{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T00:07:17Z","timestamp":1785888437141,"version":"3.56.0"},"reference-count":44,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2020,2,29]],"date-time":"2020-02-29T00:00:00Z","timestamp":1582934400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100009045","name":"Jagiellonian University Medical College","doi-asserted-by":"publisher","award":["K\/ZDS\/007832"],"award-info":[{"award-number":["K\/ZDS\/007832"]}],"id":[{"id":"10.13039\/100009045","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Standard blood cultures require at least 24\u2013120 h to be reported as preliminary positive. The objective of this study was to compare the reliability of Gram staining and fluorescent in-situ hybridization (FISH) for detecting bacteria in otherwise negative blood culture bottles. Ninety-six sets were taken from patients with a diagnosis of sepsis. Six incomplete blood culture sets and eight blood cultures sets demonstrating positive growth were excluded. We performed Gram stain and FISH on 82 sets taken from post-operative septic patients: 82 negative aerobic blood cultures, 82 anaerobic blood cultures, and 82 blood samples, as well as 57 blood samples taken from healthy volunteers. From the eighty-two blood sets analyzed from the septic patients, Gram stain visualized bacteria in 62.2% of blood samples, 35.4% of the negative aerobic bottles, and in 31.7% of the negative anaerobic bottles. Utilizing FISH, we detected bacteria in 75.6%, 56.1%, and 64.6% respectively. Among the blood samples from healthy volunteers, FISH detected bacteria in 64.9%, while Gram stain detected bacteria in only 38.6%. The time needed to obtain the study results using Gram stain was 1 h, for FISH 4 h, and for the culture method, considering the duration of growth, 5 days. Gram stain and FISH allow quick detection of bacteria in the blood taken directly from a patient. Finding phagocytosed bacteria, which were also detected among healthy individuals, confirms the hypothesis that blood microbiome exists.<\/jats:p>","DOI":"10.3390\/microorganisms8030346","type":"journal-article","created":{"date-parts":[[2020,3,2]],"date-time":"2020-03-02T06:34:23Z","timestamp":1583130863000},"page":"346","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["Classical Microbiological Diagnostics of Bacteremia: Are the Negative Results Really Negative? What is the Laboratory Result Telling Us About the \u201cGold Standard\u201d?"],"prefix":"10.3390","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7964-3911","authenticated-orcid":false,"given":"Tomasz","family":"\u0179r\u00f3d\u0142owski","sequence":"first","affiliation":[{"name":"Thoracic Anesthesia and Respiratory Intensive Care Unit, John Paul II Hospital, 31- 202 Krak\u00f3w, Poland"},{"name":"Department of Internal Medicine, St. John\u2019s Episcopal Hospital, Far Rockaway, NY 11691, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8496-4071","authenticated-orcid":false,"given":"Joanna","family":"Sobo\u0144ska","sequence":"additional","affiliation":[{"name":"Department of Molecular Medical Microbiology, Chair of Microbiology, Faculty of Medicine, Jagiellonian University Medical College, 31-121 Krakow, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5974-5520","authenticated-orcid":false,"given":"Dominika","family":"Salamon","sequence":"additional","affiliation":[{"name":"Department of Molecular Medical Microbiology, Chair of Microbiology, Faculty of Medicine, Jagiellonian University Medical College, 31-121 Krakow, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabel M.","family":"McFarlane","sequence":"additional","affiliation":[{"name":"Department of Medicine, SUNY Downstate Medical Center, Brooklyn, NY 11203, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miros\u0142aw","family":"Zi\u0119tkiewicz","sequence":"additional","affiliation":[{"name":"Thoracic Anesthesia and Respiratory Intensive Care Unit, John Paul II Hospital, 31- 202 Krak\u00f3w, Poland"},{"name":"Department of Anesthesiology and Intensive Care, Faculty of Medicine, Jagiellonian University Medical College, 31-501 Krakow, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4725-5943","authenticated-orcid":false,"given":"Tomasz","family":"Gosiewski","sequence":"additional","affiliation":[{"name":"Department of Molecular Medical Microbiology, Chair of Microbiology, Faculty of Medicine, Jagiellonian University Medical College, 