1989 · Hörnsten — Effects of Escherichia coli spheroplast formation on assays of H₂ and adenosine triphosphate based ampicillin susceptibility tests.
Super-Abstract
When the antibiotic ampicillin damages the cell walls of E. coli bacteria, forming spheroplasts, the bacteria's hydrogen production drops — but their ATP levels remain relatively normal, creating a discrepancy between two different antibiotic susceptibility testing methods. This in-vitro microbiology study investigated how spheroplast formation affects the reliability of H₂-based and ATP-based bacterial susceptibility assays. (Diagnostic Microbiology and Infectious Disease, 1989.)
Commentary
This study uses hydrogen gas production by E. coli bacteria as a metabolic marker for bacterial viability in antibiotic testing — not as a therapeutic molecule. The core finding is that ampicillin-induced spheroplast formation suppresses H₂ production disproportionately relative to cell viability, meaning H₂-based susceptibility assays may give misleadingly negative results when bacteria are not truly dead. Conversely, ATP assays overestimate viable cell density in the presence of spheroplasts. This is a methodological microbiology paper with no relevance to therapeutic molecular hydrogen use.
Key quotes
- „The metabolic changes that took place during spheroplast formation disfavored the production of molecular hydrogen.“ — spheroplast formation suppresses H₂ production, affecting assay reliability
- „The intracellular bacterial adenosine triphosphate (ATP) level remained normal or slightly elevated during spheroplast formation while viability (cfu/ml) decreased.“ — ATP and viability diverge under antibiotic pressure — the two methods give different answers
- „When using a broth with lower osmolality (50 mosm/L) no spheroplast formation occurred and a close relation between viability and intracellular ATP was observed.“ — osmolality controls whether the artefact occurs
Our assessment
This is a methodological in-vitro microbiology study. Hydrogen here is a bacterial metabolite used as a diagnostic readout — there is no connection to molecular hydrogen therapy. The paper is of interest in the context of antibiotic susceptibility testing methodology but is not relevant to the question of H₂ as a therapeutic agent in humans or animals.
Study design
- Type: in-vitro microbiology study · Organism: one strain of Escherichia coli · H₂ role: bacterial metabolic marker for antibiotic susceptibility testing (not therapeutic)
- Result: spheroplast formation at 10× MIC of ampicillin suppressed H₂ production while ATP levels remained elevated; H₂ assay and ATP assay gave discrepant results for bacterial viability; absence of spheroplasts at low osmolality restored agreement between ATP and viability
Abstract
The present study examined the effects of ampicillin on one strain of Escherichia coli in lactose peptone broth with an osmolality of 342 mosm/L under anaerobic conditions. Spheroplast formation occurred at 10 X MIC of ampicillin. The metabolic changes that took place during spheroplast formation disfavored the production of molecular hydrogen. The intracellular bacterial adenosine triphosphate (ATP) level remained normal or slightly elevated during spheroplast formation while viability (cfu/ml) decreased. Thus spheroplast formation did not interfere significantly with ampicillin susceptibility as interpreted by assaying molecular hydrogen and viability. The effect on the ATP assay was, however, pronounced. It was found that the reversion of spheroplasts to bacterial cells for this particular strain (as recorded by cfu/ml) did not occur in quantitative numbers. The ATP assay thus indicated an approximate of the density of cells, while viability studies reported a lower cell density. When using a broth with lower osmolality (50 mosm/L) no spheroplast formation occurred and a close relation between viability and intracellular ATP was observed.
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