{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,11,7]],"date-time":"2026-11-07T00:47:11Z","timestamp":1794012431314,"version":"4.8.0"},"reference-count":37,"publisher":"Springer Science and Business Media LLC","issue":"1","content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["BMC Microbiol"],"published-print":{"date-parts":[[2008,12]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background<\/jats:title><jats:p>The foodborne, gram-positive pathogen,<jats:italic>Listeria monocytogenes<\/jats:italic>, is capable of causing lethal infections in compromised individuals. In the post genomic era of<jats:italic>L. monocytogenes<\/jats:italic>research, techniques are required to identify and validate genes involved in the pathogenicity and environmental biology of the organism. The aim here was to develop a widely applicable method to tag<jats:italic>L. monocytogenes<\/jats:italic>strains, with a particular emphasis on the development of multiple strain competitive index assays.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>We have constructed a new site-specific integrative vector, pIMC, based on pPL2, for the selection of<jats:italic>L. monocytogenes<\/jats:italic>from complex samples. The pIMC vector was further modified through the incorporation of IPTG inducible markers (antibiotic and phenotypic) to produce a suite of four vectors which allowed the discrimination of multiple strains from a single sample. We were able to perform murine infection studies with up to four EGDe isolates within a single mouse and showed that the tags did not impact upon growth rate or virulence. The system also allowed the identification of subtle differences in virulence between strains of<jats:italic>L. monocytogenes<\/jats:italic>commonly used in laboratory studies.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>This study has developed a competitive index assay that can be broadly applied to all<jats:italic>L. monocytogenes<\/jats:italic>strains. Improved statistical robustness of the data was observed, resulting in fewer mice being required for virulence assays. The competitive index assays provide a powerful method to analyse the virulence or fitness of<jats:italic>L. monocytogenes<\/jats:italic>in complex biological samples.<\/jats:p><\/jats:sec>","DOI":"10.1186\/1471-2180-8-96","type":"journal-article","created":{"date-parts":[[2008,6,13]],"date-time":"2008-06-13T18:14:04Z","timestamp":1213380844000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":37,"title":["Development of multiple strain competitive index assays for Listeria monocytogenes using pIMC; a new site-specific integrative vector"],"prefix":"10.1186","volume":"8","author":[{"given":"Ian R","family":"Monk","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pat G","family":"Casey","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Cronin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cormac GM","family":"Gahan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Colin","family":"Hill","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2008,6,13]]},"reference":[{"key":"529_CR1","doi-asserted-by":"publisher","first-page":"1345","DOI":"10.1016\/S1286-4579(01)01496-4","volume":"3","author":"CR Beuzon","year":"2001","unstructured":"Beuzon CR, Holden DW: Use of mixed infections with Salmonella strains to study virulence genes and their interactions in vivo. Microbes Infect. 2001, 3: 1345-1352. 10.1016\/S1286-4579(01)01496-4.","journal-title":"Microbes Infect"},{"key":"529_CR2","doi-asserted-by":"publisher","first-page":"67","DOI":"10.1038\/nrmicro799","volume":"2","author":"S Falkow","year":"2004","unstructured":"Falkow S: Molecular Koch's postulates applied to bacterial pathogenicity\u2013a personal recollection 15 years later. Nat Rev Microbiol. 2004, 2: 67-72. 10.1038\/nrmicro799.","journal-title":"Nat Rev Microbiol"},{"key":"529_CR3","doi-asserted-by":"publisher","first-page":"12432","DOI":"10.1073\/pnas.2133653100","volume":"100","author":"LL Lenz","year":"2003","unstructured":"Lenz LL, Mohammadi S, Geissler A, Portnoy DA: SecA2-dependent secretion of autolytic enzymes promotes Listeria monocytogenes pathogenesis. Proc Natl Acad Sci USA. 2003, 100: 12432-12437. 