Microbial Pathogenesis 74 (2014) 38e41
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Short communication
Virulence determinants in Escherichia coli associated with recurrent cystitis in sexually active women Jyotsna Agarwal a, *, Bharti Mishra a, Sugandha Srivastava a, Richa Srivastava a, Amita Pandey b a b
Department of Microbiology, King George's Medical University, Lucknow, U.P 226003, India Department of Obstetrics & Gynaecology, King George's Medical University, Lucknow, U.P 226003, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 April 2014 Received in revised form 26 June 2014 Accepted 21 July 2014 Available online 10 August 2014
More than a quarter of women who experience acute cystitis develop recurrence but information on specific urovirulent genetic profile of uropathogenic Escherichia coli associated with recurrent cystitis is still limited. In this prospective cohort study, index episode E. coli from a cohort of 46 sexually active women with acute cystitis who reported recurrence during followup were grouped into repeat infection (RI) and single infection (SI) isolates, based on enterobacterial repetitive intergenic consensus (ERIC) PCR profile comparison with subsequent E. coli isolated from same women. PCR for phylogrouping and 15 virulence genes along with test for biofilm formation were done. Virulence score was calculated for each isolate as number of virulence genes detected. Among 46 index E. coli, 22 were RI, and 24 were SI isolates. RI isolates had phylogroup B2 as majority (54.5%) which is typically described as more virulent phylogroup and virulence score for RI isolates was also significantly higher compared to SI isolates. Virulence gene malX (p ¼ 0.03) was significantly associated with RI isolates. 68.2% RI isolates were strong to moderate biofilm producers in comparison to 33.3% SI isolates, an important survival strategy to reside in bladder and or vagina. Overall, E. coli associated with recurrent cystitis appear to be more virulent and malX seems to have a role in causing repeat infection. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Urinary tract infection Biofilms Phylogenetic groups of Escherichia coli Virulence factors Uropathogenic E. coli
1. Introduction Uncomplicated bladder infection (cystitis) constitute about 95% of all urinary tract infection (UTIs) and approximately 25e48% of all women with first cystitis develop a recurrence within one year of an episode despite antimicrobial treatment and anatomically normal urinary tracts [1,2]. Uropathogenic Escherichia coli (UPEC) remains the principal (~80e90%) causal pathogen [3]. UPEC possess a range of virulence genes that facilitate their adherence and survival in the urinary tract and allow them to establish infection [1,3].
Abbreviations: RI, repeat infection; SI, single infection; UPEC, uropathogenic E. coli; ERIC PCR, enterobacterial repetitive intergenic consensus PCR; MSCC, midstream clean-catch; VF, virulence factors; UTI, urinary tract infection. * Corresponding author. Tel.: þ91 522 2257569 (off); fax: þ91 522 2257539. E-mail addresses: [email protected] (J. Agarwal), bharti_micro@rediffmail. com (B. Mishra), [email protected] (S. Srivastava), [email protected] (R. Srivastava), [email protected] (A. Pandey). http://dx.doi.org/10.1016/j.micpath.2014.07.004 0882-4010/© 2014 Elsevier Ltd. All rights reserved.
Recurrent cystitis is defined as at least two symptomatic episodes of cystitis with positive urine cultures in 6 months or three episodes within 12 months (including the index infection) [4]. Recurrence may be either relapse or reinfection on the basis of the strain responsible for causation of new episode of cystitis i.e., same strain as the primary infecting strain or a different strain, respectively [5,6]. Studies done to analyze pattern of virulence gene distribution in E. coli and their association with recurrent cystitis have found varied results depending on the study settings [1,7,8]. In present study, a cohort of women with acute cystitis was followed up for recurrence and their index episode E. coli was matched with recurrent strain(s) using Enterobacterial Repetitive Intergenic Consensus (ERIC) profiles to identify those associated with recurrent infection. These E. coli were analyzed for phylogroups, virulence genotype and biofilm formation capability in order to identify virulence determinants associated with recurrent cystitis among sexually active women in the absence of anatomic abnormalities of urinary tract.
