A dynamic feedback algorithm of AFM based on cell morphology changes

AbstractThe cnidarian “planula” larva shows radial symmetry around a polarized, oral-aboral, body axis and comprises two epithelia cell layers, ectodermal and endodermal. This simple body plan is set up during gastrulation, a process which proceeds by a variety of modes amongst the diverse cnidarian species. In the hydrozoan laboratory model Clytia hemisphaerica, gastrulation involves a process termed unipolar cell ingression, in which the endoderm derives from mass ingression of individual cells via a process of epithelial-mesenchymal transition (EMT) around the future oral pole of an epithelial embryo. This contrasts markedly from the gastrulation mode in the anthozoan cnidarian Nematostella vectensis, in which endoderm formation primarily relies on cell sheet invagination. To understand the cellular basis of gastrulation in Clytia we have characterized in detail successive cell morphology changes during planula formation by Scanning and Transmission Electron Microscopy combined with confocal imaging. These changes successively accompany epithialization of the blastoderm, EMT occurring in the oral domain through the bottle cell formation and ingression, cohesive migration and intercalation of ingressed cells with mesenchymal morphology, and their epithelialization to form the endoderm. From our data, we have reconstructed the cascade of morphogenetic events leading to the formation of planula larva. We also matched the domains of cell morphology changes to the expression of selected regulatory and marker genes expressed during gastrulation. We propose that cell ingression in Clytia not only provides the endoderm, but generates internal forces that shape the embryo in the course of gastrulation. These observations help build a more complete understanding of the cellular basis of morphogenesis and of the evolutionary plasticity of cnidarian gastrulation modes.

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

10.1101/2020.01.13.904995 ◽

2020 ◽

Author(s):

Heather P. McLaughlin ◽

Julia Bugrysheva ◽

David Sue

Keyword(s):

Optical Microscopy ◽

Antimicrobial Susceptibility ◽

Cell Morphology ◽

Morphological Changes ◽

Cell Lysis ◽

Bacterial Cells ◽

Bacterial Strains ◽

Gram Negative ◽

Resistant Strains ◽

Morphology Changes

AbstractBackgroundIn 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. Length of B. pseudomallei cells was 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.

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