In the past 20–50 years, specifications of Urinary Tract Infections (UTIs) in pediatrics have changed as a result of the novel antibiotic prescription, and improvements in medicine. Therefore, management of diseases requires further investigations to provide certain information about the epidemiology and prevalence of UTIs.
UTIs are common bacterial infections in the majority of infants and children. It is difficult to diagnose UTIs in children younger than 3 years because of non-specific presentation of its symptoms.
The uropathogenic Escherichia coli (UPEC) is the most prevalent cause of UTIs in children (1, 2). It has been estimated that the UPEC is responsible for 70-90% of cases of UTIs in children (1, 2). The pathogenesis of UPEC is related to several bacterial virulence factors (3). Some of the most important uropathogenic virulence factors are aerobactin (aer), iroN, kpsMT, type 1 fimbriae, usp, fimbriae, ompT, set 1, astA, traT, F1C fimbriae, iha, iutA, and group II capsule synthesis; sfa/foc, S and serum resistance, fimH and adhesins (3-5).
Diseases caused by E. coli often require antimicrobial therapy; however, antimicrobial prescription causes turbulence in the ecological balance between host and pathogens, which might lead to antibiotic resistance in pathogenic bacteria and finally emergence of more severe infections.
Several studies have shown that antibiotic resistance in E. coli has increased over time (6, 7). Similarly, an epidemiological investigation in Iran revealed that UPEC strains were the most commonly detected pathogen in pediatric patients infected with UTIs, which showed a high incidence of resistance (20–90%) to commonly used antibiotics (8-10). Antibiotic resistance genes are known to cause antibiotic resistance in UPEC strains isolated from pediatric patients suffered from UTIs (3).
Data on the incidence of virulence genes and the antimicrobial resistance of UPEC strains isolated from pediatric patients is scarce in Iran. Therefore, the present study aimed to characterize UPEC strains isolated from Iranian pediatric patients infected with UTIs at the molecular level and to investigate their susceptibility to 15 commonly used antibiotics.
3. Patients and Methods
3.1. Samples Collection and Bacterial Identification
From July to September 2012, a total of 121 urine samples were collected from boys (n = 51) and girls (n = 70) suffered from UTIs. The patients were 1-3 years old. Diagnosis of UTIs in patients was confirmed using the ultrasound technique (11). All samples were collected from the hospitalized patients in pediatric ward of Baqiyatallah Hospital in Tehran, Iran. Most of the patients had urine catheter for a week before they got UTI.
Urinary urgengy, frequency, dysuria, cloudy, bloody, and odorous urine, as well as fever chills and nausea in some cases, are the most commonly detected symptoms in pediatric patients. In order to decrease the likelihood of bacterial, cellular, or artifactual contamination, all urine samples were collected from midstream. Urine samples were collected using the Suprapubic Aspiration (SPA) method based on the standard technique of NICE (12).
All samples were immediately transferred to the Microbiology Research Center of the Islamic Azad University while kept at 4°C. Totally, 3 mL of each sample was blended with 225 mL of nutrient broth (Merck, Germany) for 2 minutes at normal speed, using a Stomacher lab blender and then incubated at 37°C for 24 hours. Next, 1 mL sample of the nutrient broth culture was mixed with 9 mL of MacConkey broth (Merck, Germany) and further incubated at 37°C for another 24 hours.
One loop of each tube was streaked on MacConkey agar (Merck, Germany). A typical purple colony of E. coli was streaked on Eosin-Methylene Blue agar (EMB agar; Merck, Germany) plate and incubated at 37°C for 24 hours. Green colonies with a metallic luster were considered as typical E. coli colonies. Such colonies were confirmed as E. coli using standard biochemical tests (e.g., Methyl red, Voges-Proskauer, Indole, and Citrate utilization tests). E. coli isolates were stored in Tryptic soy broth (TSB, Merck, Germany) containing 20% glycerol at -70°C for further characterization.
Antimicrobial susceptibility testing pattern of antimicrobial resistance was examined using the simple disk diffusion technique. The Mueller–Hinton agar (HiMedia Laboratories, Mumbai, India, MV1084) medium was used for this purpose. Antibiotic resistance of UPEC strains against 15 commonly used antibiotics was determined using the instruction of Clinical and Laboratory Standards Institute guidelines (CLSI 2012) (13).
