The molecular-genetic characteristics of uropathogenic Escherichia coli isolated from pregnant women with asymptomatic bacteriuria

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Introduction

Urinary tract infections (UTIs) are among the most common infectious diseases. They account for up to 150 million cases worldwide each year. According to various authors, up to 50-60% of women experience an episode of UTI at least once in their lifetime [1, 2]. Clinical symptoms associated with UTIs may vary in severity depending on both the virulent properties of the pathogen and the susceptibility of the organism to infection: from asymptomatic course (asymptomatic bacteriuria) to clinically significant cystitis, pyelonephritis, up to severe urosepsis [3]. Asymptomatic bacteriuria represents bacterial colonization of the urinary tract in the absence of clinical manifestations of the disease. During pregnancy, asymptomatic bacteriuria has been considered for many years as a risk factor for pyelonephritis and adverse pregnancy outcomes (preterm labor, low birth weight, etc.) [4, 5]. Administration of antibacterial drugs for the treatment of asymptomatic bacteriuria during pregnancy may be accompanied by undesirable effects: changes in the composition of the intestinal microbiome of the pregnant woman, which subsequently determines the composition of the microbiota of the newborn; impaired development of the child's immune system [6, 7]. Furthermore, antibiotic therapy can lead to the elimination of potentially protective strains of microorganisms that prevent colonization by virulent uropathogens, thus indirectly contributing to the development of symptomatic UTIs.

The dominant bacterial pathogen among UTIs is Escherichia coli, a Gram-negative, motile, facultatively anaerobic bacillus belonging to the order Enterobacterales. E. coli is part of the commensal microbial population of the human intestine and maintains stability and homeostasis of the lumenal intestinal microbiota through symbiotic interaction with the human body. E. coli strains possessing certain virulence factors are able to adapt to new niches and cause a wide range of diseases of intestinal and extraintestinal localization.

E. coli associated with UTIs are known as uropathogenic E. coli (UPEC) [8]. UPEC possess a variety of both structural and secreted virulence factors necessary to realize their pathogenic potential in UTIs. The expression of adhesive organelles such as type 1 pili, P- and S-fimbriae allows UPEC to bind to receptors on the surface of epithelial cells in the urinary tract, colonize the uroepithelium and penetrate cells and tissues, and activate the innate immune response. In addition, S-fimbrial adhesins can be expressed by sepsis- and meningitis-associated E. coli (neonatal meningitis-associated E. coli (NMEC)). Invasins also play a significant role in the pathogenesis of neonatal meningitis, which are found predominantly in NMEC strains. An important pathogenic factor is toxins (hemolysin, cytotoxic necrotizing factor, vacuolizing vehicle toxin, secreted vehicle toxin) that damage cells and disrupt their metabolism. Production of siderophores (iron-transporting proteins) determines the ability of E. coli to capture iron, which increases viability in the urinary tract. The severity of symptomatic manifestations of UTIs is related to the acquisition and expression of virulence genes. In asymptomatic colonization of UTIs, UPEC are unable to express key virulence factors, which is probably a mechanism of adaptation to prolonged bladder persistence [9].

Based on molecular analysis, E. coli strains are divided into phylogenetic groups: A, B1, B2, C, D, E, F and G [10]. UPECs most often belong to phylogroups B2, D and to a lesser extent to groups E and F, whereas commensal strains, considered less virulent, belong predominantly to phylogroups A or B1 [11].

Numerous studies have shown an association between the presence of virulence genes and UPEC phylogroups [12–14]. However, the number of studies aimed at studying the molecular characterization and assessing the genotypic diversity of UPEC strains isolated in different manifestations of UTIs (especially in asymptomatic bacteriuria) is limited. Genetic determinants of virulence as a criterion for assessing and predicting the course of the infectious process are not currently used. Thus, an urgent direction of molecular genetic research is to study the pathogenic potential of UPEC isolated from pregnant women with asymptomatic bacteriuria to determine their clinical significance, as well as to determine the molecular basis of pathogenesis, develop new diagnostic algorithms and effective treatment methods.

The aim of the study was the molecular and genetic characterization of UPEC isolated from pregnant women with asymptomatic bacteriuria.

