{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,31]],"date-time":"2026-03-31T07:02:41Z","timestamp":1774940561889,"version":"3.50.1"},"reference-count":46,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2022,2,16]],"date-time":"2022-02-16T00:00:00Z","timestamp":1644969600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Plague, caused by the human pathogen Yersinia pestis, is a severe and rapidly progressing lethal disease that has caused millions of deaths globally throughout human history and still presents a significant public health concern, mainly in developing countries. Owing to the possibility of its malicious use as a bio-threat agent, Y. pestis is classified as a tier-1 select agent. The prompt administration of an effective antimicrobial therapy, essential for a favorable patient prognosis, requires early pathogen detection, identification and isolation. Although the disease rapidly progresses and the pathogen replicates at high rates within the host, Y. pestis exhibits a slow growth in vitro under routinely employed clinical culturing conditions, complicating the diagnosis and isolation. In the current study, the in vitro bacterial growth in blood cultures was accelerated by the addition of nutritional supplements. We report the ability of calcium (Ca+2)- and iron (Fe+2)-enriched aerobic blood culture media to expedite the growth of various virulent Y. pestis strains. Using a supplemented blood culture, a shortening of the doubling time from ~110 min to ~45 min could be achieved, resulting in increase of 5 order of magnitude in the bacterial loads within 24 h of incubation, consequently allowing the rapid detection and isolation of the slow growing Y. pestis bacteria. In addition, the aerobic and anaerobic blood culture bottles used in clinical set-up were compared for a Y. pestis culture in the presence of Ca+2 and Fe+2. The comparison established the superiority of the supplemented aerobic cultures for an early detection and achieved a significant increase in the yields of the pathogen. In line with the accelerated bacterial growth rates, the specific diagnostic markers F1 and LcrV (V) antigens could be directly detected significantly earlier. Downstream identification employing MALDI-TOF and immunofluorescence assays were performed directly from the inoculated supplemented blood culture, resulting in an increased sensitivity and without any detectable compromise of the accuracy of the antibiotic susceptibility testing (E-test), critical for subsequent successful therapeutic interventions.<\/jats:p>","DOI":"10.3390\/pathogens11020255","type":"journal-article","created":{"date-parts":[[2022,2,16]],"date-time":"2022-02-16T21:36:24Z","timestamp":1645047384000},"page":"255","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["An Improvement in Diagnostic Blood Culture Conditions Allows for the Rapid Detection and Isolation of the Slow Growing Pathogen Yersinia pestis"],"prefix":"10.3390","volume":"11","author":[{"given":"Efi","family":"Makdasi","sequence":"first","affiliation":[{"name":"Israel Institute for Biological Research, Ness-Ziona 74100, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yafit","family":"Atiya-Nasagi","sequence":"additional","affiliation":[{"name":"Israel Institute for Biological Research, Ness-Ziona 74100, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David","family":"Gur","sequence":"additional","affiliation":[{"name":"Israel Institute for Biological Research, Ness-Ziona 74100, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2750-5394","authenticated-orcid":false,"given":"Ayelet","family":"Zauberman","sequence":"additional","affiliation":[{"name":"Israel Institute for Biological Research, Ness-Ziona 74100, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6379-9292","authenticated-orcid":false,"given":"Ofir","family":"Schuster","sequence":"additional","affiliation":[{"name":"Israel Institute for Biological Research, Ness-Ziona 74100, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Itai","family":"Glinert","sequence":"additional","affiliation":[{"name":"Israel Institute for Biological Research, Ness-Ziona 74100, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shlomo","family":"Shmaya","sequence":"additional","affiliation":[{"name":"Israel Institute for Biological Research, Ness-Ziona 74100, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elad","family":"Milrot","sequence":"additional","affiliation":[{"name":"Israel Institute for Biological Research, Ness-Ziona 74100, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1902-8535","authenticated-orcid":false,"given":"Haim","family":"Levy","sequence":"additional","affiliation":[{"name":"Israel Institute for Biological Research, Ness-Ziona 