-
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 -
Journal of Clinical Microbiology Nov 1987Serratia marcescens was easily induced to form spheroplasts by beta-lactam antibiotics in the presence of Ca2+ or Mg2+ without an osmotic stabilizer such as sucrose. The...
Serratia marcescens was easily induced to form spheroplasts by beta-lactam antibiotics in the presence of Ca2+ or Mg2+ without an osmotic stabilizer such as sucrose. The spheroplasts grew in volume, although they could not divide. They were stable for more than 10 h at 37 degrees C in a medium containing a high concentration of antibiotic, and they had the ability to revert to the original bacillary form. Ca2+ was more effective in spheroplast induction than Mg2+. The effect was proportional to the concentration of cations. In 40% of 180 clinical isolates of S. marcescens, more than 40% of the original bacterial cells were induced to form spheroplasts by ceftizoxime in a medium supplemented with 40 mM Ca2+. A high spheroplast induction rate was observed even in medium with 10 mM Ca2+. Few isolates that were supersusceptible to ceftizoxime (MIC, less than 0.2 microgram/ml) were induced to form spheroplasts at a high rate. No difference in spheroplast induction rate or extent between antibiotic-resistant strains and relatively susceptible strains (MIC, greater than 0.2 microgram/ml) was found. The serotype of S. marcescens had no effect on the spheroplast induction rate. Monocations (Na+ and K+) had little effect on spheroplast induction.
Topics: Agglutination Tests; Anti-Bacterial Agents; Calcium; Culture Media; Drug Resistance, Microbial; Humans; Lactams; Magnesium; Microbial Sensitivity Tests; Microscopy, Electron, Scanning; Osmotic Pressure; Serotyping; Serratia marcescens; Spheroplasts
PubMed: 3320083
DOI: 10.1128/jcm.25.11.2154-2158.1987 -
Biophysical Journal Nov 2014We investigated the physical properties of bacterial cytoplasmic membranes by applying the method of micropipette aspiration to Escherichia coli spheroplasts. We found...
We investigated the physical properties of bacterial cytoplasmic membranes by applying the method of micropipette aspiration to Escherichia coli spheroplasts. We found that the properties of spheroplast membranes are significantly different from that of laboratory-prepared lipid vesicles or that of previously investigated animal cells. The spheroplasts can adjust their internal osmolality by increasing their volumes more than three times upon osmotic downshift. Until the spheroplasts are swollen to their volume limit, their membranes are tensionless. At constant external osmolality, aspiration increases the surface area of the membrane and creates tension. What distinguishes spheroplast membranes from lipid bilayers is that the area change of a spheroplast membrane by tension is a relaxation process. No such time dependence is observed in lipid bilayers. The equilibrium tension-area relation is reversible. The apparent area stretching moduli are several times smaller than that of stretching a lipid bilayer. We conclude that spheroplasts maintain a minimum surface area without tension by a membrane reservoir that removes the excessive membranes from the minimum surface area. Volume expansion eventually exhausts the membrane reservoir; then the membrane behaves like a lipid bilayer with a comparable stretching modulus. Interestingly, the membranes cease to refold when spheroplasts lost viability, implying that the membrane reservoir is metabolically maintained.
Topics: Cell Membrane; Elasticity; Escherichia coli; Lipid Bilayers; Osmolar Concentration; Pressure; Spheroplasts; Stress, Mechanical; Temperature
PubMed: 25418093
DOI: 10.1016/j.bpj.2014.09.034 -
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 -
Canadian Journal of Microbiology May 1973
Topics: Anaerobiosis; Animals; Bacteroides; Body Fluids; Buffers; Culture Media; Hydrogen-Ion Concentration; L Forms; Magnesium; Microscopy, Phase-Contrast; Muramidase; Rumen; Spectrophotometry; Spheroplasts; Tromethamine
PubMed: 4575450
DOI: 10.1139/m73-108 -
Journal of Bacteriology Apr 1978Heterokaryons of Mucor racemosus were produced by fusion of spheroplasts from two auxotrophic strains of the fungus. Germinated sporangiospores were converted to...
Heterokaryons of Mucor racemosus were produced by fusion of spheroplasts from two auxotrophic strains of the fungus. Germinated sporangiospores were converted to spheroplasts by using commercial chitinase and Myxobacter AL-1 chitosanase. Spheroplasts from the auxotrophic strains were mixed in a buffered Ca(NO3)2 solution and fusion occurred. After cell wall regeneration, prototrophs were isolated. The frequency of heterokaryon formation was 1.45 X 10(-4). Prototrophic isolates segregated parental nuclei at a high frequency, indicating that heterokaryons had formed.
Topics: Adenine; Cell Nucleus; Genetic Complementation Test; Leucine; Mucor; Spheroplasts; Spores, Fungal
PubMed: 649570
DOI: 10.1128/jb.134.1.349-352.1978 -
Yeast (Chichester, England) Sep 1998The rate of formation of spheroplasts of yeast can be used as an assay to study the structural integrity of cell walls. Lysis can be measured spectrophotometrically in...
