Background
Urinary tract infections (UTIs) are the most frequent human infections occurring to people of all ages, which cause morbidity and significant mortality globally [
1,
2]. UTIs are mostly (70–90%) caused by the uropathogenic
Escherichia coli (UPEC), one of the extraintestinal pathogenic
E. coli pathotypes (ExPEC) [
1,
3]. UPEC strains account for up to 90% of community-acquired UTIs and 50% of nosocomial UTIs [
4]. UPEC strains usually carry a series of virulence markers, including adhesins, toxins and iron uptake systems (siderophores) that enable them to invade, colonize, and survive in the urinary tract, and prevent them from removal during urination [
1,
4,
5]. Indeed, it is suggested that UPEC isolates usually harbor the largest number of pathogenicity-associated islands (PAIs) encoding a variety of virulence determinants involved in adhesion, invasion, and bacterial resistance to host defense and consequently influencing the pathogenicity of symptomatic or complicated UTIs [
6,
7]. Initially, UPECs colonize the bladder and cause cystitis. Then, in an ascending manner would be able to move to the kidney, causing an acute pyelonephritis or disseminates to the blood leading to urosepsis [
4,
6].
On the other hand, increasing antimicrobial resistance among UPEC isolates has been increasing dramatically and has turned out to be a serious health concern. The issue is mainly due to the emergence of multidrug-resistant (MDR) strains which contain genes encoding extended-spectrum β-lactamase (ESBLs) and resistance to sulfamethoxazole-trimethoprim (SXT) and fluoroquinolones [
4]. UTIs caused by ESBL-producing UPEC strains are associated with prolonged hospitalization and hygiene cost [
8]. The rate of MDR-UPEC strains in developed and developing countries is variable and in Iran as a developing country, has been estimated as 49.4% [
9]. Given the increased resistance to the first line antimicrobial agents used in empiric therapy, UTI treatment in clinical practice has become somehow challenging. Indeed, the study of urovirulence genes and antimicrobial resistance can serve as a key element in developing new therapeutic targets. These factors can influence and be utilized as a useful marker to predict the clinical outcomes of UTIs caused by UPECs. There have been limited published epidemiologic studies on virulence genes and antimicrobial resistance among the UPECs isolated from symptomatic patients with UTI in Iran, hence, the present study aimed to evaluate the important characteristics of UPEC isolates, as well as investigate the correlation between the urovirulence genes and the type of clinical disease or antibiotic resistance from Shiraz, Iran.
Subject characteristics
The recruited patients in our study were 50 females and 76 males aged from 1 to 100 years old with a mean age of 48.9 ± 28.8 years. There was no statistically significant difference in age and gender of subjects within the three studied disease groups. The recovered UPEC isolates from different wards were as follows: Intensive Care Unit or ICU (n = 76, 60.4%), Internal wards (n = 36, 28.6%), Surgery (n = 7, 5.6%), and Transplantation (n = 7, 5.6%). Moreover, the frequencies of cases in different wards were as follows: cystitis (ICU = 23, Internal ward = 12, Surgery = 2, Transplantation = 5), pyelonephritis (ICU = 44, Internal ward = 22, Surgery = 5, Transplantation = 2) and urosepsis (ICU = 9, Internal ward = 2, Surgery = 0, Transplantation = 0).
Distribution of virulence genes
Frequency of the studied virulence genes among clinical groups is depicted in Table
1. Overall, 14.3% (18/126) of the UPEC isolates examined were positive for at least two of virulence markers. No significant difference was observed between virulence genes and isolates from different wards (data not shown). The frequency of only 4 genes (
fimH,
sfa,
iutA, and PAI marker) was > 50% among all the isolates examined. The majority of virulence genes were determined in different proportions among the three clinical groups (Table
1). Among adhesins, the most prevalent gene in all groups was
fimH (99.2%), followed by
sfa (79.4%), and
pap GII and
afa were found 46%, equally. Neither
pap GI nor
GIII gene was detected among all of the clinical isolates. The
iutA,
pap GII, and
hlyA genes were found essentially in urosepsis isolates with significantly different (
P < 0.05) frequencies (Table
1). As shown in Table 1, among the clinical diseases, UPEC isolates recovered from urosepsis cases had the highest rate of designated genes. As for the distribution of the virulence genes, the isolates exhibited 35 distinct arrangements of virulence patterns, referred to as UPEC followed by an Arabic numeral (Table
2). The isolates recovered from pyelonephritis, cystitis, and urosepsis cases showed 27, 22, and 6 virulence patterns, respectively (Table
2). UPEC 1 was the most frequent pattern (16.7%), with the presence of
iutA-
fimH-PAI-
sfa-
afa virulence genes.
