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 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 34  |  Issue : 1  |  Page : 46-51
 

Virulence versus fitness determinants in Escherichia coli isolated from asymptomatic bacteriuria in healthy nonpregnant women


Department of Microbiology, King George's Medical University, Lucknow, Uttar Pradesh, India

Date of Submission25-May-2014
Date of Acceptance08-Aug-2014
Date of Web Publication15-Jan-2016

Correspondence Address:
Jyotsna Agarwal
Department of Microbiology, King George's Medical University, Lucknow, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.174103

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 ~ Abstract 

Purpose: Escherichia coli isolated from asymptomatic bacteriuria (ABU) correlated genotypically and phenotypically with cystitis isolates may help in distinguishing urovirulence determinants from 'fitness factors', latter necessary only for survival of E. coli in urinary tract; for gaining insight into the pathogenesis of urinary tract infection. Materials and Methods: In this cross-sectional study, we compared genotypic (phylogroups and 15 putative virulence genes), and phenotypic profiles of ABU E. coli strains with our previously genotyped collection of cystitis isolates. Virulence score was calculated for each isolate as a number of virulence genes detected. Results: Significant differences were observed in the proportion of four phylogenetic groups (P = 0.009) amongst cystitis and ABU isolates. Average virulence score was higher for ABU isolates (6.6) than cystitis strains (4.2); and hlyA (P = 0.001), cytotoxic necrotising factor 1 (P = 0.00), fyuA (P = 0.00), ibeA (P = 0.00), kpsMII (P = 0.01), and malX/pathogenicity-associated island (P = 0.01) were more frequently present in ABU strains. Conclusions: The expression of adhesins, haemolysin, aerobactin, and capsule synthesis gene were similar in two groups suggesting their role as fitness factors. ABU isolates were better biofilm producers, reflecting its importance in silent persistence. Serum resistance gene which was more expressed in cystitis isolates may represent virulence determinant. Genetic makeup of E. coli does not change much rather genes helping in survival and colonisation are expressed equally in ABU and cystitis isolates as opposed to phenotypic attenuation of those that helps in invasion or inflammation in ABU isolates.


Keywords: Fitness factors, phenotypic expression, urinary tract infection, uropathogenic Escherichia coli, virulence genes


How to cite this article:
Srivastava S, Agarwal J, Mishra B, Srivastava R. Virulence versus fitness determinants in Escherichia coli isolated from asymptomatic bacteriuria in healthy nonpregnant women. Indian J Med Microbiol 2016;34:46-51

How to cite this URL:
Srivastava S, Agarwal J, Mishra B, Srivastava R. Virulence versus fitness determinants in Escherichia coli isolated from asymptomatic bacteriuria in healthy nonpregnant women. Indian J Med Microbiol [serial online] 2016 [cited 2020 Sep 27];34:46-51. Available from: http://www.ijmm.org/text.asp?2016/34/1/46/174103



 ~ Introduction Top


Urinary tract infection (UTI) with its diverse clinical spectrum ranging from asymptomatic bacteriuria (ABU) to acute cystitis to pyelonephritis remains one of the most common but largely misunderstood bacterial infection encountered by general practitioners. The pathogenesis of UTI is complex and is influenced by host biological and behavioural factors, as well as by virulence characteristics of the infecting uropathogen.[1] Escherichia More Details coli is the most important etiological agent of UTI; responsible for >80% of all UTIs.[2] A range of virulence factors (VF) involved in the production of toxins and effectors, iron acquisition, adherence, invasion, and immune system avoidance in uropathogenic E. coli (UPEC) have been implicated in pathogenesis of UTI. Various molecular approaches have elucidated that any single gene is not sufficient to cause infection, rather it is an array of virulence genes interacting with host immune system which results in infection; although exact role of many of these virulence determinants in UTI pathogenesis is still not clearly known.[3]

