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Rev. Inst. Med. trop. S. Paulo vol.50 no.5 São Paulo Sept./Oct. 2008
Caracterização genotípica dos fatores de virulência em amostras de Escherichia coli isoladas de pacientes com cistite
Monique Ribeiro TibaI; Tomomasa YanoII; Domingos da Silva LeiteI
IDepartamento de Microbiologia e Imunologia, Laboratório de antígenos Bacterianos II, Instituto de Biologia, Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz, 13081-970 Campinas, SP, Brasil
IIDepartamento de Microbiologia e Imunologia, Laboratório de antígenos Bacterianos I, Instituto de Biologia, Universidade Estadual de Campinas, Cidade Universitária Zeferino Vaz, 13081-970 Campinas, SP, Brasil
Adhesins (P-fimbriae, S-fimbriae, type 1 fimbriae and afimbrial adhesin), toxins (α-hemolysin and cytotoxic necrotizing factor type 1), iron acquisition systems (aerobactin) and host defense avoidance mechanisms (capsule or lipopolysaccharide) have been shown to be prevalent in Escherichia coli strains associated with urinary tract infections. In this work, 162 Uropathogenic Escherichia coli (UPEC) strains from patients with cystitis were genotypically characterized by polymerase chain reaction (PCR) assay. We developed three multiplex PCR assays for virulence-related genes papC, papE/F, papG alleles, fimH, sfa/foc, afaE, hly, cnf-1, usp, cdtB, iucD, and kpsMTII, all of them previously identified in UPEC strains. The PCR assay results identified 158 fimH (97.5%), 86 kpsMTII (53.1%), 53 papC/papEF/papG (32.7%), 45 sfa (27.8%), 42 iucD (25.9%), 41 hly (25.3%), 36 usp (22.2%), 30 cnf-1(18.5%) and 10 afa (6.2%) strains. No strain was positive for cdtB. In this work, we also demonstrated that adhesins may be multiple within a single strain and that several virulence genes can occur combined in association.
Keywords: Uropathogenic Escherichia coli (UPEC); Polymerase chain reaction (PCR); Virulence factor (VF).
Adesinas (Fímbria P, fímbria S, fímbria do tipo 1 e a adesina afimbrial), toxinas (α-hemolisina e o fator necrosante citotóxico do tipo 1), sistemas de captação de ferro (aerobactina), e mecanismos de defesa do hospedeiro (cápsula ou lipopolissacarídeo) são prevalentes em amostras de Escherichia coli associadas a infecções do trato urinário. O objetivo deste trabalho foi caracterizar genotipicamente 162 amostras de Escherichia coli uropatogênica (UPEC) de pacientes com cistite através do ensaio da reação em cadeia da polimerase. Foram realizados três ensaios de PCR multiplex para os seguintes fatores de virulência: papC, papE/F, alelos de papG, fimH, sfa/foc, afaE, hly, cnf-1, usp, cdtB, iucD, e kpsMTII. Os resultados da PCR identificaram, 158 amostras fimH (97,5%), 86 amostras kpsMTII (53,1%), 53 amostras papC/papEF/papG (32,7%), 45 amostras sfa (27,8%), 42 amostras iucD (25,9%), 41 amostras hly (25,3%), 36 amostras usp (22,2%), 30 amostras cnf-1 (18,5%) e 10 amostras afa (6,2%). Nenhuma amostra foi positiva para o gene cdtB. Neste trabalho, demonstramos que podemos encontrar múltiplas adesinas em uma única amostra e que diferentes genes de fatores de virulência podem ser encontrados em associação.
Urinary Tract Infections (UTIs), including cystitis and pyelonephritis, are among the most frequent human extraintestinal infections. Escherichia coli is the major causative agent, and it also is the predominant facultative member of the normal human intestinal flora23. This agent primary of UTIs accounts for greater than 80% of these infections. The bladder is the primary site of infection in about 95% of all UTIs23. In most cases, uropathogenic clones are selected from the fecal flora and colonize the vaginal and periurethral tissue, and infected the urinary tract by the ascending manner.
Classification of UTIs depends on the part of the urinary tract which is colonized (lower or upper urinary tract), as well as on the individual predisposition. Cystitis (lower UTI) is characterized by pelvic discomfort, especially pre - and immediately post void, frequent or urgent voiding and suprapubic pain. Acute pyelonephritis (upper UTI) is, most of the times, a more serious problem and is clinically identified by the fever, flank pain, nausea and, sometimes, vomiting19,22,23.