Materials and methods

Clinical isolates of E. coli (n = 70) were isolated from the urine of pregnant women with IBU who were observed by an obstetrician-gynecologist at the D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology in the years 2018–2023.

The study was conducted with voluntary informed consent of the patients, the study protocol was approved by the local ethical committee of the D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology (protocol No. 114 of 14.12.2021).

The diagnosis of asymptomatic bacteriuria was established when the same microorganism was isolated in the amount of ≥ 10⁵ CFU/mL in 2 consecutive urine samples taken at least 24 h apart, in the absence of clinical manifestations of UTI. If more than 1 microorganism was isolated, the sample was excluded from the study. Bacteriologic examination of clinical material was performed using chromogenic nutrient medium for isolation of UTI pathogens (Brilliance UTI Clarity Agar, Oxoid). The identification results were confirmed by mass spectrometry (MALDI-TOF MS, Bruker Daltonics). The cultures were stored in trypticase-soy broth with addition of 30% glycerol at –70ºС.

DNA extraction was performed using the DNA-sorb AM reagent kit (Central Research Institute of Epidemiology).

The affiliation of E. coli strains to phylogenetic groups was determined by quadriplex-polymerase chain reaction (PCR) according to O. Clermont et al. [10].

All isolates were tested for 15 virulence markers: genes associated with adhesion (fimH, papC, sfa, afa, focG); invasion (ibeA), toxin synthesis (cnf1, hlyA, sat, vat, usp), siderophores (fyuA, iroN, iuc), and capsular antigen (kpsMII). Previously studied primers were used; PCR primers were synthesized by Syntol LLC [15-21]. For PCR amplification we used the Tersus plus PCR reagent kit (Eurogen) and Tertsik thermocycler (DNA-Technology). The amplicons were separated in 2% agarose gel. Data were visualized and documented using the Infinity gel documentation system (Vilber Lourmat).

Fisher's exact test was used to assess the statistical significance of differences in mean values. Differences with a confidence interval (CI) of 95% (p < 0.05) were considered statistically significant.

Results

Phylogenetic analysis of E. coli sequences isolated in pregnant women with asymptomatic bacteriuria showed that clinical isolates belonging to phylogroup B2 (51.4%) were significantly more frequent (p < 0.05); the remaining isolates belonged to phylogroups D, A, B1, and F (Table 1).

 

Table 1. Belonging to phylogenetic groups of E. coli isolated from pregnant women with asymptomatic bacteriuria

Indicator	Phylogroup
	А	В1	В2	D	F
Number of isolates	8	6	36	14	6
%	11,4	8,6	51,4	20,0	8,6
95% CI	5.1–21.3	3.2–17.7	39.2–63.6	11.4–31.3	3.2–17.7

 

Analysis of virulence factors associated with adhesion showed that the fimH gene was detected in 97.1% of the strains studied; rarC, in 34.3%; sfa, in 27.1%; focG, in 11.4%; and afa, in 2.9%. The ibeA gene responsible for endothelial cell invasion was not detected among UPEC isolated in pregnant women with asymptomatic bacteriuria. The most common gene encoding toxin synthesis was vat (42.9%); hlyA, cnf1, and sat genes were detected in 21.4, 22.9, and 32.9% of isolates, respectively. The uropathogen-specific usp protein was detected in 57.1% of isolates. Among the genes associated with siderophore production, the fyuA gene was detected in 78.6% of the isolates, iroN in 48.6%, and iuc in 37.1%. The gene encoding capsule antigen synthesis (kpsMII) was detected in 65.7% of the studied E. coli strains.

Between 1 and 12 virulence genes were present in UPEC clinical isolates. None of the 70 E. coli strains contained all 15 virulence markers included in the study. Phylogenetic group A was significantly more frequently represented by isolates with 1 virulence gene (75%); the remaining strains were characterized by a combination of 2 (12.5%) and 5 (12.5%) genes without statistically significant differences. Isolates belonging to phylogenetic group B1 had a combination of 2 (33.3%) and 4 (50%) virulence markers in their genome without statistically significant differences; 1 (16.7%) strain had 1 virulence gene in its genome. Phylogenetic group B2 was characterized by the highest number of genes in various combinations (from 5 to 12); isolates containing combinations of 7 (11.1%), 8 (25%), 9 (13.9%), 10 (25%) and 12 (2.8%) genes in their genome were significantly more frequent. Phylogenetic groups D and F were represented by isolates in which genes encoding virulence factors were present without significant differences in combinations of 2 to 8 and 3 to 8, respectively.

