Zdrav Vestn 2008; 77: 427–32 427 Research article/Raziskovalni prispevek PREVALENCE OF TOXIN ENCODING GENES IN ESCHERICHIA COLI ISOLATES FROM URINARY TRACT INFECTIONS IN SLOVENIA PREVALENCA GENSKIH ZAPISOV ZA TOKSINE V IZOLATIH BAKTERIJE ESCHERICHIA COLI, PRIDOBLJENIH IZ VZORCEV URINA V SLOVENIJI Marjanca Starčič-Erjavec,1 Veronika Križan-Hergouth,2 Borut Gubina,3 Darja Žgur-Bertok1 Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000 Ljubljana, Slovenia 2 Institute of Microbiology and Immunology, Medical Faculty, University of Ljubljana, Zaloška 4, 1000 Ljubljana, Slovenia 3 Department of Urology, University Clinical Center Ljubljana, Zaloška 7, 1525 Ljubljana, Slovenia Abstract Methods Results Conclusions Key words 110 uropathogenic Escherichia coli strains (UPEC) obtained from the Institute of Microbiology and Immunology of the Medical Faculty in Ljubljana were screened by PCR with primers specific for the following toxin encoding genes: hlyA (haemolysin), cnf1 (cytotoxic necrotising factor 1), usp (uropathogenic specific protein USP) and ibeA (invasin). Dot blot hybridisation experiments were performed to validate the PCR assays. In 44% of the strains usp gene sequences were detected. The prevalence of hlyA and cnf1 was 25% and 23%, respectively. Only 9% of the strains harbored ibeA. The majority of the tested toxin encoding genes was found in strains belonging to the B2 phylogenetic group. The toxin encoding genes hlyA, cnf1 and usp were strongly co-associated. Further, we found a statistically significant co-association of ibeA and usp. The prevalence of the tested toxin encoding genes in E. coli strains from urinary tract infections isolated in Slovenia is comparable to those from studies in other geographic regions. uropathogenic Escherichia coli; UPEC; toxins; hlyA; cnf1; ibeA; usp genes Izvleček Izhodišča Bakterija Escherichia coli (E. coli) je enterobakterija, ki živi v spodnjem delu prebavila človeka in toplokrvnih živali. Kljub temu da je E. coli del normalne mikrobne flore, lahko povzroči okužbe. Pestrost obolenj, v katere je vpletena E. coli, je precejšnja, saj lahko povzroča drisko, vnetje sečnika in ledvic, pljučnico, vnetje možganskih ovojnic, okužbe ran in drugo. Patogeni sevi se razlikujejo od nepatogenih po tem, da imajo v svojem genomu genske zapise za virulentne dejavnike (toksine, adhezine, kapsule ...). Okužba sečil je ena izmed najpogostejših bakterijskih infekcij in E. coli povzroča večino teh okužb. Zaradi pogostnosti pojavljanja okužb v sečilih so virulentni dejavniki sevov E. coli, ki povzročajo te okužbe (UPEC – uropatogena E. coli), za preučevanje zelo zanimivi. Kar nekaj toksinov: alfa-hemolizin (HlyA), citotoksični nekrotizirajoči dejavnik (CNF1), invazin (IbeA) in uropatogeni specifični protein (USP), povezujejo s patogenostjo sevov UPEC. Zbrane informacije o pogostnosti zapisov za toksine bi lahko bile tudi osnova za diagnostiko okužb z UPEC sevi. Corresponding author / Avtorica za dopisovanje: Doc. dr. Marjanca Starčič Erjavec, univ. dipl. biol. in prof. biol., tel. +386 1 423 33 88; e-mail: marjanca.starcic.erjavec@bf.uni-lj.si 428 Zdrav Vestn 2008; 77 Metode Proučevali smo 110 uropatogenih sevov bakterije E. coli, ki smo jih na Inštitutu za mikro- biologijo in imunologijo Medicinske fakultete v Ljubljani osamili iz diagnostičnih vzorcev urina, z namenom določiti gene, ki imajo zapise za nekatere značilne bakterijske toksine: hlyA (hemolizin), cnf1 (citotoksični nekrotizirajoči faktor 1), ibeA (invazin) in usp (uropatogeni specifični protein USP). Uporabili smo metodo verižne reakcije s polimerazo (PCR) z začetnimi oligonukleotidi, specifičnimi za posamezne proučevane genske zapise za toksine. Zanesljivost uporabljene metode PCR smo preverili z metodo hibridizacije točkovnega odtisa. Rezultati Pri 44 % sevov smo dokazali gen usp, prevalenca gena hlyA je bila 25 % in gena cnf1 23 %. Samo pri 9 % sevov smo dokazali gen ibeA za invazin. Večino najdenih genskih zapisov za toksine smo našli v sevih filogenetske skupine B2. Medsebojna povezanost pojavljanja genskih zapisov za toksine