74100, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shay","family":"Weiss","sequence":"additional","affiliation":[{"name":"Israel Institute for Biological Research, Ness-Ziona 74100, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Theodor","family":"Chitlaru","sequence":"additional","affiliation":[{"name":"Israel Institute for Biological Research, Ness-Ziona 74100, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5685-293X","authenticated-orcid":false,"given":"Emanuelle","family":"Mamroud","sequence":"additional","affiliation":[{"name":"Israel Institute for Biological Research, Ness-Ziona 74100, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3109-6554","authenticated-orcid":false,"given":"Orly","family":"Laskar","sequence":"additional","affiliation":[{"name":"Israel Institute for Biological Research, Ness-Ziona 74100, Israel"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,2,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Respicio-Kingry, L.B., Yockey, B.M., Acayo, S., Kaggwa, J., Apangu, T., Kugeler, K.J., Eisen, R.J., Griffith, K.S., Mead, P.S., and Schriefer, M.E. (2016). Two Distinct Yersinia pestis Populations Causing Plague among Humans in the West Nile Region of Uganda. PLoS Negl. Trop. Dis., 10.","DOI":"10.1371\/journal.pntd.0004360"},{"key":"ref_2","first-page":"983","article-title":"Source tracing of the Yersinia pestis strains isolated from Heqing county, Yunnan province in 2017","volume":"39","author":"Shi","year":"2018","journal-title":"Zhonghua Liu Xing Bing Xue Za Zhi"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"210","DOI":"10.3201\/eid2402.160122","article-title":"Ecologic Features of Plague Outbreak Areas, Democratic Republic of the Congo, 2004\u20132014","volume":"24","author":"Abedi","year":"2018","journal-title":"Emerg. Infect. Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1016\/S1473-3099(18)30730-8","article-title":"Epidemiological characteristics of an urban plague epidemic in Madagascar, August\u2013November, 2017: An outbreak report","volume":"19","author":"Randremanana","year":"2019","journal-title":"Lancet Infect. Dis."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2281","DOI":"10.1001\/jama.283.17.2281","article-title":"Plague as a biological weapon: Medical and public health management","volume":"283","author":"Inglesby","year":"2000","journal-title":"JAMA"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1016\/S1684-1182(10)60054-0","article-title":"Guidelines on Blood Cultures","volume":"43","author":"Towns","year":"2010","journal-title":"J. Microbiol. Immunol. Infect."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e0056421","DOI":"10.1128\/JCM.00564-21","article-title":"Improved Selective BIN Agar for a Better Rate of Yersinia pestis Isolation from Primary Clinical Specimens in Suspected Madagascar Plague Cases","volume":"59","author":"Aftalion","year":"2021","journal-title":"J. Clin. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"467","DOI":"10.1007\/0-306-48416-1_93","article-title":"A new selective medium provides improved growth and recoverability of Yersinia pestis","volume":"529","author":"Ber","year":"2003","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"5787","DOI":"10.1128\/AEM.69.10.5787-5792.2003","article-title":"Development of an improved selective agar medium for isolation of Yersinia pestis","volume":"69","author":"Ber","year":"2003","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_10","first-page":"56","article-title":"Simultaneous Immunodetection of Anthrax, Plague, and Tularemia from Blood Cultures by Use of Multiplexed Suspension Arrays","volume":"4","author":"Mechaly","year":"2018","journal-title":"J. Clin. Microbiol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Mechaly, A., Vitner, E., Levy, Y., Gur, D., Barlev-Gross, M., Sittner, A., Koren, M., Levy, H., Mamroud, E., and Fisher, M. (2021). Epitope Binning of Novel Monoclonal Anti F1 and Anti LcrV Antibodies and Their Application in a Simple, Short, HTRF Test for Clinical Plague Detection. Pathogens, 10.","DOI":"10.3390\/pathogens10030285"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Moses, S., Aftalion, M., Mamroud, E., Rotem, S., and Steinberger-Levy, I. (2021). Reporter-Phage-Based Detection and Antibiotic Susceptibility Testing of Yersinia pestis for a Rapid Plague Outbreak Response. Microorganisms, 9.","DOI":"10.3390\/microorganisms9061278"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Moses, S., Aftalion, M., Mamroud, E., Rotem, S., and Steinberger-Levy, I. (2021). Characterization of Yersinia pestis Phage Lytic Activity in Human Whole Blood for the Selection of Efficient Therapeutic Phages. Viruses, 13.","DOI":"10.3390\/v13010089"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2998","DOI":"10.1128\/JCM.00316-14","article-title":"Rapid detection and simultaneous antibiotic susceptibility analysis of Yersinia pestis directly from clinical specimens by use of reporter phage","volume":"52","author":"Vandamm","year":"2014","journal-title":"J. Clin. