RESUMEN

Con el objetivo de evidenciar diferencias en la respuesta inmunológica y bioquímica de ancianos con infección del tracto urinario (ITU) por Escherichia coli (E. coli) frente a factores de virulencia de importancia en la patogenia de la ITU, se realizó un estudio descriptivo en el cual se evaluaron 24 muestras de orina de adultos mayores con ITU provenientes de centros de reposo gerontológicos. Se determinó la concentración de hierro, TNF-α, IL-1β y la capacidad antioxidante en la orina, encontrándose una relación entre una mayor concentración de hierro y de hematíes en la orina con la presencia del gen pap GII en la E. coli. Se concluye que los adultos mayores con ITU por E. coli portadoras del gen pap GII, presentan mayor daño tisular.

Palabras clave:
Asilos de Ancianos; Ancianos; Escherichia coli Uropatogénica; Biomarcadores; Factores de Virulencia; Infecciones Urinarias; Inmunología; Orina; Antioxidantes; Citoquinas

ABSTRACT

Descriptive study in which 24 urine samples from older adults with urinary tract infection (UTI), from nursing homes, were evaluated; in order to identify differences in the immune and biochemical response from older adults with UTI by Escherichia coli (E. coli) to major virulence factors in the pathogenesis of UTI. Iron concentration, TNF-α, IL-1β and antioxidant capacity in urine were determined. A relation was found between, an increase in iron and red blood cell concentration in urine, and the presence of the pap GII gene found in E. coli. It is concluded that older adults, with UTIs by E. coli with the gene pap GII, have increased tissue damage.

Keywords:
Nursing Homes; Elderly; Uropathogenic Escherichia coli; Biomarkers; Virulence Factors, Urinary Tract Infections, Immunology, Urine, Antioxidants, Cytokines

INTRODUCTION

Escherichia coli (E. coli) is the most frequent cause of bacteremia in men and women¹1. Nicolle LE. Urinary Tract Infections in the Older Adult. Clin Geriatr Med. 2016;32(3):523-38. doi: 10.1016/j.cger.2016.03.002.
https://doi.org/10.1016/j.cger.2016.03.0... , and the urinary tract is the main way of infection in geriatric patients ²2. Mylotte JM, Tayara A, Goodnough S. Epidemiology of Bloodstream Infection in Nursing Home Residents: Evaluation in a Large Cohort from Multiple Homes. Clin Infect Dis. 2002;35(12):1484-90. doi: 10.1086/344649.
https://doi.org/10.1086/344649... . This is partially due to the fact that the older adult population (OAP) presents particular immunological characteristics, and a subclinical state of chronic inflammation, known as immunosenescence, where the polymorphonuclear lineage (main line of defense in urinary tract infections (UTIs)) ³3. Luna-Pineda VM, Ochoa S, Cruz-Córdova A, Cázares-Domínguez V, Vélez-González F, Hernández-Castro, R, et al. Infecciones del tracto urinario, inmunidad y vacunación. Bol Med Hosp Infant Mex. 2018; 75(2): 67-78. doi: 10.24875/BMHIM.M18000011.
https://doi.org/10.24875/BMHIM.M18000011... has limited function ⁽⁴4. Sauce D, Dong Y, Campillo-Gimenez L, Casulli S, Bayard C, Autran B, et al. Reduced oxidative burst by primed neutrophils in the elderly individuals is associated with increased levels of the CD16bright/CD62Ldim immunosuppressive subset. J Gerontol A Biol Sci Med Sci. 2017;72(2):163-72. doi: 10.1093/gerona/glw062.
https://doi.org/10.1093/gerona/glw062... . Several E. coli virulence factors have been described, mainly associated with bacteremia and sepsis, which include cell adhesion molecules, iron uptake systems and exotoxins that form a protein system that allows the bacteria to elude or injure the patient’s immune system ⁵5. Daga AP, Koga VL, Gabriel J, Soncini M, Matos CM De, Regina M, et al. Escherichia coli Bloodstream Infections in Patients at a University Hospital : Virulence Factors and Clinical Characteristics. Front Cell Infect Microbiol. 2019;9:191. doi: 10.3389/fcimb.2019.00191.
https://doi.org/10.3389/fcimb.2019.00191... ^,66. Johnson JR. Virulence factors in Escherichia coli urinary tract infection. Clin Microbiol Rev. 1991; 4(1):80-128. doi: 10.1128/cmr.4.1.80.
https://doi.org/10.1128/cmr.4.1.80... .