31-121 Krakow, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,2,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"326","DOI":"10.1007\/s00101-008-1345-7","article-title":"Molecular biological detection of pathogens in patients with sepsis. Potentials, limitations and perspectives","volume":"57","author":"Hunfeld","year":"2008","journal-title":"Anaesthesist"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"801","DOI":"10.1001\/jama.2016.0287","article-title":"The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3)","volume":"315","author":"Singer","year":"2016","journal-title":"JAMA"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"519","DOI":"10.5694\/j.1326-5377.2011.tb03088.x","article-title":"Sepsis in the tropical Top End of Australia\u2019s Northern Territory: Disease burden and impact on Indigenous Australians","volume":"194","author":"Davis","year":"2011","journal-title":"Med. J. Aust."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"(1990). Increase in National Hospital Discharge Survey Rates for Septicemia--United States. JAMA, 263, 937\u2013938.","DOI":"10.1001\/jama.263.7.937"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1164\/rccm.201504-0781OC","article-title":"Assessment of global incidence and mortality of hospital-treated sepsis current estimates and limitations","volume":"193","author":"Fleischmann","year":"2016","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1251","DOI":"10.1016\/j.chest.2016.08.1460","article-title":"Culture-Negative Severe Sepsis","volume":"150","author":"Gupta","year":"2016","journal-title":"Chest"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1159\/000092186","article-title":"Comparative evaluation of BacT\/ALERT 3D and BACTEC systems for the recovery of pathogens causing bloodstream infections","volume":"15","author":"Jamal","year":"2006","journal-title":"Med. Princ. Pract."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1159\/000315915","article-title":"Endotoxins and other sepsis triggers","volume":"167","author":"Opal","year":"2010","journal-title":"Contrib. Nephrol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Dickson, K., and Lehmann, C. (2019). Inflammatory Response to Different Toxins in Experimental Sepsis Models. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20184341"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"e1","DOI":"10.1093\/cid\/ciw326","article-title":"Practice Guidelines for the Diagnosis and Management of Aspergillosis: 2016 Update by the Infectious Diseases Society of America","volume":"63","author":"Patterson","year":"2016","journal-title":"Clin. Infect. Dis."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Gosiewski, T., Flis, A., Sroka, A., Kedzierska, A., Pietrzyk, A., Kedzierska, J., Drwila, R., and Bulanda, M. (2014). Comparison of nested, multiplex, qPCR; FISH; SeptiFast and blood culture methods in detection and identification of bacteria and fungi in blood of patients with sepsis. BMC Microbiol., 14.","DOI":"10.1186\/s12866-014-0313-4"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1007\/s10096-016-2805-7","article-title":"Comprehensive detection and identification of bacterial DNA in the blood of patients with sepsis and healthy volunteers using next-generation sequencing method - the observation of DNAemia","volume":"36","author":"Gosiewski","year":"2017","journal-title":"Eur. J. Clin. Microbiol. Infect. Dis."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"\u0179r\u00f3d\u0142owski, T.W., Jurkiewicz-Badacz, D., Sroka-Oleksiak, A., Salamon, D., Bulanda, M., and Gosiewski, T. (2018). Comparison of PCR, fluorescent in situ hybridization and blood cultures for detection of bacteremia in children and adolescents during antibiotic therapy. Polish J. Microbiol., 67.","DOI":"10.21307\/pjm-2018-056"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Gosiewski, T., Jurkiewicz-Badacz, D., Sroka, A., Brzychczy-W\u0142och, M., and Bulanda, M. (2014). A novel, nested, multiplex, real-time PCR for detection of bacteria and fungi in blood. BMC Microbiol., 14.","DOI":"10.1186\/s12866-014-0313-4"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"888","DOI":"10.1515\/CCLM.2008.157","article-title":"Rapid methods for diagnosis of bloodstream infections","volume":"46","author":"Klouche","year":"2008","journal-title":"Clin. Chem. Lab. Med."