24 Annales Universitatis Paedagogicae Cracoviensis Studia Naturae, 3 (supplement): 24–32, 2018, ISSN 2543-8832 DOI: 10.24917/25438832.3supp.3 Lívia Handrová*, Anna Čuvalová, Vladimír Kmeť Institute of Animal Physiology, Centre of Biosciences of the SAS, Soltesovej 4/6, 040 01 Kosice, Slovak Republic,*handrova@saske.sk The relationship between biofilm formation, genes of virulence and iron metabolism in Escherichia coli Introduction Urinary tract infections (UTIs) are the most prevalent infectious diseases, and very problematic worldwide (Navidinia et al., 2018). Uropathogenic Escherichia coli T. Es- cher. (UPEC), which can colonise successfully in the urinary tract, is the primary etiologic agents associated with UTI (Peerayeh et al., 2018). Avian pathogenic E. coli (APEC) cause septicemia, polyserositis, aerosacculitis and other mainly extraintes- tinal diseases in chickens and other avian species. APECs are found in the intestinal microbiota of healthy birds, and most of the diseases associated with them are sec- ondary to environmental and host predisposing factors (Dho-Moulin, Fairbrother, 1999). �e common presence of a set of virulence-associated genes among as well as similar disease patterns and phylogenetic background indicate a genetic relationship between APEC and UPEC isolates (Kaper et al., 2004; Moulin-Schouleur et al., 2006; Ron, 2006). �e success of E. coli in colonising such a wide range of hosts and envi- ronments is basically due to a noticeable ductility in exploiting the available resources. It is becoming increasingly clear that bio�lms have an enormous impact on medicine (Mah, O’Toole, 2001; Wang et al., 2017), since 65% of human microbial infections involve bio�lms (Labbate et al., 2004). Microbial bio�lm formation is now recognised as a principle virulence factor in many localised chronic infections (Hyun Koo et al., 2017), and their role in infecting the biological devices among hospitalised patients is a universally accepted fact (Vasudevan, 2014). In addition, recent experimental evi- dence indicates a role of bio�lm formation in acute infections (Hannan et al., 2012; Kumagai et al., 2011). Understanding bio�lm formation to �nd e�ective ways to pre- vent bio�lms is important for combating disease. �e primary aim of this study was to detect E. coli strains with a bio�lm formation from animals and the detection of APEC virulence genes presence in these strains in compared to the ability of production a bio�lm. 25 The relationship betw een biofilm form ation, genes of virulence and iron m etabolism in Escherichia coli Material and methods Escherichia coli strains were isolated from broilers rectal swab coming from farms of Eastern Slovakia. Samples were resuscitated overnight at 37°C in bu�ered peptone water (Oxoid, Basingstoke, UK) and subcultured on Mac Conkey agar (Oxoid) and UriSelect agar (Bio-Rad Laboratories, Hercules, CA, USA) again overnight at 37°C. �e colonies were isolated, identi�ed, and con�rmed as E. coli by commercial iden- ti�cation microsystem ENTEROtest24 (ErbaLachema Brno, Czech Republic) and by using the MALDI-TOF MS biotyper (Bruker Daltonics, Bremen, Germany). Nineteen strains were selected for further testing. Bio�lm formation �e ability of bio�lm formation was assessed in a quantitative assay using a micro- titer-plate test (Nunc, Roskilde, Denmark). Strains grown on BHI agar and colonies were re-suspended in BHI broth (Oxoid, UK) to reach the 0.5 suspension McFarland’s standard, and volumes of 200 μl of these cell suspensions were transferred to wells of the microplate. A�er incubation (24 h/37°C), adherent cells were washed three times using a saline solution and stained with a 0.1% crystal violet solution (Mikrochem, Pezinok, Slovakia) for 15 min. A�erwards, excess stain was rinsed o� by �lling the wells with sterile distilled water. �e adhering dye was dissolved with 30% acetic acid for 15 minutes and the optical density was measured at 570 nm in Synergy HT Mul- ti-Mode Microplate Reader (BioTek, USA) (Čuvalová, 2018). We divided isolates of E. coli into four classes based on Stepanovic et al. 2007. For classi�cation, we used average optical density (OD) value and cut-o� value (ODc) (de�ned as three standard devia- tions (SD) above the mean OD of the negative control). �e �nal OD value of a tested strain was expressed as the average OD value of the strain reduced by the ODc value. For