The colonisation of urine in the absence of clinical symptoms with >105 bacteria/ml of urine is called ABU and is reported to have a prevalence of 4–6% in healthy young adult women and up to 20% in elderly women.[4],[5] Since E. coli, associated with ABU in women resembles a commensal like carrier state, this makes it a relevant model to study the pathogenesis of UTI.[1] Some studies have suggested that ABU strains are more diverse and lacking in virulence associated genes favouring reductive evolution; however, genotypic analyses contradicted this notion as many ABU strains were shown to carry virulence genes but failed to express them suggesting that ABU strains may have arisen from virulent UPEC strains but achieved long-term persistence by phenotypic attenuation of 'VF' that provoke host response.[6],[7] Functional loss in virulence genes can be due to mutation or attenuation, resulting in signal transduction impairment leading to loss of binding to class of receptors which invoke host immune response thus helping E. coli escape immune surveillance.[7] These so-called attenuated virulence determinants in ABU E. coli which facilitate their survival and colonisation in the urinary tract in a quiescent manner, without provoking an immune response are regarded as 'fitness factors'.[8]

Putative virulence determining genes prevalent in UPEC isolated from patients with cystitis when correlated with ABU strains may help in distinguishing urovirulence determinants necessary for causing infection from the fitness determinants and to understand the adaptation strategy in respective environment. In the present study, we have compared E. coli strains associated with ABU in healthy non-pregnant women with our previously genotyped collection of UPEC, isolated during the same time period (i.e., January 2010-May 2012) from age-matched women with acute cystitis from same geographical location [9] for their genetic makeup (phylogenetic groups and presence of 15 putative virulence genes) along with biofilm formation and phenotypic expression of some of the virulence-associated genes to understand pathogenesis of UTI and cover the lacunae in knowledge in this area.


 ~ Materials and Methods Top


Study design and subjects

It was a cross-sectional study conducted in the Department of Microbiology at a Tertiary Care Center between January 2010 and July 2012. The study protocol was approved by the Institutional Ethics Committee (reference code - XL ECM/A-P8). Non-pregnant, apparently healthy, sexually active women between the age group of 18 and 50 years attending family planning/fertility clinics without any symptoms suggestive of UTI (dysuria, urine frequency >6 times/day, urgency, suprapubic pain, fever, haematuria/smoky urine, burning sensation during micturition and acute onset incontinence), consenting to participate and provide a mid-stream clean catch urine sample were invited to take part in the study. Urine cultures from women yielding >105 CFU of E. coli per millilitre were defined as ABU. Consecutive urine cultures yielding >105 CFU/ml are preferred for confirming ABU; however, it is practically impossible to get a second urine sample from an otherwise healthy woman. Therefore, single urine culture yielding >105 CFU/ml has been used to make a diagnosis of ABU by many workers world over.[5],[10] Women who were pregnant, with any underlying co-morbidity, apparent urological abnormality or a urethral catheter in place were not included in the study.

Sample size

Reported prevalence of ABU in healthy young adult women is ~5%.[5] Based on this, with a confidence interval of 95% and a probability of 5%, the sample size was estimated as 73 women with ABU. In order to get these numbers of ABU cases, we needed to enrol a minimum of 1460 healthy asymptomatic women.

Sample collection and processing

Urine samples were semi-quantitatively cultured onto cystine lactose electrolyte deficient agar plate (HIMEDIA, India). Lactose-fermenting colonies with appropriate colonial morphology were presumptively identified as E. coli and were further confirmed using standard conventional biochemical tests.[11]

Phylogenetic classification

All isolates were assigned to one of the four major E. coli phylogenetic groups (A, B1, B2, and D) using triplex polymerase chain reaction (PCR)-based phylotyping as described by Clermont et al.,[12] based on the presence of two genes (chuA and yjaA) and a DNA fragment (TSPE4.C2). Pyelonephritic isolate J96, human faecal isolate JJ055, and canine UTI isolate L31 were used as positive controls for phylogroups B2, D, and A, respectively.