Depending on the types of UPEC pathogenicity factors present, all isolates were divided into 6 clusters (Table 2).

 

Table 2. Frequency of detection of pathogenicity factors in uropathogenic E. coli isolated from pregnant women with asymptomatic bacteriuria

Pathogenicity factor	n	%	95% CI
Adhesion	7	10	4.1–19.5
Adhesion + siderophores	11	15,7	8.1–26.4
Adhesion + toxins	1	1.4	0.04–7.7
Siderophores + capsules	1	1.4	0.04–7.7
Adhesion + siderophores + capsules	2	2.9	0.03–12.6
Adhesion + siderophores + toxins	4	5.7	1.8–14.2
Adhesion + siderophores + toxins + capsules	44	62.9	50.5–74.1

 

Genetic determinants encoding 1 pathogenicity factor were identified in the genomes of 7 isolates (7%, 95% CI 4.1–19.5). The frequency of isolates containing combinations of 4 factors (62.9%, 95% CI 50.0–74.1) was statistically significantly (p < 0.0001) different from isolates characterized by the presence of combinations of genes encoding 2 and 3 pathogenicity factors.

The highest number of virulence genes was detected in strains belonging to phylogroup B2; focG, afa, cnf1, and ibeA genes were undetectable in phylogroup D; 8 virulence genes were detected in phylogroup F, except for afa, sfa, focG, cnf1, hlyA, vat, and ibeA (Table 3). The lowest gene diversity was detected in phylogroups A and B1. Virulence genes associated with adhesion (sfa, focG), encoding toxin synthesis (hlyA, cnf1, vat, usp), siderophores (fyuA, iroN, hlyA) and capsules (kps) were statistically significantly more frequent (p < 0.05) in isolates of phylogenetic group B2 compared to isolates of other phylogenetic groups. Statistically significant differences were found in the frequency of occurrence of the kps gene (p < 0.01) in UPEC isolates belonging to phylogenetic group A; vat (p < 0.05) and usp (p < 0.001) genes belonging to phylogenetic group D.

 

Table 3. Occurrence of virulence genes in E. coli of various phylogenetic groups isolated from pregnant women with asymptomatic bacteriuria

Virulence factors	Gen	Frequency of gene occurrence, n (%)
		A (n = 8)	B1 (n = 6)	B2 (n = 36)	D (n = 14)	F (n = 6)
Adhesins	papC	0	0	15 (41.7%)	5 (35.7%)	4 (66.7%)
	afa	0	0	2 (5.6%)	0	0
	fimH	8 (100.0%)	6 (10.0%)	36 (100.0%)	13 (92.8%)	6 (100.0%)
	sfa	0	0	18+*** (50.0%)	1 (7.1%)	0
	focG	0	0	8+** (22.2%)	0	0
Invasins	ibeA	0	0	0	0	0
Siderophore	fyuA	2 (25.0%)	4 (66,7%)	33+** (91.7%)	12 (85.7%)	4 (66.7%)
	iroN	0	4 (66,7%)	25+*** (69.4%)	3 (21.4%)	2 (33.3%)
	iuc	1 (12.5%)	3 (50,0%)	14 (38.9%)	4 (28.5%)	4 (66.7%)
Toxins	hlyA	0	0	14+*** (38.9%)	1 (7.1%)	0
	cnf1	0	0	16+*** (44.4%)	0	0
	sat	1 (12.5%)	0	14 (38.9%)	7 (50.0%)	2 (33.3%)
	vat	0	0	30+*** (83.3%)	2–* (14.3%)	0
	usp	0	0	36+*** (100.0%)	1–*** (7.1%)	3 (50.0%)
Capsules	kpsMII	1–** (12.5%)	0	32+*** (88.9%)	9 (64.3%)	4 (66.7%)

Note. ^– — the frequency of gene occurrence in the group is lower than the frequency of occurrence of the same gene in the overall sample; ⁺ — the frequency of gene occurrence in the group is higher than the frequency of occurrence of the same gene in the overall sample. *p < 0.05; **p < 0.01; ***p < 0.001.

