Comparison of Adhesin Genes and Antimicrobial Susceptibilities between Uropathogenic and Intestinal Commensal Escherichia coli Strains

The presence of adhesins is arguably an important determinant of pathogenicity for Uropathogenic Escherichia coli (UPEC). Antimicrobial susceptibilities were tested by agar dilution method, fifteen adhesin genes were detected by polymerase chain reaction, and multilocus sequence typing (MLST) was analyzed in 70 UPEC isolates and 41 commensal E. coli strains. Extended-spectrum β-lactamase (ESBL) was determined with confirmatory test. The prevalence of ESBL-producers in UPEC (53%, 37/70) was higher than the commensal intestinal isolates (7%, 3/41), and 97% (36/37) of the ESBL-producing UPEC harbored bla CTX-M genes. afa was present in 36% (10/28) UPEC isolates from recurrent lower urinary tract infection (UTI), and none in the acute pyelonephritis, acute uncomplicated cystitis or commensal strains (P<0.0001). papG was detected in 28% (20/70) of UPEC isolates, while 5% (2/41) of the commensal strains were papG positive (P = 0.0025), and the prevalence of papG was significantly higher in acute pyelonephritis group (71%) than the other two UTI groups (P<0.0001). The prevalence of flu, yqi, yadN and ygiL was significantly higher in UPEC isolates than in the commensal strains. ESBL-producing UPEC showed a lower prevalence of adhesin genes compared with non-ESBL-producing strains. The MLST profiles were different between UPEC and commensal strains, with ST131 (19%, 13/70) and ST10 (20%, 8/41) being the most common MLSTs, respectively. This study demonstrated that several adhesin genes were more prevalent in UPEC isolates than in commensal E. coli, and afa may be associated with recurrent lower UTI whereas papG is more frequently associated with acute pyelonephritis.


Introduction
Urinary tract infection (UTI) is one of the most common bacterial infections, and it is estimated that the overall lifetime prevalence of UTI in women is greater than 50% [1]. Further recurrent UTIs are reported in about 25% of women within 6 months of an acute UTI episode and pose a major health problem [2]. Uropathogenic Escherichia coli (UPEC) is the causative pathogen in 70-95% community-acquired UTI and over 50% nosocomial UTI [3]. Many virulence factors contribute to the pathogenesis of UPEC. The presence of adhesins is arguably the most important determinant of the pathogenicity for UPEC [4]. Adhesive organelles, including type 1, P, S/F1C, M, G and curli fimbriae, along with Afa/Dr adhesins, as well as temperature sensitive haemagglutinin (TSH), promote both bacterial attachment to and invasion of host tissues within the urinary tract [4][5][6].
E. coli expressing Dr/Afa adhesins may predispose the establishment of chronic or recurrent infections [7]. In previous studies, it was shown that UPEC entered the urinary epithelial cells through adhesins AfaD and AfaE, and therefore avoided the clearance of host immunosurveillance and antibiotic treatment. When the host cells shed, releasing a large amount of intracellular UPEC, a new round of infection could begin causing recurrence or relapse of UTI [8]. P fimbriae is encoded by the pap gene cluster, including papG encoding the tip adhesin PapG [9]. The three most studied PapG molecular variants, which are shown to bind distinct isoreceptors are PapG I, II and III [10]. Clinically the class II papG allele is primarily associated with human pyelonephritis, and class III papG allele with genitourinary infections in dogs and cats yet some cases of human cystitis [11]. In addition to P fimbriae, type 1 and S/F1C fimbriae, curli and M and G fimbriae which are encoded by the fim and sfa/foc, csg, bma and gaf gene clusters, respectively, may also be related to the adherence of UPEC [4,6,12]. More recently, it is discovered that adhesin Ag43, encoded by flu and abundant on the outer membrane of E. coli, enables bacterial cells to adhere to each other and form biofilm over the host urothelial cells, leading to poor clearance of the pathogens [13]. Other novel genes like yqi, yadN and ygiL were also found to be prevalent among UPEC strains and played an important role in colonization and pathogenesis [6,14].
Previous studies have shown that the prevalence of adhesin genes is different between UPEC and fecal commensal strains of E. coli [15]. However, few epidemiologic data about adhesin genes of UPEC are available in China. The aim of the present study was to compare the prevalence of the adhesin genes and antimicrobial susceptibilities between UPEC and intestinal colonizing strains of E. coli, and adhesin genes between strains isolated from different types of UTIs and their association with a particular type of UTIs.

