ACTA BIOLOGICA SLOVENICA LJUBLJANA 2009 Vol. 52, [t. 2: 73–83 Pathogenicity islands, plasmids and iron uptake systems in extraintestinal pathogenic Escherichia coli strains Otoki patogenosti, plazmidi in sistemi za privzem železa v zunajčrevesnih patogenih sevih bakterije Escherichia coli Marjanca Starčič ErjavEc, Tanja arbitEr, Darja Žgur bErtok Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana; E-mail: marjanca.starcic.erjavec@bf.uni-lj.si, tanja.arbiter@gmail.com, darja.zgur@bf.uni-lj.si Abstract: The aim of the presented study was to estimate the prevalence, distribution and associations of different pathogenicity islands (PAI I536 to PAI IV536, PAI IJ96, PAI IIJ96, PAI ICFT073 and PAI IICFT073), iron uptake systems (genes iutA, iucD, iroN, iroCD, fyuA, irp2, iha, ireA, and hbp) and plasmids among extraintestinal pathogenic Escherichia coli (ExPEC) strains isolated from Slovenian patients. Twenty-nine ExPEC isolates obtained from the Institute of Microbiology and Immunology, Medical Faculty, University of Ljubljana were investigated for the presence of different pathogenicity islands and iron uptake systems with PCR, the plasmid content of the investigated strains was determined by molecular biology techniques. The significance of the found associations of the studied PAIs and iron uptake systems was analyzed with the Fisher’s exact test. PAI IV536 was found in 19, PAI II CFT073 in 6, PAI ICFT073 in 4, and PAI IIJ96 in one of the studied isolates. PAI I536, PAI II536, PAI III536 and PAI IJ96 were not detected in any studied isolate. In 19 of the studied isolates plasmids were detected. The irp2 was found in 20, fyuA in 19, iucD and iutA in 12, iha in 9, iroN in 8, iroCD in 7, ireA in 7 and hbp in 4 of studied isolates. PAI IV536 was statistically significantly associated with the yersiniabactin siderophore system and PAI ICFT073 was statistically significantly associated with the aerobactin siderophore system as well as Iha. To our knowledge this is the first report on PAIs and iron uptake systems among Slovenian ExPEC isolates, as well as a first report on PAIs, iron uptake systems and plasmids among isolates from skin and soft tissue infections. Key words: extraintestinal pathogenic Escherichia coli, ExPEC, pathogenicity island, PAI, plasmid, iron uptake Izvleček: Cilj raziskave je bil oceniti prevalenco, razporeditev in asociacije različnih oto- kov patogenosti (PAI I536 do PAI IV536, PAI IJ96, PAI IIJ96, PAI ICFT073 in PAI IICFT073), sistemov za privzem železa (geni iutA, iucD, iroN, iroCD, fyuA, irp2, iha, ireA, in hbp) in plazmidov v zunaj- črevesnih patogenih sevih bakterije Escherichia coli (ExPEC) izoliranih iz slovenskih bolnikov. Devetindvajset izolatov ExPEC, ki so jih osamili na Inštitutu za mikrobiologijo in imunologijo Medicinske fakultete v Ljubljani, smo s pomočjo PCR preiskali za prisotnost različnih otokov patogenosti in sistemov za privzem železa, z molekulskobiološkimi tehnikami smo preverjali prisotnost plazmidov v preučevanih sevih. Statistično značilnost povezave preučevanih PAI in sistemov za privzem železa smo ugotavljali s Fisherjevim eksaktnim testom. PAI IV536 smo našli v 19, PAI II CFT073 v 6, PAI ICFT073 v 4 , PAI IIJ96 v enem od preučevanih izolatov. PAI I536, PAI II536, PAI III536 in PAI IJ96 nismo odkrili v nobenem izmed preučevanih izolatov. V 19 sevih smo odkrili plazmide. Gen irp2 smo našli v 20, fyuA v 19, iucD in iutA v 12, iha v 9, iroN v 8, iroCD v 7, ireA v 7 in hbp v 4 od preučevanih sevov. PAI IV536 je bil statistično značilno povezan s siderofornim sistemom jersiniabaktin in PAI ICFT073 je bil statistično značilno povezan s siderofornim sistemom aerobaktin in Iha. Kolikor nam je poznano, je to prva raziskava o PAI in sistemih za privzem železa na zbirki sevov ExPEC iz slovenskih bolnikov, in ki podaja podatke o PAI, sistemih za privzem železa in plazmidih izolatov ExPEC iz infekcij kože in podkožja. 