J. Agarwal et al. / Microbial Pathogenesis 74 (2014) 38e41
2. Materials and methods
39
[15]. All PCR were done in duplicate with appropriate positive and negative controls.
2.1. Study design and subjects 2.7. Virulence genotyping This prospective cohort study was conducted in the department of Microbiology at King George's Medical University, Lucknow, between February 2011 and May 2013. Study protocol was approved by the University Ethics Committee. Sexually active women of 18 years age with symptoms suggestive of acute cystitis (i.e., two or more of the following symptoms: dysuria, urine frequency >6 times per day, urgency, suprapubic pain, fever, haematuria/smoky urine, burning sensation during micturition and acute onset incontinence), with no prior history of similar episode in last one year, attending gynecology outpatient department, as diagnosed by the concerned gynecologist were invited to participate in the study. Women who gave written informed consent for participation, regular follow up and mid-stream clean-catch (MSCC) urine sample for examination and had positive urine culture of 102 CFU/ml E. coli, were recruited [9]. During the study, 230 women with culture proven first episode of acute cystitis with E. coli were followed up telephonically, fortnightly for at least one year for any recurrence of symptoms suggestive of cystitis. Women with recurrence of symptoms were asked to submit MSCC urine specimen for culture. Of those who came for the repeat urine culture, 46 women had culture confirmed recurrence by E. coli. Women who were pregnant or planning pregnancy in next 6 months, not willing for follow up, had urological abnormality, were recently catheterized, had signs and symptoms of acute pyelonephritis or had other comorbidities like diabetes, hypertension etc., were excluded from the study. 2.2. Bacterial strains E. coli isolates from 46 women, who had recurrent cystitis during the study period, were included in final analysis. 44 women reported one recurrence each while 2 patients had 2 recurrences each during 1 year follow up. 2.3. Sample processing Urine samples were semi-quantitatively cultured onto cysteine lactose electrolyte deficient agar plate. Lactose-fermenting colonies with appropriate colonial morphology were presumptively identified as E. coli and were further confirmed using standard conventional biochemical tests [10]. The bacterial isolates were stored in 15% glycerol at 70 C for further studies. 2.4. ERIC PCR It was done using primers ERIC1 and ERIC2 [11] and same-host isolates with 93% identical profiles or indistinguishable on visual inspection were considered similar [12]. 2.5. Biofilm assay Biofilm production was assessed using M63 minimal glucose medium as described by Danese et al. [13] in 96 well flat bottom polystyrene micro titer plates. Results were calculated as described elsewhere [14]. 2.6. Phylogenetic classification Phylotyping of E. coli was done by multiplex PCR employing three markers viz. chuA, yjaA, and TSPE4.C2 as described earlier
Isolates were screened by multiplex PCR for 15 putative virulence factors (VFs) as described previously [16]. The reactions were analyzed in five pools, as follows: in pool 1, malX (a coding region near the terminus of a pathogenicity associated island, PAI), papA (P fimbriae, structural subunit), fimH (type 1 fimbriae, adhesin molecule), and ibeA (invasin of brain endothelium); in pool 2, fyuA (yersiniabactin receptor), sfa/focDE (S/F1C fimbriae), iutA (aerobactin receptor), and papG allele III (P fimbriae, adhesin molecule); in pool 3, hlyA (a-hemolysin), papG allele I (P fimbriae, adhesin molecule), and kpsMT II (group 2 capsule lipopolysaccharide); in pool 4, traT (serum/complement resistance) and papG allele II (P fimbriae, adhesin molecule); and in pool 5, afa/draBC (Dr [blood group] fimbriae) and cnf1 (cytotoxic necrotizing factor type 1). All PCRs were done in duplicate with appropriate positive and negative controls. 2.8. Statistical method The data was entered in MSeExcel sheet. Comparisons of proportions were tested using a chi-square test and binary logistic regression analysis. The descriptive statistics for various variables were reported as percentage for qualitative variables, p value
Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Short communication