HlyA, CNF1 in USP je bila statistično značilna. Pojavljanje genskega zapisa za ibeA je bil statistično značilno povezano samo s pojavljanjem genskega zapisa za usp. Zaključki Dobljeni rezultati kažejo, da so proučevani genski zapisi za toksine pri preiskovanih UPEC sevih prisotni v podobni meri kot pri podobnih tujih raziskavah. Ker nobena izmed že objavljenih raziskav ni vključevala vseh štirih v tej raziskavi proučevanih genskih zapisov, je ta raziskava prva, ki je pokazala statistično značilno povezanost ibeA in usp. Ključne besede uropatogena Escherichia coli; UPEC; toksini; geni hlyA; cnf1; ibeA; usp Introduction Urinary tract infections (UTIs) are one of the most frequent bacterial infections especially in women. Up to one third of women will suffer from an episode of UTI during their lifetime.1 UTI is diagnosed by either clinical observation or isolation of the causative microorganism from pathologic urine.2 In Slovenia uropathogenic Escherichia coli (E. coli) strains (UPEC) cause approximately 80 % of uncomplicated urinary tract infections.3 The ability of uropathogenic strains to cause diseases is due to possession of virulence factors: adhesins, toxins, polysaccharide coatings, invasins and iron uptake systems.4 It is essential to link a pathogen’s virulence factors to clinical manifestations, especially recurrent UTIs. In pathogenic E. coli strains several important toxins have been identified. The best known are alpha hemolysin (HlyA) and cytotoxic necrotizing factor 1 (CNF1), which have been associated with UPEC strains.5 Well known toxins are also invasins, the Ibe proteins that help E. coli strains to invade the human brain microvascular endothelial cells.6 The presence of IbeA protein is statistically significantly higher in strains causing cystitis and/or pyelonephritis.7 The gene for the uropathogenic specific protein (USP) that was found as a homologue of the Vibrio cholerae zonula occludens toxin encoding gene,8 has been significantly more often detected in UPEC strains than in fecal strains from healthy individuals.9 To diminish the burden of UPEC, using effective preventive measures, data on virulence factor prevalence in different geographic regions must be assessed. Since bacterial toxins are a potentially good target for a vaccine a number of studies analysing the prevalence of virulence factors, including toxins, in different UPEC have been performed.10–12 As no such data was available for the geographic region of Slovenia, the aim of the present study was to analyse the prevalence of toxin encoding genes in 110 E. coli strains causing UTI in Ljubljana, Slovenia. Material and methods Bacterial strains A total of 110 E. coli isolates (designated DL 1 to DL 110) from diagnostic urine samples of people with urinary tract infections treated in 2002, at the Institute of Microbiology and Immunology, Medical Faculty, Ljubljana, were studied. Only one isolate from each patient was analysed. Ninety-four (86 %) of the patients were women. The serotype, phylogenetic groups and traits typical of horizontal gene transfer (traT, integrons, rep) were determined previously.13 Detection of toxin encoding genes The primers and PCR conditions used to amplify genes encoding toxins with polymerase chain reaction (PCR) are listed in Table 1. Lysates for PCR were prepared by boiling, according to Le Bouguenec et al.,17 of overnight cultures grown in liquid Luria Bertani (LB) medium. Amplification was performed in an automated thermal cycler (UNOII, Biometra, Göttingen, Germany) in a 50 µl reaction mixture containing template DNA (10 µl of boiled lysate), 20 pmol of forward and reverse primer, 0.2 mM of dNTP mixture, 1.25 U Taq DNA polymerase and 2.5 mM MgCl2 in 1× PCR buffer (Fermentas, Vilnius, Lithuania). Dot blot hybridisation experiments using the »DIG DNA Labelling and Detection kit« (Roche, Mannheim, Germany) were performed to validate the PCR assays. Probes were prepared using the same primers as for the PCR experiments and labelled with digoxigenin. The template DNA samples were the same as in the PCR experiments. Starčič Erjavec M et al. Prevalence of toxin encoding genes in E. coli isolates from urinary tract infections in Slovenia 429 Table 1. Oligonucleotide primers and PCR conditions to detect toxin encoding genes. Razpr. 