An approach to uropathogenic Escherichia coli in urinary tract infections

INTRODUCTION

Urinary tract infection (UTI) is one of the most important causes of morbidity and mortality. E.coli is the most frequent urinary pathogen isolated from 50 — 90% of all uncomplicated urinary tract infections.^[1] E.coli present in the gastrointestinal tract as commensals provide the pool for initiation of UTI. It has been traditionally described that certain serotypes of E.coli are consistently associated with uropathogenicity and are designated as Uropathogenic E.coli (UPEC). These isolates express chromosomally encoded virulence markers. These markers of UPEC are expressed with different frequencies in different disease states, ranging from asymptomatic bacteriuria to chronic pyelonephritis.

In the late 1970s, it was recognized for the first time that E.coli strains causing urinary tract infections typically agglutinate human erythrocytes, despite the presence of Mannose,^[2] and this was mediated mainly by fimbriae. Subsequently an array of virulence factors were proposed as virulence markers for uropathogenic isolates of E.coli. It is now recognized that there are a subset of fecal E.coli having the above-mentioned factors that can colonize the periurethral area, enter the urinary tract, and cause symptomatic disease. These are currently defined as UPEC.^[3]

However, most of these studies have been carried out on patient isolates and no studies have been carried out on commensal / gut isolates. In addition, the relative importance of these bacterial factors has not been validated. In the present study an attempt has been made to answer these two questions with respect to four proposed virulence factors namely hemolysin, hemagglutination, cell surface hydrophobicity, and serum resistance.

MATERIALS AND METHODS

RESULTS

A total of 220 E. coli were isolated from the urine samples, from UTI. Out of these, 191 samples had significant bacteriuria with counts 10⁵ CFU/mL and 29 had probably significant bacteriuria with counts between 10⁴ and 10⁵ CFU/mL.

DISCUSSION

Considering the high degree of morbidity and mortality of UTIs, the subject of uropathogenic E. coli was receiving increasing attention. Cell morphology and molecular biology studies have revealed that uropathogenic E.coli express several surface structures and secrete protein molecules, some of them cytotoxic and peculiar to the strains of E. coli, causing UTI.^[1] Hence, it is important to identify UPEC from non-UPEC isolates in the urinary samples. Results in this study showed that most of the urinary isolates from cases had more than one virulence marker. In this case-control study we conclude that UPEC strains are definitely associated with the aetio-pathogenesis of UTI. The occurrence of multiple virulence factors in the UPEC strains further strengthens the concept of the association of UPEC with urinary pathogenicity. It was interesting to note that UPEC with multiple virulence factors were significantly more in the cases than in the controls.

Phenotypic and biochemical characterization revealed interesting findings. A total of 18 urinary isolates were mucoid and all these were obtained from the cases only. No mucoid strains were isolated from the control group. Furthermore, 10 of these 18 were serum-resistant. The mucoid strains were capsulated^[8] and the capsule conferred serum and phagocyte resistance to some E. coli strains.^[3] This property was attributed to the content of sialic acid, which reduced the ability of the bacterial surface to activate and complement by an alternative pathway. However, eight mucoid strains were susceptible to the serum indicating that non-capsular factors had a role in serum resistance. Thus, the conventional phenotypic marker, such as the capsule, was also an important virulence factor for the UPEC. In the present study, 14 (6.36%) of the 220 isolates were atypical E. coli. Altered phenotypes could be due to an altered genetic makeup. Bhat et al^[9] studied 210 E. coli strains isolated from the urine and found 26 (12.4%) to be atypical. In their study of the 26 atypical urinary isolates, 12% were hemolytic and three had hydrophobicity (< 0.156) and were MRHA positive and all the atypical E.coli had one or the other virulence marker, indicating that an atypical phenotype probably contributed to their virulence. The cytolytic protein toxin secreted by most hemolytic E.coli strains was a-hemolysin. E.coli also produced cell-associated lysin on blood agar plates and hemolysin caused a clear zone of lysis.^[10] In the present study, although the nature of hemolysin was not further characterized it could be considered as a cytotoxic necrotizing factor (hemolysin). The difference between cases and controls for the production of hemolysin was highly significant (P<0.001). This was similar to the Johnson et al.^[2] study, where hemolysin was produced by 38% of the urinary isolates and 12% of the fecal isolates. Hemagglutination was mediated by the fimbriae.^[11] MRHA could be mediated by P fimbriae. Thus, the MRHA positive strains could be considered as UPEC, most likely having P fimbriae.^[2] In the present study there was a significant difference in MRHA between cases and controls (P<0.01). This was similar to a study by Johnson^[2] et al, where 58% of the urinary isolates and 19% of the fecal isolates showed MRHA. The expression of type 1 fimbriae was indicated by MSHA. MSHA were more in the fecal strains than in urinary isolates, in our study. More work is required to assess the role of MSHA in pathogenecity. The role of the cell-surface hydrophobicity in mediating bacterial adherence to mammalian cells was conceived by Mudd and Mudd.^[12] Crystalline surface layers: the ‘S’ layer present on both gram negative and gram positive organisms play a role in this.^[13] Hydrophobicity is a recently described novel virulence mechanism by E. coli. In the present study, although there was no significant difference in cell-surface hydrophobicity between the cases and controls (P>0.05), more isolates from the cases were hydrophobic. Taylor^[14] reviewed that bacteria were killed by normal human serum through the lytic activity of an alternative complement system. Bacterial resistance to killing by serum, results from the individual or combined effects of the capsular polysaccharide, O polysaccharide, and surface proteins.^[15] Although more E. coli isolates from the cases were serum-resistant compared to the controls, the difference was not statistically significant (P>0.05). The O group designation reveals little about a strain. The apparent virulence associated with certain groups may be mediated through other virulence factors like P fimbriae, hemolysin, and serum resistance, which is commonly associated with UTI-associated strains.^[2]

In this study, 151 out of 220 isolates had one or more virulence factors. When a comparison was done between the urinary and fecal E.coli isolates, hemolysin production, presence of capsule, and the capacity to cause MRHA, emerged as important virulence factors. The hemolysin, especially α-hemolysin, also known as cytotoxic necrotizing factor, is strongly proinflammatory leading to secretion of IL-6 and chemotaxins, which sets pace for the pathogenesis of renal disease. The capacity to cause MRHA is due to various adhesions mainly P fimbriae, P-associated fimbriae, and FIC fimbriae, seen in pyelonephritis cases. These adhere to the fibronectin on the uroepithelial cells contributing to the persistence.

We believe that the methods of detection of the above- mentioned virulence markers are reasonably easy and screening them in a clinical microbiology laboratory is a worthwhile exercise.