A Relationship Between O-Serotype,Antibiotic Susceptibility and Biofilm Formation in Uropathogenic Escherichia coli

Abstract

Uropathogenic Escherichia coli (UPEC) is a well-known pathogen that has perturbed the medical scenario because of its resistance to diverse therapeutic drugs and its ability to form a biofilm. Different O-serogroups are the prevalent cause of urinary tract infections (UTIs) along with their ability to form a biofilm. The present research aimed to assess antibiotic susceptibility, biofilm formation, and serotyping of UPEC isolates in conjunction with the demographic data. Antibiotic susceptibility was determined using the Kirby–Bauer method and biofilm formation was assessed phenotypically and at the molecular level. Serotyping was performed by multiplex PCR. A significant proportion of the total of 120 UPECs was isolated from women (p < 0.05). Most isolates were resistant to cefotaxime, ceftazidime, and tetracycline, but maintained their sensitivity to imipenem. O₂₅, O₁₅, O₈, and O₇₅ were the most commonly detected serogroups. Moreover, O₂₅, O_15, and O₈ were the highest biofilm-producing serogroups among the UPEC isolates. Serogroups O₇₅ and O₂₁ were significantly associated with diabetic patients and subjects with renal disease, respectively (p < 0.05). Overall, our results show that UTI incidence in women should be a subject of concern. The high prevalence of the O₂₅ serogroup associated with a specific antibiotic profile and a high percentage of biofilm formation suggests a close relation between serogroups and characteristic features of UPEC isolates.

Introduction

Urinary tract infection (UTI) is a well-known disorder often seen in clinics and hospitals worldwide. The concern is that, if left untreated, it can have serious consequences.^1,2 Extra-intestinal pathogenic Escherichia coli or uropathogenic E. coli (UPEC) are the most frequent organisms for 90% of community-acquired and 50% of nosocomial UTIs.^1,3,4 Because bacteriological culture modality requires 48–72 hr to provide results, but the physician cannot wait to initiate therapeutic measures, a prerequisite for surveillance of antimicrobial susceptibility of these organisms should be developed at the local, regional, or even international level as standard therapeutic protocol depending on the geographical region or hospital guidelines.⁵

E. coli has been documented to possess O (lipopolysaccharide), H (flagellar), and, in some cases, K (capsular) surface antigens that assist in serological typing. On a global scale, 174 O-serogroups have been described for E. coli, each being specifically related to a certain virulence factor profile for each strain.⁴ One mechanism that boosts the pathogenesis of the organism, assists in retaining it in the genitourinary tract, and hinders its eradication is the formation of biofilm. Different bacterial species colonize urinary catheters, leading to the formation of bacterial biofilm that eventually may lead to nosocomial UTIs.² These infections are often difficult to treat and can be a major reservoir of resistant pathogens.⁶ The genetic traits in the rpoS, sdiA, and rcsA genes have been found to be significant for influencing the ability of bacteria to form a biofilm and acquire resistance to antibiotics.⁷ To be able to reduce the rate of morbidity and begin early treatment several factors must be taken into consideration. These include geographic location, age and sex of the patient, local antimicrobial resistance, and the virulence profiles of the pathogens. There appears to be a lack of knowledge about the characteristics of common uropathogenic E. coli serogroups in Iran. We conducted this investigation to correlate uropathogenic E. coli serogroups, antimicrobial susceptibility patterns, and genes associated with biofilm formation in UPECs.

Materials and Methods

Bacteria and clinical specimens

This prospective study was done on 120 consecutive nonduplicate E. coli isolates collected from in- and outpatients of both genders, aged 1–91 years, with a history of UTI at Sina Hospital in Tabriz, Iran from October to December 2016. Midstream urine specimens were inoculated semi-quantitatively on blood agar and eosin methylene blue agar (Merck, Germany) plates with a calibrated loop. The inoculated plates were incubated at 37°C for 24 hr. Isolates were identified by colony morphology, gram staining, and standard biochemical tests as described elsewhere,⁸ followed by antimicrobial susceptibility test toward several antibiotics as described by Clinical Laboratory Standard Institute (CLSI) guidelines.⁹ These isolates were eventually stocked in tryptic soy broth (TSB; Oxoid, United Kingdom) with 20% glycerol and kept at −70°C for further study on the production of biofilm and extraction of DNA.

Antibiotic susceptibility test

The disk agar diffusion method was used to determine the susceptibility of the E. coli isolates to antibiotics using Muller–Hinton agar. The isolates were classified as susceptible, intermediate, and resistant according to CLSI.⁹ The antibiotics tested were co-trimaxazole (TS, 1.25/23.75 μg), ciprofloxacin (CIP, 5 μg), amikacin (AK, 30 μg), ceftazidime (CAZ, 30 μg), cefotaxime (CTX, 30 μg), piperacillin-tazobactam (PTZ, 100/10 μg), gentamicin (GM, 10 μg), nitrofurantoin (NI, 300 μg), tobramycin (TN, 10 μg), chloramphenicol (C, 30 μg), imipenem (IMI, 5 μg), and tetracycline (T, 10 μg). All antibiotics were purchased from Mast Diagnostics (United Kingdom). E. coli ATCC25922 was used as the standard drug-susceptible control for disk diffusion measurements.