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"FSO476","DOI":"10.2144\/fsoa-2020-0013","article-title":"A rapid high-throughput sequencing-based approach for the identification of unknown bacterial pathogens in whole blood","volume":"6","author":"Israeli","year":"2020","journal-title":"Futur. Sci. OA"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"29478","DOI":"10.1074\/jbc.M303381200","article-title":"Global iron-dependent gene regulation in Escherichia coli. A new mechanism for iron homeostasis","volume":"278","author":"McHugh","year":"2003","journal-title":"J. Biol. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1128\/CMR.6.2.137","article-title":"Role of iron in regulation of virulence genes","volume":"6","author":"Litwin","year":"1993","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"946","DOI":"10.1038\/nrmicro1046","article-title":"Iron and microbial infection","volume":"2","author":"Schaible","year":"2004","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"277","DOI":"10.3389\/fcimb.2017.00277","article-title":"Host Iron Nutritional Immunity Induced by a Live Yersinia pestis Vaccine Strain Is Associated with Immediate Protection against Plague","volume":"7","author":"Zauberman","year":"2017","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref_20","first-page":"92","article-title":"Effect of iron on the growth of virulent plague microbes","volume":"32","author":"Avanian","year":"1961","journal-title":"J. Mikrobiol. Epidemiol. Immunobiol."},{"key":"ref_21","first-page":"3","article-title":"An increase in the virulence of Yersinia pestis bacteria when the cells are incubated in hemolyzed human erythrocytes","volume":"1993","author":"Kravtsov","year":"1993","journal-title":"J. Mikrobiol. Epidemiol. Immunobiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"85","DOI":"10.7883\/yoken1952.25.85","article-title":"The effect of an iron drug on the growth of plague microorganisms in vitro and in guinea pig skin","volume":"25","author":"Tsukano","year":"1972","journal-title":"Jpn. J. Med. Sci. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1016\/0882-4010(87)90051-9","article-title":"Modulation of the low-calcium response in Yersinia pestis via plasmid-plasmid interaction","volume":"2","author":"Sample","year":"1987","journal-title":"Microb. Pathog."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1005","DOI":"10.1111\/j.1365-2958.1993.tb01644.x","article-title":"Regulation by Ca2+ in the Yersinia low-Ca2+ response","volume":"8","author":"Straley","year":"1993","journal-title":"Mol. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"5234","DOI":"10.1128\/iai.62.12.5234-5241.1994","article-title":"Physiological basis of the low calcium response in Yersinia pestis","volume":"62","author":"Fowler","year":"1994","journal-title":"Infect. Immun."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1016\/0882-4010(89)90070-3","article-title":"Expression of the low calcium response in Yersinia pestis","volume":"6","author":"Mehigh","year":"1989","journal-title":"Microb. Pathog."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2649","DOI":"10.1128\/jb.173.8.2649-2657.1991","article-title":"The Yersinia pestis V antigen is a regulatory protein necessary for Ca2(+)-dependent growth and maximal expression of low-Ca2+ response virulence genes","volume":"173","author":"Price","year":"1991","journal-title":"J. Bacteriol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1724","DOI":"10.1086\/421087","article-title":"Optimal testing parameters for blood cultures","volume":"38","author":"Cockerill","year":"2004","journal-title":"Clin. Infect. Dis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1093\/clinids\/23.1.40","article-title":"Current blood culture methods and systems: Clinical concepts, technology, and interpretation of results","volume":"23","author":"Weinstein","year":"1996","journal-title":"Clin. Infect. Dis."