Susceptibility of E. coli isolates were tested against ampicillin (10 u.disk -1), tetracycline (30 µg.disk -1), gentamycin (10 µg.disk -1), amikacin (30 u.disk -1), imipenem (30 u.disk -1), mezlocillin (30 u.disk -1), piperacillin (30 µg.disk -1), cefotaxime (30 µg.disk -1), ciprofloxacin (5 µg.disk -1), norfloxacin (30 µg.disk -1), cotrimoxazole (30 µg.disk -1), ceftazidime (30 µg.disk -1), ofloxacin (5 µg.disk -1), nitrofurantoin (300 µg.disk -1) and cephalothin (30 µg.disk -1) antibiotic agents (Oxoid).
All the inoculated plates were aerobically incubated at 37°C for 18-24 h. Results were interpreted based on the instruction provided by CLSI (2012) (13). In all reactions, the E. coli ATCC 25922 was used as quality control organism.
3.2. DNA Extraction and Bacterial Confirmation Using Polymerase Chain Reaction
Bacteria were cultured overnight on Luria-Bertani broth (Merck, Germany) and genomic DNA was extracted from typical colonies using the DNA extraction kit (DNPTM, CinnaGen, Iran) according to the manufacturer’s instruction. All E. coli colonies were also confirmed using the Polymerase Chain Reaction (PCR) technique (14).
The PCR method was done with a total volume of 50 µL, including 2 mM Mgcl2, 1 µM of forward primer, 1 µM of reverse primer (specified for the 16S rRNA gene of the E. coli) (Table 1), 5 µL PCR buffer 10X, 200 µM dNTP (Fermentas), 1 U Taq DNA polymerase (Fermentas) and 2.5 µL DNA template.
Then, the DNA was amplified by 31 successive cycles of denaturation at 95°C for 45 s, primer annealing at 59°C for 60 s, and DNA chain extension at 72°C for 60 s. The programmable thermal cycler (Eppendorf, Mastercycler® 5330, Eppendorf-Netheler-Hinz GmbH, Hamburg,Germany) PCR device was used in all PCR reaction.
3.3. Detection of UPEC Virulence Factors and Antibiotic Resistance Genes
Several PCR reactions were used for detecting virulence factors and antimicrobial resistance genes in UPEC isolates. List of primers used for detection of the virulence genes, and antibiotic resistance genes are shown in Table 1 (4, 5, 15-24). Table 2 presents PCR conditions. PCR products were electrophoresed using 2% agarose gels, which were stained with ethidium bromide at 90 V for 6 h, using 1×TBE (0.89 M Tris-Borate, 0.02 M EDTA, pH 8.3) as the running buffer. All products were examined under ultraviolet illumination. A set of molecular weight standards (Fermentas, GmbH, Germany) ranging from 100 bp to 2000 bp was included on each gel.
In order to confirm the PCR results, the sequencing method was used. For this reason, PCR products of some positive samples were purified with high pure PCR product purification kit (Roche Applied Science, Germany) according to manufacturer's recommendations. Single DNA strands were sequenced with ABI 3730 XL device and Sanger sequencing method (Macrogen, Korea). The result of the sequence of each gene was aligned with the gene sequences recorded in the GenBank database located at NCBI (National Center for Biotechnology Information).
3.4. Statistical Analysis
The data were analyzed using SPSS (Statistical Package for the Social Sciences) software and P values were calculated using chi-square and Fisher's exact tests to identify statistically significant relationships between the distribution of virulence genes and antibiotic resistance properties of the UPEC strains isolated from pediatric patients. A P value
3.5. Ethical Issues
The present study was authorized by the Ethical Committee of the Baqiyatallah hospital of Tehran, Iran, AJA University of Medical Science, Tehran Iran and the Microbiology and Infectious Diseases Center of the Islamic Azad University of Shahrekord Branch, Iran. All patients or their parents signed the written informed consent.
Out of 121 urine samples, 66 (54.54%) samples were positive for E. coli (Table 3). In addition, 19 out of 51 boys’ urine samples (37.25%) and 47 out of 70 girls’ urine samples (67.14%) were positive for E. coli (Table 3). We also found that 24 samples were positive for Kelebsiella bacterium(19.83%).
Antimicrobial resistance in the UPEC strains isolated from the pediatric patients is shown in Table 4. UPEC strains exhibited the highest level of resistance to ampicillin (69.6%), followed by tetracycline (69.6%), norfloxacin (63.6%), and ofloxacin (37.8%). Distribution of multi-drug resistant UPEC strains isolated from pediatric patients is shown in Table 5. We found that 1.66% of tested strains were resistant to more than eight antibiotics, while all the tested strains were resistant to three antibiotics.