1. Oligonukleotidni začetniki in pogoji PCR za ugotavljanje genskih zapisov za toksine. Gene Oligonucleotide sequence (5’ to 3’) Size of product (bp) PCR conditions Reference Gen Oligonukleotidno zaporedje (od 5’ do 3’) Velikost produkta (bp) Pogoji PCR Referenca hlyA aacaaggataagcactgttctggct accatataagcggtcattcccgtca 1177 95 °C 2.5 min 1× 14 94 °C 0.5 min 64 °C 0.5 min 30 × 72 °C 1.5 min 72 °C 7 min 1 × cnf1 ctgacttgccgtggtttagtcgg tacactattgacatgctgcccgga 1295 94 °C 4 min 1 × 15 94 °C 1.5 min 59 °C 1.5 min 30 × 72 °C 2 min 72 °C 5 min 1 × ibeA aggcaggtgtgcgccgcgtac tggtgctccggcaaaccatgc 170 94 °C 2.5 min 1× 11 94 °C 0.5 min 63 °C 0.5 min 25 × 72 °C 3 min 72 °C 10 min 1 × usp atgctactgtttccgggtagtgtgt catcatgtagtcggggcgtaacaat 1000, 2500, 3000 95 °C 2.5 min 1 × 16 94 °C 0.5 min 68 °C 1 min 30 × 72 °C 1 min 72 °C 7 min 1 × Statistical analysis The significance of the results was established using the Fisher’s exact test (2-tailed) available on-line on the web site http://www.matforsk.no/ola/fisher.htm and the level of significance was set at a P value < 0.05. To reveal the degree of association the Pearson’s correlation coefficient was calculated on-line on the web site http://www.quantativeskills.com/sisa/ statistics/twoby2.htm. Interpretation of the correlation coefficient was as follows: 0.1 to 0.29 – small correlation, 0.3 to 0.49 – medium correlation and 0.5 to 1 – large correlation. Results Prevalence of toxin encoding genes The presence of toxin encoding genes in the genomes of DL strains was screened by PCR and validated in the hybridization experiments. Figure 1 gives an example for ibeA detection. Among the screened toxin encoding genes the usp gene had the highest prevalence as usp specific nucleotide sequences were detected in 48 strains (44 %). The prevalence of hlyA and cnf1 was similar, 28 (25 %) and 25 strains (23 %), respectively, possessed the tested nucleotide sequences. Only 10 strains (9 %) harbored ibeA sequences (Figure 2). Distribution of toxin encoding genes among phylogenetic groups E. coli isolates can be divided into four main phylo-genetic groups A, B1, B2 and D.18 Analysis of the distribution of toxin encoding genes among the previously determined phylogenetic groups of studied strains13 revealed that the tested toxin encoding genes hlyA, cnf1, ibeA and usp are mostly harbored by UPEC strains belonging to the B2 phylogenetic group (Table 2). Table 2. Distribution of toxin encoding genes among E. coli phylogenetic groups. The prevalence of toxin encoding genes among phy-logenetic groups is given as the total number N and % of found toxin encoding gene sequences in different phylogenetic groups. Razpr. 2. Razporeditev genskih zapisov za toksine po filogenetskih skupinah E. coli. Prevalenca genskih zapisov za toksine v posameznih filogenetskih skupinah je podana kot število N in % najdenih genskih zapisov za toksine v različnih filo-genetskih skupinah. Toxin encoding Prevalence N (%) in phylogenetic group gene Prevalenca N (%) v filogenetski skupini Total Genski zapis A B1 B2 D Skupaj za toksin (N = 28) (N = 6) (N = 55) (N = 21) (N = 110) hlyA cnf1 ibeA usp 1 (4) 0 0 1 (4) 26 (47) 25 (45) 9 (16) 42 (76) 1 (5) 28 (25) 0 25 (23) 1 (5) 10 (9) 5 (24) 48 (44) Co-associations of toxin encoding genes The toxin encoding genes hlyA, cnf1 and usp were strongly co-associated (Tab. 3). These three genes often appeared concomitantly (19 of hlyA positive strains (68 %) possessed also cnf1 and usp gene sequences). The ibeA gene was associated only with the usp gene, albeit this correlation was small (Pearson’s correlation coefficient r = 0.296), it was statistically significant (P = 0.002) (Table 3). 