In vitro biofilm assay

The biofilm formation assay was performed by the micro titer plate method with some modifications using tryptic soy broth with 1% glucose.¹⁰ First, bacterial suspensions matched for equivalent with optical density of 0.5 McFarland (10⁸ colony forming unit/mL) were prepared from overnight cultures. Twenty microliters were then added to wells containing ∼180 μL of fresh TSB with 1% glucose and were eventually incubated at 37°C for 24 h. After incubation, the plate was washed with phosphate buffered saline (pH 7.4) three times to remove all nonadherent bacteria. For fixing the attached bacteria, 250 μL of methanol was added to each well and, after 15–20 min, the well was emptied and left to air dry at room temperature. The attached film was then stained with 200 μL of crystal violet for 5 min at room temperature. Extra stain was rinsed off by placing the plate under running tap water and the plates were air dried. The colorant was solubilized with 160 μL of 33% (v/v) of glacial acetic acid per well to measure the absorbance at 570 nm. The negative control consisted of a bacteria-free well. All assays were performed in triplicate, the results were analyzed as described elsewhere¹⁰ and the isolates were classified as shown in Table 1.

Table 1.

Evaluation of Biofilm Production Based on the Optical Density

Strength of biofilm formation	Average OD value
Nonbiofilm producers	OD_T ≤ OD_C
Weak biofilm producers	OD_C < OD_T ≤ 2 × OD_C
Moderate biofilm producers	2 × OD_C < OD_T ≤ 4 × OD_C
Strong biofilm producers	4 × OD_C < OD_T

OD_T, optical density of test; OD_C, optical density of control.

Polymerase chain reaction

The template DNA was prepared by the boiling method with minor modifications.¹¹ Briefly, 2–3 bacterial colonies were dissolved in 20 μL of deionized water, boiled at 95°C for 10 min, and centrifuged at 12,700 rpm for 2 min to remove cell debris. The supernatant obtained was stored at −20°C and used as template DNA for amplification of biofilm and serotyping.⁴ Detection of the uropathogenic O-serogroups (O₁, O₄, O₂, O₇, O₆, O₁₅, O₈, O₂₁, O₂₅, O₁₆, O₂₂, O₇₅, O₁₈, and O₈₃) was performed by multiplex PCR as described elsewhere.⁴ Table 2 lists the biofilm and serotype target genes and the PCR conditions. The amplified products were electrophoresed in 1.5% agarose gel (Yekta Tajhiz Azma, Iran) at 80 V for 55 min and stained with Cyber-safe stain (Yekta Tajhiz Azma, Iran).

Table 2.

Primers Used for Amplification of Biofilm-Associated Genes and Serotyping of Uropathogenic Escherichia coli

Target	Genes	Primer sequence (5′ → 3′)	Product size (bp)	Annealing temperature (°C)
Biofilm	sdiA	F-TCGCTATCTCTGCTGATGTC	239	52
	sdiA	R-TTTAATGCTGCCAAATCGGG	239	52
	rcsA	F-GTGATTCACAGCGCCCTTCA	306	54
	rcsA	R-TACTCGATTCGGTTCGGCTC	306	54
	rpoS	F-GCAGAGCATCGTCAAATGGCTGTT	120	60
	rpoS	R-ATCTTCCAGTGTTGCCGCTTCGTA	120	60
Serotype group1	O₁	GTGAGCAAAAGTGAAATAAGGAACG	1,098	62
	O₁	CGCTGATACGAATACCATCCTAC	1,098	62
	O₆	GGATGACGATGTGATTTTGGCTAAC TCTGGGTTTGCTGTGTATGAGGC	783	62
	O₇	CTATCAAAATACCTCTGCTGGAATC	610	62
	O₇	TGGCTTCGAGATTAAACCTATTCCT	610	62
	O₈	CCAGAGGCATAATCAGAAATAACAG	448	62
	O₈	GCAGAGTTAGTCAACAAAAGGTCAG	448	62
	O₁₆	GGTTTCAATCTCACAGCAACTCAG	302	62
	O₁₆	GTTAGAGGGATAATAGCCAAGCGG	302	62
	O₂₁	CTGCTGATGTCGCTATTATTGCTG	209	62
	O₂₁	TGAAAAAAAGGGAAACAGAAGAGCC	209	62
	O₇₅	GAGATATACATGGGGAGGTAGGCT	511
	O₇₅	ACCCGATAATCATATTCTTCCCAAC	511
Serotype group 2	O₂	AGTGAGTTACTTTTTAGCGATGGAC	770	58
	O₂	AGTTTAGTATGCCCCTGACTTTGAA	770	58
	O₄	TTGTTGCGATAATGTGCATGTTCC	664	58
	O₄	AATAATTTGCTATACCCACACCCTC	664	58
	O₁₅	TCTTGTTAGAGTCATTGGTGTATCG	183	58
	O₁₅	ATAAAACGAGCAAGCACCACACC	183	58
	O₁₈	GTTCGGTGGTTGGATTACAGTTAG	551	58
	O₁₈	CTACTATCATCCTCACTGACCACG	551	58
	O₂₂	TTCATTGTCGCCACTACTTTCCG	468	58
	O₂₂	GAAACAGCCCATGACATTACTACG	468	58
	O₂₅	AGAGATCCGTCTTTTATTTGTTCGC	230	58
	O₂₅	GTTCTGGATACCTAACGCAATACCC	230	58
	O₈₃	GTACACCAGGCAAACCTCGAAAGTTCTGTAAGCTAATGAATAGGCACC	362	58

Demographic data

All cases were identified from the hospital's microbiology records. The files and the electronic records of the patients were reviewed to identify infections and to retrieve the following data: demographic information, underlying conditions, type of infection (community or hospital-acquired), usage of antibiotics, and outcome.