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"2711","DOI":"10.1128\/JCM.00059-07","article-title":"Relevance of routine use of the anaerobic blood culture bottle","volume":"45","author":"Grohs","year":"2007","journal-title":"J. Clin. Microbiol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"444","DOI":"10.1128\/CMR.10.3.444","article-title":"Update on detection of bacteremia and fungemia","volume":"10","author":"Reimer","year":"1997","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Shifman, O., Aminov, T., Aftalion, M., Gur, D., Cohen, H., Bar-David, E., Cohen, O., Mamroud, E., Levy, H., and Aloni-Grinstein, R. (2021). Evaluation of the European Committee on Antimicrobial Susceptibility Testing Guidelines for Rapid Antimicrobial Susceptibility Testing of Bacillus anthracis-, Yersinia pestis and Francisella tularensis-Positive Blood Cultures. Microorganisms, 9.","DOI":"10.3390\/microorganisms9051055"},{"key":"ref_33","first-page":"292","article-title":"Role of anaerobic blood culture in the simultaneous blood culture taking for the diagnosis of bacteremia","volume":"54","year":"2016","journal-title":"Rev. Med. Inst. Mex. Seguro. Soc."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Lafaurie, M., D\u2019Anglejan, E., Donay, J.L., Glotz, D., Sarfati, E., Mimoun, M., Legrand, M., Oksenhendler, E., Bagot, M., and Valade, S. (2020). Utility of anaerobic bottles for the diagnosis of bloodstream infections. BMC Infect. Dis., 20.","DOI":"10.1186\/s12879-020-4854-x"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"285","DOI":"10.3389\/fcimb.2019.00285","article-title":"Comparison of the Performance of Three Blood Culture Systems in a Chinese Tertiary-Care Hospital","volume":"9","author":"Li","year":"2019","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"2634","DOI":"10.1128\/jcm.35.10.2634-2638.1997","article-title":"Advantage of combining resin with lytic BACTEC blood culture media","volume":"35","author":"Rohner","year":"1997","journal-title":"J. Clin. Microbiol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1590\/S0036-46651999000600002","article-title":"Diagnosis of plague and identification of virulence markers in Yersinia pestis by multiplex-PCR","volume":"41","author":"Leal","year":"1999","journal-title":"Rev. Inst. Med. Trop."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1007\/978-94-024-0890-4_11","article-title":"Plague: Clinics, Diagnosis and Treatment","volume":"918","author":"Nikiforov","year":"2016","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1111\/j.1574-695X.2010.00687.x","article-title":"The search for early markers of plague: Evidence for accumulation of soluble Yersinia pestis LcrV in bubonic and pneumonic mouse models of disease","volume":"59","author":"Flashner","year":"2010","journal-title":"FEMS Immunol. Med. Microbiol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"6025","DOI":"10.1128\/JB.00654-10","article-title":"Calcium and iron regulate swarming and type III secretion in Vibrio parahaemolyticus","volume":"192","author":"Chodur","year":"2010","journal-title":"J. Bacteriol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"220","DOI":"10.1016\/j.diagmicrobio.2018.05.021","article-title":"The impact of blood culture identification by MALDI-TOF MS on the antimicrobial management of pediatric patients","volume":"92","author":"Bhavsar","year":"2018","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1016\/j.cmi.2016.08.009","article-title":"Impact of MALDI-TOF-MS-based identification directly from positive blood cultures on patient management: A controlled clinical trial","volume":"23","author":"Osthoff","year":"2017","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_43","unstructured":"(2022, February 10). CDC Web Site, Available online: https:\/\/www.cdc.gov\/plague\/healthcare\/clinicians.html."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1016\/j.trstmh.2008.11.026","article-title":"Epidemiological features of pneumonic plague outbreak in Himachal Pradesh, India","volume":"103","author":"Joshi","year":"2009","journal-title":"Trans. R. Soc. Trop. Med. Hyg."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1007\/s10895-009-0546-z","article-title":"Application of fluorescent nanocrystals (q-dots) for the detection of pathogenic bacteria by flow-cytometry","volume":"20","author":"Zahavy","year":"2010","journal-title":"J. Fluoresc."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Levy, Y., Flashner, Y., Zauberman, A., Tidhar, A., Aftalion, M., Lazar, S., Gur, D., Cohen, S., Shafferman, A., and Mamroud, E. (2010). Protection against Plague Afforded by Treatment with Polyclonal \u03b1LcrV and \u03b1F1 Antibodies, Springer.","DOI":"10.1007\/978-90-481-9054-6_29"}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/11\/2\/255\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:20:42Z","timestamp":1760134842000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/11\/2\/255"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,2,16]]},"references-count":46,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2022,2]]}},"alternative-id":["pathogens11020255"],"URL":"https:\/\/doi.org\/10.3390\/pathogens11020255","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,2,16]]}}}