Bacterial Cytological Profiling (BCP) as a Rapid and Accurate Antimicrobial Susceptibility Testing Method for Staphylococcus aureus

Successful treatment of bacterial infections requires the timely administration of appropriate antimicrobial therapy. The failure to initiate the correct therapy in a timely fashion results in poor clinical outcomes, longer hospital stays, and higher medical costs. Current approaches to antibiotic susceptibility testing of cultured pathogens have key limitations ranging from long run times to dependence on prior knowledge of genetic mechanisms of resistance. We have developed a rapid antimicrobial susceptibility assay for Staphylococcus aureus based on bacterial cytological profiling (BCP), which uses quantitative fluorescence microscopy to measure antibiotic induced changes in cellular architecture. BCP discriminated between methicillin-susceptible (MSSA) and -resistant (MRSA) clinical isolates of S. aureus (n = 71) within 1–2 h with 100% accuracy. Similarly, BCP correctly distinguished daptomycin susceptible (DS) from daptomycin non-susceptible (DNS) S. aureus strains (n = 20) within 30 min. Among MRSA isolates, BCP further identified two classes of strains that differ in their susceptibility to specific combinations of beta-lactam antibiotics. BCP provides a rapid and flexible alternative to gene-based susceptibility testing methods for S. aureus, and should be readily adaptable to different antibiotics and bacterial species as new mechanisms of resistance or multidrug-resistant pathogens evolve and appear in mainstream clinical practice.

[1]  R. Humphries,et al.  A Current Perspective on Daptomycin for the Clinical Microbiologist , 2013, Clinical Microbiology Reviews.

[2]  H. Sahl,et al.  Mechanisms of daptomycin resistance in Staphylococcus aureus: role of the cell membrane and cell wall , 2013, Annals of the New York Academy of Sciences.

[3]  K. Pogliano,et al.  Bacterial cytological profiling rapidly identifies the cellular pathways targeted by antibacterial molecules , 2013, Proceedings of the National Academy of Sciences.

[4]  M. Kawakami,et al.  Autolysis of methicillin-resistant Staphylococcus aureus is involved in synergism between imipenem and cefotiam , 1995, Antimicrobial agents and chemotherapy.

[5]  T. Nakae,et al.  Characterization of oxacillin-susceptible mecA-positive Staphylococcus aureus: a new type of MRSA , 2007, Journal of infection and chemotherapy : official journal of the Japan Society of Chemotherapy.

[6]  D. Levine,et al.  Vancomycin: a history. , 2006, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[7]  Tatsuo Yamamoto,et al.  A New Local Variant (ST764) of the Globally Disseminated ST5 Lineage of Hospital-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) Carrying the Virulence Determinants of Community-Associated MRSA , 2013, Antimicrobial Agents and Chemotherapy.

[8]  Mary Jane Ferraro,et al.  Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically : approved standard , 2000 .

[9]  A. Wollam,et al.  Whole-Genome Analyses of Enterococcus faecium Isolates with Diverse Daptomycin MICs , 2014, Antimicrobial Agents and Chemotherapy.

[10]  Michael D. Burkart,et al.  Azithromycin Synergizes with Cationic Antimicrobial Peptides to Exert Bactericidal and Therapeutic Activity Against Highly Multidrug-Resistant Gram-Negative Bacterial Pathogens , 2015, EBioMedicine.

[11]  Alex van Belkum,et al.  The role of nasal carriage in Staphylococcus aureus infections. , 2005, The Lancet. Infectious diseases.

[12]  Teruyo Ito,et al.  Cloning and Nucleotide Sequence Determination of the Entire mec DNA of Pre-Methicillin-Resistant Staphylococcus aureus N315 , 1999, Antimicrobial Agents and Chemotherapy.

[13]  L. Rice.  Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. , 2008, The Journal of infectious diseases.

[14]  C. Nast,et al.  Phenotypic and Genotypic Characterization of Daptomycin-Resistant Methicillin-Resistant Staphylococcus aureus Strains: Relative Roles of mprF and dlt Operons , 2014, PloS one.

[15]  G. K. Best,et al.  Evidence for Participation of Autolysins in Bactericidal Action of Oxacillin on Staphylococcus aureus , 1974, Antimicrobial Agents and Chemotherapy.

[16]  Dhanalakshmi R. Nair,et al.  Role of ArlRS in Autolysis in Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus Strains , 2011, Journal of bacteriology.

[17]  R. Kullar,et al.  Early use of daptomycin versus vancomycin for methicillin-resistant Staphylococcus aureus bacteremia with vancomycin minimum inhibitory concentration >1 mg/L: a matched cohort study. , 2013, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[18]  M. Zervos,et al.  Daptomycin versus vancomycin for bloodstream infections due to methicillin-resistant Staphylococcus aureus with a high vancomycin minimum inhibitory concentration: a case-control study. , 2012, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[19]  D. Stevens.  The role of vancomycin in the treatment paradigm. , 2006, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[20]  B. Kreiswirth,et al.  Global epidemiology of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA). , 2012, Current opinion in microbiology.

[21]  F. Lowy,et al.  Pathogenesis of methicillin-resistant Staphylococcus aureus infection. , 2008, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[22]  Daniel N. Wilson.  Ribosome-targeting antibiotics and mechanisms of bacterial resistance , 2013, Nature Reviews Microbiology.

[23]  J. C. Lee,et al.  A rapid antimicrobial susceptibility test based on single-cell morphological analysis , 2014, Science Translational Medicine.

