Uropathogenic Escherichia coli causing urinary tract infections

Uropathogenic Escherichia coli causing urinary tract infections

Mandal, P

Background & objectives: The information on the characteristics of Escherichia coli causing urinary tract infections is limited. We have characterised the urovirulence factors of Esch. coli isolated from symptomatic patients of urinary tract infections (UTI) in order to determine their pathogenic potential and the antibiotic sensitivity profile.

Methods: Semi-quantitative urine culture was done on 370 symptomatic patients suffering from urinary tract infections. Phenotypic characterization of the urovirulence factors of Esch. coli was undertaken and the antibiotic susceptibility was determined.

Results: Esch. coli was responsible for 45.5 per cent of infections. Resistance to amoxycillin, cotrimoxazole, nalidixic acid, norfloxacin and ciprofloxacin among Esch. coli isolates ranged from 70-95 per cent. Serotype 0101 was found to be the commonest serotype (7/26). The virulence factors associated with uropathogenic Esch. coli were haemolysin production (5/30), presence of mannose resistant P-fimbriae (5/30), presence of mannose sensitive type 1 fimbriae (6/30) and the presence of mannose resistant F-fimbriae (2/30). Siderophores production was seen in all the isolates causing UTI.

Interpretation & conclusion: Esch. coli was found to be the commonest cause of UTI in our study population. Antibiotic resistance was high among the strains circulating which emphasises the need for judicious use of antibiotics. Certain virulence factors like haemolysin production and presence of fimbriae in the Esch. coli may be associated with the urovirulence.

Key words Escherichia coli – urinary tract infection (UTI) – uropathogenic – urovirulence

The organisms infecting the urinary tract and their antibiotic sensitivity patterns differ from place to place. Escherichia coli is the most common urinary pathogen, accounting for 85 per cent of community acquired urinary tract infections (UTIs) and 50 per cent of hospital acquired infections1. Earlier hospital based studies had reported Esch. coli to be the causative agent in 30-40 per cent of UTIs2-4, more so among diabetics (64.3%)5. In contrast to the diarrhoeagenic Esch. coli where the virulence factors or toxins are well defined, the factors responsible for determining the uropathogenic potential of Esch. coli remain ill defined. However, it has been suggested that the virulence results from the cumulative impact of one or several virulence factors which serve to distinguish these strains from the commensal intestinal strains6. Expression of certain 0: K: H serotypes, different adhesions like P, F, type -I, S and X- adhesins, haemolysin production and siderophore production are some documented virulence factors of uropathogenic Esch. colic. There has been no study from India on urovirulence factors of Esch. coli.

The present study was designed to determine the urovirulence factors of Esch. coli isolated from the patients of UTI and to study their antimicrobial susceptibility pattern.

Material & Methods

Sample size: The sample size was decided on the basis of availability of patients and feasibility of investigations. The patients included in the study were those suspected of UTI based upon clinical criteria8,9. All patients gave informed consent to participate in the study.

Urine specimen: Mid stream urine samples were collected from patients residing in the urban slums of south Delhi, and presenting with complaints suggestive of UTI at mobile health clinics conducted by the Center for Community Medicine and out patients departments of All India Institute of Medical Sciences, New Delhi during July 1998 to June 1999. The sample was immediately transported to the bacteriology laboratory or refrigerated if delay was anticipated up to 4 h, for processing.

Conventional culture: All the samples were cultured by the semi quantitative method10. The specimens yielding colony counts >= 10^sup 4^/ml were interpreted as diagnostic of UTI as only symptomatic patients had been included in the study”. Bacterial counts less than this were considered insignificant and growth of more than 2 types of organisms was considered as contamination.

Identification of isolates was done using standard microbiological techniques12. Antibiotic sensitivity testing was done on Mueller Hinton agar (Hi-Media, India) by disc diffusion method using NCCLS guidelines13. The concentrations ((mu)g) of various antibiotics used per disc were: amoxycillin (10), cephalexin (30), gentamicin (10), co-trimoxazole (125/23.75), nalidixic acid (30), nitrofurant (300), norfloxacin (10), ciprofloxacin (5). The Esch. coli isolates obtained were preserved in glycerol at -70 deg C till further characterised.

Phenotypic characterization of Each. coli isolates: (i) Serotyping – Serotyping was carried out at the

National Salmonella and Escherichia Center, Central Research Institute, Kasauli, H.P. based on ‘O’ antigen of Esch. coli.

(ii) Haemolysin production assay — Haemolysin production assay was done as described by Stapleton et a14. The strains showing haemolysin production were confirmed by repeating the experiments twice.

(iii) Mannose resistant haemagglutination assay (MRHA)- (a) Mannose resistant haemagglutination assay for the P. fimbriae14. The agglutination of 3 per cent of ‘O’ group human RBCs (provided by the Blood Bank, AIIMS, New Delhi) in the presence of 2 per cent mannose (E-merk, India) was indicative of the presence of P. fimbriae.