Uropathogenic Escherichia coli (UPEC) isolates are a genetically heterogeneous group that exhibit several virulence factors associated with colonization and persistence of the bacteria in the urinary tract. The virulent strains of UPEC that cause cystitis typically produce, at least, one adhesion system. Adhesins can also contribute to virulence, promoting colonization, invasion and replication within uroepithelial cells22,23. Besides bacterial adherence, several virulence factors may contribute to the pathogenicity of UPEC, facilitating the ability to adhere specifically to uroepithelial cells and the expression of other bacterial products to host tissues such as toxins, iron acquisition systems and host defense avoidance mechanisms26.
Virulence factors (VFs) associated with UPEC include adhesins (P fimbriae, type 1 fimbriae, S and F1C fimbriae, afimbrial adhesin), toxins (hemolysin, and cytotoxic necrotizing factor), siderophores (the aerobactin system) and polysaccharide coatings (group II capsules)1,29. Recently, a new urovirulence factor gene, which was more frequently found in UPEC strains, was reported. This gene encoding a protein designated usp (uropathogenic-specific protein) was demonstrated significantly enhancing the infectivity of E. coli in the mouse UTI model15,18,28.
The aim of this study was to detect the virulence genes of E.coli strains isolated from patients with clinical signs of cystitis in Brazil. PCR method was used for this purpose.
MATERIAL AND METHODS
Bacterial strains: A total of 162 E. coli strains collected in the mid-1990s, were isolated from each woman patient with recurrent UTI, attended at the Ambulatory sector of Medical School Hospital of the Campinas State University (UNICAMP), Campinas, São Paulo. Subjects had no clinical history of severe complications such as vesicoureteral reflux, neurogenic bladder, or malignant neoplasm. The diagnosis of E. coli UTI was characterized by typical clinical symptoms of cystitis such dysuria, frequency, urgency, small-volume voids or lower abdominal pain and more than 105cfu E. coli per mL urine sample. All isolates were stored at room temperature in NB (Nutrient broth) 0.75% agar.
The reference E. coli strains FVL2 (sfa, pap, iucD, hly, cnf-1), FV35 (afa, iucD, cnf-1), ORN115 (fimH), FVL8 (sfa, pap, papGII, hly, usp, iucD, cnf1), J96 (papGI and papGIII), U9-41 (O2:K1:H4, kpsMTII), and CLDT7 (cdtB) belong to Laboratório de Antígenos Bacterianos II (Campinas State University) and were used as positive control. E. coli strain DH5α was used as a negative control.
Polymerase Chain Reaction (PCR): Primers for each VF genes were first established individually by the use of template DNA from appropriate positive and negative control strains. We examined through PCR, the presence of the following groups of genes: colonization factors, such as P fimbriae (papC, papE/F and papG alleles), type 1 fimbriae (fimH), S fimbriae (sfaC/D), afimbrial adhesin (afaB/C), group II capsule (kpsMTII), aerobactin (iucD), cytotoxic necrotizing factor type 1 (cnf-1), α-hemolysin (hly), cytolethal distending toxin (cdtB), and uropathogenic specific protein (usp). The appropriate primers sequences, annealing temperature, and size of amplified fragment (base pairs - bp) for the characteristics studied are shown in Table 1.
Bacterial DNA to be amplified was released from whole organisms by boiling, and a multiplex PCR assay was carried out in a total volume of 25 µL, containing 2 µL of template DNA, each of the primers at 60 ng, the four deoxynucleotide triphosphates (each at 200 µM), PCR buffer 1X and 1.5 U of Taq DNA polymerase3,10. For Multiplex PCR, amplifications consisted of 25 cycles of 94 ºC for one min, specific annealing temperature for each primer for one min (Table 1), 68 ºC for three min, and a final extension at 72 ºC for seven min in a Thermal Cycler (Gene Amp PCR System 9700/Perkin Elmer Corporation, Norwal CT/USA). Primers were then sorted in three pools according to annealing temperature and amplicon size: Multiplex A: papC, papGI, papGII, papGIII; Multiplex B: kpsMTII, papE/F, iucD, hlyA; Multiplex C: sfaD/E, cnf-1. The primers for fimH, cdtB, usp and afaB/C genes were used individually.
The amplified DNA product was visualized by standard submarine gel electrophoresis using 10 µL of the final reaction mixture on a 2% agarose gel in TAE buffer (1,6 M Tris-EDTA, 0,025 M acetic acid). Amplified DNA fragments of specific sizes were located by UV fluorescence, after staining with ethidium bromide. The 100-bp ladder was used as a standard for determining molecular size of PCR products.
A total of 162 UPEC strains from patients with cystitis were genotypically characterized by the use of PCR assay. The PCR assay results identified 158 fimH (97.5%), 86 kpsMTII (53.1%), 53 papC/papEF/papG (32.7%), 45 sfa (27.8%), 42 iucD (25.9%), 41 hly (25.3%), 36 usp (22.2%), 30 cnf-1 (18.5%) and 10 afa (6.2%) strains (Table 2). All strains were negative for cdtB and papGI.