Virulence determinants in Escherichia coli associated with recurrent cystitis in sexually active women Jyotsna Agarwal a, *, Bharti Mishra a, Sugandha Srivastava a, Richa Srivastava a, Amita Pandey b a b
Department of Microbiology, King George's Medical University, Lucknow, U.P 226003, India Department of Obstetrics & Gynaecology, King George's Medical University, Lucknow, U.P 226003, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 April 2014 Received in revised form 26 June 2014 Accepted 21 July 2014 Available online 10 August 2014
More than a quarter of women who experience acute cystitis develop recurrence but information on specific urovirulent genetic profile of uropathogenic Escherichia coli associated with recurrent cystitis is still limited. In this prospective cohort study, index episode E. coli from a cohort of 46 sexually active women with acute cystitis who reported recurrence during followup were grouped into repeat infection (RI) and single infection (SI) isolates, based on enterobacterial repetitive intergenic consensus (ERIC) PCR profile comparison with subsequent E. coli isolated from same women. PCR for phylogrouping and 15 virulence genes along with test for biofilm formation were done. Virulence score was calculated for each isolate as number of virulence genes detected. Among 46 index E. coli, 22 were RI, and 24 were SI isolates. RI isolates had phylogroup B2 as majority (54.5%) which is typically described as more virulent phylogroup and virulence score for RI isolates was also significantly higher compared to SI isolates. Virulence gene malX (p ¼ 0.03) was significantly associated with RI isolates. 68.2% RI isolates were strong to moderate biofilm producers in comparison to 33.3% SI isolates, an important survival strategy to reside in bladder and or vagina. Overall, E. coli associated with recurrent cystitis appear to be more virulent and malX seems to have a role in causing repeat infection. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Urinary tract infection Biofilms Phylogenetic groups of Escherichia coli Virulence factors Uropathogenic E. coli
1. Introduction Uncomplicated bladder infection (cystitis) constitute about 95% of all urinary tract infection (UTIs) and approximately 25e48% of all women with first cystitis develop a recurrence within one year of an episode despite antimicrobial treatment and anatomically normal urinary tracts [1,2]. Uropathogenic Escherichia coli (UPEC) remains the principal (~80e90%) causal pathogen [3]. UPEC possess a range of virulence genes that facilitate their adherence and survival in the urinary tract and allow them to establish infection [1,3].
Abbreviations: RI, repeat infection; SI, single infection; UPEC, uropathogenic E. coli; ERIC PCR, enterobacterial repetitive intergenic consensus PCR; MSCC, midstream clean-catch; VF, virulence factors; UTI, urinary tract infection. * Corresponding author. Tel.: þ91 522 2257569 (off); fax: þ91 522 2257539. E-mail addresses: [email protected] (J. Agarwal), bharti_micro@rediffmail. com (B. Mishra), [email protected] (S. Srivastava), [email protected] (R. Srivastava), [email protected] (A. Pandey). http://dx.doi.org/10.1016/j.micpath.2014.07.004 0882-4010/© 2014 Elsevier Ltd. All rights reserved.
Recurrent cystitis is defined as at least two symptomatic episodes of cystitis with positive urine cultures in 6 months or three episodes within 12 months (including the index infection) [4]. Recurrence may be either relapse or reinfection on the basis of the strain responsible for causation of new episode of cystitis i.e., same strain as the primary infecting strain or a different strain, respectively [5,6]. Studies done to analyze pattern of virulence gene distribution in E. coli and their association with recurrent cystitis have found varied results depending on the study settings [1,7,8]. In present study, a cohort of women with acute cystitis was followed up for recurrence and their index episode E. coli was matched with recurrent strain(s) using Enterobacterial Repetitive Intergenic Consensus (ERIC) profiles to identify those associated with recurrent infection. These E. coli were analyzed for phylogroups, virulence genotype and biofilm formation capability in order to identify virulence determinants associated with recurrent cystitis among sexually active women in the absence of anatomic abnormalities of urinary tract.