74 Acta Biologica Slovenica, 52 (2), 2009 Ključne besede: Zunajčrevesna patogena Escherichia coli, ExPEC, otoki patogenosti, PAI, plazmid, sistemi za privzem železa Introduction Escherichia coli (E. coli) is the most abundant facultative anaerobe of the human intestinal micro- flora. Despite the fact that it is a commensal bac- terium, some E. coli strains have acquired specific virulence attributes that confer an increased ability to adapt to new niches and allow them to cause a broad spectrum of disease at either intestinal or extraintestinal sites (kapEr & al. 2004). Often the virulence attributes are genetically linked – located in a subgroup of genomic islands, in the so called pathogenicity islands (PAI) (Schmidt & hEnSEl 2004). Typical characteristics of PAIs (Tab. 1), apart from encoding virulence genes, are: presence in pathogenic strains but absence or rareness in nonpathogenic strains of the same or related species; size ranging from 10 kb up to 200 kb; relative instability, different G+C content than the core genome, association with tRNA genes, presence of mobile genetic elements (insertion sequences, transposons, integrases, and bacteri- ophage DNA), flanked by direct repeat sequences and due to different episodes of horizontal gene transfer a mosaic-like structure (hackEr & kapEr 2000, Schmidt & hEnSEl 2004). In addition to PAIs, plasmids, extrachromo- somal DNA elements that range in size from approximately 300 bp to 2400 kbp, can carry genes encoding virulence factors (kado 1998). Based on the overall genetic content, two types of plasmids are distinguished. One, designated as non-conjugative or non-transmissible, har- bour genes for the initiation and regulation of its replication but do not possess genes required for conjugal transfer. The second type are conjugative or self-transmissible that also carry genes involved in conjugation (hElinSki & al. 1996). Iron is an essential cofactor for many basic metabolic pathways and bacteria have developed specialized iron uptake systems to capture iron. The most prominent are the siderophores, iron- binding molecules that are taken up by special siderophore receptors and ATP-consuming porin- Table 1: Characteristics of studied PAIs Tabela 1: Značilnosti preučevanih PAI PAI name Size (kbp) Insertion position Identified carried virulence (associated) factors Ref. PAI I536 76,8 selC alpha-hemolysin, F17-like fimbriae, and CS12-like fimbriae (Schmidt & hEnSEl 2004) PAI II536 102,2 leuX P-related fimbriae, alpha-hemolysin, Hek adhesin, hemagglutinin-like adhesins (Schmidt & hEnSEl 2004) PAI III536 68,1 thrW S-fimbriae, iro siderophore system, a HmuR-like heme receptor, a Sap adhesin, a TSH-like hemoglobin protease (Schmidt & hEnSEl 2004) PAI IV536 = HPI 30,2 asnT yersiniabactin siderophore system (Schmidt & hEnSEl 2004) PAI IJ96 > 170 pheV alpha-hemolysin, P- fimbriae (Schmidt & hEnSEl 2004) PAI IIJ96 110 pheU alpha-hemolysin, Prs- fimbriae, cytotoxic necrotizing factor I (Schmidt & hEnSEl 2004) PAI ICFT073 = PAI- CFT073-pheV 123 pheV alpha-hemolysin, P-fimbriae, Iha, autotransporter Sat, aerobactin siderophore system, antigen 43 precursor, capsule gene kpsTM (lloyd & al. 2007) PAI IICFT073 = PAI- CFT073-pheU 52 pheU P-fimbriae (lloyd & al. 2007) 75M. Starčič Erjavec, T. Arbiter, D. Žgur Bertok: Pathogenicity islands, plasmids and iron uptake systems … like transporters in the bacterial outer membrane (SchaiblE & kaufmann 2004). Siderophores can be classified into three groups: (i) the catecholate type (enterobactin, salmochelin = enterochelin), (ii) hydroxamate type (aerobactin) and (iii) a mixed type – a combination of both (yersiniabac- tin) (graSS 2006, SchaiblE & kaufmann 2004). In addition to siderophore synthesis strains can use siderophores produced and released into the extracellular medium by other bacteria and even fungi. In the host, bacteria may use iron sources such as