10.1073\/pnas.2133653100.","journal-title":"Proc Natl Acad Sci USA"},{"key":"529_CR4","doi-asserted-by":"publisher","first-page":"1255","DOI":"10.1128\/IAI.01484-06","volume":"75","author":"BA Lowe","year":"2007","unstructured":"Lowe BA, Miller JD, Neely MN: Analysis of the polysaccharide capsule of the systemic pathogen Streptococcus iniae and its implications in virulence. Infect Immun. 2007, 75: 1255-1264. 10.1128\/IAI.01484-06.","journal-title":"Infect Immun"},{"key":"529_CR5","doi-asserted-by":"publisher","first-page":"12860","DOI":"10.1074\/jbc.M611593200","volume":"282","author":"JT Pratt","year":"2007","unstructured":"Pratt JT, Tamayo R, Tischler AD, Camilli A: PilZ domain proteins bind cyclic diguanylate and regulate diverse processes in Vibrio cholerae. J Biol Chem. 2007, 282: 12860-12870. 10.1074\/jbc.M611593200.","journal-title":"J Biol Chem"},{"key":"529_CR6","doi-asserted-by":"publisher","first-page":"584","DOI":"10.1128\/CMR.14.3.584-640.2001","volume":"14","author":"JA Vazquez-Boland","year":"2001","unstructured":"Vazquez-Boland JA, Kuhn M, Berche P, Chakraborty T, Dominguez-Bernal G, Goebel W, Gonzalez-Zorn B, Wehland J, Kreft J: Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev. 2001, 14: 584-640. 10.1128\/CMR.14.3.584-640.2001.","journal-title":"Clin Microbiol Rev"},{"key":"529_CR7","doi-asserted-by":"publisher","first-page":"923","DOI":"10.1016\/S0092-8674(00)81070-3","volume":"84","author":"J Mengaud","year":"1996","unstructured":"Mengaud J, Ohayon H, Gounon P, Mege R-M, Cossart P: E-cadherin is the receptor for internalin, a surface protein required for entry of L. monocytogenes into epithelial cells. Cell. 1996, 84: 923-932. 10.1016\/S0092-8674(00)81070-3.","journal-title":"Cell"},{"key":"529_CR8","doi-asserted-by":"publisher","first-page":"457","DOI":"10.1111\/j.1462-5822.2005.00634.x","volume":"8","author":"N Khelef","year":"2006","unstructured":"Khelef N, Lecuit M, Bierne H, Cossart P: Species specificity of the Listeria monocytogenes InlB protein. Cell Microbiol. 2006, 8: 457-470. 10.1111\/j.1462-5822.2005.00634.x.","journal-title":"Cell Microbiol"},{"key":"529_CR9","doi-asserted-by":"crossref","first-page":"849","DOI":"10.1126\/science.1063447","volume":"294","author":"P Glaser","year":"2001","unstructured":"Glaser P, Frangeul L, Buchrieser C, Rusniok C, Amend A, Baquero F, Berche P, Bloecker H, Brandt P, Chakraborty T, Charbit A, Chetouani F, Couve E, de Daruvar A, Dehoux P, Domann E, Dominguez-Bernal G, Duchaud E, Durant L, Dussurget O, Entian KD, Fsihi H, Garcia-del Portillo F, Garrido P, Gautier L, Goebel W, Gomez-Lopez N, Hain T, Hauf J, Jackson D, Jones LM, Kaerst U, Kreft J, Kuhn M, Kunst F, Kurapkat G, Madueno E, Maitournam A, Vicente JM, Ng E, Nedjari H, Nordsiek G, Novella S, de Pablos B, Perez-Diaz JC, Purcell R, Remmel B, Rose M, Schlueter T, Simoes N, Tierrez A, Vazquez-Boland JA, Voss H, Wehland J, Cossart P: Comparative genomics of Listeria species. Science. 2001, 294: 849-852.","journal-title":"Science"},{"key":"529_CR10","doi-asserted-by":"publisher","first-page":"2386","DOI":"10.1093\/nar\/gkh562","volume":"32","author":"KE Nelson","year":"2004","unstructured":"Nelson KE, Fouts DE, Mongodin EF, Ravel J, DeBoy RT, Kolonay JF, Rasko DA, Angiuoli SV, Gill SR, Paulsen IT, Peterson J, White O, Nelson WC, Nierman W, Beanan MJ, Brinkac LM, Daugherty SC, Dodson RJ, Durkin AS, Madupu R, Haft DH, Selengut J, Van Aken S, Khouri H, Fedorova N, Forberger H, Tran B, Kathariou S, Wonderling LD, Uhlich GA, Bayles DO, Luchansky JB, Fraser CM: Whole genome comparisons of serotype 4b and 1\/2a strains of the food-borne pathogen Listeria monocytogenes reveal new insights into the core genome components of this species. Nucleic Acids Res. 2004, 32: 2386-2395. 