Statistical analyses

Chi-square statistics were performed in SPSS (version 20). Differences at p ≤ 0.05 were considered statistically significant.

Results

Out of 235 urine specimens sent to the microbiology laboratory during the period of study, 120 E. coli isolates were detected from 84 (70%) women and 36 (30%) men (p < 0.05). Women between the ages of 41 and 60 years had comparatively higher prevalence (41.7%) than men aged 61–80 years (36.1%) (Table 3).

Table 3.

Frequency of Uropathogenic Escherichia coli in Various Age Groups in Both Genders

Age (in years)	Female (%)	Male (%)
<20	5 (6)	1 (2.8)
21–40	16 (19)	5 (13.9)
41–60	35 (41.7)	10 (27.8)
61–80	22 (26.2)	13 (36.1)
>81	6 (7.1)	7 (19.4)
Total	84 (100)	36 (100)

Most isolates (75%) were resistant to cefotaxime. Antibiotic resistance to the tested antibiotics was as follows: ceftazidime 74.2%, tetracycline 68.3%, co-trimoxazole 62.5%, ciprofloxacin 54%, gentamicin 34.2%, tobramycin 19.2%, chloramphenicol 10%, nitrofurantoin 6.7%, piperacillin-tazobactam 5.8%, and amikacin 0.8%. All E. coli isolates retained their susceptibility to imipenem.

The distribution of O-serogroups in the UPEC strains isolated from male and female patients is shown in Table 4. Overall, serotype O₂₅ had the highest prevalence (n = 67; 55.8%) in both groups of patients, followed by O₁₅ (n = 22; 18.3%), O₈ (n = 13; 10.8%), O₇₅ (n = 12; 10%), and O₆ (n = 5; 4.2%). An additional 19 (15.8%) UPEC isolates that could not be allotted to a serogroup were categorized as nondetected serogroups. The O₂₅ serogroup had the highest incidence in UPEC from women, followed by O₁₅, O₈, O₇₅, and nondetected serogroups. We found that the O₂₅ serogroup had the highest incidence in urine samples of men, followed by nondetected O₁₅, O₂, O₈, and O₁₅. The results showed significant differences (p < 0.05) between the overall prevalence of O₂₅ and O₁₅ and between the incidence of O₁ and O₆, O₈ and O₇₅, O₂₁ and O₈₃ with O₂ and O₁₈.

Table 4.

Distribution of Uropathogenic Escherichia coli Serogroups in Both Genders

Gender	No. (%) of O-serogroups
Gender	O₁	O₆	O₇	O₈	O₁₆	O₂₁	O₇₅	O₂	O₄	O₁₅	O₁₈	O₂₂	O₂₅	O₈₃	UDS
Female (n = 84)	3 (3.6)	3 (3.6)	1 (1.2)	10 (11.9)	1 (1.2)	2 (2.4)	9 (10.7)	4 (4.8)	3 (3.6)	18 (21.4)	3 (3.6)	1 (1.2)	45 (53.6)	1 (1.2)	13 (15.5)
Male (n = 36)	1 (2.8)	2 (5.6)	0 (0)	3 (8.3)	0 (0)	2 (5.6)	3 (8.3)	0 (0)	0 (0)	4 (11.1)	0 (0)	1 (2.8)	22 (61.1)	1 (2.8)	6 (16.7)
Total (n = 120)	4 (3.3)	5 (4.2)	1 (0.8)	13 (10.8)	1 (0.8)	4 (3.3)	12 (10)	4 (3.3)	3 (2.5)	22 (18.3)	3 (2.5)	2 (1.7)	67 (55.8)	2 (1.7)	19 (15.8)

UDS, undetected serotypes.

All UPEC serogroups showed maximum resistance to ciprofloxacin, tetracycline, cefotaxime, and ceftazidime and minimum resistance to imipenem, amikacin, and nitrofurantoin. The results showed significant differences (p < 0.05) between the O₆ serogroup with ciprofloxacin and ceftazidime and the O₂ serogroup with ceftazidime and cefotaxime (Table 5).

Table 5.