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"O468","DOI":"10.1111\/1469-0691.12490","article-title":"Comparison of peptide nucleic acid fluorescence in situ hybridization assays with culture-based matrix-assisted laser desorption\/ionization-time of flight mass spectrometry for the identification of bacteria and yeasts from blood cultures and cerebrospina","volume":"8","author":"Calderaro","year":"2014","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1111\/j.1439-0507.2011.02166.x","article-title":"Evaluation of the peptide nucleic acid fluorescence in situ hybridisation technology for yeast identification directly from positive blood cultures: An Italian experience","volume":"55","author":"Farina","year":"2012","journal-title":"Mycoses"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1016\/j.mimet.2013.09.004","article-title":"V PNA-FISH assays for early targeted bacteraemia treatment","volume":"95","author":"Parcell","year":"2013","journal-title":"J. Microbiol. Methods"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1687","DOI":"10.1007\/s10096-014-2153-4","article-title":"Developments for improved diagnosis of bacterial bloodstream infections","volume":"33","author":"Loonen","year":"2014","journal-title":"Eur. J. Clin. Microbiol. Infect. Dis."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"122","DOI":"10.20452\/pamw.3949","article-title":"Fluorescent in situ hybridization and Gram-stained smears of whole blood as complementary screening tools in the diagnosis of sepsis","volume":"127","author":"Flis","year":"2017","journal-title":"Polish Arch. Intern. Med."},{"key":"ref_21","unstructured":"Gosiewski, T., and Brzychczy, M. (2018). Gosiewski Method for efficient isolation of microbial dna from blood. (US9879311 (B2)), United States Patent."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"149","DOI":"10.1007\/s00284-013-0451-1","article-title":"Comparison of Methods for Isolation of Bacterial and Fungal DNA from Human Blood","volume":"68","author":"Gosiewski","year":"2014","journal-title":"Curr. Microbiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1919","DOI":"10.1128\/aem.56.6.1919-1925.1990","article-title":"Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations","volume":"56","author":"Amann","year":"1990","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"830","DOI":"10.1128\/JCM.38.2.830-838.2000","article-title":"Fluorescent In situ hybridization allows rapid identification of microorganisms in blood cultures","volume":"38","author":"Kempf","year":"2000","journal-title":"J. Clin. Microbiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1046\/j.1462-2920.2003.00397.x","article-title":"Microbial community and physicochemical analysis of an industrial waste gas biofilter and design of 16S rRNA-targeting oligonucleotide probes","volume":"5","author":"Friedrich","year":"2003","journal-title":"Environ. Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1620","DOI":"10.1007\/s00134-015-3906-y","article-title":"The Surviving Sepsis Campaign bundles and outcome: Results from the International Multicentre Prevalence Study on Sepsis (the IMPreSS study)","volume":"41","author":"Rhodes","year":"2015","journal-title":"Intensiv. Care Med."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"567","DOI":"10.1093\/femsre\/fuv013","article-title":"The dormant blood microbiome in chronic, inflammatory diseases","volume":"39","author":"Potgieter","year":"2015","journal-title":"FEMS Microbiol. Rev."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1126","DOI":"10.1111\/j.1469-0691.2011.03690.x","article-title":"Detection and identification of plasma bacterial and viral elements in HIV\/AIDS patients in comparison to healthy adults","volume":"18","author":"Li","year":"2012","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"5","DOI":"10.3389\/fcimb.2018.00005","article-title":"Identification and Characterization of Blood and Neutrophil-Associated Microbiomes in Patients with Severe Acute Pancreatitis Using Next-Generation Sequencing","volume":"8","author":"Li","year":"2018","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"621","DOI":"10.1128\/iai.15.2.621-627.1977","article-title":"Novel bacterial structures in human blood: Cultural isolation","volume":"15","author":"Domingue","year":"1977","journal-title":"Infect. Immun."