Table 1
Distribution of virulence genes among different clinical diseases
fimH
| 41 (97.6) | 73 (100) | 11 (100) |
PAI | 36 (85.7) | 61 (83.6) | 11 (100) |
sfa
| 33 (78.6) | 58 (79.5) | 9 (81.8) |
iutA
a
| 24 (57.1) | 52 (71.2) | 10 (90.9) |
pap GII
b
| 15 (35.7) | 34 (46.6) | 9 (81.8) |
afa
| 19 (45.2) | 36 (49.3) | 3 (27.3) |
hlyA
b
| 10 (23.8) | 19 (26) | 7 (63.6) |
Table 2
Virulence patterns identified among UPEC isolates
UPEC1 | iutA-fimH-PAI-sfa-afa | 21 | 16.7 | 9 | 12.3 | 10 | 23.8 | 2 | 18.2 |
UPEC2 | papGII-iutA-fimH-PAI-hlyA-sfa-afa | 11 | 8.7 | 7 | 9.6 | 1 | 2.4 | 3 | 27.3 |
UPEC3 | papGII-iutA-fimH-PAI-hlyA-sfa | 11 | 8.7 | 4 | 5.5 | 4 | 9.5 | 3 | 27.3 |
UPEC4 | iutA-fimH-PAI-sfa | 9 | 7.1 | 6 | 8.2 | 3 | 7.1 | 0 | 0 |
UPEC5 | papGII-iutA-fimH-PAI-sfa-afa | 9 | 7.1 | 6 | 8.2 | 3 | 7.1 | 0 | 0 |
UPEC6 |
fimH-sfa
| 8 | 6.3 | 6 | 8.2 | 2 | 4.8 | 0 | 0 |
UPEC7 | papGII-iutA-fimH-PAI-sfa | 6 | 4.8 | 5 | 6.8 | 0 | 0 | 1 | 9.1 |
UPEC8 | fimH-PAI | 4 | 3.2 | 1 | 1.4 | 3 | 7.1 | 0 | 0 |
UPEC9 | iutA-fimH-PAI | 4 | 3.2 | 4 | 5.5 | 0 | 0 | 0 | 0 |
UPEC10 | fimH-PAI-sfa | 3 | 2.4 | 2 | 2.7 | 1 | 2.4 | 0 | 0 |
UPEC11 | papGII-iutA-fimH-PAI | 3 | 2.4 | 2 | 2.7 | 0 | 0 | 1 | 9.1 |
UPEC12 | iutA-fimH-PAI-afa | 3 | 2.4 | 2 | 2.7 | 1 | 2.4 | 0 | 0 |
UPEC13 | fimH-PAI-hlyA-sfa | 3 | 2.4 | 2 | 2.7 | 1 | 2.4 | 0 | 0 |
UPEC14 | papGII-fimH-PAI-sfa-afa | 3 | 2.4 | 1 | 1.4 | 2 | 4.8 | 0 | 0 |
UPEC15 |
papGII-fimH
| 2 | 1.6 | 2 | 2.7 | 0 | 0 | 0 | 0 |
UPEC16 |
fimH-afa
| 2 | 1.6 | 1 | 1.4 | 1 | 2.4 | 0 | 0 |
UPEC17 |
papGII-fimH-sfa
| 2 | 1.6 | 1 | 1.4 | 1 | 2.4 | 0 | 0 |
UPEC18 |
iutA-fimH-sfa
| 2 | 1.6 | 1 | 1.4 | 1 | 2.4 | 0 | 0 |
UPEC19 | papGII-fimH-PAI-sfa | 2 | 1.6 | 1 | 1.4 | 1 | 2.4 | 0 | 0 |
UPEC20 | papGII-fimH-PAI-hlyA-sfa | 2 | 1.6 | 2 | 2.7 | 0 | 0 | 0 | 0 |
UPEC21 | iutA-fimH-PAI-hlyA-sfa | 2 | 1.6 | 2 | 2.7 | 0 | 0 | 0 | 0 |
UPEC22 |
iutA-fimH
| 1 | 0.8 | 1 | 1.4 | 0 | 0 | 0 | 0 |
UPEC23 |
fimH-hlyA
| 1 | 0.8 | 0 | 0 | 1 | 2.4 | 0 | 0 |
UPEC24 | papGII-fimH-PAI | 1 | 0.8 | 0 | 0 | 1 | 2.4 | 0 | 0 |
UPEC25 | fimH-PAI-afa | 1 | 0.8 | 0 | 0 | 1 | 2.4 | 0 | 0 |
UPEC26 | PAI-sfa-afa | 1 | 0.8 | 0 | 0 | 1 | 2.4 | 0 | 0 |
UPEC27 | papGII-fimH-PAI-afa | 1 | 0.8 | 0 | 0 | 1 | 2.4 | 0 | 0 |
UPEC28 | iutA-fimH-PAI-hlyA | 1 | 0.8 | 1 | 1.4 | 0 | 0 | 0 | 0 |
UPEC29 | fimH-PAI-sfa-afa | 1 | 0.8 | 1 | 1.4 | 0 | 0 | 0 | 0 |
UPEC30 | papGII-iutA-fimH-PAI-afa | 1 | 0.8 | 1 | 1.4 | 0 | 0 | 0 | 0 |
UPEC31 |
papGII-iutA-fimH-sfa-afa
| 1 | 0.8 | 1 | 1.4 | 0 | 0 | 0 | 0 |