Antimicrobial Resistance Features in Uropathogenic Escherichia coli Serogroups

	Antibiotics
Serogroups	TS	NI	AK	IMI	PTZ	CIP	CAZ	CTX	GM	C	TN	T
O₁ (4)	2	0	0	0	0	3	3	3	0	1	1	4
O₆ (5)	1	0	0	0	0	2	1	2	0	0	0	4
O₇ (7)	1	0	0	0	0	1	1	1	1	0	0	1
O₈ (13)	7	1	1	0	3	12	7	7	4	1	3	9
O₁₆ (1)	1	0	0	0	0	1	1	1	1	0	0	1
O₂₁ (4)	3	0	0	0	2	4	4	4	0	1	2	3
O₇₅ (12)	8	0	0	0	1	11	7	7	4	1	5	7
O₂ (4)	1	0	0	0	0	1	1	1	1	0	0	3
O₁₅ (22)	15	1	0	0	7	18	17	18	8	3	7	16
O₁₈ (3)	1	0	0	0	0	0	0	0	0	0	0	2
O₂₅ (67)	43	5	3	0	10	56	53	54	26	11	27	51
O₄ (3)	1	0	0	0	0	2	3	3	2	0	0	1
O₂₂ (2)	1	0	0	0	1	2	2	2	1	1	0	2
O₈₃ (2)	2	1	0	0	1	2	1	1	1	0	0	2
Nondetected group (19)	13	2	0	0	5	18	12	12	7	3	8	10
Total (120)	100	10	4	0	30	133	113	116	56	22	53	116

TS, co-trimaxazole; NI, nitrofurantoin; AK, amikacin; IMI, imipenem; PTZ, piperacillin-tazobactam; CIP, ciprofloxacin; CAZ, ceftazidime; CTX, cefotaxime; GM. gentamicin; C, chloramphenicol; TN, tobramycin; T, tetracycline.

The biofilm production assay showed that 102 (85%) of all UPEC isolates were biofilm producers. The results of micro titer plate method showed that, among the 120 E. coli investigated for biofilm production, 18 (15%) isolates were strong producers, 14 (11.7%) showed moderate biofilm production, 70 (58.3%) isolates showed weak biofilm production, and 18 (15%) were nonbiofilm producers (Table 6). A high distribution of UPEC biofilm-associated genes was observed in our study. Overall, sdiA, rcsA, and rpoS had the highest prevalence (76.7%, 85.8%, and 87.7%, respectively) among the biofilm genes studied (Table 6). Biofilm production was significantly associated with the sdiA, rcsA, and, rpoS genes (p < 0.05).

Table 6.

Distribution of Biofilm Genes in Uropathogenic Escherichia coli Isolates

Biofilm genes	Biofilm-producing abilities
Biofilm genes	Strong	Moderate	Weak	Nonbiofilm producers
sdi A (n = 92)	18 (p ≤ 0.05)	12	59 (p < 0.05)	3 (p < 0.05)
rcs A (n = 103)	18	14	64 (p < 0.05)	7 (p < 0.05)
rpo S (n = 104)	18	14	66 (p < 0.05)	6 (p < 0.05)

We also found that O₂₅ serogroup had the highest prevalence of biofilm-associated genes in the UPEC isolates, followed by O₁₅ and O₈. The results showed significant differences (p = 0.032) between the prevalence of the O₇₅ serogroup and rpoS biofilm-associated gene, O₂ serogroup and rcsA biofilm-associated gene, and serogroup O₁₅ and biofilm-associated rpoS gene (Table 7). Table 8 lists the UPEC abilities of biofilm production by serogroup. Overall, O₂₅ (55.6%), O₁₅ (55.6%), and O₈ (11.1%) were the highest biofilm-producing serogroups, while O₆, O₇₅, and O₄ were the lowest biofilm-producing serogroups.

Table 7.

Relation Between Uropathogenic Escherichia coli Serogroups and Biofilm-Associated Genes

Biofilm genes	Number of UPEC isolates in various serogroups possessing the respective biofilm-associated genes
Biofilm genes	O₁	O₆	O₇	O₈	O₁₆	O₂₁	O₇₅	O₂	O₄₄	O₁₅	O₁₈	O₂₂	O₂₅	O₈₃	NBP
sdiA (n = 92)	2	4	0	10	1	3	7	2	3	18	0	2	53	2	14
rcsA (n = 106)	4	5	1	11	1	4	10	3 (p < 0.05)	3	22	2	1	59	2	13
rpoS (n = 104)	4	5	0	10	1	4	8 (p < 0.05)	4	3	22 (p < 0.05)	2	2	57	2	16

NBP, nonbiofilm producers; UPEC, uropathogenic Escherichia coli.

Table 8.

Biofilm Production in Various Serogroups by Uropathogenic Escherichia coli

Biofilm-producing abilities	Prevalence of serogroups (%) with respective ability of biofilm production
Biofilm-producing abilities	O₁	O₆	O₇	O₈	O₁₆	O₂₁	O₇₅	O₂	O₄	O₁₅	O₁₈	O₂₂	O₂₅	O₈₃	NBP
Strong	0	1 (5.6)	0	2 (11.1)	0	1 (5.6)	1 (5.6)	0	1 (5.6)	10 (55.6)p < 0.05	0	0	10 (55.6)	0	2
Moderate	0	1 (7.1)	0	2 (14.3)	1 (7.1)	1 (7.1)	2 (14.3)	0	0	4 (28.6)	0	0	8 (57.1)	0	2
Weak	4 (5.7)	3 (4.3)	0	6 (8.6)	0	2 (2.9)	5 (7.1)	3 (4.3)	2 (2.9)	8 (11.4)	1 (1.4)	1 (1.4)	40 (57.1)	2 (2.9)	11
Nonbiofilm producer	0	0	1 (5.6)	3 (16.7)	0	0	4 (22.2)	1 (5.6)	0	0	2 (11.1)p < 0.05	1 (5.6)	9 (50)	0	4

