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Antimicrobial Agents and Chemotherapy Sep 2019Antibiotic tolerance, the ability to temporarily sustain viability in the presence of bactericidal antibiotics, constitutes an understudied and yet potentially...
Antibiotic tolerance, the ability to temporarily sustain viability in the presence of bactericidal antibiotics, constitutes an understudied and yet potentially widespread cause of antibiotic treatment failure. We have previously shown that the Gram-negative pathogen can tolerate exposure to the typically bactericidal β-lactam antibiotics by assuming a spherical morphotype devoid of detectable cell wall material. However, it is unclear how widespread β-lactam tolerance is. Here, we tested a panel of clinically significant Gram-negative pathogens for their response to the potent, broad-spectrum carbapenem antibiotic meropenem. We show that clinical isolates of , , and , but not , exhibited moderate to high levels of tolerance of meropenem, both in laboratory growth medium and in human serum. Importantly, tolerance was mediated by cell wall-deficient spheroplasts, which readily recovered wild-type morphology and growth upon removal of antibiotic. Our results suggest that carbapenem tolerance is prevalent in clinically significant bacterial species, and we suggest that this could contribute to treatment failure associated with these organisms.
Topics: Amdinocillin; Anti-Bacterial Agents; Drug Tolerance; Enterobacter aerogenes; Enterobacter cloacae; Escherichia coli; Gram-Negative Bacterial Infections; Klebsiella pneumoniae; Meropenem; Microbial Sensitivity Tests; Spheroplasts
PubMed: 31285232
DOI: 10.1128/AAC.00756-19 -
Scanning 2017The preparation and observations of spheroplast W303 cells are described with Environmental Scanning Electron Microscope (ESEM). The spheroplasting conversion was...
The preparation and observations of spheroplast W303 cells are described with Environmental Scanning Electron Microscope (ESEM). The spheroplasting conversion was successfully confirmed qualitatively, by the evaluation of the morphological change between the normal W303 cells and the spheroplast W303 cells, and quantitatively, by determining the spheroplast conversion percentage based on the OD absorbance data. From the optical microscope observations as expected, the normal cells had an oval shape whereas spheroplast cells resemble a spherical shape. This was also confirmed under four different mediums, that is, yeast peptone-dextrose (YPD), sterile water, sorbitol-EDTA-sodium citrate buffer (SCE), and sorbitol-Tris-Hcl-CaCl (CaS). It was also observed that the SCE and CaS mediums had a higher number of spheroplast cells as compared to the YPD and sterile water mediums. The OD absorbance data also showed that the whole W303 cells were fully converted to the spheroplast cells after about 15 minutes. The observations of the normal and the spheroplast W303 cells were then performed under an environmental scanning electron microscope (ESEM). The normal cells showed a smooth cell surface whereas the spheroplast cells had a bleb-like surface after the loss of its integrity when removing the cell wall.
Topics: Culture Media; Microscopy; Microscopy, Electron, Scanning; Spheroplasts; Surface Properties; Yeasts
PubMed: 29109826
DOI: 10.1155/2017/8393578 -
Nature Communications Oct 2022Squalene-hopene cyclases are a highly valuable and attractive class of membrane-bound enzymes as sustainable biotechnological tools to produce aromas and bioactive...
Squalene-hopene cyclases are a highly valuable and attractive class of membrane-bound enzymes as sustainable biotechnological tools to produce aromas and bioactive compounds at industrial scale. However, their application as whole-cell biocatalysts suffer from the outer cell membrane acting as a diffusion barrier for the highly hydrophobic substrate/product, while the use of purified enzymes leads to dramatic loss of stability. Here we present an unexplored strategy for biocatalysis: the application of squalene-hopene-cyclase spheroplasts. By removing the outer cell membrane, we produce stable and substrate-accessible biocatalysts. These spheroplasts exhibit up to 100-fold higher activity than their whole-cell counterparts for the biotransformations of squalene, geranyl acetone, farnesol, and farnesyl acetone. Their catalytic ability is also higher than the purified enzyme for all high molecular weight terpenes. In addition, we introduce a concept for the carrier-free immobilization of spheroplasts via crosslinking, crosslinked spheroplasts. The crosslinked spheroplasts maintain the same catalytic activity of the spheroplasts, offering additional advantages such as recycling and reuse. These timely solutions contribute not only to harness the catalytic potential of the squalene-hopene cyclases, but also to make biocatalytic processes even greener and more cost-efficient.