The distribution of virulence genes within the UPEC strains isolated from pediatric patients is shown in Table 6. Generally, fim (71.2%), set-1 (66.6%), iha (62.1%), papGI (59%), and usp (56%) were the most common virulence genes in pediatric patients (Figures 1-7). The distribution of antibiotic resistance genes within the UPEC strains isolated from pediatric patients is shown in Table 7. Genes that encode resistance to beta-lactams, tetracycline and cephalothin antibiotics, i.e., blaSHV (45.4%), tetA (43.9%) and CITM (37.87%) were the most common antibiotic resistance genes in the pediatric patients (Figure 8).
Relationship between the presence of antibiotic resistance genes and distribution of virulence factors in UPEC strains isolated from pediatric patients is shown in Table 8. We found that virulence factors had low distribution in samples with positive results for antibiotic resistance genes. The most prevalent virulence genes in aac(3)-IV+, sul1+, blaSHV+, CITM+, tetA+, tetB+ and dfrA1+ strains were fim (17.64%), fim (13.33%), papGI (16.66%), fim and papGI (12%), papGI and usp (10.34%), fim, iha and papGi (5.55%) and fim and usp (7.69%), respectively.
To the best of our knowledge, this is the most comprehensive report on virulence genes and antimicrobial resistance properties in UPEC strains isolated from pediatric patients in Iran. In the present study, all isolates that had UPEC virulence factors were identified as UPEC strains. This principle has been confirmed in previous studies (25, 26). Our work has identified the high presence of UPEC strains in the urine samples of pediatric patients in Iran. Totally, 37.25% of boys and 67.14% of girls were infected with UPEC strains (P < .05).
One possible explanation for the high prevalence of UPEC strains in girls is that they have a relatively short and wide urethra. Also, host factors such as changes in normal vaginal flora may put girls at higher risk for UTIs. The effects of genetic factors, including expression of Lewis blood group Le (a+b-) and Le (a-b-) and HLA-A3 should not be overlooked. Our results are in agreement with the results of Jadhav et al. (27) and Vollmerhausen et al. (28) which were conducted in India and Australia, respectively.
One possible explanation for the low prevalence of UPEC strains in boys of our study is that all of them were circumcised. Also, they were 1-3 years old. During the first year of life, boys have a higher incidence of UTIs; in all other age groups, girls are more prone to develop UTIs (29). Higher incidence of UTIs in uncircumcised boys has been reported previously (30).
Another important finding of our investigation relates to the distribution of antibiotic resistance pattern in UPEC strains. Totally, bacterial strains of our study had the lowest resistance against imipenem (1.5%) and amikacin (4.5%), while resistance to ampicillin (69.6%), tetracycline (69.6%) and norfloxacin (63.6%) were high. We found statistically significant (P < .05) association between the incidence of antibiotic resistance to ampicillin, tetracycline and norfloxacin and imipenem and amikacin. All the studies that have been conducted in this field (7, 31, 32), have shown a high distribution of antibiotic resistance against ampicillin, tetracycline and norfloxacin. High efficacy of imipenem and amikacin for treatment of the cases of UTIs due to UPEC strains has been reported previously from Iran (31, 33, 34).
The results of our study showed that considerable numbers of isolates were resistant to more than three antibiotics. Similar investigation had been reported previously (3, 31-34). Pyelonephritis, urethritis, cystitis, urethral infections and other clinical complications of infection with UPEC strains are serious among children, compelling clinicians to consider the provision of early, and empirical antibiotic therapy.
However, current recommendations and the available data (although limited in scope and only formally studied for UPEC-related infections in children) suggest that antibiotics should be withheld if UPEC infection is suspected, given the concerns that antibiotics may trigger the release of virulence factors, antibiotic resistance genes and progression to pyelonephritis, urethritis and cystitis, resulting in worse clinical outcomes.
Our results indicated that UPEC strains with positive antibiotic resistance genes had lower incidence of virulence factors. It seems that the aac(3)-IV, sul1, CITM, dfrA1 and tetB induced the higher presence of fim gene; blaSHV, CITM and tetA induced the higher presence of papGI gene; tetB induced the higher presence of iha gene; and finally dfrA1 induced the higher presence of usp gene.
