Abstract
BackgroundIn 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.ResultsHere, 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. B. pseudomallei cell lengths were strain-, drug- and drug concentration-dependent.ConclusionsBoth 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.
Highlights
In Gram-negative species, β-lactam antibiotics target penicillin binding proteins (PBPs) resulting in morphological alterations of bacterial cells
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
We identify PBP homologs encoded in the B. pseudomallei genome which may represent the potential targets for β-lactams antibiotics and better explicate the antibacterial mechanisms of action
Summary
In Gram-negative species, β-lactam antibiotics target penicillin binding proteins (PBPs) resulting in morphological alterations of bacterial cells. Burkholderia pseudomallei is a Gram-negative, biothreat pathogen and the causative agent of melioidosis, an often fatal infectious disease for humans. Β-lactams are the most common treatment for bacterial infections and the class accounts for 70% of antibiotic prescriptions in the United States [5]. Melioidosis is a life-threatening human infection with case fatality rates that may exceed 70% as a result of ineffective antimicrobial therapy [7,8,9]. Naturally-acquired melioidosis infections are caused by inhalation, ingestion or exposure of broken skin to the pathogen Burkholderia pseudomallei. This disease is endemic across tropical areas and is estimated to account for ~ 165,000 human cases per year worldwide, ~ 89,000 of which result in death [8]. Awareness of melioidosis and research into B. pseudomallei is increasing due to the heavy disease burden and the biothreat potential [10]
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