heme, hemoglobin, hemopexin, and iron bound to transferrin and lactoferrin (braun & braun 2002). Apart from the siderophores and their receptors, autotransporters, virulence-asso- ciated proteins in gram-negative bacteria, can also play a role in obtaining iron for example, the hemoglobin protease Hbp (otto & al. 2002). All autotransporter proteins are energy-independent secreted via a type 5 secretion system and possess an overall unifying structure, comprising (i) an amino-terminal leader peptide (for secretion across the inner membrane), (ii) the secreted mature pro- tein (or passenger domain), and (iii) a dedicated C-terminal domain, which forms a pore in the outer membrane through which the passenger domain passes to the cell surface (hEndErSon & nataro 2001). Hbp, after it is autotransported out of the bacterial cell, interacts specifically with human hemoglobin, degrades it, and subsequently binds the released heme (otto & al. 1998). E. coli isolates capable of causing disease outside the intestinal tract, e. g., uropathogenic E. coli (UPEC), sepsis-associated E. coli, and neonatal meningitis-associated E. coli, are classified as extraintestinal pathogenic E. coli (ExPEC) (ruSSo & al. 2000). Within the human intestinal tract, ExPEC may colonize without causing disease, but when they disseminate to other body sites, they elicit, due to encoded viru- lence factors, pathogenesis (WilES & al. 2008). In ExPEC isolates many pathogenicity islands were found, among the best known and studied are pathogenicity islands PAI I536 to PAI IV536 from the uropathogenic E. coli strain 536, PAI IJ96 and PAI IIJ96 from the uropathogenic E. coli strain J96 and PAI ICFT073 and PAI IICFT073 from uropathogenic E. coli strain CFT073 (Schmidt & hEnSEl 2004). Further, among ExPEC strains many iron uptake systems were found, characteriz ed and associ- ated with pathogenesis, among them aerobactin, salmochelin, yersiniabactin, Iha, IreA and Hbp. The aim of the present study was to characterize 29 ExPEC strains isolated from Slovenian patients suffering from extraintestinal E. coli infections for the presence of the best characterised PAIs and iron uptake systems. In addition, we aimed to estimate the prevalence of PAIs, iron uptake systems and plasmids, and to analyse the distribution and as- sociations of PAIs and iron uptake systems among ExPEC strains isolated in Slovenia. Material and methods Bacterial strains and media Twenty-nine randomly collected E. coli iso- lates from humans with extraintestinal infections isolated at the Institute of Microbiology and Im- munology, Medical Faculty, Ljubljana, Slovenia were studied. Only one isolate from each patient was analyzed. Nineteen isolates were from patients with a urinary tract infection, 3 isolates were from decubiti, 2 isolates were from wound infections, 2 isolates were from surgical wound infections, 2 isolates were from foot ulci and one isolate was from a genital tract infection. All patients were older than 14 years. For cultivation of strains Luria Bertani medium or agar were used. Detection of PAIs and iron uptake systems The primers and PCR conditions used to am- plify PAI markers; PAI I536, PAI II536, PAI III536, PAI IV536, PAI IJ96, PAI IIJ96, PAI ICFT073 and PAI IICFT073, and iron uptake genes; iutA – ferric aerobactin receptor gene, iucD – lysine: N6-hydroxylase gene (aerobactin biosynthesis), iroN – catecholate siderophore (ferric salmochelin, ferric 2,3-dihy- droxybenzoic acid, ferric 2,3-dihydroxybenzoyl- D-ornithine) receptor gene, iroCD – salmochelin ATP-binding cassette ABC transporter gene (iroC), ferric salmochelin esterase gene (iroD), fyuA – fer- ric yersiniabactin receptor gene, irp2 – yersini- abactin synthetase (yersiniabactin biosynthesis), iha – gene for a bifunctional protein: catecholate siderophore (ferric enterobactin, ferric 2,3-dihy- droxybenzoylserine) receptor and adhesin, ireA – putative TonB-dependent siderophore receptor, 76 Acta Biologica Slovenica, 52 (2), 2009 Target Oligonucleotide name and sequence (5’ to 3’) Size of product (kbp) PCR conditions Reference PAI I536 I.9 TAATGCCGGAGATTCATTGTC I.10 AGGATTTGTCTCAGGGCTTT 1,8 94°C 5 min 1× 94°C 1 min 56°C 1 min 30× 72°C 1 min 72°C 10 min 1× (SabatE & al. 2006) PAI II536 orfIup CATGTCCAAAGCTCGAGC orfIdown CTACGTCAGGCTGGCTTT 1,0 94°C 5 min 1× 94°C 1 min 62°C 1 min 30× 72°C 1 min 72°C 10 min 1× (SabatE & al. 2006) PAI III536 sfaAI1 CGGGCATGCATCAATTATCTTTG sfaAI2 TGTGTAGATGCAGTCACTCCG 0,2 94°C 5 min 1× 94°C 1 min 63°C 1 min 30× 72°C 1 min 72°C 10 min 1× (SabatE & al. 2006) PAI IV536 IRP2FP AAGGATTCGCTGTTACCGGAC IRP2RP TCGTCGGGCAGCGTTTCTTCT 0,3 94°C 5 min 1× 94°C 1 min 61°C 1 min 30× 72°C 1 min 72°C 10 min 1× (SabatE & al. 2006) PAI IJ96 papGIf TCGTGCTCAGGTCCGGAATTT papGIr TGGCATCCCACATTATCG 0,4 94°C 5 min 1× 94°C 0,5 min 57°C 0,5 min 30× 72°C 1 min 72°C 10 min 1× (SabatE & al. 2006) PAI IIJ96 Hlyd GGATCCATGAAAACATGGTTAATG cnf GATATTTTTGTTGCCATTGGTTACC 2,3 94°C 5 min 1× 94°C 1 min 61°C 1 min 30× 72°C 2,5 min 72°C 10 min 1× (SabatE & al. 2006) PAI ICFT073 RPAi GGACATCCTGTTACAGCGCGCA RPAf TCGCCACCAATCACAGCGAAC 0,93 94°C 5 min 1× 94°C 1 min 63°C 1 min 30× 72°C 1 min 72°C 10 min 1× (SabatE & al. 2006) PAI IICFT073 Cft073.2Ent1 ATGGATGTTGTATCGCGC Cft073.2Ent2 ACGAGCATGTGGATCTGC 0,4 94°C 5 min 1× 94°C 0,5 min 56°C 0,5 min 30× 72°C 1 min 72°C 10 min 1× (SabatE & al. 2006) iutA iutA f GGCTGGACATCATGGGAACTGG iutA r CGTCGGGAACGGGTAGAATCG 0,3 94°C 4 min 1× 94°C 1 min 68°C 1 min 35× 72°C 1 min 72°C 10 min 1× (johnSon & al. 1997) Table 2: Oligonucleotide primers and PCR conditions to detect PAIs and iron uptake systems Tabela 2: Oligonukleotidni začetniki in pogoji PCR za ugotavljanje PAI in genov za sisteme za privzem železa 77M. Starčič Erjavec, T. Arbiter, D. Žgur Bertok: Pathogenicity islands, plasmids and iron uptake systems … Target Oligonucleotide name and sequence (5’ to 3’) Size of product (kbp) PCR conditions Reference iucD Aer 1 TACCGGATTGTCATATGCAGACCGT Aer 2 AATATCTTCCTCCAGTCCGGAGAAG 0,6 94°C 4,5 min 1× 94°C 0,5 min 62°C 0,5 min 35× 72°C 50 sec 72°C 10 min 1× (yamamoto & al. 1995) iroN iroN f AAGTCAAAGCAGGGGTTGCCCG iroN r GACGCCGACATTAAGACGCAG 0,7 94°C 2,5 min 1× 94°C 0,5 min 68°C 0,5 min 25× 72°C 2 min 72°C 10 min 1× (johnSon & al. 2000) iroCD P52-A GGCTGAGAAATATCAACATCCG P52-B ATCGCACATCCGAAGAACGACT 1,0 94°C 2,5 min 1× 94°C 0,5 min 63°C 1 min 30× 72°C 1 min 72°C 10 min 1× This study fyuA fyuA 1 TGATTAACCCCGCGACGGGAA fyuA 2 CGCAGTAGGCACGATGTTGTA 0,8 94°C 2,5 min 1× 94°C 0,5 min 63°C 0,5 min 25× 72°C 3 min 72°C 10 min 1× (johnSon & StEll 2000, SchubErt & al. 1998) irp2 Irp2 f AAGGATTCGCTGTTACCGGAC Irp2 r TCGTCGGGCAGCGTTTCTTCT 0,3 94°C 5 min 1× 94°C 1 min 61°C 1 min 35× 72°C 1 min 72°C 8 min 1× (SchubErt & al. 1998) iha iha f CTGGCGGAGGCTCTGAGATCA iha r TCCTTAAGCTCCCGCGGCTGA 0,8 94°C 4 min 1× 94°C 0,5 min 58°C 0,5 min 30× 72°C 1 min 72°C 8 min 1× (johnSon & al. 2000) ireA ireA f TGGTCTTCAGCTATATGG ireA r ATCTATGATTGTGTTGGT 0,4 94°C 2,5 min 1× 94°C 0,5 min 55°C 1 min 25× 72°C 0,5 min 72°C 7 min 1× (ruSSo T. A. & al. 2001) hbp Hbp f GGTGAAGGTACGCTGACGGT Hbp r GCGTGACGCTGGAGTTATCT 0,9 94°C 4,5 min 1× 94°C 0,5 min 65°C 1 min 35× 72°C 1 min 72°C 10 min 1× This study 78 Acta Biologica Slovenica, 52 (2), 2009 and hbp – hemoglobin protease autotransporter gene with polymerase chain reaction (PCR) are listed in Tab. 2. DNA to be amplified was released from whole organisms by boiling according to lE bouguEnEc & al. (1992). Amplification was performed in an automated thermal cycler (UN- OII, 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). General molecular biology DNA techniques Plasmid isolation, DNA digestion and agarose gel electrophoresis were performed by standard methods (Sambrook & ruSSEll 2001). 