Escherichia coli from Crohn’s disease patient displays virulence features of enteroinvasive (EIEC), enterohemorragic (EHEC), and enteroaggregative (EAEC) pathotypes

Gut Pathogens

Josias Rodrigues¹  Ligia Yukie Sassaki²  Fernando Gomes Romeiro²  Ana Carolina da Silva Santos¹ 

[1] Laboratory of Medical Bacteriology, Department of Microbiology and Immunology, Institute of Biosciences of the State University of São Paulo (UNESP), Brazil, Distrito de Rubião Junior, Botucatu, CEP 18618-970, SP, Brazil;Department of Internal Medicine, Botucatu Medical School of the State University of São Paulo (UNESP), Brazil, Distrito de Rubião Junior, Botucatu, CEP 18618-970, SP, Brazil

关键词: Virulence;    Adherence;    Serotype;    MLST;    Invasive;    Crohn’s disease;    Escherichia coli;   

Others  :  1137553
DOI  :  10.1186/s13099-015-0050-8

 received in 2014-10-20, accepted in 2015-01-06,  发布年份 2015

【 摘 要 】

Background

Escherichia coli is a normal inhabitant of the gut which upon acquiring virulence factors becomes potentially able to cause diseases. Although E. coli population augments in Crohn’s disease (CD), the reason of this proliferation is not yet clear. CD associated E. coli shows features of extraintestinal pathogenic categories (ExPEC), and eventually the ability to invade cultured epithelial cells, a property observed among diarrheagenic E. coli (DEC). In this work, data on the characterization of an E. coli isolate from a CD patient reveal that, besides invasiveness, CD associated E. coli may harbor other typical DEC markers, namely those defining enterohemorragic (EHEC) and enteroaggregative (EAEC) pathotypes.

Results

The studied strain, detected both in an ileum biopsy and stools, belonged to the B2 E. coli reference collection (EcoR) phylogroup and harbored the intimin, Shiga cytotoxin 1, and AggR transcriptional activator encoding genes (eae, stx1, aggR, respectively); displayed aggregative adherence to Hep-2 cells and an ability to enter Caco-2 cells four times as high as that of EIEC reference strain and half of invasiveness of AIEC LF82. It was able to enter and replicate in J774 macrophages with invasiveness 85 times as high as that of LF82, but with only one sixth of the intracellular proliferation ability of the later. Extracellular products with cytotoxic activity on Vero cells were detected in strain’s cultures. Preliminary analysis indicated similarity of this strain’s genome with that of O104:H4/2011C-3493.

Methods

Following its isolation from a resected CD patient, the strain was characterized by in vitro adhesion and invasion assays to Hep-2, invasion to Caco-2 cells and to J774 macrophages and tested for the ability to form biofilm and to produce Shiga cytotoxins. PCRs were carried out to identify virulence genetic markers and for EcoR phylogrouping. The strain’s genome was sequenced by means of Ion torrent PGM platform.

Conclusion

The detection, in a CD patient, of an E. coli combining virulence features of multiple DEC pathotypes seems not only to stress the relevance of E. coli to CD etiopathogenesis but also to indicate the existence of new and potentially more virulent strains putatively associated with this disease.