UPEC32 | papGII-fimH-PAI-hlyA-afa | 1 | 0.8 | 0 | 0 | 0 | 0 | 1 | 9.1 |
UPEC33 | fimH-PAI-hlyA-sfa-afa | 1 | 0.8 | 0 | 0 | 1 | 2.4 | 0 | 0 |
UPEC34 | papGII-iutA-fimH-PAI-hlyA-afa | 1 | 0.8 | 1 | 1.4 | 0 | 0 | 0 | 0 |
UPEC35 | iutA-fimH-PAI-hlyA-sfa-afa | 1 | 0.8 | 0 | 0 | 1 | 2.4 | 0 | 0 |
Antimicrobial resistance among UPEC isolates
The highest resistance rate was observed against ampicillin (88.9%), followed by nalidixic acid (81%), sulfamethoxazole-trimethoprim (72.2%), ceftazidime (65.1%), ciprofloxacin (55.6%), cefoxitin (20.6%), gentamicin (19.8%), amikacin (7.9%), nitrofurantoin (4.8%), and imipenem (0.8%). The majority of isolates (n = 98, 77.8%) were MDR with predominant patterns for ampicillin, sulfamethoxazole-trimethoprim, nalidixic acid, ceftazidime, ciprofloxacin (15.1%), followed by ampicillin, sulfamethoxazole-trimethoprim, nalidixic acid, and ceftazidime with the frequency of 11.1%. Antibiotic susceptibility patterns were different depending on the place recovery of isolates. The frequencies of MDR isolates from ICU, Internal, Surgery, and Transplantation wards were 81.6, 69.4, 71.4, and 85.7%, respectively. Moreover, the isolates from urosepsis cases were more resistant than those recovered from cystitis and pyelonephiritis cases (81.8% vs. 78.6, and 76.7%, respectively, P > 0.05). Analysis of antibiotic resistance in terms of the gender and age of participants revealed no statistically significant differences among them (P = 0.82).
Relationship between the distribution of virulence genes and resistance to multiple drugs was also investigated. Among the studied genes, 100 and 78.6% of UPEC isolates harboring
fimH and
sfa were MDR, respectively. On the other hand, 60.7, 53.6, and 60.7% of the isolates carrying
iutA,
pap GII, and
hlyA found to be susceptible to antimicrobial agents, respectively (data not shown). Further analysis revealed that the rate of ESBL-producing isolates was 54.8% (69/126). There was a significant correlation (
P < 0.05) between ESBL-producing isolates and antibiotic resistance to all the antibiotics tested, except for amikacin, nitrofurantoin, and imipenem (Table
3). A significant difference was also observed between ESBL production and MDR positive isolates (97.1% ESBL producers vs. 54.4% non-ESBL producers,
P < 0.001). Among the seven evaluated genes, a statistically significant difference was determined between ESBL production with
pap GII,
iutA, and PAI marker genes, and ESBL-negative isolates with
afa gene (Table
4).