The interaction between the immune system of patients with UTIs and the genetic ability of the bacteria to form virulence factors determines the bacterial clearance in the urinary tract ⁶6. Johnson JR. Virulence factors in Escherichia coli urinary tract infection. Clin Microbiol Rev. 1991; 4(1):80-128. doi: 10.1128/cmr.4.1.80.
https://doi.org/10.1128/cmr.4.1.80... , so it is necessary to explore the difference in the immune and biochemical response of older adults with UTIs to the various virulence factors associated with sepsis in uropathogenic E. coli (UPEC).

THE STUDY

Between April and July 2018, the urine of 24 older adults with UTIs, of both sexes, residing in private gerontological nursing homes in Lima, was evaluated. The diagnostic criteria for UTIs were defined as: a microscopic count of >5 leukocytes per field of 400x magnification, the conversion of nitrites by the Griess method and a count of more than 100,000 colony-forming units in the blood agar culture medium. Urine sediment analysis was standardized according to the recommendations of the Chilean Health Institute ⁷7. Gómez R, Pellegrini P. Recomendaciones para el Ánalisis del Sedimento urinario. Departamento Laboratorio Biomédico Nacional y de Referencia. Instituto Nacional de Salud Pública de Chile. 2013 [citado el 03 de mayo de 2020]. Disponible en: https://www.araucaniasur.cl/wp-content/uploads/2019/11/sedimento-urinario 10052013A.pdf.
https://www.araucaniasur.cl/wp-content/u... .

The etiological agent was identified by using traditional biochemical methods and the bacteria identified as E. coli were preserved in tryptic soy broth and 20% glycerol. Polymicrobial cultures were excluded from the study. Subsequently, the bacterial DNA was extracted using the GeneJet Genomic kit (Thermo Scientific®, Massachusetts, USA) and the presence of 11 virulence genes was evaluated: aer, α-hly, cnf-1, sfa, chuA, TcpC, nanA, pap GI, GII, GIII and iucC by end-point polymerase chain reaction.

The urine samples were centrifuged at 3,000 g (Sigma, 3-30KS) for 10 minutes, preserving the supernatant. Concentrations of TNF-α, IL-1β and iron were determined; besides, the total antioxidant capacity in urine was evaluated by ABTS^•+ and FRAP methods, which acted as immune response markers through the increase of reactive oxygen substances (ROS). All analytes were measured using the Multiskan Go spectrophotometer (Thermo Scientific®, Massachusetts, USA).

The statistical analysis was carried out with the Epidat version 4.1 program. The description of the qualitative variables was made through frequency tables. Shapiro Wilk’s normality test was applied to determine the distribution of quantitative variables and Levene’s test was used to evaluate the variance homogeneity of the variables. The quantitative variables with normal distribution were analyzed by means of the Student’s T test. p values <0.05 were considered significant.

This study was approved by the Ethics Committee of the Faculty of Medicine of the Universidad Nacional Mayor de San Marcos, act 1812 with project code 0013.

FINDINGS

Twenty-four urine samples from older adults with UTIs by E. coli, from gerontological nursing homes, were analyzed. The most frequent virulence genes were nanA, pap GII, aer, chuA and iucC. Genes α-hly and cnf-1 were found in low proportion and the following genes were not found: TcpC, pap GI, pap GIII and sfa (Figure 1).

Regarding the immune and biochemical response of older adults to the various virulence genes evaluated, a concentration of 37.6 red blood cells/μL and 193.4 μg/L of iron was observed in patients carrying the pap GII gene, values were significantly higher in patients infected with E. coli, but without this gene. In addition, patients with strains carrying the pap GII gene showed a positive tendency towards having a higher urine antioxidant capacity regarding the ABTS^•+ test (1348.7 vs. 634.8 Eq-real Vit C μg/mL) (Table 1). On the other hand, a higher concentration of leukocytes (p = 0.070) was observed in patients infected with E. coli carriers of the three genes related to iron metabolism evaluated (iucC, aer and chuA) (Table 2).