【 授权许可】

   
2015 da Silva Santos et al.; licensee Biomed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Winter SE, Winter MG, Xavier MN, Thiennimitr P, Poon V, Keestra AM, et al.: Host-derived nitrate boosts growth of E. coli in the inflamed gut. Science 2013, 339(6120):708-11.
• [2]de Souza HL, de Carvalho VR, Romeiro FG, Sassaki LY, Keller R, Rodrigues J: Mucosa-associated but not luminal Escherichia coli is augmented in Crohn’s disease and ulcerative colitis. Gut Pathogens 2012, 4(1):21. BioMed Central Full Text
• [3]Chassaing B, Rolhion N, de Vallee A, Salim SY, Prorok-Hamon M, Neut C, et al.: Crohn disease--associated adherent-invasive E. coli bacteria target mouse and human Peyer’s patches via long polar fimbriae. J Clin Invest 2011, 121(3):966-75.
• [4]Prorok-Hamon M, Friswell MK, Alswied A, Roberts CL, Song F, Flanagan PK, et al.: Colonic mucosa-associated diffusely adherent afaC+ Escherichia coli expressing lpfA and pks are increased in inflammatory bowel disease and colon cancer. Gut 2014, 63(5):761-70.
• [5]Dogan B, Scherl E, Bosworth B, Yantiss R, Altier C, McDonough PL, et al.: Multidrug resistance is common in Escherichia coli associated with ileal Crohn’s disease. Inflamm Bowel Dis 2013, 19(1):141-50.
• [6]Kotlowski R, Bernstein CN, Sepehri S, Krause DO: High prevalence of Escherichia coli belonging to the B2 + D phylogenetic group in inflammatory bowel disease. Gut 2007, 56(5):669-75.
• [7]Darfeuille-Michaud A, Neut C, Barnich N, Lederman E, Di Martino P, Desreumaux P, et al.: Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn’s disease. Gastroenterology 1998, 115(6):1405-13.
• [8]Darfeuille-Michaud A: Adherent-invasive Escherichia coli: a putative new E. coli pathotype associated with Crohn’s disease. IJMM 2002, 292(3–4):185-93.
• [9]Boudeau J, Glasser AL, Masseret E, Joly B, Darfeuille-Michaud A: Invasive ability of an Escherichia coli strain isolated from the ileal mucosa of a patient with Crohn’s disease. Infect Immun 1999, 67(9):4499-509.
• [10]Glasser AL, Boudeau J, Barnich N, Perruchot MH, Colombel JF, Darfeuille-Michaud A: Adherent Invasive Escherichia coli strains from patients with Crohn’s disease survive and replicate within macrophages without inducing host cell death. Infect Immun 2001, 69(9):5529-37.
• [11]Martin HM, Campbell BJ, Hart CA, Mpofu C, Nayar M, Singh R, et al.: Enhanced Escherichia coli adherence and invasion in Crohn’s disease and colon cancer. Gastroenterology 2004, 127(1):80-93.
• [12]Eaves-Pyles T, Allen CA, Taormina J, Swidsinski A, Tutt CB, Jezek GE, et al.: Escherichia coli isolated from a Crohn’s disease patient adheres, invades, and induces inflammatory responses in polarized intestinal epithelial cells. IJMM 2008, 298(5–6):397-409.
• [13]Nash JH, Villegas A, Kropinski AM, Aguilar-Valenzuela R, Konczy P, Mascarenhas M, et al.: Genome sequence of adherent-invasive Escherichia coli and comparative genomic analysis with other E. colipathotypes. BMC Genomics 2010, 11:667. BioMed Central Full Text
• [14]Darfeuille-Michaud A, Boudeau J, Bulois P, Neut C, Glasser AL, Barnich N, et al.: High prevalence of adherent-invasive Escherichia coli associated with ileal mucosa in Crohn’s disease. Gastroenterology 2004, 127(2):412-21.
• [15]Toma C, Lu Y, Higa N, Nakasone N, Chinen I, Baschkier A, et al.: Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli. J Clin Microbiol 2003, 41(6):2669-71.
• [16]Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, et al.: Multilocus sequence typing of total-genome-sequenced bacteria. J Clin Microbiol 2012, 50(4):1355-61.
• [17]Kaper JB, Nataro JP, Mobley HL: Pathogenic Escherichia coli. Nature Rev Microbiol 2004, 2(2):123-40.
