problem is more severe in countries like Bangladesh due to an improper tendency of frequently prescribing antibiotics for the treatment of UTIs. The multidrug-resistant strains of E. coli have been reported to further add to the complications in the UTI patients and decrease the effectiveness of the treatment. 

 

In addition, patients with RUTIs have been reported to have a higher prevalence of antimicrobial resistance due to the evolution and spread of more virulent strains by various genetic mechanisms. The frequency of RUTIs caused by the multidrug resistant strains of E. coli has increased recently and has sparked strong attention from the government, medical practitioners, and health agencies.

 

Therefore, regional studies on the patterns of antibiotic sensitivity are much needed for selecting proper treatment strategies to overcome the massive problem of drug resistance. Virulence factors play a major role in the pathogenicity of E.coli associated RUTI infections. The S- fimbriae adhesin (sfa) genes, encoded by sfa operon are common in all the types of UTIs found to be strongly associated with E.coli pathogenicity. The sfa gene is associated with UTIs, gestational pyelonephritis, recurring cystitis, pregnancy complications, and diarrhea. 

 

The isolates of E. coli, especially from the uropathogenic (UPEC) and the diffusely adhering (DAEC) groups are known to produce this virulence factor that mediates the host-pathogen interactions. Recent development in molecular techniques, especially the 16S rRNA gene sequencing tool has been extensively used for the analysis of bacterialspecies in clinical samples (16, 17). In addition, the introduction of computer-aided bioinformatics tools in sequence analysis has simplified the understanding of the strains that are poorly characterized and rarely isolated. Therefore, the aim of the present study was the characterization and phylogenetic positioning of the isolates of E. coli from women with RUTIs, by using the computer-aided bioinformatic analysis of their 16S rRNA gene sequences.

 

MATERIALS AND METHODS 
Ethics statement 
This work has been conducted in accordance with“The Code of Ethics of the World Medical Association”. The Graduate Research Ethics Committee (GREC) of the School of Life Sciences, Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh, approved and monitored the study. The patients consent data were handled according to human privacy rights. The collection and culture of the bacterial isolates. A total of 15 isolates of E. coli (E1-E15) were collected from three different hospitals: the Popular Medical Diagnostic Centre (PMDC), Sylhet, Bangladesh, the Jalalabad Ragib-Rabeya 
Medical College and Hospital (JRRMCH), Sylhet, Bangladesh, and the MAG Osmani Medical College and Hospital (MOMCH), Sylhet, Bangladesh from January, 2017 to December, 2017. 

 

After screening the patient data, samples were collected from only those women, who were having a history of RUTIs. In addition, other information about the patients, including patient ID (PID), age, and the types of infection were also recorded from the patient consent forms (Table 1).  Primarily, all the patient samples were inoculated onto a chromogenic medium, containing Eosin Methylene Blue (EMB) and incubated at 37 °C for 24 h at the Biochemistry and Microbiology laboratory of PMDC, Sylhet, Bangladesh. The samples were then transported, at a low temperature, to the laboratory for further studies. 

 

The biochemical characterizations 
All the isolates of E. coli were subjected to several biochemical and microbiological tests, following the Bergey’s manual for the presumptive identification of E. coli (18). The isolates were assayed for Gram staining, catalase test, oxidase test, oxidative-fermentative (OF) test, H2S production test, Methyl Red (MR) test, VogesProskauer (VP) test, citrate test, urease test, gelatin test, gas production test, etc.

 

Following these biochemical tests, the positive isolates of E. coli were preserved and cultured for further identifications through the PCR technique. The extraction and quantification of genomic DNA 
Using a commercial bacterial genomic DNA extraction kit (Bio Basic Inc., Markham, Ontario, Canada), genomic DNA from the isolates of E. coli was extracted. Following the manufacturer’s instructions, Proteinase K and RNase A were added to remove impurities from the DNA samples. Using a lambda (λ) DNA molecular weight marker, the quantification of the extracted genomic DNA samples was done on an agarose gel and considering protein-DNA absorbance recorded using NanoDrop UV-Visible measurements for nucleic acid (ThermoFisher Scientific, 2000c). The DNA was then diluted accordingly to make the final concentration 30 ng/µl. The extracted genomic DNA samples were then preserved in an ultrafreezer at –20° C for further use.

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3-[(2-methoxyethyl)amino]propanoic acid Catalog No.:AA01AGNP CAS No.:1040073-81-1 MDL No.:MFCD11174804 MF:C6H13NO3 MW:147.1723	1,1,3-Trioxo-2-(oxolan-2-ylmethyl)-2,3-dihydro-1,2-benzothiazole-6-carboxylic acid Catalog No.:AA019W4K CAS No.:1040074-58-5 MDL No.:MFCD11580046 MF:C13H13NO6S MW:311.3104
2-Bromo-1-(4-chlorobenzyloxy)-4-fluorobenzene Catalog No.:AA007WQB CAS No.:1040075-19-1 MDL No.:MFCD11125986 MF:C13H9BrClFO MW:315.5654	2-chloro-N-(2-chloro-4-methylphenyl)propanamide Catalog No.:AA019KMS CAS No.:1040079-49-9 MDL No.:MFCD11183593 MF:C10H11Cl2NO MW:232.1064
4-Chloro-2-[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]aniline Catalog No.:AA019YN6 CAS No.:1040079-81-9 MDL No.:MFCD11124605 MF:C11H12ClN3O MW:237.6855	4-chloro-1-N-propylbenzene-1,2-diamine Catalog No.:AA019VC6 CAS No.:1040083-67-7 MDL No.:MFCD11186901 MF:C9H13ClN2 MW:184.6659