The exclusive use of flow cytometry to evaluate the antibiotic-susceptibility

• Published in: Biochimica et Biophysica Acta (BBA) - General Subjects Vol. 1820; no. 12; pp. 1980 - 1986
• Main Authors: Soejima, Takashi, Minami, Jun-ichi, Iwatsuki, Keiji
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2012; Elsevier BV
• Subjects: Affinity Labels; Affinity Labels - pharmacology; Anti-Bacterial Agents; Anti-Bacterial Agents - pharmacology; antibiotics; Azides; Azides - pharmacology; Bacteremia; Bacteremia - drug therapy; Bacteremia - metabolism; Bacteremia - microbiology; blood; cell membranes; Diagnosis; DNA; DNA, Bacterial; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; EMA; Erythrocytes; Erythrocytes - drug effects; Erythrocytes - metabolism; Erythrocytes - microbiology; ethidium; FCM; Flow Cytometry; Flow Cytometry - methods; Gram-positive bacteria; Granulocytes; Granulocytes - drug effects; Granulocytes - metabolism; Granulocytes - microbiology; Humans; Infant; infants; Injured; Light; Listeria monocytogenes; Listeria monocytogenes - drug effects; Listeria monocytogenes - growth & development; Live; Lymphocytes; Lymphocytes - drug effects; Lymphocytes - metabolism; Lymphocytes - microbiology; Microbial Viability; Microbial Viability - drug effects; Monocytes; Monocytes - drug effects; Monocytes - metabolism; Monocytes - microbiology; pathogens; patients; Photoaffinity Labels; rapid methods; Live; EMA; FCM; Injured; Diagnosis; Bacteremia
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2012.09.003

Live and injured bacteria cannot be successfully discriminated using flow cytometric methods (FCM) with commercial live/dead staining agents because injured cells have intact cell membranes and are counted as live cells. We previously reported that photoactivated ethidium monoazide (EMA) directly cleaves bacterial DNA both in vivo and in vitro (Microbiol. Immunol. 51:763-775, 2007). We report that EMA cleaves the chromosomal DNA of antibiotic-injured, but not live, Listeria monocytogenes. The combination of FCM and EMA treatment was evaluated as a rapid method to discriminate between live and antibiotic-injured L. monocytogenes. Additionally, we evaluated our methodology using blood from pediatric patients infected with other gram-negative and gram-positive bacteria. For antibiotic-injured, but not live, L. monocytogenes in blood, photoactivated EMA suppressed SYTO9 staining, as the SYTO9 staining of the antibiotic-injured L. monocytogenes was weak compared with that of live cells. Similarly, the rapid and clear discrimination between live and injured bacteria (gram-negative and gram-positive) was performed using the blood of pediatric patients administered antibiotics. The combination of FCM with EMA treatment is a rapid method for evaluating the susceptibility of live pathogens in infants with bacteremia without the need for bacterial culture. This assay is more rapid than other currently available techniques due to the elimination of the time-consuming culture step and could be used in clinical settings to rapidly determine the success of antibiotic treatment in pediatric bacteremia through the discrimination of injured (i.e., susceptible to the administered antibiotics) and live pathogens. ► Live and injured bacteria cannot be evaluated by a routine flow cytometry alone. ► EMA cleaves the DNA of antibiotic-injured, but not live, Listeria monocytogenes. ► EMA-FCM distinguishes live and injured pathogen in pediatric bacteremia patient. ► EMA-FCM allows physicians to rapidly evaluate antibiotic-susceptibility. ► Physicians can rapidly determine if antibiotic treatment is successful.