Topics: Spheroplasts; Squalene; Farnesol; Acetone; Intramolecular Transferases; Terpenes
PubMed: 36271006
DOI: 10.1038/s41467-022-34030-0 -
Bioengineered Bugs 2010Transformation (i.e., genetic modification of a cell by the incorporation of exogenous DNA) is indispensable for manipulating fungi. Here, we review the transformation... (Review)
Review
Transformation (i.e., genetic modification of a cell by the incorporation of exogenous DNA) is indispensable for manipulating fungi. Here, we review the transformation methods for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris and Aspergillus species and discuss some common modifications to improve transformation efficiency. We also present a model of the mechanism underlying S. cerevisiae transformation, based on recent reports and the mechanism of transfection in mammalian systems. This model predicts that DNA attaches to the cell wall and enters the cell via endocytotic membrane invagination, although how DNA reaches the nucleus is unknown. Polyethylene glycol is indispensable for successful transformation of intact cells and the attachment of DNA and also possibly acts on the membrane to increase the transformation efficiency. Both lithium acetate and heat shock, which enhance the transformation efficiency of intact cells but not that of spheroplasts, probably help DNA to pass through the cell wall.
Topics: Acetates; Cell Wall; DNA, Fungal; Electroporation; Endocytosis; Fungi; Polyethylene Glycols; Saccharomyces cerevisiae; Spheroplasts; Transformation, Genetic
PubMed: 21468206
DOI: 10.4161/bbug.1.6.13257 -
Applied and Environmental Microbiology Oct 2019A viability quantitative PCR (qPCR) utilizing propidium monoazide (PMA) is presented for rapid quantification of viable cells using the foodborne pathogen as a...
A viability quantitative PCR (qPCR) utilizing propidium monoazide (PMA) is presented for rapid quantification of viable cells using the foodborne pathogen as a bacterial model. It includes optimized spheroplast formation via lysozyme and EDTA, induction of a mild osmotic shock for enhancing the selective penetration of PMA into dead cells, and exploitation of an internal sample process control (ISPC) involving cell inactivation to assess residual false-positive signals within each sample. Spheroplasting of bacteria in exponential phase did not permit PMA entrance into viable cells since a strong linear relationship was detected between simple qPCR and PMA-qPCR quantification, and no differences were observed regardless of whether spheroplasting was utilized. The PMA-qPCR signal suppression of dead cells was elevated using spheroplast formation. With regard to the ISPC, cell inactivation by hydrogen peroxide resulted in higher signal suppression during qPCR than heat inactivation did. Viability quantification of cells by optimized spheroplasting-PMA-qPCR with ISPC was successfully applied in an aging pure culture under aerobic conditions and artificially inoculated meat. The same method exhibited a high linear range of quantification (1.5 to 8.5 log viable cells ml), and results were highly correlated with culture-based enumeration. PMA-qPCR quantification of viable cells can be affected by their rigidity, age, culture media, and niches, but spheroplast formation along with osmotic shock and the use of a proper ISPC can address such variations. The developed methodology could detect cells in a viable-but-nonculturable state and might be utilized for the quantification of other Gram-negative bacteria. There is need for rapid and accurate methods to detect viable bacterial cells of foodborne pathogens. Conventional culture-based methods are time-consuming and unable to detect bacteria in a viable-but-nonculturable state. The high sensitivity and specificity of the quantitative PCR (qPCR) are negated by its inability to differentiate the DNAs from viable and dead cells. The combination of propidium monoazide (PMA), a DNA-intercalating dye, with qPCR assays is promising for detection of viable cells. Despite encouraging results, these assays still encounter various challenges, such as false-positive signals by dead cells and the lack of an internal control identifying these signals per sample. The significance of our research lies in enhancing the selective entrance of PMA into dead cells via spheroplasting and in developing an internal sample process control, thus delivering reliable results in pure cultures and meat samples, approaches that can be applicable to other Gram-negative pathogens.
Topics: Azides; Campylobacter coli; Food Microbiology; Meat; Microbial Viability; Propidium; Real-Time Polymerase Chain Reaction; Spheroplasts
PubMed: 31420339
DOI: 10.1128/AEM.01499-19 -
Viruses Apr 2021Sindbis virus (SINV), a positive-sense single stranded RNA virus that causes mild symptoms in humans, is transmitted by mosquito bites. SINV reverse genetics have many...
Sindbis virus (SINV), a positive-sense single stranded RNA virus that causes mild symptoms in humans, is transmitted by mosquito bites. SINV reverse genetics have many implications, not only in understanding alphavirus transmission, replication cycle, and virus-host interactions, but also in biotechnology and biomedical applications. The rescue of SINV infectious particles is usually achieved by transfecting susceptible cells (BHK-21) with SINV-infectious mRNA genomes generated from cDNA constructed via in vitro translation (IVT). That procedure is time consuming, costly, and relies heavily on reagent quality. Here, we constructed a novel infectious SINV cDNA construct that expresses its genomic RNA in yeast cells controlled by galactose induction. Using spheroplasts made from this yeast, we established a robust polyethylene glycol-mediated yeast: BHK-21 fusion protocol to rescue infectious SINV particles. Our approach is timesaving and utilizes common lab reagents for SINV rescue. It could be a useful tool for the rescue of large single strand RNA viruses, such as SARS-CoV-2.
