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Antimicrobial Susceptibility of Persister Biofilm Cells of Bacillus cereus and Pseudomonas fluorescens.

ABSTRACT: The present study evaluates the antimicrobial susceptibility of persister cells of Bacillus cereus and Pseudomonas fluorescens after their regrowth in suspension and as biofilms. Two conventional (benzalkonium chloride-BAC and peracetic acid-PAA) and two emerging biocides (glycolic acid-GA and glyoxal-GO) were selected for this study. Persister cells resulted from biofilms subjected to a critical treatment using the selected biocides. All biocide treatments developed B. cereus persister cells, except PAA that effectively reduced the levels of vegetative cells and endospores. P. fluorescens persister cells comprise viable and viable but non-culturable cells. Afterwards, persister cells were regrown in suspension and in biofilms and were subjected to a second biocide treatment. In general, planktonic cultures of regrown persister cells in suspension lost their antimicrobial tolerance, for both bacteria. Regrown biofilms of persister cells had antimicrobial susceptibility close to those regrown biofilms of biocide-untreated cells, except for regrown biofilms of persister P. fluorescens after BAC treatment, which demonstrated increased antimicrobial tolerance. The most active biocide against persister cells was PAA, which did not promote changes in susceptibility after their regrowth. In conclusion, persister cells are ubiquitous within biofilms and survive after critical biocide treatment. The descendant planktonic and biofilms populations showed similar properties as the original ones.

SUBMITTER: Fernandes S

PROVIDER: S-EPMC8779418 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Biofilms are commonly recalcitrant to antibiotics, through incompletely elucidated mechanisms such as tolerance and persistence. We aimed at investigating how a Pseudomonas aeruginosa biofilm escapes ciprofloxacin treatment. P. aeruginosa PA14 in vitro mature biofilms were challenged with supra-MIC ciprofloxacin concentrations. Cell viability was quantified by fluorescein diacetate assay. Population dynamics were determined by counts of surviving culturable cells. Biofilms were analyzed using confocal laser scanning microscopy (CLSM), and the expression of genes involved in stringent response, toxin-antitoxin HigB/HigA, and type 3 secretion system (T3SS) was quantified by RT-qPCR in untreated and treated biofilms. Ciprofloxacin exposure resulted in an initial reduction of bacterial counts following a biphasic time-kill curve. After 24 h of treatment, the overall cell activity and the density of culturable cells significantly decreased as compared to untreated biofilm. No resistant mutant was isolated among the <1% surviving cells. Phenotypic adaptation toward persistence appeared to start after only 1 h of antibiotic exposure, by an overexpression of the genes involved in stringent response and in the toxin-antitoxin system, whereas the expression of genes encoding for the T3SS remained unchanged. After 4 h of ciprofloxacin exposure, stringent response genes returned to their basal level of expression. After a prolonged ciprofloxacin exposure, a deep alteration in the matrix structure that became thinner and lost mushroom-like aggregates was observed, in relation with reduced biovolumes of exopolysaccharides and extracellular DNA. These results support that ciprofloxacin might first induce the bacterial killing of most bacterial cells, but simultaneously activate stringent response mechanisms contributing to the switch of a subpopulation toward a persister phenotype. Once the persister phenotype is expressed, and despite an unexpected alteration of the biofilm matrix, ciprofloxacin fails to eradicate biofilm.

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Antimicrobial Susceptibility of Persister Biofilm Cells of Bacillus cereus and Pseudomonas fluorescens.

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