[24]  G. Nimmo.  USA300 abroad: global spread of a virulent strain of community-associated methicillin-resistant Staphylococcus aureus. , 2012, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[25]  P. Ward,et al.  Clinical features associated with bacteremia due to heterogeneous vancomycin-intermediate Staphylococcus aureus. , 2004, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[26]  M. G. Pinho,et al.  Staphylococcus aureus PBP4 Is Essential for β-Lactam Resistance in Community-Acquired Methicillin-Resistant Strains , 2008, Antimicrobial Agents and Chemotherapy.

[27]  N. McCallum,et al.  Fitness Cost of SCCmec and Methicillin Resistance Levels in Staphylococcus aureus , 2004, Antimicrobial Agents and Chemotherapy.

[28]  M. Ferraro.  Performance standards for antimicrobial susceptibility testing , 2001 .

[29]  Vance G. Fowler,et al.  Staphylococcus aureus Infections: Epidemiology, Pathophysiology, Clinical Manifestations, and Management , 2015, Clinical Microbiology Reviews.

[30]  V. Nizet,et al.  Differential Effects of Penicillin Binding Protein Deletion on the Susceptibility of Enterococcus faecium to Cationic Peptide Antibiotics , 2015, Antimicrobial Agents and Chemotherapy.

[31]  F. Tenover,et al.  Using Rapid Diagnostic Tests to Optimize Antimicrobial Selection in Antimicrobial Stewardship Programs , 2012, Pharmacotherapy.

[32]  S. Metzger,et al.  Rapid antibiotic susceptibility phenotypic characterization of Staphylococcus aureus using automated microscopy of small numbers of cells. , 2014, Journal of microbiological methods.

[33]  Remy Elbez,et al.  Self-assembled magnetic bead biosensor for measuring bacterial growth and antimicrobial susceptibility testing. , 2012, Small.

[34]  Raoul Kopelman,et al.  Asynchronous magnetic bead rotation microviscometer for rapid, sensitive, and label-free studies of bacterial growth and drug sensitivity. , 2012, Analytical chemistry.

[35]  Andre Sharon,et al.  A microfluidic platform for rapid, stress-induced antibiotic susceptibility testing of Staphylococcus aureus. , 2012, Lab on a chip.

[36]  V. Nizet,et al.  Heterogeneity of Genetic Pathways toward Daptomycin Nonsusceptibility in Staphylococcus aureus Determined by Adjunctive Antibiotics , 2015, Antimicrobial Agents and Chemotherapy.

[37]  V. Nizet,et al.  Examining the Use of Ceftaroline in the Treatment of Streptococcus pneumoniae Meningitis with Reference to Human Cathelicidin LL-37 , 2015, Antimicrobial Agents and Chemotherapy.

[38]  M. Bonora,et al.  Evaluation of the VITEK 2 System for Identification and Antimicrobial Susceptibility Testing of Medically Relevant Gram-Positive Cocci , 2002, Journal of Clinical Microbiology.

[39]  L. Cui,et al.  Mutation of RNA Polymerase β-Subunit Gene Promotes Heterogeneous-to-Homogeneous Conversion of β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus , 2013, Antimicrobial Agents and Chemotherapy.

[40]  V. Nizet,et al.  In Vitro Activity of Daptomycin in Combination with β-Lactams, Gentamicin, Rifampin, and Tigecycline against Daptomycin-Nonsusceptible Enterococci , 2015, Antimicrobial Agents and Chemotherapy.

[41]  Yinduo Ji.  Methicillin-Resistant Staphylococcus aureus (MRSA) Protocols , 2007, Methods in Molecular Biology.

[42]  F. Vandenesch,et al.  MRSA Harboring mecA Variant Gene mecC, France , 2012, Emerging infectious diseases.

[43]  Anne E Carpenter,et al.  Improved structure, function and compatibility for CellProfiler: modular high-throughput image analysis software , 2011, Bioinform..

[44]  P. Pancholi,et al.  An antimicrobial stewardship program's impact with rapid polymerase chain reaction methicillin-resistant Staphylococcus aureus/S. aureus blood culture test in patients with S. aureus bacteremia. , 2010, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[45]  A. Tomasz,et al.  Complementation of the Essential Peptidoglycan Transpeptidase Function of Penicillin-Binding Protein 2 (PBP2) by the Drug Resistance Protein PBP2A in Staphylococcus aureus , 2001, Journal of bacteriology.

[46]  David J Payne,et al.  Challenges of antibacterial discovery revisited , 2010, Annals of the New York Academy of Sciences.

[47]  Pieter C Dorrestein,et al.  The Bacillus subtilis cannibalism toxin SDP collapses the proton motive force and induces autolysis , 2012, Molecular microbiology.

[48]  B. Levin,et al.  The biological cost of antibiotic resistance. , 1999, Current opinion in microbiology.

[49]  J. Pogliano,et al.  Defining Daptomycin Resistance Prevention Exposures in Vancomycin-Resistant Enterococcus faecium and E. faecalis , 2014, Antimicrobial Agents and Chemotherapy.

[50]  A. Rubio,et al.  Frequency and Distribution of Single-Nucleotide Polymorphisms within mprF in Methicillin-Resistant Staphylococcus aureus Clinical Isolates and Their Role in Cross-Resistance to Daptomycin and Host Defense Antimicrobial Peptides , 2015, Antimicrobial Agents and Chemotherapy.

[51]  Sunghoon Kwon,et al.  Rapid antibiotic susceptibility testing by tracking single cell growth in a microfluidic agarose channel system. , 2013, Lab on a chip.

[52]  Julian Parkhill,et al.  Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study , 2011, The Lancet. Infectious diseases.