430 Zdrav Vestn 2008; 77 Figure 1. An example of detection of toxin encoding genes – detection of the ibeA gene. (A) Visualization of PCR products obtained in PCR reactions on lysates of DL strains with primers specific for the ibeA gene (1 % agarose gel, stained with ethidium bromide). M: marker –1 kb DNA ladder (Fermentas, Vilnius, Lithuania); 1: strain DL 3 (ibeA-); 2: strain DL 9 (ibeA-); 3: strain DL 23 (ibeA-); 4: strain DL 29 (ibeA+); 5: strain DL 30 (ibeA-); 6: strain DL 49 (ibeA-); 7: strain DL 54 (ibeA+); 8: strain DL 88 (ibeA+); 9: strain DL 89 (ibeA+); 10: strain DL 90 (ibeA-); 11: laboratory strain DH5? (ibeA-) and 12: negative control – a PCR reaction with sterile water instead of a lysate. (B) Validation of the PCR assay with DIG hybridization of a ibeA specific probe on ibeA PCR products (10 µl) bound to a nylone membrane. 1: strain DL 3 (ibeA-); 2: strain DL 9 (ibeA-); 3: strain DL 23 (ibeA-); 4: strain DL 29 (ibeA+); 5: strain DL 30 (ibeA-); 6: strain DL 49 (ibeA-); 7: strain DL 54 (ibeA+); 8: strain DL 88 (ibeA+); 9: strain DL 89 (ibeA+); 10: strain DL 90 (ibeA-). Sl. 1. Primer detekcije genskega zapisa za toksin – de-tekcija gena ibeA. (A) Prikaz pridelkov PCR, dobljenih v reakcijah PCR na lizatih sevov DL z začetnimi oligonukleotidi specifičnimi za gen ibeA (1 % agarozni gel, obarvan z eti-dijevim bromidom). M: standard – 1 kb DNK-lestvica (Fermentas, Vilnius, Litva); 1: sev DL 3 (ibeA-); 2: sev DL 9 (ibeA-); 3: sev DL 23 (ibeA-); 4: sev DL 29 (ibeA+); 5: sev DL 30 (ibeA-); 6: sev DL 49 (ibeA-); 7: sev DL 54 (ibeA+); 8: sev DL 88 (ibeA+); 9: sev DL 89 (ibeA+); 10: sev DL 90 (ibeA-); 11: laboratorijski sev DH5? (ibeA-) in 12: negativna kontrola – reakcija PCR s sterilno vodo, namesto li-zata. (B) Preverjanje PCR z DIG-hibridizacijo z vezavo sonde specifične za ibeA na produkte ibeA iz PCR (10 µl), vezane na najlonski membrani. 1: sev DL 3 (ibeA-); 2: sev DL 9 (ibeA-); 3: sev DL 23 (ibeA-); 4: sev DL 29 (ibeA+); 5: sev DL 30 (ibeA-); 6: sev DL 49 (ibeA-); 7: sev DL 54 (ibeA+); 8: sev DL 88 (ibeA+); 9: sev DL 89 (ibeA+); 10: sev DL 90 (ibeA-). 44% 25% 23% 9% r 1 1 1 1 hlyA enfi usp Figure 2. Prevalence (in % of the total 110 studied E. coli strains) of tested toxin encoding gene sequences. Sl. 2. Prevalenca (v % od 110 preučevanih sevov E. coli) genskih zapisov za toksine. Table 3. Co-association of tested toxin encoding genes. Numbers below the diagonal are the r value of Pearson’s correlation coefficient. Numbers above the diagonal are P values of Fisher’s exact test. Razpr. 3. Vezanost genskih zapisov za toksine. Števila pod diagonalo so vrednosti r Pearsonovega korelacijskega koeficienta. Števila nad diagonalo so vrednosti P Fisherjevega eksaktnega testa. Toxin encoding gene Genski zapis za toksin hlyA cnf1 ibeA usp hlyA cnf1 ibeA ups <0.001 0.729 0.040 0.021 0.496 0.529 NS/NZ NS/NZ 0.296 <0.001 <0.001 0.002 NS/NZ = not significant / ni značilno Discussion The repertoire of virulence genes present in a certain strain determines the severity of disease.4 Toxins are well established virulence factors that are often responsible for the major symptoms of a bacterial infection. In the presented study 110 UTI E. coli strains were characterized using PCR with primers specific for four toxin encoding genes: hlyA, cnf1, ibeA and usp. Analysis of the prevalence of hlyA, cnf1, usp and ibeA sequences among strains from our study (25, 23, 44, and 9 %, respectively) compared to studies of uro-pathogenic E. coli isolates from other geographic regions (Tab. 4) showed a comparable prevalence of ibeA Starčič Erjavec M et al. Prevalence of toxin encoding genes in E. coli isolates from urinary tract infections in Slovenia 431 Table 4. Comparison of results from different studies of UTI toxin encoding genes. Razpr. 