(b) Mannose resistant haemagglutination assay for F. fimbriae15. The agglutination of 3 per cent sheep RBCs in the presence of 2 per cent mannose was indicative of the presence of F. fimbriae.

(iv) Mannose sensitive haemagglutination assay (MSHA) – This assay was done to look for the presence of type I fimbriaeI5. The agglutination of 3 per cent guinea pig RBCs which was inhibited by 2 per cent mannose was indicative of type I fimbriae.

(v) Siderophore production assay – The production of siderophore by Esch. coli isolates in the external milieu was analyzed by the modified universal chemical assay16 as standardised in our laboratory17.

Results & Discussion

A total of 370 urine samples were collected from 370 patients with signs and symptoms suggestive of UTI. Of these, 205 were from adult women, 115 from adult men and 50 from children under the age of 12 yr.

Significant bacteriuria was found in 61, patients (16.5%) indicating UTI while 240 patients (64.9%) were culture negative. In 18 patients (4.9%) there was insignificant bacteriuria and samples related to 51 patients (13.7%) were contaminated. The male: female ratio of patients with significant bacteriuria was 1:1.03. Among the 61 culture positive patients, the number of bacterial isolates obtained were 66 (56 had a single pathogen and 5 had 2 types of bacteria grown in culture). Esch. coli was responsible for 30 (45.5%) of the 66 infections which is higher compared to all other infections considered together. The other bacteria isolated were Proteus sp. (8), Klebsiella sp. (5), Enterobacter sp. (5), Citrobacter sp. (4), Pseudomonas aeruginosa (4), Esch. faecalis (4), coagulase negative Staphylococcus (3), Staphylococcus aureus (2), Acinetobacter (1).

Antibiotic susceptibility of the Esch. coli isolates to various antibacterial agents is shown in the Fig. A high per cent of the isolates were resistant to most of the commonly used antibiotics in the community ranging from 70-95 per cent to amoxicillin, cotrimoxazole, nalidixic acid, norfloxacin and ciprofloxacin. Resistance to cefotaxime (a second line drug) was found to be the least. Among the oral first line drugs, nitrofurantoin was the least resistant being found in 30 per cent of the isolates only. Esch. coli isolates were further characterized by serotyping. All the 30 isolates were sent to CRI, Kasauli of which the results were available for 26 isolates. Seven of these 26 isolates were type 0101. The other serotypes were 025 (4), 058 (2) and 04, OIQ, 015, 016, 079, 0107, 0136, 0153 were I each (5 strains were rough and could not be serotyped).

Among the virulence factors, haemolysin production was seen in 5 of 30 isolates, presence of mannose resistant P. fimbriae in 5, presence of mannose sensitive type 1 fimbriae in 6 and the presence of mannose resistant F. fimbriae in 2 of the 30 isolates. Siderophores production was seen in all the isolates causing UTI as it is required for the iron uptake by the bacteria inside the human host and is essential for the survival of the organism.

We conclude that Esch. coli remains the commonest cause of UTI. There are some uropathogenic strains of Esch. coli that have virulence factors which enable them to colonize the urinary tract by helping them to adhere to the uroepithelium and production of haemolysin which causes local tissue damage and helps in invasion6,7,15.

We observed a high incidence of drug resistance in our isolates to the commonly used oral antibiotics. Similar observation was made by earlier workers from Mumbai where resistance was reported to be increasing over a 5 yr period4. The isolates showed decreased sensitivity. to some new drugs like norfloxacin, ciprofloxacin, cephalosporins and cefuroxime, which is alarming. We found resistance to cefotaxime to be the least but suggest that third generation cephalosporin should be reserved as second line therapy. Nitrofurantoin still continues to be good drug with resistance being least among the first line oral drugs possibly because of its limited use in other infections.

The limitation of our study is that it was done in a limited population and the numbers are small. However, it suggests a need to carry out further studies in a larger population to study the association of virulence factors of uropathogenic Esch, coli.

Acknowledgment

Authors are grateful to the Director, CRI, Kasauli (HP) for undertaking the serotyping of the isolates. Authors thank Sh. Bhagwan, Center for Community Medicine for assistance in the collection and transport of the urine samples.

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Reprint requests: Dr Arti Kapil, Additional Professor, Department of Micorbiology, All India Institute of Medical Sceinces Ansari Nagar, New Delhi 110029 , India.

Uropathogenic Escherichaia coli causing urinary tract infections

P. Madal, A. Kapil, K, Goswami*, B. Das & S.N. Dwivedi**

Department of Micorbiology, *Centre for Community Medicine & **Department of Biostatics All India Insititute of Medical Sciences, New Delhi, India

Received May 17, 2001

Copyright Indian Council of Medical Research Dec 2001

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