Comparison of the UPEC serotypes with underlying conditions

Table 9 shows the demographic and clinical data of the 120 UPEC isolated patients. Most episodes of infection (n = 16; 36.4%) were from patients admitted to internal medicine wards, followed by urology wards (n = 12; 27.3%), infectious disease wards (n = 8; 18.2%), intensive care units (n = 5; 11.4%), and surgical wards (n = 3; 6.8%). Of the total number of patients from whom UPEC were isolated, 54 had a history of undergoing surgery before being afflicted with UPEC. Of these, 27 (22.5%) had recently undergone urological surgery and 27 (22.5%) had recently undergone nonurological surgery.

Table 9.

Demographic and Clinical Data of the Patients

Variables	Number (%)	p-Value
Accommodation
Urban	72 (60%)	NSD
Rural	48 (40%)	NSD
Underlying medical condition
Recent surgery	27 (22.5%)	NSD
ICU stay	13 (10.8%)	NSD
Renal disease other than stone formation	34 (28.3%)	p < 0.05
Urinary catheter	20 (16.7%)	NSD
Calculus of kidney	10 (8.3%)	NSD
Urological operation	27 (22.5%)	NSD
Insulin-dependent diabetes mellitus	1 (0.8%)	p < 0.05
Insulin nondependent diabetes mellitus	10 (8.3%)	p < 0.05
Prostate disease	3 (2.5%)	p < 0.05
Received antibiotic including ciprofloxacin more than once in the last year	26 (21.7%)	NSD
Received antibiotic other than ciprofloxacin	29 (24.2%)	p < 0.05
Types of disease
Complicated	43 (35.8%)	NSD
Uncomplicated	77 (64.2%)	NSD
Wards
ICU	5 (11.4%)	NSD
Internal	16 (36.4%)	NSD
Surgery	3 (6.8%)	p < 0.05
Infectious	8 (18.2%)	p < 0.05
Urology	12 (10%)	NSD

NSD, nonsignificant difference; ICU, intensive care unit.

Of the 120 E. coli isolates from 84 women and 36 men, 64.2% were from uncomplicated UTI (59 women aged 41–60 [39%] and 18 men aged 41–60 [38.9%]) and 43 (35.8%) were from complicated UTI (25 women aged 61–80 [48%] and 18 men aged 61–80 [50%]). Of the 43 patients with complicated UTIs, 10 (8.3%) presented with calculus of the kidney, 34 (28.3%) used urinary catheters, 34 (28.3%) were afflicted with renal disease, 10 (8.3%) had diabetes mellitus independent of insulin, 1 (0.8%) had insulin-dependent diabetes mellitus, and 3 (2.5%) had prostate disorders.

The sero-epidemiological study in this investigation identified a total of 14 O-serotypes. Approximately 98.5% of the isolates were typable. Serotypes O₂₅, O₁₅, O₈, and O₇₅ predominated and accounted for 55.8%, 18.3%, 10.8%, and 10% of all isolates, respectively. The most common serotype of UPEC isolated from diabetic patients was O₇₅ (p < 0.05), and O₂₁ (p < 0.05) was found to be common in patients afflicted with renal disease. Biofilm-associated genes such as rpoS were present in isolates obtained from the infectious and surgical wards (p < 0.05).

Discussion

UTI is a common infectious disease with a high prevalence. If left untreated it may become a chronic infection. In accordance with previously published studies,^4,5,12–15 E. coli was the principle pathogen responsible for UTIs in our patients, with a higher prevalence in women (>85%) in comparison with men (36%). The higher incidence of women reported to be afflicted with UTI can be explained by the fact that young, healthy premenopausal women with acute cystitis tend to treat themselves or are treated with antimicrobials empirically prior attaining a laboratory urine culture report,^16,17 which can lead to repeated infections.

One possible reason for men being less prone to UTIs is their longer urethra and the presence of antimicrobial substances in prostatic fluid.^18,19 It has been extensively reported that a the prevalence of UTI is higher in adult women than in men, principally owing to the short, straight urethral anatomy of the female and other physical factors.^20,21 Elderly men (over 61 years of age) had a higher incidence of UTI (55.5%) than elderly women (33.3%) in our investigation. This may be because prostate enlargement and neurogenic bladder problems increase as men age.^20,21

Antibiotic resistance in UPEC isolates is of increasing public health concern.²¹ Trimethoprim-sulfamethoxazole, fluoroquinolone, and nitrofurantoin are predominantly suggested for empirical treatment of uncomplicated UTI. However, studies in the United States of America and worldwide have demonstrated a high resistance to trimethoprim-sulfamethoxazole in E. coli UTI isolates.^22–24 In this research, the highest percentages of resistance were found for cefotaxime (75%), ceftazidime (74.2%), tetracycline (68.3%), and co-trimoxazole (62.5%) and the highest percentages of susceptibility were found for nitrofurantoin (93.3%), piperacillin-tazobactam (94.2%), amikacin (99.2%), and imipenem (100%).