E. coli isolates
A total of 70 clinical isolates of UPEC were collected from clean midstream urine specimens of inpatients (63 female and 7 male) with urinary tract infections at Huashan Hospital, Shanghai from January 2008 to December 2010. The exclusion criteria were: 1) patients with indwelling catheter; 2) patients with urinary calculus or structural abnormalities. Isolation was performed according to standard laboratory protocols and UPEC were isolated from urine with .10 5 colony-forming units/ml.
Among the 70 UPEC isolates, 28 were from patients with recurrent lower UTIs. The recurrent lower UTIs were defined as at least two episodes of UTI within 6 month. Each time the patients had: a) typical clinical symptoms as urinary urgency, pain, and frequent voiding, b) pyuria, c) without fever, chill or other systematic symptoms. All of the 28 patients in recurrent lower UTI group had recurrences caused by UPEC. MLST typing of the UPEC strains from 11 randomly selected patients showed that the strains isolated from different episodes of each patient had the same MLST type.
Seventeen strains were isolated from acute pyelonephritis patients by the following criteria: a) the temperature .38.5uC, b) positive percussion pain on kidney area, c) an elevated leukocyte count (.11610 9 /L) and elevated percentage of neutrophils (.70%) in the blood routine test. The remaining 25 strains were from patients with acute uncomplicated cystitis.
The intestinal commensal E. coli isolates (n = 41) were collected from anal swab specimens of healthy volunteers. The inclusion criteria emphasized: 1) without diarrhea or abdominal pain, 2) without antibiotic administration within the three months before taking the specimen. After isolation, all isolates were kept frozen at 270uC in 20% glycerol.

Antimicrobial susceptibility testing
The minimum inhibitory concentrations (MICs) of imipenem, cefepime, ceftazidime, cefotaxime, cefuroxime, cefazolin, piperacillin/tazobactam, piperacillin, gentamicin, amikacin, ciprofloxacin, and nitrofurantoin for both UPEC and commensal isolates were determined by agar dilution according to CLSI standards [16]. The antimicrobial concentrations were from 0.06 to 128 mg/ ml. E. coli ATCC 25922 was used as a quality control strain.

Detection of ESBL-producing bacterial strains and ESBL genes
Extended-spectrum b-lactamase (ESBL) was determined with the previously recommended CLSI disk diffusion ESBL screening and confirmatory test [17]. CTX-M-specific genes in ESBLproducing strains were identified by polymerase chain reaction (PCR) with the primers reported previously [18].

Analysis of UPEC by multilocus sequence typing (MLST)
All of the 70 clinical UPEC and 41 intestinal commensal isolates were analyzed by MLST. The seven housekeeping genes (adk, fumC, icd, purA, gyrB, recA and mdh) for typing [23], as well as the primer sets and PCR conditions were according to the MLST website for E. coli (mlst.ucc.ie/mlst/dbs/Ecoli/documents/ primersColi_html). The based upon related sequence types (BURST) clustering algorithm (eburst.mlst.net) was used to analyze the allelic profiles and define clonal complexes (CCs). CCs were identified according to the number of single-locus variants (SLVs) and double-locus variants (DLVs) shared between isolates, where only STs that shared six or more loci were assigned to a defined CCs.

Statistical analysis
The comparison of prevalence rates was performed using Pearson Chi-square test or Fisher's exact test (two-tailed) with the software SPSS 16.0, and the level of significance was set at P,0.05.