 

Discussion

Studies on the molecular and genetic characterization of uropathogenic E. coli, the results of which are currently available for analysis, were based on the assessment of the pathogenic potential of strains isolated in UTIs. Given the significant impact of asymptomatic bacteriuria on the development of pregnancy complications, we conducted a study aimed at investigating E. coli strains isolated from patients with this pathology.

Clinical isolates of E. coli isolated from pregnant women with asymptomatic bacteriuria belonged to 5 phylogenetic groups (A, B1, B2, D and F). It was shown that the dominant phylogroups were B2 and D (to a lesser extent) with 51.4 and 20%, respectively, which is consistent with the results of other studies [11]. According to the extended classification by O. Clermont (2013), phylogroup F is a subgroup of phylogroup B2, and E. coli belonging to this group are also considered uropathogenic. In our study, 8.6% of the isolates in asymptomatic bacteriuria were attributed to this phylogroup. E. coli belonging to phylogenetic groups A (11.4%) and B1 (8.6%) were also found to be associated with commensal isolates; this suggests that the main reservoir of E. coli that are able to colonize the urinary tract is the intestine.

Adhesion factors play a key role in the pathogenesis of UTIs by facilitating the attachment of E. coli to the uroepithelium. Overall, adhesion factors in our study were detected in isolation or in various combinations in 69 (98.6%) isolates, confirming the role of adhesins as a major urovirulence factor. According to numerous studies, the fimH gene is the most common adhesion gene encoding type 1 fimbriae, which was confirmed by the results of our study: the fimH gene was detected in the majority of isolates (97.1%). Virulence factors associated with the development of ascending infection (pyelonephritis) play an important role in the pathogenesis of UTIs, especially in pregnant women. The rarC gene (pyelonephritis-associated pili) and afa-adhesins (associated with the development of gestational pyelonephritis) were found in 34.3 and 2.9% of isolates in our study, respectively. The prevalence of genes encoding fimbrial adhesins sfa and focG, which are expressed by strains causing meningitis, sepsis and pyelonephritis, was 34.7 and 12.9%, respectively. The results of similar studies on the genetic determinants of virulence of UPEC isolated in asymptomatic bacteriuria show a lower prevalence of rarC (12.9% to 20.6%), sfa (8.1% to 16.9%), but a higher frequency of afa (34.9%) and focG (35.1%) genes detected [20, 22, 23].

The urinary tract can be a source of NMEC, which is one of the most common infections with high morbidity and mortality in the neonatal period [24]. The ibeA gene, which is one of the important virulence factors of NMEC and is responsible for endothelial cell invasion, was not detected among E. coli isolated from pregnant women with asymptomatic bacteriuria.

UPEC is characterized by the presence of a uropathogen-specific protein, bacteriocin-like toxin, associated with the development of pyelonephritis and bacteremia. The appearance of this virulence marker is often associated with an increase in the virulence of the strain and its survival in the urinary tract. The results of our study showed a higher detection rate of the usp gene (57.1%) compared to the results of similar studies, where the detection rate of the usp gene in UPEC isolated in asymptomatic bacteriuria was 22.6–34.4% [20, 22].

Toxin formation is characteristic of E. coli strains responsible for more severe forms of disease (pyelonephritis, urosepsis). The hlyA and cnf1 genes associated with toxin synthesis were found in 68.2 and 63.6% of isolates from pyelonephritis cases; in 19.4 and 25.8% of those isolated in asymptomatic bacteriuria [22]. The results of our study showed a similar pattern — hlyA and cnf1 genes were detected in 21.4 and 22.9% of isolates from pregnant women with asymptomatic bacteriuria. Secreted autotransport toxin (sat) is also a virulence factor characteristic of UPEC isolated in pyelonephritis. In our study, the detection rate of sat gene was 32.9%, which is significantly higher than that of L. Maniam et al. (7.5%) [20]. The vat gene is found in more than half of E. coli isolates from patients cystitis and pyelonephritis [25]. In our study, the vat gene was detected in 42.9% of E. coli isolates isolated in asymptomatic bacteriuria.