The higher resistance observed for some antibiotics in comparison with others in Iranian studies²⁵ may be due to excessive use of antibiotics without a prescription in recent years. The overall resistance to ciprofloxacin (54%) among our isolates was high. Fluoroquinolones have a wide variety of indications, permeate most body compartments, and are ubiquitously prescribed, which accounts for the emergence of resistance. These findings indicate that the empiric use of fluoroquinolones should be seriously reconsidered or that strategies to counteract increased resistance to these antibiotics must be developed. Nitrofurantoin demonstrated better activity against UPEC isolates (93.3% susceptible), but this drug is not recommended for serious upper UTIs or for those cases with systemic involvement. Our findings are similar to other Indian and Iranian studies^20,26 that have recommended nitrofurantoin as a first-line drug for community-acquired UTIs. Given this fact, nitrofurantoin has no role in the treatment of other infections, it can be administered orally and is highly concentrated in the urine; it may therefore be the most appropriate agent for empirical use in uncomplicated UTIs.²⁰

Resistance to aminoglycosides (amikacin and gentamicin) was not a prime feature in this investigation. Aminoglycosides are injectable and used restrictively in the community-care setting; hence, they have shown better sensitivity rates.^26,27 Resistance to piperacillin/tazobactam and tobramycin for Enterobactericeae was low, probably reflecting their lower usage for treatment of community-acquired infections and this is similar to the results of a previous study.²⁸ Our findings indicate that urgent strategies are required to counteract increased resistance to these drugs or that their use in uncomplicated infections should be strictly reduced.

Several O-serogroups were detected in our UPEC isolates. Overall, O₂₅, O₁₅, O₈, and O₇₅ were the most commonly detected O-serogroups among the UPEC isolates of this study. Similar results have been reported previously^4,14,29 from Iran and Mexico. A statistically significant difference was observed between the presence of O₂₅ and O₁₅ serogroups (p < 0.01), O₁ and O₆, O₈ and O₇₅, and O₂₁ and O₈₃ with O₂ and O₁₈ (p < 0.05). On the whole, O₂₅ and O₁₅ were the most prevalent serogroups, which is similar to a recent study⁴ that reported a high distribution of UPEC in serogroups O₂₅ (26.66%), O₁₅ (20.0%), and O₁₆ (13.33%). Momtaz et al.¹⁴ found that O₂₅ (26.01%), O₁₅ (21.13%), and O₁₆ (10.56%) were the most prevalent serogroups. Many investigators have reported several O-serogroups to be present at different frequencies in UTI patients.¹⁴

In this study, O₂₅ (55.6%), O₁₅ (55.6%), and O₈ (11.1%) were the highest biofilm-producing serogroups, while O₄, O₆, O₂₁, and O₇₅ were the lowest biofilm producers with only 5.6% prevalence among uropathogenic E. coli isolates. Among the 120 E.coli isolates investigated for biofilm production, 18 (15%) isolates showed strong positivity, 14 (11.7%) showed moderate positivity, 70 (58.3%) isolates showed weak biofilm productivity, and 18 (15%) showed no biofilm productivity using the tissue culture plate method. The findings of these investigations are in agreement with a report by Tajbakhsh et al.⁴ that showed UPEC serogroups O₂₅ (26.66%), O₁₅ (20%), and O₁₆ (13.33%) as the highest biofilm producers and O₂, O₄, O₆, O₈, O₂₁, and O₂₂ as the lowest biofilm producers.

Previous studies have shown that rpoS, sdiA, and rcsA genes are involved in biofilm formation.^7,30–32 In the present investigation, we attempted to find a link between the presence of particular genes and the bacterial ability to form a biofilm. The gene rpoS is an alternative sigma transcription factor that controls the expression of a large number of genes involved in the cellular response to stress.^7,32,33 Similarly, rcsA is a regulatory gene that belongs to the complex Rcs system for the regulation of cell wall integrity, cell division, stationary phase sigma factor activity, motility, and virulence. It has been shown that this gene has a role in adhesion to eukaryotic cells using curli.^7,34 The sdiA gene of E. coli is homologous to luxR in other bacteria and is the activator of quorum sensing in biofilm.^7,35,36

In this study, the prevalence of sdiA, rcsA, and rpoS genes in uropathogenic E. coli was determined and the results showed that the sdiA gene had the highest prevalence. Moreover, it was shown that biofilm production was significantly associated with the sdiA, rcs, and rpo biofilm genes. Similar findings had been observed by Adamus-Białek et.al.⁷ Ours is perhaps the first research of its kind from Iran to assess the relationship between serotype and biofilm-associated genes. In the present investigation, the O₂₅ serogroup had the highest incidence of biofilm-associated genes in the UPECs, followed by O₁₅ and O₈. Our results also show significant differences (p < 0.05) between the prevalence of O₇₅ and O₁₅ with rpoS and serogroup O₂ with rcsA.