interpretation of the results, strains were divided into the following categories: OD ≤ ODc = non-bio�lm producer; ODc < OD ≤ 2 x ODc = weak bio�lm producer; and, 2 x ODc < OD ≤ 4 x ODc = moderate and 4 x ODc < OD = strong bio�lm producers. Detection of genes by PCR Screening of E. coli isolates for APEC virulence genes were carried out by polymerase chain reactions with the ampli�cation of the following: the receptor for aerobactin – iutA (Johnson, Stell, 2000); colicin V – cvaC (Johnson, Stell, 2000); increased serum survival – iss (Foley et al., 2000); temperature sensitive haemagglutinin – tsh (Dozois et al., 2000); P �mbrial adhesion – papC (Le Bouguénec et al., 1992); capsular polysial- ic acid virulence factor – kps (Johnson, Stell, 2000); iron-regulated gene a homologue adhesion – Iha (Johnson et al., 2000) and genes of iron metabolism – putative iron transport gene – sitA (Rodrigues-Siek et al., 2005); iron-related genes – gene which Lí vi a H an dr ov á, A nn a Č uv al ov á, V la di m ír K m eť 26 mediates ferric iron uptake feoB (Rodrigues-Siek et al., 2005), encodes an iron-re- sponsive element and putative sideropohore receptor gene – IreA (Russo et al., 2001) and iron repressible gene associated with yersiniabactin synthesis – irp2 (Schubert et al., 1998), yersiniabactin receptor for ferric yersiniabactin uptake – fyuA (Schubert et al., 1998), and the catecholate siderophore receptor gene – IroN (Johnson, Stell, 2000), and primers are listed in table 1. Tab. 1. Primers used for detection of virulence genes and genes of iron metabolism Gene Primer sequences (5’–3’) Annealing [°C] Size [bp] iutA F: GGCTGGACATGGGAACTGGR: CGTCGGGAACGGGTAGAATCG 63 300 cvaC F: CACACACAAACGGGAGCTGTTR: CACACACAAACGGGAGCTGTT 63 680 iss F: ATCACATAGGATTCTGCCGR: ACAAAAAGTTCTATCGCTTCC 61 700 tsh F: GGTGGTGCACTGGAGTGGR: AGTCCAGCGTGATAGTGG 55 620 papC F: GACGGCTGTACTGCAGGGTGTGGCGR: ATATCCTTTCTGCAGGGATGCAATA 61 328 kps F: GCGCATTTGCTGATCGTTGR: CATCCAGACGATAAGCATGAGCA 63 272 Iha F: CTGGCGGAGGCTCTGAGATCA R: TCCTTAAGCTCCCGCGGCTGA 60 827 sitA F: AGGGGGCACAACTGATTCTCGR: TACCGGGCCGTTTTCTGTGC 59 608 feoB F: AATTGGCGTGCATGAAGATAACTGR: AGCTGGCGACCTGATAGAACAATG 59 470 IreA F: TGGTCTTCAGCTATATGGR: ATCTATGATTGTGTTGGT 55 415 irp2 F: AAGGATTCGCGTGACR: TCGTCGGGCAGCGTTTCTTCT 59 287 fyuA F: TGATTAACCCCGCGACGGGAAR: CGCAGTAGGCACGATGTTGTA 55 880 IroN F:AAGTCAAAGCAGGGGTTGCCCGR: GACGCCGACATTAAGACGCAG 60 655 Results �e interpretation of obtained results requires a de�nition of the cut-o� value that separates bio�lm producing from non-bio�lm-producing strains. We divided isolates based upon the previously calculated OD values, which was a modi�cation of method and classi�cation described by Stepanović et al. (2007): very weak 12/19 (63.0% of strains), weak 2/19 (10.5%), moderate 2/19 (10.5%) and strong 3/19 (16.0%) bio�lm producers. �e occurrences of 13 detected genes are presented in �gure 1. Among 19 E. coli isolates, all isolates contained the feoB gene, 16 isolates contained the sitA 27 gene, 13 isolates contained the iss and iroN genes, 12 isolates contained the iutA gene, 11 isolates contained the fyuA gene, 6 isolates contained the papC and IreA genes, 5 isolates contained the cvaC and tsh genes, 4 isolates contained the irp2 gene, and 2 isolates contained the kps gene. For better comparison of our results, we created two groups of strains (Fig. 2). �e �rst of the two groups represented very weak bio�lm producers and the second group represented weak, moderate, and strong formers. Representation genes of virulence were high in isolates from the �rst group – from seven genes were six highly, only papC was low. Genes of iron metabolism were di�erent. Genes sitA, fyuA, and ireA were higher in the second group, and feoB, irp2 and iroN were higher in the �rst group. Discussion Genes coding adhesins, toxins, or iron acquisition systems have been described to be of particular importance during the pathogenesis of septicemia (Gyles, 1994; Babai et al., 1997; Terlizzi et al., 2017; Robinson et al., 2018), and iron acquisition is a require- ment for UPEC survival in an environment that is as iron-limited as the urinary tract (Skaar, 2010). Isolated E. coli strains were investigated for the presence of thirteen virulence genes that are associated with colibacillosis and iron metabolism. Two genes, fyuA and irp2, coding proteins involved in iron acquisition, were de- scribed in Yersinia