• [18]Conte MP, Longhi C, Marazzato M, Conte AL, Aleandri M, Lepanto MS, et al.: Adherent-invasive Escherichia coli (AIEC) in pediatric Crohn’s disease patients: phenotypic and genetic pathogenic features. BMC Res Notes 2014, 7:748. BioMed Central Full Text
• [19]Kostic AD, Xavier RJ, Gevers D: The microbiome in inflammatory bowel disease: current status and the future ahead. Gastroenterology 2014, 146(6):1489-99.
• [20]Rhodes JM: The role of Escherichia coli in inflammatory bowel disease. Gut 2007, 56(5):610-2.
• [21]Momtaz H, Karimian A, Madani M, Safarpoor Dehkordi F, Ranjbar R, Sarshar M, et al.: Uropathogenic Escherichia coli in Iran: serogroup distributions, virulence factors and antimicrobial resistance properties. Ann Clin Microbiol Antimicrob 2013, 12:8. BioMed Central Full Text
• [22]Clarke DJ, Chaudhuri RR, Martin HM, Campbell BJ, Rhodes JM, Constantinidou C, et al.: Complete genome sequence of the Crohn’s disease-associated adherent-invasive Escherichia coli strain HM605. J Bacteriol 2011, 193(17):4540.
• [23]Miquel S, Peyretaillade E, Claret L, de Valle´e A, Dossat C, Vacherie B, et al.: Complete genome sequence of Crohn’s disease-associated adherent-invasive E. coli strain LF82. PloS ONE 2010, 5(9):e12714. doi:10.1371/journal.pone.0012714
• [24]Boyd EF, Hartl DL: Chromosomal regions specific to pathogenic isolates of Escherichia coli have a phylogenetically clustered distribution. J Bacteriolol 1998, 180(5):1159-65.
• [25]MLST database at UoW. [http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/Downloads_HTML]
• [26]Sjogren R, Neill R, Rachmilewitz D, Fritz D, Newland J, Sharpnack D, et al.: Role of Shiga-like toxin I in bacterial enteritis: comparison between isogenic Escherichia coli strains induced in rabbits. Gastroenterology 1994, 106(2):306-17.
• [27]Sears CL, Kaper JB: Enteric bacterial toxins: mechanisms of action and linkage to intestinal secretion. Microbiol Rev 1996, 60(1):167-215.
• [28]Farina C, Caprioli A, Luzzi I, Sonzogni A, Goglio A: Ulcerative colitis precipitated by a verocytotoxin-producing Escherichia coli infection. Ital J Gastroenterol 1995, 27(9):498-500.
• [29]von Wulffen H, Russmann H, Karch H, Meyer T, Bitzan M, Kohrt TC, et al.: Verocytotoxin-producing Escherichia coli O2:H5 isolated from patients with ulcerative colitis. Lancet 1989, 1(8652):1449-50.
• [30]Ljungh A, Eriksson M, Eriksson O, Henter JI, Wadstrom T: Shiga-like toxin production and connective tissue protein binding of Escherichia coli isolated from a patient with ulcerative colitis. Scand J Infect Dis 1988, 20(4):443-6.
• [31]Dogan B, Suzuki H, Herlekar D, Sartor RB, Campbell BJ, Roberts CL, et al.: Inflammation-associated adherent-invasive Escherichia coli are enriched in pathways for use of propanediol and iron and M-cell translocation. Inflamm Bowel Dis 2014, 20(11):1919-32.
• [32]Yam WC, Robins-Browne RM, Lung ML: Genetic relationships and virulence factors among classical enteropathogenic Escherichia coli serogroup O126 strains. J Med Microbiol 1994, 40(4):229-35.
• [33]Cortes C, De la Fuente R, Blanco J, Blanco M, Blanco JE, Dhabi G, et al.: Serotypes, virulence genes and intimin types of verotoxin-producing Escherichia coli and enteropathogenic E. coliisolated from healthy dairy goats in Spain. Vet Microbiol 2005, 110(1–2):67-76.
• [34]Rich C, Favre-Bonte S, Sapena FDR, Joly B, Forestier C: Characterization of enteroaggregative Escherichia coli isolates. FEMS Microbiol Lett 1999, 173(1):55-61.
• [35]Orden JA, Cortes C, Horcajo P, De la Fuente R, Blanco JE, Mora A, et al.: A longitudinal study of verotoxin-producing Escherichia coli in two dairy goat herds. Vet Microbiol 2008, 132(3–4):428-34.
• [36]Moyenuddin M, Wachsmuth IK, Moseley SL, Bopp CA, Blake PA: Serotype, antimicrobial resistance, and adherence properties of Escherichia coli strains associated with outbreaks of diarrheal illness in children in the United States. J Clin Microbiol 1989, 27(10):2234-9.