Prevalence of adhesin genes
(1) Afa/Dr adhesin family member genes. afa was present only in 10 UPEC isolates that belonged to the recurrent lower UTIs group (10/28, 36%). None of the isolates from the acute pyelonephritis group or the acute uncomplicated cystitis group carried afa. No afa was found in 41 intestinal commensal E. coli isolates. Neither draE nor daaE was detected among all the strains we screened.
(5) Other adhesin genes. sfaD was present in 5 isolates and focG in 2 isolates among the UPEC group. One of the sfaD carrying isolates was also detected focG positive. None of the isolates in the intestinal commensal group were sfaD/focG positive. No more than 5 isolates in UPEC or commensal groups produced amplicons with the bmaE, gafD or tsh primers. (Table 2) Comparison of the prevalence of fimbrial genes between ESBL-producing and non-ESBL-producing UPEC The prevalence of fimH (97%, 32/33) in the non-ESBLproducing UPEC was significantly higher than that in the ESBLproducing UPEC (76%, 28/37, P = 0.0110). All the six strains carrying sfaD/focG were from the non-ESBL-producing UPEC group. The prevalence of the other fimbrial genes in the non-ESBL-producing UPEC group seemed a little higher than the ESBL-producing UPEC strains, however, there were no statistically significant differences (P.0.05, Table 3)
The 41 intestinal commensal isolates analyzed by MLST were grouped into 32 different STs. 28 were already included in the E. coli website database and 4 were new STs. ST10 (n = 8, 20%) was the most common group. A new ST had 2 strains, while the other 29 STs contained a single strain each. As analyzed by eBURST, ST10, ST34, ST167, ST48, ST559 and ST832 belonged to CC10 ( Figure 1B).