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.langsrud.com/ fisher.htm and the level of significance was set at a P value < 0.05. Results Prevalence of PAIs, plasmids and iron uptake systems The PAI with the highest prevalence among the studied ExPEC isolates was PAI IV536, also designated HPI (high-pathogenicity island), which was found in 19 studied strains. PAI II CFT073 was found in 6 strains, PAI ICFT073 in 4 strains and PAI IIJ96 in only one isolate. The PAIs: PAI I536, PAI II536, PAI III536 and PAI IJ96 were not detected in any studied isolate. In 19 strains plasmids were detected, among them 9 harboured a plasmid larger than 30 kbp. The iron uptake system with the highest prevalence was yersiniabactin, the irp2 gene coding for the yersiniabactin synthetase was found in 20 strains and the fyuA gene coding for the ferric yersiniabactin receptor was found in 19 strains. The aerobactin iron uptake system genes iucD, coding for lysine: N6-hydroxylase needed in aerobactin biosynthesis, and iutA, encoding the ferric aerobactin receptor, were detected in 12 strains. The iha gene was found in 9 strains. The salmochelin uptake system genes iroN, coding for the catecholate siderophore receptor, and iroCD coding for proteins needed in salmochelin trans- port, were found in 8 and 7 isolates, respectively. The ireA gene was harboured by 7 studied strains and the hbp gene by 4 isolates. Distribution of PAIs and iron uptake systems among strains As seen from Tab. 3 most of the strains har- bouring PAI possessed one PAI (12 strains) and 7 strains harboured 2 or 3 PAIs. In 10 strains none of tested PAIs could be detected. The average PAI number per strain was 1.03. The majority of tested strains possessed 1 to 3 iron uptake systems (22 strains). In 4 strains none of the tested iron uptake systems could be detected. Three strains possessed four or five different iron uptake systems. The average iron uptake system number per strain was 1.97. Associations of PAIs and iron uptake systems Since many PAIs are known to carry iron uptake systems (see Tab. 1), associations of PAIs and iron uptake systems were analyzed. As seen from Tab. 4, only 3 statistically significant asso- ciations of PAIs with iron uptake systems could be determined: the yersiniabactin siderophore system was associated with PAI IV536, the aero- bactin siderophore system and Iha were associated with PAI ICFT073. Discussion Our findings showed that PAIs, plasmids and iron uptake systems are abundant, as the majority of the tested isolates harboured PAIs, plasmids and iron uptake systems. Since we analysed a relatively small number of isolates it is difficult to compare our results with results obtained by other authors on larger collec- tions of strains, such as the study of SABATE & al. (2006) on the prevalence of PAIs among 100 UPEC strains and 50 commensal strains and the 79M. Starčič Erjavec, T. Arbiter, D. Žgur Bertok: Pathogenicity islands, plasmids and iron uptake systems … Ta bl e 3: PA Is , i ro n up ta ke sy st em s a nd p la sm id s i n st ud ie d st ra in s Ta be la 3 : P A I, si st em i z a pr iv ze m ž el ez a in p la zm id i v p re uč ev an ih se vi h St ra in Pa tie nt ’s di ag no si sa Pa tie nt ’s ge nd er b PA I Ir on u pt ak e sy st em Pl as m id (b p) I 5 36 II 53 6 II I 5 36 IV 53 6 I J 96 II J9 6 I C FT 07 3 II C FT 07 3 iu tA iu cD ir oN ir oC D fy uA ir p2 ih a ir eA hb p D L2 U TI F – – – + – – – + – – + + + + – + – >3 0. 00 0 D L6 U TI M – – – – – – – – – – – – – – – – – – D L7 U TI F – – – – – – – – + + – – – – – – – <3 0. 00 0 D L8 U TI F – – – – – – – – – – – – – – + – – – D L1 4 U TI F – – – + – – – – + + + + + + – + + >3 0. 00 0 D L1 7 U TI F – – – + – – – – + – – – + + – – – <3 0. 00 0 D L2 2 U TI M – – – + – – – – – – – – + + – – + <3 0. 00 0 D L3 7 U TI F – – – – – – – – – – + + – – – – – <3 0. 00 0 D L4 1 U TI F – – – + – – – – – – – – + + – – – – D L4 3 U TI F – – – + – – – – – – – – + + – + – – D L4 6 U TI F – – – + – – – – + + – – + + + – – <3 0. 