INTRODUCTION:

Of all Urinary Tract Infections (UTIs), cystitis and pyelonephritis are the most frequently encountered human extra intestinal infections. Escherichia coli is the major causative agent for this, and is also a predominant facultative member of the normal human intestinal flora. This agent primary of UTIs accounts for greater than 80% of these infections. The bladder is the primary site of infection in about 95% of all UTIs [1]. In most cases, uropathogenic clones are selected from the fecal flora and colonize the vaginal and periurethral tissue, and infected the urinary tract by the ascending manner.

Virulence factors (VFs) associated with UPEC include adhesins (P fimbriae, type 1 fimbriae, S and F1C fimbriae, afimbrialadhesin), toxins (hemolysin, and cytotoxic necrotizing factor), siderophores (the aerobactin system) and polysaccharide coatings (group II capsules)[2]. Recently, a new virulence factor gene associated with uropathogenesis was more frequently found in UPEC strains have been reported. This gene encoding a protein designated usp (uropathogenic-specific protein) was demonstrated significantly enhancing the infectivity of E. coli in the mouse UTI model. [3] Based on this background, we have taken this objective to detect the papC gene among uropathogenic E. coli.

MATERIALS AND METHODS:

Bacterial isolates:

A total of 20 non repetitive urinary isolates of E. coli were collected from Saveetha Medical College and Hospitals, Chennai. They were processed for a battery of standard biochemical tests and confirmed. Isolates were preserved in semisolid trypticase soy broth stock and were stored at 4 ºC until further use.

Antibiotic susceptibility testing:

Antibiotic susceptibility test was determined for these isolates to routinely used antibiotics such as ampicillin, amoxicillin, amikacin, norfloxacin, ceftazimide, cefo taxime, ciprofloxacin and gentamicin, imipenem as by Kirby Bauer disc diffusion method ^[4]

Detection of papC gene in E.Coli:

E.coli isolates were detected for the presence of papC gene by PCR analysis. Detection of the gene was carried out using primer as depicted in table 1. Bacterial DNA was extracted by boiling lysis method. 1 µL of DNA extract was used as template for PCR reaction. The reaction mixture contained 1mM of Mgcl₂0.2mM dNTP mix and 0.8µM of bla_CTX-Mgene with 0.5U of Taq polymerase (New England Biolabs) in a 1x PCR buffered reaction. A positive control of E.coli with papC gene was also included in this study. PCR amplification was carried out using thermal cycler (Eppendorf) with the following cycling condition. Initial denaturation at 97°C for 3 minutes, 40 cycles of denaturation at 95°C for 1 minute, primer annealing at 54°C for 40 seconds and primer extension at 72°C for 1 minute and final extension at 72°C for 5 minutes were used. PCR products were resolved in 1.5% agarose gel. A 100bp ladder was including in all the gel analysis.^[5]

Table 1: Primer sequence of papC gene

Primer

Primer sequence

Product size

papC

GTG GCA GTA TGA GTA ATG ACC GTT A

ATA TCC TTT CTG CAG GGA TGC AAT A

198 bp

RESULTS:

Sample wise distribution of clinical isolates of E.coli:

Of the 20 clinical isolates of E.coli, 12/20 (60%) were from acute urinary tract infections and 8/20 (40%) were from chronic urinary tract infections. Figure 1 depicts the sample wise distribution of clinical isolates of E.coli.

Figure 1: Sample wise distribution of urinary isolates of E.coli

Antibiotic susceptibility testing:

In our isolates, we have found increased percentage 14/20 (70%) of isolates showed sensitivity to amikacin followed by gentamicin, which showed sensitivity of 9/20 (45%). 80- 90% of E.coli isolates showed resistance to cephalosporin group of drugs. 6/20 (30%) were found to be resistant to imipenem. However, we have observed an elevated level of resistance to other routinely used antibiotics. The detailed resistant pattern of E.coli isolates were showed in table 1.

Table 2: showing antibiotic sensitivity pattern of E.coli

Antibiotics	Sensitivity (20)(%)	Intermediate (20)(%)	Resistant (20) (%)
Ampicillin	5	0	95
Amoxicillin	5	0	95
Ceftazidime	10	10	80
Cefotaxime	5	5	90
Amikacin	70	10	20
Gentamicin	45	20	35
Norfloxacin	15	15	70
Ciprofloxacin	20	5	75
Imipenem	70	0	30

Result of papCgene in E.coli:

Of 20 clinical isolates of E. coli, we have observed 8/20 (40%) isolates were found to harbor papCgene by PCR.

L3-100bp ladder; L2,L4-papC gene positive

Figure 2: Representative gel picture shows the papC

DISCUSSION:

E. coli is the major causative agent in human UTIs, one of the most common bacterial infections. In most cases, uropathogenic clones are selected from the fecal flora, and the pathogenic potential of E. coli strains is thought to be dependent on the presence of VFs [6]. Based on their components and products called virulence factors, E. coli cells attach selectively to the mucosa uro-epithelium, promoting colonization and persisting in the urinary tract, inducing, then, a local inflammatory response and sometimes to promote tissue lesions.[7]

In our study, we have observed 8/20 (40%) of uropathogenic E. coli were harboring papC gene. Similar kind of study conducted by Ribeiro and colleagues in 2008, have studied a wide array of uropathogenic virulence genes by PCR such as papC, papE/F, papG alleles, fimH, sfa/foc, afaE, hly, cnf-1, usp, cdtB, iucD, and kpsMTII genes, wherein 32.7% positivity was documented for papC.[8] This indicates the possible correlation of papC gene in causing chronic urinary tract infections.

CONCLUSION:

We conclude that papC gene is one of the important putative virulent factor for causing urinary tract infection. However, more number of isolates have to be included to validate the proper conclusion.

REFERENCES:

1. Mulvey, M.A. - Adhesion and entry of uropathogenic Escherichia coli. Cell. Microbiol., 4: 257-271, 2002

2. Arisoy, M.; Aysev, D.; Ekim, M. et al. - Detection of virulence factors of Escherichia coli from children by multiplex polymerase chain reaction. Int. J. Clin. Pract., 60: 170-173, 2006.

3. Kanamaru, S; Kurazono, H; Nakano, M. et al. - Subtyping of uropathogenic Escherichia coli according to the pathogenicity island encoding uropathogenic specific protein: comparison with phylogenetic groups. Int. J. Urol., 13: 754-760, 2006.

4. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk Tests; Approved Standards; Doucement M2-A9, 9^th ed., Vol 26. Wayne, PA: CLSI; 2015.

5. Lopez-Banda DA, Carrillo-Casas EM et al. Identification of Virulence Factors Genes in Escherichia coliIsolates from Women with Urinary Tract Infection in Mexico. BioMed Research International. 10; 2014:1-10.

6. Lane, M.C.; Simms, A.N. and Mobley, H.L. - Complex interplay between type 1 fimbrial expression and flagellum-mediated motility of uropathogenic Escherichia coli. J. Bact., 189: 5523-5533, 2007.

7. Mulvey, M.A. - Adhesion and entry of uropathogenic Escherichia coli. Cell. Microbiol., 4: 257-271, 2002.

8. Ribeiro M, Yano T, da Silva Leite D. Genotypic characterization of virulence factors in Escherichia coli strains from patients with cystitis. Rev. Inst. Med. trop. S. Paulo 2008; 50(5):255-260.

Received on 10.08.2016 Modified on 23.08.2016

Research J. Pharm. and Tech 2016; 9(12):2183-2185.

DOI: 10.5958/0974-360X.2016.00442.X