The rate of formation of spheroplasts of yeast can be used as an assay to study the structural integrity of cell walls. Lysis can be measured spectrophotometrically in hypotonic solution in the presence of Zymolyase, a mixture of cell wall-digesting enzymes. The optical density of the cell suspension decreases as the cells lyse. We optimized this assay with respect to enzyme concentration, temperature, pH, and growth conditions for several strains of Saccharomyces cerevisiae. The level of variability (standard deviation) was 1-5% between trials where the replications were performed on the same culture using enzyme prepared from the same lot, and 5-15% for different cultures of the same strain. This assay can quantitate differences in cell wall structure (1) between exponentially growing and stationary phase cells, (2) among different S. cerevisiae strains, (3) between S. cerevisiae and Candida albicans, (4) between parental and mutated lines, and (5) between drug- or chemically-treated cells and controls.
Topics: Anti-Bacterial Agents; Bleomycin; Candida albicans; Cell Wall; Ferrous Compounds; Fungal Proteins; Hot Temperature; Hydrogen-Ion Concentration; Hydrolases; Membrane Glycoproteins; Quaternary Ammonium Compounds; Reproducibility of Results; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Spectrophotometry; Spheroplasts; Time Factors
PubMed: 9791887
DOI: 10.1002/(SICI)1097-0061(19980930)14:13<1159::AID-YEA317>3.0.CO;2-3 -
Journal of Bacteriology Aug 1982Cell wall-deficient forms (spheroplasts) of Mycobacterium smegmatis strain P53 were prepared by combined treatment with glycine, lysozyme, and lytic enzyme no. 2 as the...
Cell wall-deficient forms (spheroplasts) of Mycobacterium smegmatis strain P53 were prepared by combined treatment with glycine, lysozyme, and lytic enzyme no. 2 as the spheroplasting agents. Quantitative mass conversion to spherical forms was effected by pretreatment of the intact cells with 1.2% glycine in nutrient broth, followed by transfer to spheroplasting medium containing the above agents. Two apparent modes of reversion to the bacillary form were observed under electron microscopy. The first one was initiated by budding from the spheroplasts. The buds gradually elongated to become the mycelial form, which showed branching, septation, and fragmentation. The second resulted from the intracellular formation of tiny cells, possibly the elementary bodies, and their release from the spheroplasts.
Topics: Cell Membrane; Cell Wall; Glycine; Microscopy, Electron, Scanning; Muramidase; Mycobacterium; Spheroplasts
PubMed: 7096262
DOI: 10.1128/jb.151.2.1035-1039.1982 -
Journal of Bacteriology Nov 1978Homogenous pesticin, a bacteriocin produced by Yersinia pestis, promoted rapid dose-dependent killing of Escherichia coli phi but permitted residual generation of cell...
Homogenous pesticin, a bacteriocin produced by Yersinia pestis, promoted rapid dose-dependent killing of Escherichia coli phi but permitted residual generation of cell mass. Both growing cells and those blocked in net synthesis of nucleic acids or protein were converted by pesticin to osmotically stable spheroplast-like forms. Morphology and viability of cells starved for fermentable carbohydrate were not affected by pesticin. Similar spheroplast-like structures were formed from sensitive cells of Yersinia pseudotuberculosis, Yersinia enterocolitica, and Y. pestis.
Topics: Bacterial Proteins; Bacteriocins; Culture Media; DNA, Bacterial; Escherichia coli; RNA, Bacterial; Spheroplasts; Yersinia; Yersinia pestis
PubMed: 361722
DOI: 10.1128/jb.136.2.786-789.1978 -
PloS One 2023Vibrio cholerae, the causative agent of cholera epidemics, is a rod-shaped bacterium with a highly polarized cellular organization. It can survive harmful growth...
Vibrio cholerae, the causative agent of cholera epidemics, is a rod-shaped bacterium with a highly polarized cellular organization. It can survive harmful growth conditions by entering a non-proliferating spheroplast state, which involves loss of the cell envelope and polarity. How polarized rod organization cells are formed when the spheroplasts exit the non-proliferating state remains largely uncharacterized. To address this question, we investigated how L-arabinose-induced V. cholerae spheroplasts return to growth. We found that de novo morphogenesis started with the elimination of an excess of periplasm, which was immediately followed by cell elongation and the formation of cell branches with a diameter similar to that of normal V. cholerae cells. Periplasm elimination was driven by bifunctional peptidoglycan synthases involved in cell-wall maintenance, the aPBPs. Elongation and branching relied on the MreB-associated monofunctional peptidoglycan synthase PBP2. The cell division monofunctional peptidoglycan synthase FtsI was not involved in any of these processes. However, the FtsK cell division protein specifically targeted the sites of vesicle extrusion. Genetic material was amplified by synchronous waves of DNA replication as periplasmic elimination began. The HubP polarity factor targeted the tip of the branches as they began to form. However, HubP-mediated polarization was not involved in the efficiency of the recovery process. Finally, our results suggest that the positioning of HubP and the activities of the replication terminus organizer of the two V. cholerae chromosomes, MatP, are independent of cell division. Taken together, these results confirm the interest of L-arabinose-induced V. cholerae spheroplasts to study how cell shape is generated and shed light on the de novo establishment of the intracellular organization and cell polarization in V. cholerae.
Topics: Humans; Vibrio cholerae; Spheroplasts; Peptidoglycan; Arabinose; Cholera; Bacterial Proteins
PubMed: 37883451
DOI: 10.1371/journal.pone.0293276