DISCUSSION

This study evaluated the immune and biochemical response in the urine of hospitalized older patients infected by UPEC (the main etiological agent of UTIs). One of the main findings was that the presence of the pap GII gene induced hematuria and a higher iron concentration in the urine. The frequency of virulence genes important in the pathogenicity process of UTIs was also evaluated. Another important finding was that 95.8% of UPEC presented the nanA gene.

N-acetylneuraminic acid, an important substrate in the energetic production of E. coli, is split by the N-acetylneuraminic lyase (nanA) enzyme into pyruvate and N-acetyl-D-manosamine ⁸8. Smith SN, Hagan EC, Lane MC, Mobley HLT. Dissemination and Systemic Colonization of Uropathogenic Escherichia coli in a Murine Model of Bacteremia. MBio. 2010;1(5):1-9. doi: 10.1128/mBio.00262-10.
https://doi.org/10.1128/mBio.00262-10... . The presence of nanA enzyme generates high competitiveness in the bacteremia-producing UPEC; and although its function in UTI pathogenesis in murine models is less relevant ⁸8. Smith SN, Hagan EC, Lane MC, Mobley HLT. Dissemination and Systemic Colonization of Uropathogenic Escherichia coli in a Murine Model of Bacteremia. MBio. 2010;1(5):1-9. doi: 10.1128/mBio.00262-10.
https://doi.org/10.1128/mBio.00262-10... , results indicate the existence of a high risk of developing sepsis in older adults with bacteremia.

On the other hand, exotoxins have a role in the pathogenesis of UTIs. Neutrophils, the main line of defense in UTIs, are lysed by high doses of α-hemolysin (HlyA) and, in addition, bladder cells in low doses are exfoliated ⁹9. Lüthje P, Brauner A. Virulence Factors of Uropathogenic E. coli and Their Interaction with the Host. Adv Microb Physiol. 2014;65:337-72. doi: 10.1016/bs.ampbs.2014.08.006.
https://doi.org/10.1016/bs.ampbs.2014.08... , which deteriorates the body’s two main lines of defense ³3. Luna-Pineda VM, Ochoa S, Cruz-Córdova A, Cázares-Domínguez V, Vélez-González F, Hernández-Castro, R, et al. Infecciones del tracto urinario, inmunidad y vacunación. Bol Med Hosp Infant Mex. 2018; 75(2): 67-78. doi: 10.24875/BMHIM.M18000011.
https://doi.org/10.24875/BMHIM.M18000011... . Nevertheless, we obtained a frequency of 8.3% in the studied UPEC, which is lower than what was found in other studies carried out in geriatric population ¹⁰10. Santo, E, Macedo C, Marin JM. Virulence factors of uropathogenic Escherichia coli from a University Hospital in Ribeirão Preto, São Paulo, Brazil. Rev Inst Med Trop Sao Paulo. 2006;48(4):185-88. doi: 10.1590/s0036-46652006000400002.
https://doi.org/10.1590/s0036-4665200600... ^,1111. Croxall G, Hale J, Weston V, Manning G, Cheetham P, Achtman M, et al. Molecular epidemiology of extraintestinal pathogenic Escherichia coli isolates from a regional cohort of elderly patients highlights the prevalence of ST131 strains with increased antimicrobial resistance in both community and hospital care settings. J Antimicrob Chemother. 2011;66(11):2501-8. doi: 10.1093/jac/dkr349.
https://doi.org/10.1093/jac/dkr349... . Likewise, the exotoxins, such as the necrotizing cytotoxic factor type 1 (CNF1) genetically related to HlyA, induce a rearrangement of the neutrophilic cytoskeleton through the activation of the Rho GTPase type enzymes ⁹9. Lüthje P, Brauner A. Virulence Factors of Uropathogenic E. coli and Their Interaction with the Host. Adv Microb Physiol. 2014;65:337-72. doi: 10.1016/bs.ampbs.2014.08.006.
https://doi.org/10.1016/bs.ampbs.2014.08... . In accordance with what was reported by α-hly, we found a UPEC carrier of the cnf-1 gene.