Virulence genotyping

E. coli isolates were tested for the presence of 15 virulence genes of various functional categories using multiplex PCR assay with appropriate positive and negative controls.[13] VF studied were: Adhesins papA (P fimbrial structural subunit), papG alleles I, II, and III (P fimbrial adhesin varients), fimH (type 1 fimbriae), afa/draBC (Dr-binding adhesin), and sfa/focDE (S and FIC fimbriae); toxins hlyA (haemolysin) and cytotoxic necrotising factor 1 (cnf1); siderophores iutA (aerobactin) and fyuA ( Yersinia More Detailsbactin); capsule synthesis specific for group II (K1, K5, K12, etc.) kpsMII; serum resistance-associated traT; invasion of brain endothelium ibeA; and malX, a coding region near the terminus of a pathogenicity-associated island (PAI). J96 pyelonephritis isolate, 2H25 urosepsis isolate, V27 urosepsis isolate, L31 canine UTI isolate, and 2H16 urosepsis isolate were used as positive controls while human faecal isolate JJ055 was used as negative control. PCR conditions have been described in [Table 1]. UPEC control strains used in the current study were kindly provided by Dr. J. R. Johnson, VA Medical Centre and the University of Minnesota, Minneapolis, MN, USA.
Table 1: PCR conditions for multiplex PCR for virulence genotyping

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The virulence p elements (papA, papGI, papGII, and papGIII) were considered as a single pap factor. Thus, if a strain was positive for at least one or more of the studied pap markers, it was regarded as pap- positive and given a score of 1. The results of such in vitro testing predict experimental virulence in vivo.[9]

Biofilm production

This was assessed using M63 minimal glucose medium (Ameresco, Ohio, USA) in 96-well flat bottom polystyrene microtiter plates (Axiva Sichem Biotech, India) under static conditions. E. coli strain ATCC 25922 and JJ055, a faecal E. coli isolate, were used as positive and negative controls, respectively. The results were analysed as described elsewhere.[9]

Phenotypic assays

Type 1 fimbriae was detected by agglutination of Saccharomyces cerevisiae cells with aliquots of overnight bacterial cultures in Luria Broth (LB) as described previously by Hancock et al.[14] For cellulose production, the bacterial strain was streaked onto LB-agar plates containing 0.02% calcofluor and incubated overnight. Fluorescence of the colonies was checked at 360 nm.[14] For detection of P fimbriae, E. coli strain grown overnight on LB media at 37°C were incubated with human erythrocytes. Haemagglutination was compared after incubation for 5 min on ice.[7]

Haemolysin was detected by clear zone observed around colonies, after overnight growth of E. coli at 37°C on sheep blood agar plates.[7] Siderophore production was checked by measuring the colour changes of the chrome azurol sulphonate medium from blue to orange as described by Pérez-Miranda et al.[15] Serum resistance assay was done as previously described by Jadhav et al.[16] Bacteria was mixed with human serum and inoculated on nutrient agar plates at difference of 0 h, 1 h, and 2 h. If >90% of the E. coli survived after 2 h of incubation, the organism was considered resistant.

Statistical analysis

Data were entered in Microsoft Excel spreadsheet software (Microsoft Corp., Redmond, WA, USA). Prevalence ratios and comparisons of proportions between ABU and cystitis isolates were tested using the Chi-square test by statistical software Stata version 11.1 (Stata Inc., College Station, TX, US). The descriptive statistics for various variables were reported as a percentage for qualitative variables, and P < 0.05 was considered significant.


 ~ Results Top


During the study period, urine specimens from 1680 apparently healthy women were cultured, of whom 39 (2.3%) yielded >105 CFU/ml of E. coli, 1483 (88.3%) were sterile, 130 (7.7%) contaminated, while 28 (1.7%) showed growth of other bacteria. The mean age of women with ABU was 31.98 ± 7.73.