4. Primerjava rezultatov različnih raziskav genskih zapisov za toksine pri okužbi sečil. Toxin encoding gene prevalence (%) Prevalenca genskih zapisov za toksine (%) Study Raziskava hlyA/D cnf1 ibeA usp Reference Referenca 76 pyelonephritis strains (Japan) 36 93 19, 20 76 pielonefritičnih sevov (Japonska) 74 cystitis strains (USA) 74 cistitičnih sevov (ZDA) 36 34 24 N 21 170 pyelonephritis strains (USA) 39 170 pielonefritičnih sevov (ZDA) 194 cystitis strains (Japan) 194 cistitičnih sevov (Japonska) 41 48 N 79 19, 20 100 cystitis strains (Israel) 34 100 cistitičnih sevov (Izrael) 78 UTI strains (Romania) 23 13 23 78 sevov iz okužb sečil (Romunija) 243 UTI strains (Italy) 243 sevov iz okužb sečil (Italija) 21 19 N N 24 110 UTI strains (Slovenia) 25 23 9 44 this study 110 sevov iz okužb sečil (Slovenija) ta raziskava N = not available / ni navedeno toxin encoding genes. However, it has to be noted that among E. coli collections of strains causing either cystitis19–22 or pyelonephritis7, 19, 20 a higher prevalence of toxin encoding genes can be found than among collections of mixed UTI E. coli isolates.23, 24, this study The tested toxin encoding genes were mostly found in strains belonging to the B2 phylogenetic group (Table 2). Picard et al.25 established the link between phylogeny and extraintestinal virulence in the E. coli species. They showed that the strains of the B2 group represent a divergent lineage of highly virulent strains that possess the highest level of virulence determinants. Therefore the observed distribution of tested toxin encoding genes in our study is not surprising. Uropathogenic strains are known to carry large chromosomal regions, pathogenicity islands (PAI), encoding several virulence factors required for virulence. A number of PAIs have been defined in the archetypal uropathogenic strains 536, J96 and CFT073.4 The high associations found between hlyA, cnf1 and usp that are known to be carried on PAIs4, 8 are therefore expected. When Marrs et al. were trying to define pathotypes of E. coli strains causing UTI, similar to pathotypes defined for E. coli isolates causing enteric/diar-rheal diseases, they also found a strong association between cnf1 and hlyA and these two genes were used as key genes to determine the two most virulent UTI pathotypes, pathotype 1 (E. coli isolates with HlyA, CNF1, class III P-fimbriae) and pathotype 2 (E. coli isolates with HlyA, CNF1, S-fimbriae).26 Further, a statistically significant co-association of ibeA and usp was ascertained. To our knowledge in no previously published study all four toxin coding genes were screened for, so this is the first report of a statistically significant co-association of these two genes. It should be noted, that some of UTIs have an organic cause, for example an obstruction of urinary tract in the form of urinary stones, or a tumor, which predisposes a patient to an infection. Further, the pre- sentation of the infection is sometimes misapprehended as urinary tract irritation like urinary stone passage, overactive bladder or prostatism.2 Therefore, further studies of the prevalence of toxin and other virulence factors encoding genes among UPEC strains isolated from patients with different clinical pictures would be interesting, especially as these results could build the basis for diagnostics of UPEC strains. In summary, we have found among the UTI E. coli strains isolated in Slovenia a prevalence of common virulence toxin encoding genes comparable to those obtained in studies in different geographic regions. Acknowledgements The authors are thankful to Ms. Darja Lončar and Mr. Matija Rijavec for their help in performing some preliminary testing of the UPEC strains, to prof. dr. Andrej Blejec for his help with statistical analysis and to prof. dr. Marija Gubina for fruitful discussion and her help in preparing the manuscript. This research was supported by Grant PO-0508-0487 of the Ministry of Education, Science and Technology, Slovenia and by a personal donation from Farmadent, d.o.o., Maribor, Slovenia. References 1. Griebling TL. Urologic diseases in America project: trends in resource use for urinary tract infections in women. J Urol 2005; 173: 1281–7. 2. Weiss RM, George NJR, O’Reilly PO. Urinary tract infections. Comprehensive Urology 2001; 2: 295–312. 3. Lindič J. Pristop k bolniku z okužbo sečil. Accession to a patient with urinary tract infection]. In: Fras Z, Poredoš P, eds. 47. Tavčarjevi dnevi [47th Tavčar’s days]. Zbornik prispevkov [Proceedings]. 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