sp., and this iron acquisition determinant has been found in human septicemic and enteroaggregative E. coli isolates (Karch et al., 1999; Pelludat et al., 1998; Schubert et al., 1998). �e sequences of the irp2 and fyuA genes in E. coli are al- Fig. 1. Distribution of genes virulence in Escherichia coli strains: iutA (12/19), cvaC (5/19), iss (13/19), tsh (5/19), papC (6/19), kps (2/19), Iha (1/19), sitA (16/19), feoB (19/19), Irp-2 (4/19), fyuA (11/19), IroN (13/19) and IreA (6/19) 0 4 8 12 16 20 iutA cvaC iss tsh papC kps Iha sitA feoB irp2 fyuA IroN IreA D is tr ib ut io n of g en es v ir ul en ce Escherichia coli strains The relationship betw een biofilm form ation, genes of virulence and iron m etabolism in Escherichia coli Lí vi a H an dr ov á, A nn a Č uv al ov á, V la di m ír K m eť 28 most identical to those of Yersinia spp. (Germon et al., 2005) and have been described in APEC isolates by Gophna et al. (2001), Subedi et al. (2018). From nineteen isolates in our study, we detected irp-2 gene in four and fyuA in eleven strains. To increase survival and resistance, E. coli strains also form bio�lm, but published data is variable, depending on the strain origin, di�erent types of surfaces, culture medium, and the methodology used for quantifying bio�lm. In our study, the presence of genes of viru- lence was low in second group – better bio�lm formers and only papC was higher. To compare with another study (Naves et al., 2008; Pavlickova et al., 2017), papC was also determined in a strain with better forming bio�lm, and tsh, which was similar to our results, was detected in weak bio�lm formers. IutA was represented in both groups of strains with weak and strong production of bio�lm. Genes of iron metabolism shows that sitA, fyuA, and ireA were represented higher in the second group (weak, moder- ate and strong). Naves et al. (2008) recorded the presence of fyuA in all strong bio�lm producers, but iroN, unlike our study, was higher in low bio�lm producers. �e liter- ature contains only a few papers correlating the virulence factors investigated in this study with the ability of pathogenic E. coli to form bio�lms in vitro. Further studies involving larger numbers of clinical strains are needed to corroborate our data con- cerning the interaction between bio�lm formation and virulence factors. Conclusion Bio�lms are of particular interest in the poultry industry and public health, because these �lms can harbour pathogenic microorganisms. In this study, Escherichia coli strains were identi�ed and analysed for the presence of genes of iron metabolism, Fig. 2. Presence of detected genes expressed as a percentage; divided in two groups: �rst group = �rst column of data (very weak producers) and second group = second column of data (weak, moderate and strong bio�lm producers) 0 20 40 60 80 100 iutA cvaC iss tsh papC kps Iha sitA feoB irp2 fyuA IroN IreA [% ] Escherichia coli genes very weak producers (1. group) better producers (2,3,4 group) 29 virulence-associated genes, and bio�lm-forming abilities. All 19 E. coli strains evalu- ated were able to form bio�lms, with the majority exhibiting very weak bio�lm-form- ing potential. �e prevalence of the virulence-related genes was higher in low bio�lm producers, where the presence of the siderophore-related genes was variable, but no signi�cant di�erences were observed between strong and weak bio�lm producers. Re- sults provide a basis for the further study of the pathogenesis of APEC and its abilities of formation bio�lms. 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Journal of Microbiology Experi- mental, 1–14. DOI: 10.15406/jmen.2014.01.00014 Wang, Y., Jayan, G., Patwardhan, D., Phillips, K.S. (2017). Antimicrobial and Anti-Bio�lm Medical De- vices: Public Health and Regulatory Science Challenges. In: Z. Zhang, V. Wagner (eds.), Antimicrobial Coatings and Modi�cations on Medical Devices. Springer, Cham. DOI: 10.1007/978-3-319-57494-3_2 Abstract Escherichia coli is known as one of the bacterial species with the widest adaptability to a variety of niches either within organisms or outside in environment. Most strains of E. coli are of low virulence and associated with opportunistic infections, whereas others are highly virulent. �e success of E. coli in colonising such a wide range of hosts and environments is basically due to a noticeable ductility in exploiting the available resources. It is becoming increasingly clear that bio�lms have an enormous impact on medicine, because 65% of animal and human bacterial infections involve bio�lms. In the present study, we isolated strains of E. coli from animals. 