Topics: Alphavirus Infections; Animals; COVID-19; Cell Fusion; DNA, Complementary; Host Microbial Interactions; RNA, Viral; SARS-CoV-2; Saccharomyces cerevisiae; Sindbis Virus; Spheroplasts; Yeasts
PubMed: 33916100
DOI: 10.3390/v13040603 -
International Journal of Molecular... Sep 2020Cell enlargement is essential for the microinjection of various substances into bacterial cells. The cell wall (peptidoglycan) inhibits cell enlargement. Thus, bacterial... (Review)
Review
Cell enlargement is essential for the microinjection of various substances into bacterial cells. The cell wall (peptidoglycan) inhibits cell enlargement. Thus, bacterial protoplasts/spheroplasts are used for enlargement because they lack cell wall. Though bacterial species that are capable of gene manipulation are limited, procedure for bacterial cell enlargement does not involve any gene manipulation technique. In order to prevent cell wall resynthesis during enlargement of protoplasts/spheroplasts, incubation media are supplemented with inhibitors of peptidoglycan biosynthesis such as penicillin. Moreover, metal ion composition in the incubation medium affects the properties of the plasma membrane. Therefore, in order to generate enlarged cells that are suitable for microinjection, metal ion composition in the medium should be considered. Experiment of bacterial protoplast or spheroplast enlargement is useful for studies on bacterial plasma membrane biosynthesis. In this paper, we have summarized the factors that influence bacterial cell enlargement.
Topics: Bacteria; Cell Enlargement; Cell Membrane; Cell Wall; Culture Media; Ions; Metals; Osmotic Pressure; Penicillins; Peptidoglycan; Protein Biosynthesis; Protoplasts; Spheroplasts
PubMed: 32992574
DOI: 10.3390/ijms21197131 -
Journal of Bacteriology Jun 2013
Topics: Cell Wall; Escherichia coli; Escherichia coli Proteins; Peptidoglycan; Spheroplasts; Stress, Physiological
PubMed: 23543717
DOI: 10.1128/JB.00306-13 -
Thorax Aug 1997
Review
Topics: Animals; DNA, Bacterial; Disease Models, Animal; Humans; Mycobacterium; Mycobacterium Infections; Sarcoidosis; Spheroplasts
PubMed: 9381427
DOI: 10.1136/thx.52.2008.s47 -
Scandinavian Journal of Immunology Jul 2015Various strategies adapted to develop an efficient vaccine against foodborne pathogen, Listeria monocytogenes, have met with little success. Spheroplasts (bacterial cell... (Comparative Study)
Comparative Study
Various strategies adapted to develop an efficient vaccine against foodborne pathogen, Listeria monocytogenes, have met with little success. Spheroplasts (bacterial cell devoid of cell wall) are likely to undergo membrane-membrane fusion, leading to the delivery of their content to the cytosol of antigen-presenting cells, thus facilitating MHC class I antigen processing and presentation. In this study, we evaluated the prophylactic potential of Listeria spheroplast-based vaccine against experimental murine listeriosis in comparison with heat-killed Listeria (HKL) and archaeosome-entrapped Listeria whole-cell protein (LWCP). Compared with HKL, the spheroplast-based vaccine was found to evoke better Th1 response as exhibited by the presence of type 1 cytokines in the host (interferon-γ and IL-12) and a high IgG2a /IgG1 ratio. Robust lympho-proliferative efficacy was apparent in both spheroplast-immunized and archaeosome-entrapped LWCP-immunized groups. The upregulation of costimulatory and effector memory markers upon immunization with spheroplasts was found to be at par with that evoked by archaeosome-entrapped LWCP-immunized group. Central memory response in gated CD8(+) T cell was much higher in spheroplast-immunized animals when compared with archaeosome-entrapped LWCP group. The data presented here clearly demonstrate that spheroplasts evoked a robust immune response and offer better prophylactic potential against L. monocytogenes.
Topics: Animals; Antigens, Bacterial; Bacterial Vaccines; CD8-Positive T-Lymphocytes; Cell Wall; Disease Models, Animal; Female; Hypersensitivity, Delayed; Immunization; Immunoglobulin Class Switching; Immunoglobulin G; Interferon-gamma; Interleukin-12; Interleukin-4; Listeria monocytogenes; Listeriosis; Mice; Mice, Inbred BALB C; Spheroplasts; Th1 Cells; Vaccines, Inactivated
PubMed: 25833403
DOI: 10.1111/sji.12296