10.1093\/nar\/gkh562.","journal-title":"Nucleic Acids Res"},{"key":"529_CR11","doi-asserted-by":"publisher","first-page":"4177","DOI":"10.1128\/JB.184.15.4177-4186.2002","volume":"184","author":"P Lauer","year":"2002","unstructured":"Lauer P, Chow MY, Loessner MJ, Portnoy DA, Calendar R: Construction, characterization, and use of two Listeria monocytogenes site-specific phage integration vectors. J Bacteriol. 2002, 184: 4177-4186. 10.1128\/JB.184.15.4177-4186.2002.","journal-title":"J Bacteriol"},{"key":"529_CR12","doi-asserted-by":"publisher","first-page":"157","DOI":"10.1016\/S0378-1119(96)00420-9","volume":"179","author":"PS Lovett","year":"1996","unstructured":"Lovett PS: Translation attenuation regulation of chloramphenicol resistance in bacteria\u2013a review. Gene. 1996, 179: 157-162. 10.1016\/S0378-1119(96)00420-9.","journal-title":"Gene"},{"key":"529_CR13","doi-asserted-by":"publisher","first-page":"3091","DOI":"10.1128\/AEM.02940-06","volume":"73","author":"CU Riedel","year":"2007","unstructured":"Riedel CU, Monk IR, Casey PG, Morrissey D, O'Sullivan GC, Tangney M, Hill C, Gahan CG: Improved luciferase tagging system for Listeria monocytogenes allows real-time monitoring in vivo and in vitro. Appl Environ Microbiol. 2007, 73: 3091-3094. 10.1128\/AEM.02940-06.","journal-title":"Appl Environ Microbiol"},{"key":"529_CR14","doi-asserted-by":"publisher","first-page":"143","DOI":"10.1111\/j.1365-2958.1993.tb01211.x","volume":"8","author":"A Camilli","year":"1993","unstructured":"Camilli A, Tilney LG, Portnoy DA: Dual roles of plcA in Listeria monocytogenes pathogenesis. Mol Microbiol. 1993, 8: 143-157. 10.1111\/j.1365-2958.1993.tb01211.x.","journal-title":"Mol Microbiol"},{"key":"529_CR15","doi-asserted-by":"publisher","first-page":"6686","DOI":"10.1128\/AEM.70.11.6686-6694.2004","volume":"70","author":"IR Monk","year":"2004","unstructured":"Monk IR, Cook GM, Monk BC, Bremer PJ: Morphotypic conversion in Listeria monocytogenes biofilm formation: biological significance of rough colony isolates. Appl Environ Microbiol. 2004, 70: 6686-6694. 10.1128\/AEM.70.11.6686-6694.2004.","journal-title":"Appl Environ Microbiol"},{"key":"529_CR16","doi-asserted-by":"publisher","first-page":"2876","DOI":"10.1128\/AEM.72.4.2876-2884.2006","volume":"72","author":"PA Bron","year":"2006","unstructured":"Bron PA, Monk IR, Corr SC, Hill C, Gahan CG: Novel luciferase reporter system for in vitro and organ-specific monitoring of differential gene expression in Listeria monocytogenes. Appl Environ Microbiol. 2006, 72: 2876-2884. 10.1128\/AEM.72.4.2876-2884.2006.","journal-title":"Appl Environ Microbiol"},{"key":"529_CR17","doi-asserted-by":"publisher","first-page":"12907","DOI":"10.1073\/pnas.0705589104","volume":"104","author":"K Sjostrom","year":"2007","unstructured":"Sjostrom K, Blomberg C, Fernebro J, Dagerhamn J, Morfeldt E, Barocchi MA, Browall S, Moschioni M, Andersson M, Henriques F, Albiger B, Rappuoli R, Normark S, Henriques-Normark B: Clonal success of piliated penicillin nonsusceptible pneumococci. Proc Natl Acad Sci USA. 2007, 104: 12907-12912. 10.1073\/pnas.0705589104.","journal-title":"Proc Natl Acad Sci USA"},{"key":"529_CR18","doi-asserted-by":"publisher","first-page":"5953","DOI":"10.1128\/IAI.69.9.5953-5957.2001","volume":"69","author":"V Auerbuch","year":"2001","unstructured":"Auerbuch V, Lenz LL, Portnoy DA: Development of a competitive index assay to evaluate the virulence of Listeria monocytogenes actA mutants during primary and secondary infection of mice. Infect Immun. 2001, 69: 5953-5957. 10.1128\/IAI.69.9.5953-5957.2001.","journal-title":"Infect Immun"},{"key":"529_CR19","doi-asserted-by":"publisher","first-page":"3948","DOI":"10.1128\/IAI.70.7.3948-3952.2002","volume":"70","author":"CA Nadon","year":"2002","unstructured":"Nadon CA, Bowen BM, Wiedmann M, Boor KJ: Sigma B contributes to PrfA-mediated virulence in Listeria monocytogenes. Infect Immun. 2002, 70: 3948-3952. 