In conclusion, this study showed that UTIs, especially in women, should be a subject of concern. Serogroups O₂₅, O₁₅, and O₈ and the sdiA, rcsA, and rpoS biofilm genes showed the highest frequencies in UPEC isolates. The high prevalence of the O₂₅ serogroup in our patients with UTIs with their antibiotic profiles and the high percentage of biofilm formation suggest a close relation between serogroups and the characteristic features of UPEC isolates.

Footnotes

Acknowledgments

Ms. Leila Dehghani and Dr. Khalil Aziziyan are thanked for their technical assistance in the collection of clinical isolates, phenotypic and molecular workup. This work was supported by Infectious and Tropical Diseases Research Center, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, I.R. Iran. This is a report of a database from MSc thesis of the first author registered in the Tabriz University of Medical Sciences (Thesis No-58558).

Disclosure Statement

No competing financial interests exist.

References

, Liu

, Chen

, Guo

, Liu

, Feng

, and Wang

. 2010. A multiplex PCR method to detect 14 Escherichia coli serogroups associated with urinary tract infections. J. Microbiol. Methods, 82:71–77.

Longhi

, Cossu

, Iebba

, Massaro

, Cipriani

, Chiarini

, Conte

, Seganti

, Osborn

, and Schippa

. 2008. Virulence traits in Escherichia coli strains isolated from outpatients with urinary tract infections. Int. J. Immunopathol. Pharmacol. 21:715–723.

Mittal

, Sharma

, and Chaudhary

. 2015. Biofilm and multidrug resistance in uropathogenic Escherichia coli. Pathog. Glob. Health, 109:26–29.

Tajbakhsh

, Ahmadi

, Abedpour-Dehkordi

, Arbab-Soleimani

, and Khamesipour

. 2016. Biofilm formation, antimicrobial susceptibility, serogroups and virulence genes of uropathogenic E. coli isolated from clinical samples in Iran. Antimicrob. Resist. Infect. Control, 5:11.

Kurutepe

, Surucuoglu

, Sezgin

, Gazi

, Gulay

, and Ozbakkaloglu

. 2005. Increasing antimicrobial resistance in Escherichia coli isolates from community-acquired urinary tract infections during 1998–2003 in Manisa, Turkey. Jpn. J. Infect. Dis. 58:159–161.

Kot

, Wicha

, Gruzewska

, Piechota

, Wolska

, and Obrebska

. 2016. Virulence factors, biofilm-forming ability, and antimicrobial resistance of urinary Escherichia coli strains isolated from hospitalized patients. Turk. J. Med. Sci. 46:1908–1914.

Adamus-Bialek

, Kubiak

, and Czerwonka

. 2015. Analysis of uropathogenic Escherichia coli biofilm formation under different growth conditions. Acta Biochim. Pol. 62:765–771.

Mahon,

C.R.

, D.C.

Lehman

, and Mahon

G.M.

. 2011. Textbook of Diagnostic Microbiology. 4th ed. Maryland Heights, MO: W.B. Saunders Company, Elsevier, pp. 111–182.

Clinical and Laboratory Standards Institute (CLSI). 2010. Performance Standards for Antimicrobial Susceptibility Testing: 20th Informational Supplement. CLSI Document M100-S20. Clinical and Laboratory Standards Institute, Wayne, PA.

10.

Stepanović

, Vuković

, Dakić

, Savić

, and Švabić-Vlahović

. 2000. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J. Microbiol. Methods, 40:175–179.

11.

Aher

, Roy

, and Kumar

. 2012. Molecular detection of virulence genes associated with pathogenicity of Klebsiella spp. isolated from the respiratory tract of apparently healthy as well as sick goats. Isr. J. Vet. Med., 67:249–252.

12.

Dormanesh

, Dehkordi

F.S.

, Hosseini

, Momtaz

, Mirnejad

, Hoseini

M.J.

, Yahaghi

, Tarhriz

, and Darian

E.K.

. 2014. Virulence factors and o-serogroups profiles of uropathogenic Escherichia coli isolated from Iranian pediatric patients. Iran. Red Crescent Med. J. 16:e14627.

13.

Hagan

E.C.

, and Mobley

H.L.

. 2007. Uropathogenic Escherichia coli outer membrane antigens expressed during urinary tract infection. Infect. Immun. 75:3941–3949.

14.

Momtaz

, Karimian

, Madani

, Dehkordi

F.S.

, Ranjbar

, Sarshar

, and Souod

. 2013. Uropathogenic Escherichia coli in Iran: serogroup distributions, virulence factors and antimicrobial resistance properties. Ann. Clin. Microbiol. Antimicrob. 12:8.

15.

Soto

S.M.

, Smithson

, Martinez

J.A.

, Horcajada

J.P.

, Mensa

, and Vila

. 2007. Biofilm formation in uropathogenic Escherichia coli strains: relationship with prostatitis, urovirulence factors and antimicrobial resistance. J. Urol. 177:365–368.

16.

Warren

J.W.

, Abrutyn

, Hebel

J.R.

, Johnson

J.R.

, Schaeffer

A.J.

, and Stamm

W.E.

. 1999. Guidelines for antimicrobial treatment of uncomplicated acute bacterial cystitis and acute pyelonephritis in women. Clin. Infect. Dis. 29:745–759.