19 interesting isolates were selected and tested by PCR ampli�cation to virulence – iutA, cvaC, iss, tsh, papC, kps, iha and iron metabolism genes – sitA, feoB, irp2, fyuA, iroN, and ireA. �e ability of bio�lm formation was assessed in a quantitative assay using microtiter-plate tests. Bacterial strains were grown on BHI. We divided isolates of E. coli into four classes: very weak (63.0%), weak (10.5%), mod- erate (10.5%), and strong (16.0%) bio�lm producers. Representation genes of virulence were high in isolates from very weak bio�lm producers – from 7 genes were 6 highly and only papC (P �mbrial adhesin) was low. Genes of iron metabolism were di�erent. Genes – sitA, fyuA, and ireA in strong isolates producing bio�lm and feoB, irp2, and iroN in weak producers were most represented. �e results show a possible relation between presence virulence factors and low bio�lm formation. Key words: bio�lm, virulence genes, iron metabolisms genes Received: [2018.05.30] Accepted: [2018.12.29] The relationship betw een biofilm form ation, genes of virulence and iron m etabolism in Escherichia coli Lí vi a H an dr ov á, A nn a Č uv al ov á, V la di m ír K m eť 32 Związek pomiędzy tworzeniem się biofilmu, genami wirulencji i metabolizmem żelaza u Escherichia coli Streszczenie Escherichia coli znana jest jako jeden z gatunków bakterii o najszerszej zdolności adaptacji do różnych nisz w organizmach lub w środowisku zewnętrznym. Większość szczepów E. coli ma niską wirulencję i wiąże się z infekcjami oportunistycznymi, podczas gdy pozostałe szczepy są wysoce wirulentne. Sukces E. coli w kolo- nizowaniu tak szerokiego zakresu żywicieli i środowisk wynika przede wszystkim z zauważalnej ciągliwości w wykorzystywaniu dostępnych zasobów. Staje się jasne, że bio�lmy mają ogromny wpływ na medycynę, ponieważ 65% zakażeń bakteryjnych zwierząt i ludzi dotyczy bio�lmów. W obecnych badaniach izolowano szczepy E. coli ze zwierząt. Wybrano 19 interesujących izolatów i testowano je przez ampli�kację PCR pod względem wirulencji – geny metabolizmu iutA, cvaC, iss, tsh, papC, kps, iha oraz żelaza – sitA, feoB, irp2, fyuA, iroN, ireA. Zdolność tworzenia bio�lmu oceniano w teście ilościowym, stosując test płytki mikroti- tracyjnej. Szczepy bakteryjne hodowano na BHI. Izolaty E. coli podzielono na cztery klasy producentów bio�lmu: bardzo słabe (63,0%), słabe (10,5%), umiarkowane (10,5%) i silne (16,0%). Geny reprezentacyjne wirulencji były w większości izolowane od bardzo słabych producentów bio�lmu – z 7 genów było 6 wyso- ko wirulentnych; tylko papC (adhezyna �mbrialna) była niska. Geny metabolizmu żelaza były różne pod względem wirulencji. Najbardziej reprezentowane były geny – sitA, fyuA, ireA w silnych izolatach produku- jących bio�lm oraz feoB, irp2, iroN u słabych producentów. Wyniki pokazują możliwą zależność pomiędzy obecnym czynnikiem zjadliwości, a niską formacją bio�lmu. Słowa kluczowe: bio�lm, geny wirulencji, geny metabolizmu żelaza Information on the authors Lívia Handrová http://orcid.org/0000-0002-0985-1771 �e main area of her interest is genetic ecology and the spread of antibiotic resistance genes. She studies the resistance occurrence in small mammals, which could serve as a reservoir of antibiotic resistance (ESBL, plasmid encoded chinolone resistance, carbapenemases) in indicator bacteria Escherichia coli, Pseudomonas aeruginosa and Staphylococcus spp. Anna Čuvalová She is interested in anti-bio�lm activities of natural compounds using the static and dynamic bio�lm mo- dels with resistant staphylococci (MRSA and MRCoNS), Escherichia coli (ESBL and cefotaximases) and Pseudomonas aeruginosa on various surfaces (plastics, catheters and food grade stainless sheet). Vladimír Kmeť http://orcid.org/0000-0002-8081-8579 He is interested in bio�lm and anti-bio�lm activities of natural compounds using the static and dyna- mic bio�lm models with Escherichia coli, resistant staphylococci (MRSA and MRCoNS). �e area of his interest is genetic ecology and gene encoding factors of virulence, metabolism, and the spreading of these genes. He studies the resistance occurrence in animal, which could serve as a reservoir of antibiotic resistance in indicator bacteria.