10.1128\/IAI.70.7.3948-3952.2002.","journal-title":"Infect Immun"},{"key":"529_CR20","doi-asserted-by":"publisher","first-page":"449","DOI":"10.1084\/jem.20051866","volume":"203","author":"H Xu","year":"2006","unstructured":"Xu H, Chun T, Choi HJ, Wang B, Wang CR: Impaired response to Listeria in H2-M3-deficient mice reveals a nonredundant role of MHC class Ib-specific T cells in host defense. J Exp Med. 2006, 203: 449-459. 10.1084\/jem.20051866.","journal-title":"J Exp Med"},{"key":"529_CR21","doi-asserted-by":"publisher","first-page":"6721","DOI":"10.1128\/IAI.71.12.6721-6727.2003","volume":"71","author":"SE D'Orazio","year":"2003","unstructured":"D'Orazio SE, Velasquez M, Roan NR, Naveiras-Torres O, Starnbach MN: The Listeria monocytogenes lemA gene product is not required for intracellular infection or to activate fMIGWII-specific T cells. Infect Immun. 2003, 71: 6721-6727. 10.1128\/IAI.71.12.6721-6727.2003.","journal-title":"Infect Immun"},{"key":"529_CR22","doi-asserted-by":"publisher","first-page":"6675","DOI":"10.1128\/IAI.00886-06","volume":"74","author":"HS O'Neil","year":"2006","unstructured":"O'Neil HS, Marquis H: Listeria monocytogenes flagella are used for motility, not as adhesins, to increase host cell invasion. Infect Immun. 2006, 74: 6675-6681. 10.1128\/IAI.00886-06.","journal-title":"Infect Immun"},{"key":"529_CR23","doi-asserted-by":"publisher","first-page":"86","DOI":"10.1186\/1471-2180-6-86","volume":"6","author":"JB Andersen","year":"2006","unstructured":"Andersen JB, Roldgaard BB, Lindner AB, Christensen BB, Licht TR: Construction of a multiple fluorescence labelling system for use in co-invasion studies of Listeria monocytogenes. BMC Microbiol. 2006, 6: 86-10.1186\/1471-2180-6-86.","journal-title":"BMC Microbiol"},{"key":"529_CR24","doi-asserted-by":"crossref","first-page":"482","DOI":"10.4315\/0362-028X-70.2.482","volume":"70","author":"KK Nightingale","year":"2007","unstructured":"Nightingale KK, Milillo SR, Ivy RA, Ho AJ, Oliver HF, Wiedmann M: Listeria monocytogenes F2365 carries several authentic mutations potentially leading to truncated gene products, including inlB, and demonstrates atypical phenotypic characteristics. J Food Prot. 2007, 70: 482-488.","journal-title":"J Food Prot"},{"key":"529_CR25","doi-asserted-by":"publisher","first-page":"5976","DOI":"10.1073\/pnas.89.13.5976","volume":"89","author":"C Ginocchio","year":"1992","unstructured":"Ginocchio C, Pace J, Galan JE: Identification and molecular characterization of a Salmonella typhimurium gene involved in triggering the internalization of salmonellae into cultured epithelial cells. Proc Natl Acad Sci USA. 1992, 89: 5976-5980. 10.1073\/pnas.89.13.5976.","journal-title":"Proc Natl Acad Sci USA"},{"key":"529_CR26","doi-asserted-by":"publisher","first-page":"1990","DOI":"10.1093\/nar\/23.11.1990","volume":"23","author":"Y Sheng","year":"1995","unstructured":"Sheng Y, Mancino V, Birren B: Transformation of Escherichia coli with large DNA molecules by electroporation. Nucleic Acids Res. 1995, 23: 1990-1996. 10.1093\/nar\/23.11.1990.","journal-title":"Nucleic Acids Res"},{"key":"529_CR27","volume-title":"Appl Environ Microbiol","author":"IR Monk","year":"2008","unstructured":"Monk IR, Gahan CG, Hill C: Tools for functional post-genomic analysis of Listeria monocytogenes. Appl Environ Microbiol. 2008, doi:10.1128\/AEM.00314-08"},{"key":"529_CR28","doi-asserted-by":"publisher","first-page":"705","DOI":"10.1007\/s00253-005-0107-6","volume":"68","author":"I Mierau","year":"2005","unstructured":"Mierau I, Kleerebezem M: 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl Microbiol Biotechnol. 2005, 68: 705-717. 10.1007\/s00253-005-0107-6.","journal-title":"Appl Microbiol Biotechnol"},{"key":"529_CR29","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1128\/AEM.64.1.82-87.1998","volume":"64","author":"PR Jensen","year":"1998","unstructured":"Jensen PR, Hammer K: The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl Environ Microbiol. 