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Optical microscopy reveals the dynamic nature of B. pseudomallei morphology during β-lactam antimicrobial susceptibility testing

View ORCID ProfileHeather P. McLaughlin, Julia Bugrysheva, View ORCID ProfileDavid Sue
doi: https://doi.org/10.1101/2020.01.13.904995
Heather P. McLaughlin
Laboratory of Preparedness and Response Branch, Division of Preparedness and Emerging Infections, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, MS: H17-5, Atlanta, GA 30333, USA
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  • ORCID record for Heather P. McLaughlin
  • For correspondence: hmclaughlin@cdc.gov
Julia Bugrysheva
Laboratory of Preparedness and Response Branch, Division of Preparedness and Emerging Infections, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, MS: H17-5, Atlanta, GA 30333, USA
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David Sue
Laboratory of Preparedness and Response Branch, Division of Preparedness and Emerging Infections, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE, MS: H17-5, Atlanta, GA 30333, USA
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Abstract

Background In Gram-negative species, β-lactam antibiotics target penicillin binding proteins (PBPs) resulting in morphological alterations of bacterial cells. Observations of antibiotic-induced cell morphology changes can rapidly and accurately differentiate drug susceptible from resistant bacterial strains; however, resistant cells do not always remain unchanged. Burkholderia pseudomallei is a Gram-negative, biothreat pathogen and the causative agent of melioidosis, an often fatal infectious disease for humans.

Results Here, we identified β-lactam targets in B. pseudomallei by in silico analysis. Ten genes encoding putative PBPs, including PBP-1, PBP-2, PBP-3 and PBP-6, were detected in the genomes of susceptible and resistant strains. Real-time, live-cell imaging of B. pseudomallei strains demonstrated dynamic morphological changes in broth containing clinically relevant β-lactam antibiotics. At sub-inhibitory concentrations of ceftazidime (CAZ), amoxicillin-clavulanic acid (AMC), and imipenem (IPM), filamentation, varying in length and proportion, was an initial response of the multidrug-resistant strain Bp1651 in exponential phase. However, a dominant morphotype reemerged during stationary phase that resembled cells unexposed to antibiotics. Similar morphology dynamics were observed for AMC-resistant strains, MSHR1655 and 724644, when exposed to sub-inhibitory concentrations of AMC. For all B. pseudomallei strains evaluated, increased exposure time and exposure to increased concentrations of AMC at and above minimal inhibitory concentrations (MICs) in broth resulted in cell morphology shifts from filaments to spheroplasts and/or cell lysis. B. pseudomallei morphology changes were more consistent in IPM. Spheroplast formation followed by cell lysis was observed for all strains in broth containing IPM at concentrations greater than or equal to MICs, however, the time to cell lysis was variable. Length of B. pseudomallei cells was strain-, drug- and drug concentration-dependent.

Conclusions Both resistant and susceptible B. pseudomallei strains exhibited filamentation during early exposure to AMC and CAZ at concentrations used to interpret susceptibility (based on CLSI guidelines). While developing a rapid β-lactam antimicrobial susceptibility test based on cell-shape alone requires more extensive analyses, optical microscopy detected B. pseudomallei growth attributes that lend insight into antibiotic response and antibacterial mechanisms of action.

  • List of Abbreviations

    (AST)
    Antimicrobial susceptibility test,
    (BMD)
    broth microdilution,
    (CLSI)
    Clinical and Laboratory Standards Institute,
    (IPM)
    imipenem,
    (CAZ)
    ceftazidime,
    (AMC)
    amoxicillin-clavulanic acid,
    (MDR)
    multidrug-resistant,
    (R)
    resistant,
    (I)
    intermediate,
    (S)
    susceptible,
    (SBA)
    Trypticase Soy Agar II with 5% sheep blood,
    (CAMHB)
    cation-adjusted Mueller Hinton broth,
    (MIC)
    minimal inhibitory concentrations,
    (TES)
    N-tris(hydroxymethyl) methyl-2-aminoethanesulfonic acid,
    (SESA)
    Segmentation and Extraction Surface Area,
    (SD)
    standard deviation,
    (PBPs)
    penicillin-binding proteins.
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.
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    Posted January 14, 2020.
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    Optical microscopy reveals the dynamic nature of B. pseudomallei morphology during β-lactam antimicrobial susceptibility testing
    Heather P. McLaughlin, Julia Bugrysheva, David Sue
    bioRxiv 2020.01.13.904995; doi: https://doi.org/10.1101/2020.01.13.904995
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    Optical microscopy reveals the dynamic nature of B. pseudomallei morphology during β-lactam antimicrobial susceptibility testing
    Heather P. McLaughlin, Julia Bugrysheva, David Sue
    bioRxiv 2020.01.13.904995; doi: https://doi.org/10.1101/2020.01.13.904995

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