00 0 D L4 8 U TI F – – – – – – – – – – – – – – – – – – D L5 6 U TI F – – – + – – – – + + – – + + + + – >3 0. 00 0 D L7 6 U TI F – – – + – – – – – – – – + + + – – >3 0. 00 0 D L8 1 U TI F – – – + – – – – – – – – + + – – – <3 0. 00 0 D L8 4 U TI F – – – – – – – – – – – – – + + + – >3 0. 00 0 D L1 08 U TI F – – – + – – – + – – + + + + – + – <3 0. 00 0 D L1 09 U TI F – – – + – – – – + + – – + + – – + >3 0. 00 0 D L1 10 U TI F – – – + – + – – + + + + + + – + – >3 0. 00 0 TA 10 W I F – – – + – – – – – + + – + + – – + – TA 49 W I F – – – + – – + + + + – – + + + – – – TA 50 D M – – – + – – + + + + – – + + + – – >3 0. 00 0 TA 71 SW I F – – – + – – + + + + – – + + + – – – TA 74 G TI M – – – + – – – – – – – – + + – – – – TA 10 3 SW I M – – – – – – – – – – – – – – – – – >3 0. 00 0 TA 16 0 SU M – – – – – – – – – – – – – – – – – <3 0. 00 0 TA 17 1 D F – – – – – – – – + + + + – – – – – <3 0. 00 0 TA 17 4 U C M – – – – – – – – – – + + – – – – – – TA 21 2 D M – – – + – – + + + + – – + + + – – <3 0. 00 0 a U TI – u rin ar y tra ct in fe ct io n, W I – w ou nd in fe ct io n; D – d ec ub itu s; S W I – su rg ic al w ou nd in fe ct io n; S U – sk in u lc us ; U C – u lc us c ru ris b F – fe m al e; M – m al e 80 Acta Biologica Slovenica, 52 (2), 2009 Table 4: Associations of PAIs with iron uptake systems Tabela 4: Povezave PAI in sistemov za privzem železa PAI (no. of strains) Iron uptake system IV536 +(19) IV536 – (10) IIJ96 +(1) IIJ96 –(28) ICFT073 +(4) ICFT073 –(25) IICFT073 +(6) IICFT073 –(23) Aer 9 2 1 10 4 7* 4 7 Sal 4 3 1 6 0 7 2 5 Yer 19 0*** 1 18 4 15 6 13 Iha 7 2 0 9 4 5* 4 5 IreA 6 1 1 6 0 7 2 5 Hbp 4 0 0 4 0 4 0 4 P values (Fisher’s exact test) of statistically significant associations (P<0.05) are indicated by asterisks. Symbols: *, P < 0.05; **, P < 0.005; ***P < 0.0005. Aer, aerobactin siderophore system; Sal, salmochelin siderophore system; Yer, yersiniabactin siderophore system. Statično značilna vrednost P (Fisherjev eksaktni test) < 0,05 je nakazana z zvezdicami: simboli *, P < 0,05; **, P ≤ 0,01; ***, P ≤ 0,001. Aer, aerobaktinski sideroforni sistem; Sal, salmohelinski sideroforni sistem; Yer, jersiniabaktinski sideroforni sistem. study of JOHNSON & al. (2005) on the prevalence of several virulence factors, including iha, fyuA and iutA, among 83 cystitis and 170 pyelonephritis E. coli. A further difficulty for comparison of our results with others’ is the fact that differences might also be due to geographical differences. Differences in virulence factor profiles between distinct populations have previously been reported among cat populations from distant locations on feline uropathogenic E. coli strains from the United Kingdom and feline uropathogenic E. coli strains from New Zealand (frEitag & al 2005). However, it is interesting that among all our studied strains none carried PAI I536, PAI II536, or PAI III536 and that among the UTI strains included in this study no strain carrying PAI ICFT073 could be detected, while SabatE & al. (2006) reported detection of all PAIs except PAI IJ96. The overall lower prevalence in our study could be due to the fact, that our study incorporated only 29 strains. In our study 19 ExPEC strains harboured at least one plasmid. Since it is commonly known that plasmids are abundant in all bacterial species, the obtained prevalence is of no surprise. It is also not unexpected, that 9 strains in our study har- boured large plasmids (>30 kbp), since virulence factors enabling a strain to cause pathogenicity as well as antibiotic resistances can be encoded on large plasmids. The prevalence of iron uptake system in our study is comparable to data presented in studies on cystitis E. coli strains (johnSon & al. 2005, kanamaru & al. 2003). However, uroseptic and pyelonephritic E. coli isolates have a higher prevalence of iron uptake systems (johnSon & StEll 2000), (johnSon & al. 2005), also this is not