Phylogenetic groups

About 41% ABU strains belonged to phylogroup B2 followed by phylogroup B1 (25.6%), D (23.1%) and A [10.3%; [Table 2] and [Figure 1].
Table 2: Phylogenetic distribution, genotypic prevalence and phenotypic expression of virulence determinants in Escherichia coli isolate associated with ABU and cystitis

Click here to view
Figure 1: Triplex polymerase chain reaction profiles for Escherichia coli phylogenetic groups. Lane M: 100 bp molecular weight marker, lanes 1 and 2: Phylogroup D; lanes 3, 4, and 6: Phylogroup B2; lane 5: Phylogroup B1; and lane 7: Phylogroup A

Click here to view


Virulence genes distribution

The most prevalent gene was fimH (97%), followed by fyuA (84.6%), traT (79.5%), kpsMII (64.1%), cnf1 ( 61.5%), and iutA (56.4), whereas papG allele I was absent in ABU isolates [Table 2] and [Figure 2]. Most of the VF were confined to phylogenetic group B2 (data not shown in details). Mean virulence score was 6.6, and higher mean score were observed for phylogroups B2 and D (8.56 and 5.66, respectively; data not shown in detail).
Figure 2: Multiplex polymerase chain reaction profiles specific for Escherichia coli virulence genes; (a) pool 1: MalX (930 bp), papA (720 bp), fimH (508 bp); (b) pool 2: FyuA (880 bp), sfa/focDE (410 bp), papG all III (258 bp), iutA (300 bp); (c) pool 3: HlyA (1177 bp), papG all I (461 bp), kpsMII (272 bp); (d) pool 4: TraT (290 bp), papG allII (190 bp); and (e) pool 5: Afa/draBC (594 bp), cytotoxic necrotising factor 1 (498 bp);

Click here to view


Biofilm production

25.6% ABU isolates had a strong capacity to form biofilm while 35.9% and 30.8% were moderate and weak biofilm producers, respectively. Remaining 7.7% were unable to form a biofilm under static in vitro conditions.

Phenotypic expression

Phenotypic profiles of ABU and previously genotyped collection of cystitis isolates showed statistically non-significant differences in the expression of the genes required for the production of adhesins (type 1 fimbriae andPfimbriae), haemolysin, capsule, and siderophore aerobactin; though phenotypic expression was higher in cystitis isolates [Table 2]. Interestingly, the expression of serum resistance traT gene was more in cystitis isolates relative to ABU isolates (P = 0.009).


 ~ Discussion Top


The clinically benign nature of ABU was initially explained by a lack of virulence coding genes and reductive evolution was thought to be responsible for converting virulent UPEC to asymptomatic carrier strains.[7] However, a recent molecular analysis of the ABU prototype E. coli strain 83972 supports the hypothesis that many ABU E. coli strains carry virulence genes but fail to express the associated phenotype.[8] This makes ABU an appropriate model to understand the pathogenesis of UTI by characterising fitness factors that promote colonisation and survival of E. coli as opposed to virulence determinants needed to cause cystitis.

The majority of the pathogenic strains of E. coli associated with symptomatic UTI generally belong to phylogroup B2 and D worldwide.[12],[17] The prototype ABU isolate E. coli 83972 also belongs to the phylogenetic lineage B2, which indicates its close relation with pathogenic strains. In the present study, we also found predominance of phylogroup B2 in our ABU isolates but along with relatively higher prevalence of phylogroup B1, difference was statistically significant in the proportion of four phylogenetic groups (P = 0.009). Some of the previous studies, however, found this difference to be statistically not significant.[10],[18]hlyA (0.001), cnf1 (0.00), fyuA (0.00), ibeA (0.00), kpsMII (0.01), and PAI (0.01) genes were more frequently present in ABU strains which is in accordance with the findings reported by Salvador et al.[7] Average virulence score was higher for ABU isolates (6.6) than cystitis strains (4.2). Upon genotypic profiling, our ABU isolates thus appeared to be more virulent than some of the earlier reported ABU and even cystitis isolates.[9],[19] However, when we looked at the corresponding phenotypic expression for some of these genes, we did not find any significant difference in expression.