17.

Zhanel

G.G.

, Hisanaga

T.L.

, Laing

N.M.

, DeCorby

M.R.

, Nichol

K.A.

, Palatnick

L.P.

, Johnson

, Noreddin

, Harding

G.K.

, and Nicolle

L.E.

. 2005. Antibiotic resistance in outpatient urinary isolates: final results from the North American Urinary Tract Infection Collaborative Alliance (NAUTICA). Int. J. Antimicrob. Agents, 26:380–388.

18.

Fair

W.R.

, Couch

, and Wehner

. 1976. Prostatic antibacterial factor identity and significance. Urology, 7:169–177.

19.

Farajnia

, Alikhani

M.Y.

, Ghotaslou

, Naghili

, and Nakhlband

. 2009. Causative agents and antimicrobial susceptibilities of urinary tract infections in the northwest of Iran. Int. J. Infect. Dis. 13:140–144.

20.

Kashef

, Djavid

G.E.

, and Shahbazi

. 2010. Antimicrobial susceptibility patterns of community-acquired uropathogens in Tehran, Iran. J. Infect. Dev. Ctries. 4:202–206.

21.

Zaki

, and Elewa

. 2015. Evaluation of uropathogenic virulence genes in Escherichia coli isolated from children with urinary tract infection. Int. J. Adv. Res. 3:165–173.

22.

Al-Tawfiq

J.A.

2006. Increasing antibiotic resistance among isolates of Escherichia coli recovered from inpatients and outpatients in a Saudi Arabian hospital. Infect. Control Hosp. Epidemiol. 27:748–753.

23.

Dias Neto

J.A.

, Martins

A.C.P.

, L.D.M.d. Silva, Tiraboschi

R.B.

, Domingos

A.L.A.

, Cologna

A.J.

, Paschoalin

E.L.

, and Tucci

Jr.

2003. Community acquired urinary tract infection: etiology and bacterial susceptibility. Acta Cir. Bras. 18:33–36.

24.

Karlowsky

J.A.

, Jones

M.E.

, Thornsberry

, Critchley

, Kelly

L.J.

, and Sahm

D.F.

. 2001. Prevalence of antimicrobial resistance among urinary tract pathogens isolated from female outpatients across the US in 1999. Int. J. Antimicrob. Agents, 18:121–127.

25.

Babypadmini

, and Appalaraju

. 2004. Extended spectrum-lactamases in urinary isolates of Escherichia coli and Klebsiella pneumoniae-prevalence and susceptibility pattern in a tertiary care hospital. Indian J. Med. Microbiol. 22:172–174.

26.

Kothari

, and Sagar

. 2008. Antibiotic resistance in pathogens causing community-acquired urinary tract infections in India: a multicenter study. J. Infect. Dev. Ctries. 2:354–358.

27.

Biswas

, Gupta

, Prasad

, Singh

, Arya

, and Kumar

. 2006. Choice of antibiotic for empirical therapy of acute cystitis in a setting of high antimicrobial resistance. Indian J. Med. Microbiol. 60:53–58.

28.

Sood

, and Gupta

. 2012. Antibiotic resistance pattern of community acquired uropathogens at a tertiary care hospital in Jaipur, Rajasthan. Indian J. Community Med. 37:39–44.

29.

Paniagua-Contreras

G.L.

, Monroy-Pérez

, Rodríguez-Moctezuma

J.R.

, Domínguez-Trejo

, Vaca-Paniagua

, and Vaca

. 2017. Virulence factors, antibiotic resistance phenotypes and O-serogroups of Escherichia coli strains isolated from community-acquired urinary tract infection patients in Mexico. J. Microbiol. Immunol. Infect. 50:478–485.

30.

Corona-Izquierdo

F.P.

, and Membrillo-Hernández

. 2002. A mutation in rpoS enhances biofilm formation in Escherichia coli during exponential phase of growth. FEMS Microbiol. Lett. 211:105–110.

31.

Collet

, Vilain

, Cosette

, Junter

G.A.

, Jouenne

, Phillips

R.S.

, and Di Martino

. 2007. Protein expression in Escherichia coli S17-1 biofilms: impact of indole. Antonie Van Leeuwenhoek, 91:71–85.

32.

Ito

, May

, Kawata

, and Okabe

. 2008. Significance of rpoS during maturation of Escherichia coli biofilms. Biotechnol. Bioeng. 99:1462–1471.

33.

Adams

J.L.

, and McLean

R.J.

. 1999. Impact of rpoS deletion on Escherichia coli biofilms. Appl. Environ. Microbiol. 65:4285–4287.

34.

Ferrières

, and Clarke

D.J.

. 2003. The RcsC sensor kinase is required for normal biofilm formation in Escherichia coli K‐12 and controls the expression of a regulon in response to growth on a solid surface. Mol. Microbiol., 50:1665–1682.

35.

Firouzi

, Derakhshandeh

, and Khoshbakht

. 2014. Distribution of sdiA quorum sensing gene and its two regulon among Salmonella serotypes isolated from different origins. Comp. Clin. Pathol. 23:1435–1439.

36.

Miller

M.B.

, and Bassler

B.L.

. 2001. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55:165–199.