The effect of antibiotic therapy on the release of bacterial genes has been also reported by Farshad et al. (35) and Rijavec et al. (36). Farshad et al. (35) reported that the prevalence of all virulent genes was lower in resistant groups of UPEC strains but not statistically significant except for pap and cnf1 with nalidixic acid (8% and 4.5% positive in resistant vs. 29.4% and 29.6% positive in susceptible groups, respectively, P < .05) was observed. Rijavec et al. (2008) showed that susceptible strains of uropathogenic E. coli exhibited a higher virulence factor score. The results of both Rijavec et al. (2008) (36) and Farshad et al. (2010) (35) were similar to our findings, which showed that susceptible UPEC strains had the highest distribution of papG, usp and fim virulence factors. Both quinolone and fluroquinolone resistant strains of previous study harbored lower distribution of pap, hly, cnf1 and sfa virulence genes (37).
It seems that the susceptible strains of uropathogenic E. coli have higher incidence of uropathogenic virulence factors. Furthermore, because inappropriate prescriptions of antibiotics causes antibiotic resistance, it is not surprising that our study found that the distribution of blaSHV, tetA, CITM, tetB and aac(3)-IV antibiotic resistance genes were 45.4%, 43.9%, 37.8%, 27.2%, and 25.7%, respectively. There were statistically significant differences (P < .05) amongst the incidences of tetA and tetB and blaSHV and dfrA1 genes. Incidence of sul1 gene which encodes resistance against sulfonamides was 22.7% in our study while, its prevalence in the study of Gundogdu et al. (38) and Momtaz et al. (3) was 72% and 36.58%, respectively. Momtaz et al. (3) reported that aadA1 (52.84%) and qnr (46.34%) had the highest incidence of antibiotic resistance genes while, cat1 (15.44%), cmlA (15.44%) and dfrA1 (21.95%) had the lowest incidence.
All of the UPEC strains harbored various virulence factors. The most commonly detected virulence factors in UPEC strains of our study were fim (71.2%), set-1 (66.6%), iha (62.1%), papG1 (59%), and usp (56%). Significant statistical differences (P < .05) were seen between the incidence of virulence factors in girls and boys and also between the incidence of fim, set-1 and iha and finally between astA, sigA, sap, kpsMT, iss, irp2, tsh and vat genes. High presence of fim, set-1 and iha genes have been reported previously by Momtaz et al. (3) (86.17% for fim, 79.67% for set-1 and 17.88% for iha) and Momtaz et al. (3) (69.67% for fim and 17.88% for iha) (both from Iran). The distribution of fim gene in UPEC strains of the Arabi et al. (Iran) (39), Ananias and Yano, (Brazil) (40) and Qin et al. (China) (41) were 87.7%, 75% and 86%, respectively. Fim gene is involved in adhesion, invasion, and apoptosis of urothelial cells and initiates bladder pathology by binding to the uroplakin receptor complex (25).
The above-mentioned data highlight large differences in the prevalence of UPEC strains in the different studies, as well as differences in virulence genes and antibiotic resistance properties in the clinical samples. This could be related to differences in the type of sample (stool, blood, urine and other clinical samples) tested, number of samples, method of sampling, experimental methodology, geographical area, antibiotic prescription preference among clinicians, antibiotic availability, and climate differences in the areas where the samples were collected, in each study.
In conclusion, we identified a large number of virulence factors and antibiotic resistance genes and resistance to more than one antibiotic in the UPEC strains isolated from Iranian patients. Our data indicate that resistance against tetracycline and ampicillin, fim virulence factors and blaSHV antibiotic resistance gene were the most commonly detected characteristics of the UPEC strains isolated from Iranian pediatric patients with UTIs. Hence, judicious use of antibiotics is required by clinicians. It is compulsory to evaluate the prevalence of virulence factors, antibiotic resistance genes and pattern of antibiotic resistance among clinical isolates of UPEC strains.
Furthermore, because of the variation of resistance pattern in each hospital, it is important for each region and even hospital to formulate their own antibiotic policy according to their local resistance pattern. We recommended managing the children affected with community-acquired UTIs initially with imipenem and amikacin. It seems that various antibiotic resistance genes can induce the presence of certain virulence factors, but further studies must be done to prove this finding.
The authors would like to thank Professor H. Momtaz at the Microbiology Research Center of the Islamic Azad University of Shahrekord Branch and Dr. M. J. Hosseini at Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran for their important technical and clinical support.
All authors participated equally in the present study.
All authors declared that they had no conflict of interest.
All financial supports of this study have been made by the AJA University of Medical Science, Tehran, Iran.