Most of the intestinal commensal isolates tested in this study
were susceptible to all the tested antimicrobial agents, while the UPEC isolates were characterized by a varying degree of susceptibilities to different antimicrobials. Of 70 UPEC strains, 37 (53%) produced extended-spectrum b-lactamase (ESBL). In a previous study, 2.9% of E. coli isolates recovered by outpatient urine cultures in 2008 harbored ESBL in USA [24], which is much higher than previously reported but far lower than the rate in our study. The annual percentage of ESBL-producing UPEC with bla CTX-M genes changed from 35% in 2003 to 64% in 2008 with the average rate of 56% in that study [24]. However, in this study, bla CTX-M ESBL genes were detected in 36 (97%) of 37 ESBL positive strains, which is much higher than in the USA.
All 10 UPEC isolates harboring the afa belonged to recurrent lower UTI group, none was afa positive in isolates from other UTIs or intestinal carriage, indicating that UPEC carrying afa might correlate with the recurrence of UTI episodes. Blanco and colleagues screened UPEC for afa and found no afa positive strain in acute pyelonephritis isolates, however, afa was detected in 5 of the 116 isolates (4.3%) from cystitis patients and 1 of the 42 (2.4%) isolates from asymptomatic bacteriuria [19]. Previous studies showed that Afa/Dr fimbrial adhesins contributed to the ability of UPEC isolates to colonize and persist long term within the urinary tract and therefore more likely to cause the recurrence of UTI episodes [7][8].
The isoreceptors of adhesin PapG primarily are scattered within the kidney, including glomerulus, proximal tubulus, distal tubulus, and collecting duct. Therefore sitting on the top of type P fimbriae, adhesin PapG is thought to be associated with bacterial adhesion to the kidney [9]. The prevalence of papG in acute pyelonephritis isolates was significantly higher (71%) than that in recurrent lower UTIs (18%) and acute uncomplicated cystitis (12%) in the present study, illustrating high correlation between the papG and pyelonephritis. Our findings are consistent with some previous studies. Johnson et al found 69% (118/170) acute pyonephritis strains and 25% (21/83) of cystitis strains were papG positive [15]. Encoded by flu, Ag43 is a self-recognizing adhesin that is associated with cell aggregation and biofilm formation of UPEC [13]. We found a high prevalence (77%) of flu in UPEC isolates, while flu was detected in 41% of the intestinal commensal isolates. The finding is consistent with the results of Ulett and colleagues. They reported 83% (30/36) of the UPEC isolates and 56% (35/ 62) of the commensal strains carried flu [22]. However, there was no statistically significant difference in flu gene prevalence between recurrent lower UTI and non-recurrent lower UTI in the present study.
It was reported that the adhesin encoding gene yqi was prevalent among UPEC by more than 50% while absent in all the intestinal pathogenic E. coli [6]. In this study, yqi was detected in 7% of commensal isolates, but the prevalence was significantly lower than that in UPEC strains (27%, P = 0.0114).
yadN and ygiL were found to be more prevalent in UPEC (54% and 44%, respectively) than the intestinal commensal strains (32% and 17%) in the present study (P,0.05), which coincides with Mobley's work. It was reported that fimbriae related structure Yad and Ygi could enhance the virulence-related phenotypes, including biofilm formation and adherence to immortalized human epithelial cells [14]. In addition, we did not detect any significant prevalence difference of yadN or ygiL between UPEC strains causing recurrent lower UTI, acute pyelonephritis and uncomplicated cystitis.  Table 3. Comparison of the prevalence of fimbrial genes between ESBL-producing and non-ESBL-producing UPEC strains.  The results showed that 86% of UPEC isolates and 73% of commensal isolates carried fimH, encoding the adhesin FimH as the tip fimbria for type 1 fimbriae. Wang et al detected 91% (72/ 79) fimH positive in UPEC isolates [25]. On the other hand, Schlager found that 81% (74/91) strains isolated from fecal specimen of healthy girls carried fimH [26]. A high prevalence of fimH was found in both UPEC and commensal isolates. However, the transcription and expression level of fimH might be different between UPEC and commensal strains, which made type 1 fimbriae an important virulence factor for UPEC [27].
In the present study, we found that ESBL-producing UPEC isolates showed a lower prevalence of fimbrial genes, fimH and sfaD/focG, compared with non-ESBL-producing ones. The results coincided with the previous findings that fimbrial genes were of reduced prevalence among UPEC resistant to extended-spectrum cephalosporins. A possible explanation is that: virulence traits like the fimbrial genes, as well as resistance genes, can be carried on conjugative plasmids; therefore, the incompatible resistanceencoding plasmids are outcompeting fimbrial factor encoding plasmids [28]. It is reported that the acquisition of antibiotic resistance may cause alterations in phenotypic and physiological characteristics, which are referred to as ''biological fitness cost''. The biological fitness cost on antibiotics resistance generally results in reduced growth rates. More dramatic phenotypic changes, including poor fimbrial expression, were reported in an ampicillinresistant mutant Acinetobacter sp. strain DR1A comparing with the wild type strain DR1 [29]. Therefore, the decreased fimbrial genes and adherence capability in ESBL-producing UPEC might also be a fitness trade-off for the ESBL to survive antibiotics exposure. The exact explanation for the lower incidence of fimbrial genes among resistant UPEC isolates needs additional study.
This study indicated that afa of Afa/Dr adhesin family might be associated with lower UTI recurrence, while papG might be correlated with acute pyelonephritis. However, we only focused on detecting the prevalence of adhesin genes by PCR, instead of the mechanism of the related adhesins of UPEC. Therefore, we could not prove the correlation between the adhesins and the pathogenesis of the UPEC. Moreover, because of the ''cross-talk'' among the regulators of different adhesin systems of UPEC and their functional redundancy, when a certain type of adhesin is lost, it may be quite difficult to study the relationship between the UTI recurrence and a specific kind of adhesin. Studies on other factors and mechanisms involved in pathogenesis and persistence of UPEC are required.