Table 3
Distribution of antibiotic resistant UPEC isolates according to ESBL production
Co-trimoxazole | 36 (63.2) | 55 (79.7) | 0.039 |
Ampicillin | 43 (75.4) | 69 (100) | < 0.001 |
Nalidixic acid | 35 (61.4) | 67 (97.1) | < 0.001 |
Amikacin | 2 (3.5) | 8 (11.6) | 0.095 |
Nitrofurantoin | 2 (3.5) | 4 (5.8) | 0.54 |
Ceftazidime | 13 (22.8) | 69 (100) | < 0.001 |
Imipenem | 0 | 1 (1.4) | 0.36 |
Gentamicin | 5 (8.8) | 20 (29) | 0.005 |
Ciprofloxacin | 25 (43.9) | 45 (65.2) | 0.016 |
Cefoxitin | 17 (29.8) | 9 (13) | 0.021 |
Table 4
Distribution of virulence genes among UPEC isolates according to ESBL production
pap GII
| 20 (35.1) | 38 (55.1) | 0.03 |
iutA
| 31 (54.4) | 55 (79.7) | 0.004 |
fimH
| 56 (98.2) | 69 (100) | 0.45 |
PAI | 42 (73.7) | 66 (95.7) | 0.001 |
hlyA
| 15 (26.3) | 21 (30.4) | 0.69 |
sfa
| 42 (73.7) | 58 (84.1) | 0.19 |
afa
| 35 (61.4) | 23 (33.3) | 0.002 |
According to disk diffusion results, 60 (47.6%) isolates were high-level quinolone-resistant bacteria with ciprofloxacin MIC ≥6 μg/mL, while 42 (33.3%) isolates were identified as low level quinolone-resistant bacteria (MIC ≤1). In overall, the MIC range of all 70 ciprofloxacin-resistant isolates was between 6 and > 32 μg/mL, and both MIC50 and MIC90 were estimated > 32 μg/mL.
Discussion
A better knowledge of the virulence markers of UPEC strains, especially in hospitalized patients allows the physicians to follow up the trend of pathogenicity of strains causing the urinary tract infections. The studied samples in the present investigation were originated from 126 inpatients with pyelonephritis, cystitis and urosepsis, which were evaluated for the presence of nine urovirulence genes and their corresponding antibiotic susceptibility patterns.
Genes encoding adhesins are the most frequently occurring virulence factors in UPECs [
15]. Fimbriae are important to establish the UTI and probably in the progression to urosepsis [
16]. It is suggested that type 1 and P fimbriae are common among cystitis and pyelonephritis-associated UPEC strains, respectively [
5]. As we expected, in our study almost all the isolates (99.2%) carried the
fimH gene, encoding of the type 1 fimbriae, consistent with some previous reports [
1,
5,
7,
17,
18]. Conversely, in a recent study from Mexico, the prevalence of
fimH was reported 61.3% [
15], which was in agreement with some other published data [
19,
20]. Recently, in an investigation on 183 UPEC isolates [
5], the
fimH was found to be associated with cystitis cases, but in the current work no correlation was found with clinical manifestations. The
sfa was the second most prevalent adhesion gene (79.4%) in our isolates, consistent with a study conducted in South Korea with frequency of 100% [
1]. On the contrary, in some reports the prevalence of less than 50% [
7,
21,
22] or even 0% [
23] was cited for this gene. Although the exact role of S-fimbriae is not identified; however, the dissemination of bacterium within the host tissue is suggested for this adhesin [
1]. In the present study, the frequency of
pap GII and
afa were found to be 46%. It was reported that class II
pap G allele is related to pyelonephritis cases, while
pap GIII is primarily associated with UTIs in dogs and cats [
18], which is in agreement with our findings. In two studies from South Korea and China [
7,
18], no
pap GI gene was identified in their isolates, similar to our findings, either. Indeed, it has been suggested that there is a possibility of mutation at the level of a specific gene, resulting in the absence of the corresponding gene in PCR method [
19]. The role of
afa afimbrial adhesin is mentioned in the development of chronic nephritis [
19]. Our findings revealed that 49.3% of isolates from pyelonephritis cases were
afa PCR-positive which is higher than those reported by other investigators [
1,
15,
17‐
19,
22‐
24]. This discrepancy could be due to differences in type disease (symptomatic or asymptomatic bacteriuria) or geographic region.
The second most common gene in this study was found to be PAI marker (85.7%). The determinants such as toxins, siderophores, and protectins are encoded on UPEC PAIs [
15]. This frequency was higher than those previously reported for UPEC isolates [
15,
23,
24], but lower than that reported in other studies from Iran [
25‐
27].
The
iutA as a siderophore marker donates the potency of resistance against serum killing to the UPEC strains, thereby enabling them to persist in body fluids such as the blood [
24]. The attributed characteristic is important for the pathogenesis of isolates causing urosepsis. As indicated by present findings, 90.9% of UPEC were carrying the
iutA gene, suggesting the isolates are invasive and a significant association between this gene and clinical groups. According to the obtained data, the frequency in the current study was higher than those previously reported [
7,
15], indicating the genes codifying siderophore vary depending on geographic areas and hosts [
15]. However, of the three clinical complications, isolates recovered from urosepsis cases had the highest frequency among the studied genes.