The production of siderophores, which play an important role in iron capture, increases the viability of microorganisms within the urethral tract. The presence of these virulence factors in UPEC appears to compensate for the absence of other virulence genes related to adhesion and toxin formation, and thus promotes prolonged colonization in the urinary tract without inducing an inflammatory response in the host. In the study performed, genes responsible for the synthesis of yersinebactin (fyuA), salmochelin (iroN) and aerobactin (iuc) were identified in 78.6%, 48.6% and 37.1% of isolates, respectively.

The capsule has a protective role against the host immune system, which promotes long-term persistence in the urinary tract. We detected the kpsMII gene in 65.7% of the isolates from patients with asymptomatic bacteriuria. Similar studies have shown that the frequency of this gene ranged from 38 to 73% [20, 23].

The presence of individual virulence genes in the genome is not sufficient for the realization of uropathogenic potential. Many studies suggest that several virulence factors are present in bacteria at once [22, 26]. Our data also indicate the genetic diversity of UPEC isolated in asymptomatic bacteriuria. The results of quantitative distribution of virulence factors in phylogenetic groups showed that 90% of E. coli isolates had two or more virulence markers. E. coli belonging to phylogroups B2, D, E, and F associated with UPEC contained more virulence genes than phylogroups A and B1 associated with commensal strains. Furthermore, E. coli belonging to phylogenetic group B2 possessed the highest number of genes in various combinations (5 to 12); genes associated with adhesion (sfa, focG), synthesis of siderophores (fyuA, iroN, iuc), toxins (hlyA, cnf1, vat, usp) and capsules (kpsMII) were found significantly more frequently (p < 0.05).

Numerous factors (immune response, ability to form biofilms) may influence the expression of virulence factors and the ability of UPEC isolates to adapt to persistence in UTIs. When comparing the virulence properties of UPEC isolates in asymptomatic bacteriuria and symptomatic UTIs (cystitis, pyelonephritis) in pregnant women, it was shown that E. coli isolates in asymptomatic bacteriuria and cystitis showed comparable virulence rates [23]. The determination of a large number of virulence genes in the isolate, according to the researchers, may indicate the uropathogenic potential of the pathogen, and the ability to manifest depends on the expression of this gene or a set of genes.

Conclusion

The molecular characterization and genotypic diversity of E. coli strains are essential for better understanding of their role in the pathogenesis of UTIs. Identification of key virulence determinants and/or virulence gene combinations may be a marker for predicting the course of UTIs, especially in pregnant women, and will enhance the diagnostic capabilities based on the virulence properties of the uropathogen

About the authors

Tatiana A. Khusnutdinova

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Author for correspondence.
Email: husnutdinovat@yandex.ru
ORCID iD: 0000-0002-2742-2655

Cand. Sci. (Med.), senior researcher, Department of medical microbiology

Russian Federation, Saint Petersburg

Olga V. Budilovskaya

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: husnutdinovat@yandex.ru
ORCID iD: 0000-0001-7673-6274

Cand. Sci. (Med.), senior researcher, Department of medical microbiology

Russian Federation, Saint Petersburg

Anna A. Krysanova

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: husnutdinovat@yandex.ru
ORCID iD: 0000-0003-4798-1881

Cand. Sci. (Med.), senior researcher, Department of medical microbiology

Russian Federation, Saint Petersburg

Kira V. Shalepo

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: husnutdinovat@yandex.ru
ORCID iD: 0000-0002-3002-3874

Cand. Sci. (Med.), senior researcher, Department of medical microbiology

Russian Federation, Saint Petersburg

Anna A. Sinyakova

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: husnutdinovat@yandex.ru
ORCID iD: 0000-0003-3094-665X

Cand. Sci. (Med.), senior researcher, Department of obstetrics and perinatology

Russian Federation, Saint Petersburg

Alevtina M. Savicheva

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: husnutdinovat@yandex.ru
ORCID iD: 0000-0003-3870-5930

D. Sci. (Med.), Professor, Head, Department of medical microbiology

Russian Federation, Saint Petersburg

Igor Yu. Kogan

D.O. Ott Research Institute of Obstetrics, Gynecology and Reproductology

Email: husnutdinovat@yandex.ru
ORCID iD: 0000-0002-7351-6900

D. Sci. (Med.), Professor, RAS Corresponding Member, Director

Russian Federation, Saint Petersburg

References