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"458","DOI":"10.1007\/BF01935925","article-title":"Electron microscopical evidence of the evolution of corynebacteria-like microorganisms within human erythrocytes","volume":"34","author":"Tedeschi","year":"1978","journal-title":"Experientia"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1111\/j.1365-2958.2011.07922.x","article-title":"Bacterial L-forms on tap: An improved methodology to generate Bacillus subtilis L-forms heralds a new era of research","volume":"83","author":"Devine","year":"2012","journal-title":"Mol. Microbiol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e00089-17","DOI":"10.1128\/CMR.00089-17","article-title":"Emerging technologies for molecular diagnosis of sepsis","volume":"31","author":"Sinha","year":"2018","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1172\/JCI82224","article-title":"Sepsis-induced immune dysfunction: Can immune therapies reduce mortality?","volume":"126","author":"Delano","year":"2016","journal-title":"J. Clin. Investig."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1174","DOI":"10.1097\/TA.0b013e31824d1085","article-title":"Risk factors for central line-associated bloodstream infections in the era of best practice","volume":"72","author":"Lissauer","year":"2012","journal-title":"J. Trauma Acute Care Surg."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"577","DOI":"10.1016\/j.ajic.2017.12.012","article-title":"Healthcare-associated infections studies project: An American Journal of Infection Control and National Healthcare Safety Network data quality collaboration: Location mapping","volume":"46","author":"Wright","year":"2018","journal-title":"Am. J. Infect. Control"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1539","DOI":"10.1007\/s10096-015-2383-0","article-title":"Performance of Gram staining on blood cultures flagged negative by an automated blood culture system","volume":"34","author":"Peretz","year":"2015","journal-title":"Eur. J. Clin. Microbiol. Infect. Dis."},{"key":"ref_38","first-page":"257","article-title":"Evaluation of negative results of BacT\/Alert 3D automated blood culture system","volume":"43","author":"Kocoglu","year":"2005","journal-title":"J. Microbiol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1016\/j.ajic.2018.09.013","article-title":"Role of transfusions in the development of hospital-acquired urinary tract-related bloodstream infection among United States Veterans","volume":"47","author":"Greene","year":"2019","journal-title":"Am. J. Infect. Control"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Watanabe, N., Kryukov, K., Nakagawa, S., Takeuchi, J.S., Takeshita, M., Kirimura, Y., Mitsuhashi, S., Ishihara, T., Aoki, H., and Inokuchi, S. (2018). Detection of pathogenic bacteria in the blood from sepsis patients using 16S rRNA gene amplicon sequencing analysis. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0202049"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"R60","DOI":"10.1186\/cc4894","article-title":"Plasma DNA concentration as a predictor of mortality and sepsis in critically ill patients","volume":"10","author":"Rhodes","year":"2006","journal-title":"Crit. Care"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"R116","DOI":"10.1186\/cc13908","article-title":"Prognostic and diagnostic value of eosinopenia, C-reactive protein, procalcitonin, and circulating cell-free DNA in critically ill patients admitted with suspicion of sepsis","volume":"18","author":"Macher","year":"2014","journal-title":"Crit. Care"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s13073-016-0326-8","article-title":"Next-generation sequencing diagnostics of bacteremia in septic patients","volume":"8","author":"Grumaz","year":"2016","journal-title":"Genome Med."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Ben\u00edtez-P\u00e1ez, A., \u00c1lvarez, M., Belda-Ferre, P., Rubido, S., Mira, A., and Tom\u00e1s, I. (2013). Detection of transient bacteraemia following dental extractions by 16S rDNA pyrosequencing: A pilot study. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0057782"}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/8\/3\/346\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:02:46Z","timestamp":1760173366000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/8\/3\/346"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,2,29]]},"references-count":44,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2020,3]]}},"alternative-id":["microorganisms8030346"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms8030346","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,2,29]]}}}