• [37]Ishiguro F, Nakamura M, Asada T: [Four serotypes highly positive for pathogenic factor-related genes found among Escherichia coli isolates from sporadic diarrheal cases in Fukui Prefecture]. J Jap Assoc Infect Dis 2002, 76(9):730-7.
• [38]Nataro JP, Kaper JB: Diarrheagenic Escherichia coli. Clin Microbiol Rev 1998, 11(1):142-201.
• [39]Thomazini CM, Samegima DA, Rodrigues MA, Victoria CR, Rodrigues J: High prevalence of aggregative adherent Escherichia coli strains in the mucosa-associated microbiota of patients with inflammatory bowel diseases. IJMM 2011, 301(6):475-9.
• [40]Watanabe E, Romeiro F, Sassaki L, Keller R, Rodrigues J: Enteroaggregative Escherichia coli seems to proliferate in the terminal portion of the colon of IBD patients. Inflam Bowel Dis 2012, 18:S112.
• [41]Nataro JP, Steiner T, Guerrant RL: Enteroaggregative Escherichia coli. Emerg Infect Dis 1998, 4(2):251-61.
• [42]Steiner TS, Lima AA, Nataro JP, Guerrant RL: Enteroaggregative Escherichia coli produce intestinal inflammation and growth impairment and cause interleukin-8 release from intestinal epithelial cells. J Infect Dis 1998, 177(1):88-96.
• [43]Ahmed SA, Awosika J, Baldwin C, Bishop-Lilly KA, Biswas B, Broomall S, et al.: Genomic comparison of Escherichia coli O104:H4 isolates from 2009 and 2011 reveals plasmid, and prophage heterogeneity, including shiga toxin encoding phage stx2. PLoS One 2012, 7(11):e48228.
• [44]Bielaszewska M, Mellmann A, Zhang W, Kock R, Fruth A, Bauwens A, et al.: Characterisation of the Escherichia coli strain associated with an outbreak of haemolytic uraemic syndrome in Germany, 2011: a microbiological study. Lancet Infect Dis 2011, 11(9):671-6.
• [45]Ogura Y, Ooka T, Iguchi A, Toh H, Asadulghani M, Oshima K, et al.: Comparative genomics reveal the mechanism of the parallel evolution of O157 and non-O157 enterohemorrhagic Escherichia coli. Proc Natl Acad Sci U S A 2009, 106(42):17939-44.
• [46]Neut C, Bulois P, Desreumaux P, Membre JM, Lederman E, Gambiez L, et al.: Changes in the bacterial flora of the neoterminal ileum after ileocolonic resection for Crohn’s disease. Am J Gast 2002, 97(4):939-46.
• [47]Nikolaus S, Schreiber S: Diagnostics of inflammatory bowel disease. Gastroenterology 2007, 133(5):1670-89.
• [48]Toledo MRF, Fontes CF, Trabulsi LR: EPM - Modificação do meio de Rugai e Araújo para a realização do teste de produção de gás a partir de glucose, H2S, urease e triptofano desaminase. Rev Microbiol São Paulo 1982, 12:309-15.
• [49]Martinez-Medina M, Naves P, Blanco J, Aldeguer X, Blanco JE, Blanco M, et al.: Biofilm formation as a novel phenotypic feature of adherent-invasive Escherichia coli (AIEC). BMC Microbiol 2009, 9:1-16. BioMed Central Full Text
• [50]Paton AW, Paton JC: Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. colihlyA, rfbO111, and rfbO157. J Clin Microbiol 1998, 36(2):598-602.
• [51]Clermont O, Bonacorsi S, Bingen E: Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol 2000, 66(10):4555-8.
• [52]Doumith M, Day MJ, Hope R, Wain J, Woodford N: Improved multiplex PCR strategy for rapid assignment of the four major Escherichia coli phylogenetic groups. J Clin Microbiol 2012, 50(9):3108-10.
• [53]Karmali MA, Petric M, Lim C, Fleming PC, Arbus GS, Lior H: The association between idiopathic hemolytic uremic syndrome and infection by verotoxin-producing Escherichia coli. J Infect Dis 1985, 151(5):775-82.
• [54]Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, et al.: Artemis: sequence visualization and annotation. Bioinformatics 2000, 16(10):944-5.
• [55]Lowe TM, Eddy SR: tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 1997, 25(5):955-64.