A total of 76 strains shared DNA sequences related to, at least, two operons of adhesins, encoding fimH and other genes associated as pap, sfa, and/or afa (Table 3).
FimH adhesin was the most prevalent virulence factor detected, having occurred in 97.5% of strains, and it was detected in 100% of operon pap (papC/papEF/papG alleles). Eighty two strains of the 158 fimH studied have only adhesin-encoding operon fimH, and in 48 of them it was not possible to detect any other VF gene.
Among the 53 papC/papE/FpapG strains, 29 were papGII, 23 were papGIII, one was papGII/papGIII and none was papGI (Table 2). Although papG alleles II and III were both associated with sfa, they exhibited associations with other VF genes. PapGII allele was associated with iucD, kpsMTII, hly, and negatively associated with cnf-1, whereas papG allele III was associated with hly, cnf-1 and kpsMTII. S fimbrial adhesin was associated with papG alleles, capsule, cnf-1 and α-hly toxins. Table 3 shows the association between virulence factors and adhesins genes.
E. coli is the major causative agent in human UTIs, one of the most common bacterial infections. In most cases, uropathogenic clones are selected from the fecal flora, and the pathogenic potential of E. coli strains is thought to be dependent on the presence of VFs19,23. Based on their components and products called virulence factors, E. coli cells attach selectively to the mucosa uro-epithelium, promoting colonization and persisting in the urinary tract, inducing, then, a local inflammatory response and sometimes to promote tissue lesions23,29.
We used, to detect and identify the presence of potential uropathogenic Escherichia coli virulence factors, a genotypic assay. PCR method is highly specific, informative and a powerful genotypic assay, used for detection of adhesin-encoding operons and other virulence factors that can also contribute to virulence in UTI. In this study, we confirmed the prevalence of fimH among UPEC strains10,21,23. FimH occurred in 158 strains. Eighty two strains presented only adhesin-encoding operon fim, and in 48 of these strains no other VF gene was detected. This result demonstrated that type 1 fimbriae is an important and relevant VF, and that it can also contribute to virulence in E.coli strains. Type 1-mediated adherence has been proposed to play a role in the induction of inflammation, enhancing E. coli virulence for the urinary tract. CONNELL et al., (1996)4 reported that mice infected with a type 1-positive 01:K1:H7 isolate showed a higher urinary neutrophil influx into the urine than type 1- negative isolates. Furthermore, we demonstrated that 76 fimH strains, occurred with at least one adhesin, such as pap, sfa, or afa. This result corroborates the occurrences presented in the literature, where fimH was found in associations with P fimbriae and S fimbriae in UPEC11.
P fimbriae, the principal mannose - resistant adherence organelles of extraintestinal pathogenic Escherichia coli, are known to contribute to pathogenesis by promoting bacterial colonization of host tissues and by stimulating an injurious host inflammatory response17. E. coli strains were examined for pap genotype and specific primers were utilized to detect genes associated with outer membrane protein (papC), minor structural subunit (papE/F) and papG allele's adhesin (papGI/papGII/papGIII).
The classes of papG genotype differed according to the type of receptor specificity and infection22,23. The class II G adhesin is associated with pyelonephritis and bacteremia, while the class III G adhesin sequence is associated with cystitis, although they have been found in pyelonephritis and bacteremia6,9. PapGI strains might have a larger prevalence among fecal isolates.
Fifty three strains (32.7%) were papC/papE/F/papG, demonstrating the presence of the intact operon, responsible for adhesin assembly. This frequency of pap in a cystitis strain agrees with literature data26. JOHNSON et al., 200511, reported that cystitis isolates differed from pyelonephritis and prostatitis isolates by their lower prevalence of papA/C/EF/G, papG allele II and the papGII/III alleles combination. We found 53 strains papC and all of them presented papG, demonstrated by PCR. There were 29 papGII strains, 23 papGIII strains and one strain carrying both sequences. The result that papGIII was less prevalent than papGII was not expected, since the strains were all derived from patients with cystitis.
PapGII and papGIII alleles are found in association with hly2. E. coli strains that produce Hly, frequently produce other two urovirulent factors: CNF-1 and P fimbriae. These three genes are found in the same pathogenicity island (PAI)2,10. In this study we detected 1 papGII/GIII/hly strain, 11 papGII/hly strains and 19 papGIII/hly strains. Our results are similar to that described in the literature2, where the hemolytic gene (hlyA) is present in both papG alleles.
While hly was found in association with P fimbriae, cnf1 was detected in 27 sfa strains. This result corroborates the occurrences presented in literature where, cnf-1 was found in association with sfa10,23.