J. Agarwal et al. / Microbial Pathogenesis 74 (2014) 38e41
2. Materials and methods
39
[15]. All PCR were done in duplicate with appropriate positive and negative controls.
2.1. Study design and subjects 2.7. Virulence genotyping This prospective cohort study was conducted in the department of Microbiology at King George's Medical University, Lucknow, between February 2011 and May 2013. Study protocol was approved by the University Ethics Committee. Sexually active women of 18 years age with symptoms suggestive of acute cystitis (i.e., two or more of the following symptoms: dysuria, urine frequency >6 times per day, urgency, suprapubic pain, fever, haematuria/smoky urine, burning sensation during micturition and acute onset incontinence), with no prior history of similar episode in last one year, attending gynecology outpatient department, as diagnosed by the concerned gynecologist were invited to participate in the study. Women who gave written informed consent for participation, regular follow up and mid-stream clean-catch (MSCC) urine sample for examination and had positive urine culture of 102 CFU/ml E. coli, were recruited [9]. During the study, 230 women with culture proven first episode of acute cystitis with E. coli were followed up telephonically, fortnightly for at least one year for any recurrence of symptoms suggestive of cystitis. Women with recurrence of symptoms were asked to submit MSCC urine specimen for culture. Of those who came for the repeat urine culture, 46 women had culture confirmed recurrence by E. coli. Women who were pregnant or planning pregnancy in next 6 months, not willing for follow up, had urological abnormality, were recently catheterized, had signs and symptoms of acute pyelonephritis or had other comorbidities like diabetes, hypertension etc., were excluded from the study. 2.2. Bacterial strains E. coli isolates from 46 women, who had recurrent cystitis during the study period, were included in final analysis. 44 women reported one recurrence each while 2 patients had 2 recurrences each during 1 year follow up. 2.3. Sample processing Urine samples were semi-quantitatively cultured onto cysteine lactose electrolyte deficient agar plate. Lactose-fermenting colonies with appropriate colonial morphology were presumptively identified as E. coli and were further confirmed using standard conventional biochemical tests [10]. The bacterial isolates were stored in 15% glycerol at 70 C for further studies. 2.4. ERIC PCR It was done using primers ERIC1 and ERIC2 [11] and same-host isolates with 93% identical profiles or indistinguishable on visual inspection were considered similar [12]. 2.5. Biofilm assay Biofilm production was assessed using M63 minimal glucose medium as described by Danese et al. [13] in 96 well flat bottom polystyrene micro titer plates. Results were calculated as described elsewhere [14]. 2.6. Phylogenetic classification Phylotyping of E. coli was done by multiplex PCR employing three markers viz. chuA, yjaA, and TSPE4.C2 as described earlier
Isolates were screened by multiplex PCR for 15 putative virulence factors (VFs) as described previously [16]. The reactions were analyzed in five pools, as follows: in pool 1, malX (a coding region near the terminus of a pathogenicity associated island, PAI), papA (P fimbriae, structural subunit), fimH (type 1 fimbriae, adhesin molecule), and ibeA (invasin of brain endothelium); in pool 2, fyuA (yersiniabactin receptor), sfa/focDE (S/F1C fimbriae), iutA (aerobactin receptor), and papG allele III (P fimbriae, adhesin molecule); in pool 3, hlyA (a-hemolysin), papG allele I (P fimbriae, adhesin molecule), and kpsMT II (group 2 capsule lipopolysaccharide); in pool 4, traT (serum/complement resistance) and papG allele II (P fimbriae, adhesin molecule); and in pool 5, afa/draBC (Dr [blood group] fimbriae) and cnf1 (cytotoxic necrotizing factor type 1). All PCRs were done in duplicate with appropriate positive and negative controls. 2.8. Statistical method The data was entered in MSeExcel sheet. Comparisons of proportions were tested using a chi-square test and binary logistic regression analysis. The descriptive statistics for various variables were reported as percentage for qualitative variables, p value