The
hlyA toxin is involved in tissue damage and impairment of local immune responses [
19]. There was a significant association between
hlyA gene and urosepsis isolates, which is consistent with invasive nature of UPECs isolated from urosepsis cases. Our results (28.6%) are in agreement with those found by other studies [
7,
19,
28].
On the other hand, in the present investigation, 35 patterns of combinations of the urovirulence markers were characterized. The UPEC 1 pattern with
iutA-
fimH-PAI-
sfa-
afa template was the most frequently present. According to different geographic regions and disease status, different patterns of combinations of the virulence genes and antimicrobial resistance phenotypes have been reported in previous studies [
7,
15,
23].
According to our data, all UPECs contained at least two virulence genes. This is in contrast to Oliveira et al. [
24], who reported 90% UPECs showed at least one of the eight virulence genes. It was shown in a study [
19], that UPECs isolated from hospitalized patients offered a great diversity of gene associations, in agreement with our data, indicating heterogeneity in the distribution of virulence genes among UPEC strains in different regions [
24].
Increased antibiotic resistance, particularly for third-generation cephalosporins and fluoroquinolones among UPEC isolates has created challenges in clinical practice [
29]. Majority of our isolates were remarkably resistant to the most of the tested antibiotics, with 77.8% of strains showing multi-drug resistance, making them the causative agent of an important health problem in our area, in agreement with previous works from different regions [
15,
20,
25,
30]. As empirical antimicrobial therapy is usually the first conventional treatment for UTIs, awareness of the local epidemiological data for an efficient therapy is necessary and useful [
31]. According to our investigation, 88.9, 81, 72.2, and 55.6% isolates were resistant to ampicillin, nalidixic acid, sulfamethoxazole-trimethoprim, and ciprofloxacin, respectively, the therapeutic agents used as the first-line empirical treatments for UTIs [
24,
32]. Ciprofloxacin is the most common fluroquinolone used to treat UTIs. However, due to its overuse in the last decade, the resistance rate of UPECs to that antibiotic has markedly increased [
33]. In comparison to other studies from different countries [
18,
20,
24,
28], our rate (55.6%) is high, but it is consistent with another investigation from Iran with frequency of 61.3% [
30]. Our findings regarding the MIC study of ciprofloxacin showed that the MIC
50 and MIC
90 ranges are higher than their corresponding maximum values in the E-test strip (32 μg/mL). The MIC values of clinical isolates vary based on geographic area and time. In a study from Algeria [
28], ciprofloxacin MIC range was mentioned between 0.5 to > 128 μg/mL. It seems that in a clinical setting, we cannot surmount this level of resistance even by using the manifold dosages of MIC
50 and MIC
90 values. One explanation for our observed high rate in our region could be the wide use of antibiotics for bacterial infections, for example prescription of ciprofloxacin by clinicians in the first visit of patients with uncomplicated UTIs.
Similar to the other studies from Iran and other countries [
1,
18,
20,
34,
35], the majority of isolates in the present study were susceptible to imipenem (99.2%) and nitrofurantoin (95.2%). Because of the emergence of antibiotic resistance in Gram-negative rods, imipenem is not preferably included in the first line therapy of UTIs and is recommended as a choice agent for only ESBL-producers [
28,
32]. Additionally, in spite of good activity of nitrofurantoin against UPEC isolates, but due to numerous side effects, its application is limited. Nevertheless, because of increasing resistance rate of UPEC strains to sulfamethoxazole-trimethoprim and quinolones, rational use of nitrofurantoin has been recommended again for the re-infection prophylaxis of recurrent non complicated UTIs [
36,
37].
Appropriate diagnosis and treatment of UTIs caused by ESBL-producing phenotypes is important for the prevention of long-term clinical outcomes [
8]. One of our major concerns is about the observed high rates of MDR among UPEC ESBL-producers (97.1%). The data from a multi-centric study revealed that the rates of UPEC ESBL-producers among Iranian isolates were 42, and 44% of isolates which were MDR [
38]. In the present study, the rate of ESBL-positive isolates was 54.8%, which was similar to some other studies [
18,
34]. In contrary, in three studies from Iran a range of 22.3–35.7% was observed for UPEC ESBL-producers [
20,
39,
40]. Such discrepancies might be due to the differences in epidemiology of isolates or sample size of studies.