Cdt-producing E. coli strains were first isolated from children with enteritis12. In other studies, cdt genes were detected in urosepsis and fecal E. coli isolates and from patients with a variety of diarrhea symptoms and encephalopathy10,13. CDTs produce giant mononucleated cells caused by an irreversible block in the cell cycle at the G2/M, occurring cellular death. In this study, cdtB was not detected by the PCR assay, indicating their relative unimportance in our E.coli collection. This finding suggests that cystitis can be caused by strains that are less virulent than those isolates that had cdtB and typically cause pyelonephritis, urosepsis and diarrhea.
Other VF found in association with pyelonephritis, meningitis and sepsis is S fimbriae23. S fimbrial adhesin recognizes surface sialic acid content, on receptors expressed by kidney epithelial and vascular endothelial cells, mediating bacterial adherence. Recently, it was identified sialic acid residues in the UP3, one of the four integral membrane uroplakin proteins, expressed on the bladder luminal surface, suggesting that S fimbriae may also have a role in cystitis20. We detected 45 sfa strains. Sfa was found in positive cnf-1, hly and papG alleles strains (Table 3). These results were similar to the ones described in the literature2,10.
The concentration of iron (Fe) is limited in sites of extraintestinal infection, in large part, due to host factors that reduce its availability. As a result, Fe acquisition is of critical need for pathogens that must grow within a host. One of the iron acquisition mechanisms is the biosynthesis of siderophore7. In the literature was reported that aerobactin system was found associated with papG allele II2. In this study, we detected 19 papGII/iucD strains and three papGIII/iucD strains, then, these results were similar to the ones described in the literature2.
The majority of the UPEC strains present group II capsules (K1, K5) determined by kps operon8. Capsule is common in UPEC and is better known for contributing with pyelonephritis than other urinary tract infections8. In this study were detected 86 (53.1%) kpsMTII strains. All of them were associated with other VF searched. The literature reported the association among the VFs present in UPEC: capsule, papG allele II and aerobactin. An association of kpsMTII and hlyA was also observed. Our results corroborate those present in the literature10, once we found 19 papGII/kps/iucD strains and only two papGIII/kps/iucD strains.
Kurazono et al. (2000)18 reported a putative pathogenicity island (PAI), which was more frequently found in UTI collections than in fecal E. coli. This PAI contains usp that encodes a 346-amino acid protein, which was designated as uropathogenic specific protein (usp). Parret & De Mot (2002)25 hypothesized that the usp protein may represent a novel type of E. coli bacteriocin, acting against competing E. coli strains that occupy the same niche, thereby enhancing their infectivity in the urinary tract environment. Recently, it was demonstrated that usp significantly enhanced the infectivity of E. coli in the mouse UTI model29.
In the study proceeding from Japan, usp gene occurred in 80% of 195 cystitis isolates, and 93% of the 76 pyelonephritis isolates18. It was noted that usp occurred more frequently in pyelonephritis than cystitis14. Herein, we detected 36 usp (22.2%) strains, and the reason for differences between these studies could be interpreted as being due to distinct women population analyzed or from the existence of different bacterial clones distributed between women from Brazil and Japan.
The VFs displayed distinctive and complex associations with each other. We demonstrated that papG allele II was found in positive kps, iucD and hly strains and was not found in positive cnf1 strain, whereas papG allele III was associated with kps, hly and cnf-1 genes. Both papG alleles were associated with S fimbriae. S fimbriae were positively associated with cnf-1 and hly, kpsMTII and papG alleles.
The present study provides molecular epidemiological information about VF genes found in lower UTI (cystitis). We have rigorously established a multiplex PCR assay, capable of detecting VF highly prevalent among UTI isolates. We confirmed the prevalence of fimH genes among E. coli strains from cystitis. We showed that P and type 1 fimbriae are not sole adhesins on uropathogenic E. coli and that these adhesions, may be multiple within a single strain with several virulence genes occurring together in association.
We are grateful to Mr. Erivaldo José da Silva and Mrs Mirtis Ferraz for technical support. This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
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Received: 3 March 2008
Accepted: 6 August 2008
Abbreviations: aerobactin (iucD), afimbrial adhesin (afaB/C), cytolethal distending toxin (cdtB), cytotoxic necrotizing factor type 1 (cnf-1), G adhesin classes of P fimbriae (papG alleles), group II capsule (kpsMTII), α-hemolysin (hly), minor structural subunits of P fimbriae (papE/F), outer membrane protein of P fimbrae (papC), polymerase chain reaction (PCR), S fimbriae (sfaC/D), type 1 fimbriae (fimH), urinary tract infection (UTI), uropathogenic specific protein (usp), uropathogenic Escherichia coli (UPEC), virulence factors (VFs).