unexpected since uroseptic and pyelonephritic strains are in general more virulent and possess more virulence factors. It is known that iron uptake systems can be encoded either chromosomally, sometimes as a part of a genomic island, or on plasmids. The yersiniabactin siderophore system is known to be carried on the HPI (=PAI IV536) (Schmidt & hEnSEl 2004) and in our study the association of PAI IV536 with this siderophore system was highly statistically significant, all 19 strains encoding the yersiniabactin siderophore system also harboured PAI IV536. The aerobactin siderophore system is known to be part of the PAI ICFT073 (Schmidt & hEnSEl 2004), but it was also found to be carried on a plasmid (carbonEtti & WilliamS 1984). In our study all 4 strains that possessed the PAI ICFT073 also harboured the aerobactin siderophore genes, and their association proved to be statistically significant. However, in 7 strains that encoded the aerobactin siderophore system, PAI ICFT073 could not be detected, but all 7 strains harboured plasmids large enough to carry the aerobactin siderophore 81M. Starčič Erjavec, T. Arbiter, D. Žgur Bertok: Pathogenicity islands, plasmids and iron uptake systems … system therefore, we could assume that the aero- bactin siderophore system is more often encoded on plasmids than chromosomally. The same was found for the salmochelin siderophore system. This siderophore system can also be carried by a PAI, the PAI III536 (Schmidt & hEnSEl 2004), but also on a plasmid (SorSa & al. 2003). In our study in 7 strains we detected the salmochelin siderophore system however, none of these strains harboured the PAI III536 and 6 strains carried plasmids large enough to encode the salmochelin siderophore system. The iha is known to be part of the PAI ICFT073 (Schmidt & hEnSEl 2004) and in our study 4 out of 9 strains possessing iha also possessed the PAI ICFT073 (the association was statistically significant). However, the other 5 iha encoding strains did not harbour the PAI ICFT073, in 4 of them plasmids large enough to encode iha were found. To our knowledge it has not yet been reported that iha could be plasmid encoded. The ireA and hbp were never associated with PAIs, ireA was found to be chromosomally encoded (ruSSo & al. 2001) and hbp was found to be plasmid encoded (otto & al. 1998). In 6 from 7 ireA-encoding strains and in 3 from 4 hbp-encoding strains plasmids were detected, so we might assume that both ireA as well as hbp could be either chromosomally encoded or carried by plasmids however, further studies to confirm the location of iha, ireA and hbp genes are needed. To our knowledge this is the first study of the prevalence of PAIs in a collection of ExPEC strains that included not only UPEC isolates, but also isolates from other extraintestinal infections. Thus, this is the first report of UPEC associated PAIs, PAI ICFT073 and PAI IICFT073, that were origi- nally found in UPEC strain CFT073 isolated from blood and urine of a woman with pyelonephritis (Schmidt & hEnSEl 2004), in strains isolated from skin and soft tissue infections. Further, to our knowledge this is the first study on molecular epidemiology including more than 4 iron uptake systems in ExPEC strains and it is worth to be emphasized that this is the first report on the as- sociation of iron uptake systems with PAIs and plasmids. However, since in this study only 29 ExPEC isolates were investigated, further stu- dies examining a large number of ExPEC strains should be performed. Conclusions To summarize and conclude: 1. 