The TcpC protein recently described as interfering in the production of proinflammatory cytokines in the UTI ¹²12. Xiao T, Waldhuber A, Svanborg C, Snyder G, Römmler F, Miethke T, et al. A Comparative Analysis of the Mechanism of Toll-Like Receptor-Dis-ruption by TIR-Containing Protein C from Uropathogenic Escherichia coli. Pathogens. 2016;5(1):25. doi: 10.3390/pathogens5010025.
https://doi.org/10.3390/pathogens5010025... was not found in any of the evaluated UPEC, which may reflect the absence of the type IV pathogenicity island, dependent on its horizontal transfer, or the recent evolutionary acquisition of the gene in extra-intestinal E. coli¹²12. Xiao T, Waldhuber A, Svanborg C, Snyder G, Römmler F, Miethke T, et al. A Comparative Analysis of the Mechanism of Toll-Like Receptor-Dis-ruption by TIR-Containing Protein C from Uropathogenic Escherichia coli. Pathogens. 2016;5(1):25. doi: 10.3390/pathogens5010025.
https://doi.org/10.3390/pathogens5010025... .

Regarding the immunological and biochemical response observed in the older adults, iron is scarce in the urinary fluid and is indispensable for bacterial metabolism ¹³13. Pfrimer K, Micheletto RF, Marchini JS, Padovan GJ, Moriguti J, Ferriolli E. Impact of Aging on Urinary Excretion of Iron and Zinc. Nutr Metab Insights. 2014;7:47-50 doi: 10.4137/NMI.S12977.
https://doi.org/10.4137/NMI.S12977... ^,1414. Alteri CJ, Smith SN, Mobley HLT. Fitness of Escherichia coliduring urinary tract infection requires gluconeogenesis and the TCA cycle. PLoS Pathog. 2009;5(5):e1000448. doi: 10.1371/journal.ppat.1000448.
https://doi.org/10.1371/journal.ppat.100... , the microorganisms that infect the urinary tract must have the capacity to capture and compete for iron assimilation ¹⁴14. Alteri CJ, Smith SN, Mobley HLT. Fitness of Escherichia coliduring urinary tract infection requires gluconeogenesis and the TCA cycle. PLoS Pathog. 2009;5(5):e1000448. doi: 10.1371/journal.ppat.1000448.
https://doi.org/10.1371/journal.ppat.100... . UPECs have three systems of iron uptake (siderophores, hemophores and direct iron uptake in its ferrous state), which are reported to have increased expression in vivo¹⁵15. Hagan EC, Lloyd AL, Rasko DA, Faerber GJ, Mobley HLT. Escherichia coli global gene expression in urine from women with urinary tract infection. PLoS Pathog. 2010; 11;6(11): e1001187. doi: 10.1371/journal.ppat.1001187.
https://doi.org/10.1371/journal.ppat.100... . On the other hand, iron restriction is an immune defense mechanism used by the host to limit bacterial proliferation ¹⁶16. Bullen JJ. The Significance of Iron in Infection. Clin Infect Dis. 1981;3(6):1127-38. doi: 10.1093/clinids/3.6.1127.
https://doi.org/10.1093/clinids/3.6.1127... . Although we could not find significant differences between immunological and biochemical markers and genes associated with iron metabolism, it can be observed that patients with UPEC, carriers of the iucC, aer and chuA genes, tend to have higher leukocyte concentrations, which may indicate a greater immune stress. Besides, the presence of chuA gene and other siderophore systems are highly concentrated in strains that cause recurrent UTI ¹⁷17. Ejrnæs K, Stegger M, Reisner A, Ferry S, Monsen T, Holm SE, et al. Characteristics of Escherichia coli causing persistence or relapse of urinary tract infections: Phylogenetic groups, virulence factors and biofilm formation. Virulence. 2011;2(6):528-37. doi: 10.4161/viru.2.6.18189.
https://doi.org/10.4161/viru.2.6.18189... , a common situation in gerontological resting centers.