Haemolysin (hlyA) gene was expressed equally among the ABU and cystitis isolates, and increased expression of aerobactin (iutA) in cystitis isolates was not statistically significant. Both haemolysin and aerobactin have an essential role in obtaining other nutrients for bacterial growth and iron acquisition during or after colonisation.[20.21] Capsule synthesis that promotes bacterial antiphagocytic and anticomplement effect, serum resistance and evasion of immune recognition although were expressed more by cystitis isolates, the difference was statistically non-significant; thus seem to help in survival rather than in causing infection.[20],[22] On the other hand, traT gene which confers resistance to killing of bacteria by serum and is known to be associated with pyelonephritis, cystitis, and bacteremia had a significantly higher expression in cystitis isolates than in ABU isolates (52.3% vs. 25.8%).[20] There was no significant difference in overall presence or expression of adhesin genes between ABU and cystitis isolates as also reported earlier, reflecting its role in the survival of both.[17] We were not able to look at the expression of invasive genes ibeA.

The ability to form biofilm is often considered to be a virulence-associated trait and persistence strategy for UPEC to reside in bladder and or vagina in a quiescent state that significantly enhances resistance towards removal by both natural defence mechanism and antibiotics.[16],[23] An overwhelming majority (~61%) of the ABU isolates had strong to moderate biofilm formation capability in comparison to cystitis isolates where most (60.7%) were weak biofilm producers.[9] Similar findings were also reported previously by Hancock et al.[14] for ABU isolates. Mabbett et al.[10] also suggested that ABU strains grow as a biofilm in the urinary tract leading to reduced virulence gene expression and decreased interaction with the host.


 ~ Conclusion Top


ABU E. coli isolates showed functional but not the anatomic loss of virulence determining genes when compared with cystitis E. coli isolates. Many of the so-called virulence determinants described for UPEC (e.g., adhesins, alpha-haemolysin, aerobactin, and capsule synthesis genes); were similarly expressed in both, the ABU and cystitis E. coli isolates suggesting their role as 'fitness factors' helping in mere survival of the organism in urinary tract without actually initiating infection. On the other hand serum resistance traT gene which was significantly more expressed in cystitis isolates may represent the true virulence determinant needed for the pathogenesis of UTI. Further studies with expanded virulence gene repertoire of the pathogen and host immune response are needed to gain more insights into the exact role of various virulence determinants.

Financial support and sponsorship

Department of Biotechnology, New Delhi, India vide grant number BT/PR 13637/MED/29/172/2010.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

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Hancock V, Ferrières L, Klemm P. Biofilm formation by asymptomatic and virulent urinary tract infectious Escherichia coli strains. FEMS Microbiol Lett 2007;267:30-7.  Back to cited text no. 14
    
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Pérez-Miranda S, Cabirol N, George-Téllez R, Zamudio-Rivera LS, Fernández FJ. O-CAS, a fast and universal method for siderophore detection. J Microbiol Methods 2007;70:127-31.  Back to cited text no. 15
    
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Abraham S, Chapman TA, Zhang R, Chin J, Mabbett AN, Totsika M, et al. Molecular characterization of Escherichia coli strains that cause symptomatic and asymptomatic urinary tract infections. J Clin Microbiol 2012;50:1027-30.  Back to cited text no. 18
    
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Tiba MR, Yano T, Leite Dda S. Genotypic characterization of virulence factors in Escherichia coli strains from patients with cystitis. Rev Inst Med Trop Sao Paulo 2008;50:255-60.  Back to cited text no. 19
    
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22.
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