29 ExPEC strains were screened with PCR for the presence of well characterized PAIs and iron uptake systems as well as with mo- lecular biology techniques for the presence of plasmids; 2. the prevalence, the distribution and the genetic associations of the tested PAIs and iron uptake systems were determined; 3. PAI IV536 was found in 19, PAI IICFT073 in 6, PAI ICFT073 in 4, PAI IIJ96 in 1 of the studied isolates, while PAI I536, PAI II536, PAI III536 and PAI IJ96 were not detected in any studied isolate; 4. in 19 of the studied isolates plasmids were detected; 5. irp2 was found in 20, fyuA in 19, iucD and iutA in 12, iha in 9, iroN in 8, iroCD in 7, ireA in 7 and hbp in 4 of the studied isolates; 6. PAI IV536 was statistically significantly associ- ated with the yersiniabactin siderophore system and PAI ICFT073 was statistically significantly associated with the aerobactin siderophore system and Iha; 7. to our knowledge this is the first report on PAIs and iron uptake systems in an ExPEC collection including isolates from skin and soft tissue infections. Povzetek Bakterija Escherichia coli (E. coli) je najpo- gostejši fakultativni anaerob med človeškimi čre vesnimi mikrobioti. Kljub temu, da je E. coli komenzalna bakterija, lahko določeni sevi, ki so pridobili genske zapise za virulentne dejavnike, povzročajo zelo širok spekter okužb, tako črevesnih kot zunajčrevesnih. Genski zapisi za virulentne dejavnike so pogosto vezani – umeščeni v otoke patogenosti (PAI), ki predstavljajo podskupino genomskih otokov. Genski zapisi za virulentne dejavnike pa se pogosto nahajajo tudi v plaz- midih, v izvenkromosomskih molekulah DNA. Železo je pomemben element, saj nastopa kot kofaktor v mnogih metabolnih poteh tako go- stitelja kot mikroorganizma. Ker je v gostitelju malo prostega železa, imajo mikrobi različne 82 Acta Biologica Slovenica, 52 (2), 2009 sisteme za privzem železa, med najbolj znanimi so t. i. sideroforji. Naša raziskava je vključevala 29 sevov zunajčrevesnih patogenih E. coli (Ex- PEC), osamljenih na Inštitutu za mikrobiologijo in imunologijo Medicinske fakultete v Ljubljani iz diagnostičnih vzorcev 19 bolnikov z urinar- nimi infekcijami, 9 bolnikov z infekcijami kože in podkožja in 1 bolnika z infekcijo spolovil. S pomočjo PCR smo preučevane seve preiskali za prisotnost različnih otokov patogenosti, ki so jih prvotno našli in opisali v uropatogenih sevih E. coli (PAI I536 do PAI IV536, PAI IJ96, PAI IIJ96, PAI ICFT073 in PAI IICFT073) in sisteme privzema železa (gena iutA in iucD aerobaktinskega siderofornega sistema, geni iroN, in iroCD salmohelinskega siderofornega sistema, gena fyuA in irp2 jersi- niabaktinskega siderofornega sistema, gen iha receptorja kateholatnega sideroforja Iha, gen ireA od TonB odvisnega siderofornega receptorja IreA in gen hbp hemoglobinske proteaze Hbp). Nadalje, smo z molekulskobiološkimi tehnikami preverjali prisotnost plazmidov v preučevanih sevih. Ugotavljali smo prevalenco in povezave preučevanih PAI in sistemov za privzem železa. PAI IV536 smo našli v 19, PAI II CFT073 v 6, PAI ICFT073 v 4 , PAI IIJ96 v 1 od preučevanih izolatov. PAI I536, PAI II536, PAI III536 in PAI IJ96 nismo odkrili v nobenem izmed preučevanih izolatov. V 19 sevih smo odkrili plazmide, v 9 sevih smo našli plazmide, ki so bili večji od 30 kb. Gen irp2 smo našli v 20, fyuA v 19, iucD in iutA v 12, iha v 9, iroN v 8 , iroCD v 7, ireA v 7 in hbp v 4 izmed preučevanih sevov. PAI IV536 je bil statistično značilno povezan s siderofornim sistemom jersi- niabaktin in PAI ICFT073 je bil statistično značilno povezan s siderofornim sistemom aerobaktin in Iha. Kolikor nam je poznano je to prva raziskava o PAI in sistemih za privzem železa na zbirki sevov ExPEC, ki vključuje tudi izolate iz infekcij kože in podkožja. Acknowledgements The authors thank Veronika Križan-Hergouth, M.D., M.Sc. and Marija Gubina, M.D., Ph.D. from the Institute of Microbiology and Immunology, Medical Faculty, University of Ljubljana for providing the investigated strains. This research was supported by Grant PO-0508-0487 of the Ministry of Education, Science and Technology, Slovenia. Literature braun V. & M. braun 2002: Iron transport and signaling in Escherichia coli. 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