1998, 64: 82-87.","journal-title":"Appl Environ Microbiol"},{"key":"529_CR30","doi-asserted-by":"publisher","first-page":"1460","DOI":"10.1111\/j.1365-2958.2005.04780.x","volume":"57","author":"A Shen","year":"2005","unstructured":"Shen A, Higgins DE: The 5' untranslated region-mediated enhancement of intracellular listeriolysin O production is required for Listeria monocytogenes pathogenicity. Mol Microbiol. 2005, 57: 1460-1473. 10.1111\/j.1365-2958.2005.04780.x.","journal-title":"Mol Microbiol"},{"key":"529_CR31","unstructured":"Scalable Vector graphics: plasmid map. [http:\/\/bioinformatics.org\/savvy\/]"},{"key":"529_CR32","doi-asserted-by":"crossref","first-page":"1230","DOI":"10.1128\/aem.54.5.1230-1236.1988","volume":"54","author":"C Hill","year":"1988","unstructured":"Hill C, Venema G, Daly C, Fitzgerald GF: Cloning and characterization of the tetracycline resistance determinant of and several promoters from within the conjugative transposon Tn919. Appl Environ Microbiol. 1988, 54: 1230-1236.","journal-title":"Appl Environ Microbiol"},{"key":"529_CR33","doi-asserted-by":"publisher","first-page":"3177","DOI":"10.1093\/nar\/15.7.3177","volume":"15","author":"J Brehm","year":"1987","unstructured":"Brehm J, Salmond G, Minton N: Sequence of the adenine methylase gene of the Streptococcus faecalis plasmid pAM beta 1. Nucleic Acids Res. 1987, 15: 3177-10.1093\/nar\/15.7.3177.","journal-title":"Nucleic Acids Res"},{"key":"529_CR34","doi-asserted-by":"crossref","first-page":"4166","DOI":"10.1128\/jb.178.14.4166-4175.1996","volume":"178","author":"JA Gutierrez","year":"1996","unstructured":"Gutierrez JA, Crowley PJ, Brown DP, Hillman JD, Youngman P, Bleiweis AS: Insertional mutagenesis and recovery of interrupted genes of Streptococcus mutans by using transposon Tn917: preliminary characterization of mutants displaying acid sensitivity and nutritional requirements. J Bacteriol. 1996, 178: 4166-4175.","journal-title":"J Bacteriol"},{"key":"529_CR35","doi-asserted-by":"crossref","first-page":"587","DOI":"10.1128\/aem.60.2.587-593.1994","volume":"60","author":"C Platteeuw","year":"1994","unstructured":"Platteeuw C, Simons G, de Vos WM: Use of the Escherichia coli beta-glucuronidase (gusA) gene as a reporter gene for analyzing promoters in lactic acid bacteria. Appl Environ Microbiol. 1994, 60: 587-593.","journal-title":"Appl Environ Microbiol"},{"key":"529_CR36","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1128\/aem.53.1.74-78.1987","volume":"53","author":"C Hill","year":"1987","unstructured":"Hill C, Daly C, Fitzgerald GF: Development of High-Frequency Delivery System for Transposon Tn919 in Lactic Streptococci: Random Insertion in Streptococcus lactis subsp. diacetylactis 18\u201316. Appl Environ Microbiol. 1987, 53: 74-78.","journal-title":"Appl Environ Microbiol"},{"key":"529_CR37","doi-asserted-by":"crossref","first-page":"2005","DOI":"10.4049\/jimmunol.139.6.2005","volume":"139","author":"DK Bishop","year":"1987","unstructured":"Bishop DK, Hinrichs DJ: Adoptive transfer of immunity to Listeria monocytogenes. The influence of in vitro stimulation on lymphocyte subset requirements. J Immunol. 1987, 139: 2005-2009.","journal-title":"J Immunol"}],"container-title":["BMC Microbiology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/link.springer.com\/content\/pdf\/10.1186\/1471-2180-8-96.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,2,27]],"date-time":"2024-02-27T09:53:06Z","timestamp":1709027586000},"score":1,"resource":{"primary":{"URL":"https:\/\/bmcmicrobiol.biomedcentral.com\/articles\/10.1186\/1471-2180-8-96"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2008,6,13]]},"references-count":37,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2008,12]]}},"alternative-id":["529"],"URL":"https:\/\/doi.org\/10.1186\/1471-2180-8-96","relation":{},"ISSN":["1471-2180"],"issn-type":[{"value":"1471-2180","type":"electronic"}],"subject":[],"published":{"date-parts":[[2008,6,13]]},"assertion":[{"value":"14 March 2008","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"13 June 2008","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"13 June 2008","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}}],"article-number":"96"}}