Finally, the pap GII gene is a virulence factor whose function is to ensure the adhesion of UPEC to renal tissue. The association of pap GII gene with pyelonephritis stages has been documented ⁶6. Johnson JR. Virulence factors in Escherichia coli urinary tract infection. Clin Microbiol Rev. 1991; 4(1):80-128. doi: 10.1128/cmr.4.1.80.
https://doi.org/10.1128/cmr.4.1.80... , besides, it has been proposed that it confers competitive advantage during bacteremia stages by UTI ⁸8. Smith SN, Hagan EC, Lane MC, Mobley HLT. Dissemination and Systemic Colonization of Uropathogenic Escherichia coli in a Murine Model of Bacteremia. MBio. 2010;1(5):1-9. doi: 10.1128/mBio.00262-10.
https://doi.org/10.1128/mBio.00262-10... . Some evidence indicates that its presence is not determinant in stages of pyelonephritis, therefore, its function is not yet conclusive in the process of infection in the urinary tract ¹⁸18. Mobley HLT, Jarvis KG, Elwood JP, Whittle DI, Lockatell CV, Russell RG, et al. Isogenic P-fimbrial deletion mutants of pyelonephritogenic Escherichia coli: the role of alpha Gal(1-4) beta Gal binding in virulence of a wild-type strain. Mol Microbiol. 1993;10(1):143-55. doi: 10.1111/j.1365-2958.1993.tb00911.x.
https://doi.org/10.1111/j.1365-2958.1993... .

Results obtained show that patients with UPEC positive for the pap GII gene had higher iron concentration compared to patients with UPEC negative for the pap GII gene. It has been found that the interaction of pap GII with its receptor induces rapid transcription of the airS gene in UPEC in urine, which has a fundamental role in the activation of the siderophore systems (aerobactin/enterobactin) ¹⁹19. Zhao R, Shi J, Shen Y, Li Y, Han Q, Zhang X, et al. Phylogenetic distribution of virulence genes among ESBL-producing uropathogenic escherichia coli isolated from long-term hospitalized patients. J Clin Diagnostic Res. 2015;9(7):1-4. doi: 10.7860/JCDR/2015/13234.6157.
https://doi.org/10.7860/JCDR/2015/13234.... by decreasing the concentration of iron in urine. In the results obtained we did not find this association. However, this could be explained by the higher concentration of red blood cells and hemolysis in this group of patients. The adhesion of the pap GII protein activates the synthesis of cytokines ²⁰20. Svensson M, Duan R, Svanborg C. Role of the Ceramide-sig-naling Pathway in Cytokinr Responses to P-fimbriated. J Exp Med. 1996;183(3):1037-44. doi: 10.1084/jem.183.3.1037.
https://doi.org/10.1084/jem.183.3.1037... , causing the rupture of blood vessels located in the lamina itself. On the other hand, the ABTS^•+ test describes that in patients with positive pap GII UPEC there is a tendency to have greater antioxidant capacity, which could be due to higher iron concentration, which interferes with the assay, although, this tendency could also be explained by the deficiency of neutrophils to produce reactive oxygen substances. Recent studies have described an increase in the neutrophil subpopulation CD16/CD62L in the OAP, which presents a lower level of response to cytokine stimuli ⁴4. Sauce D, Dong Y, Campillo-Gimenez L, Casulli S, Bayard C, Autran B, et al. Reduced oxidative burst by primed neutrophils in the elderly individuals is associated with increased levels of the CD16bright/CD62Ldim immunosuppressive subset. J Gerontol A Biol Sci Med Sci. 2017;72(2):163-72. doi: 10.1093/gerona/glw062.
https://doi.org/10.1093/gerona/glw062... .

This study has several limitations. First, it was not possible to record information from the patients regarding their clinical condition, infection stage, use of antibiotics, infection recurrence. Second, the low number of urine samples analyzed, which increases the probability of obtaining a type II error. Third, we do not complement our studies with gene expression analysis, so we do not know if the genes evaluated are indeed expressed by the bacteria.

In conclusion, E. coli carrying the pap GII gene can induce greater tissue damage that possibly favors a higher iron concentration in the urine, which stimulates its increase. In addition, patients infected with E. coli with pap GII show a positive tendency to have greater antioxidant capacity, which may be due to the deficiency of neutrophils recruited in the OAP during the production of ROS. Finally, the generalized presence of nanA gene is important due to its high relevance in sepsis stages.

References

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