Abstract
Most antimicrobials currently used in the clinical practice are tested as growth inhibitors against free-floating microorganisms in a liquid suspension, rather than against sessile cells constituting biofilms. Hence, reliable, fast, and reproducible methods for assessing biofilm susceptibility to antimicrobials are strongly needed. Isothermal microcalorimetry (IMC) is a nondestructive sensitive technique that allows for the real-time monitoring of microbial viability in the presence or absence of antimicrobial compounds. Therefore, the efficacy of specific antimicrobials, alone or in combination, may be promptly validated supporting the development of new drugs and avoiding the administration of ineffective therapies. Furthermore, the susceptibility of both planktonic and biofilm cells to antimicrobials can be conveniently assessed without the need for elaborated staining procedures and under nontoxic working conditions. Quantitative data regarding the antimicrobial effect against different strains might be collected by monitoring the microbial cell replication, and, more importantly, a dose-dependent activity can be efficiently detected by measuring the delay and decrease in the heat flow peak of the treated samples. A limitation of IMC for anti-biofilm susceptibility test is the inability to directly quantify the non-replicating cells in the biofilm or the total biomass. However, as IMC is a nondestructive method, the samples can be also analyzed by using different techniques, acquiring more information complementary to calorimetric data. IMC finds application also for the investigation of antibiotic eluting kinetics from different biomaterials, as well as for studying bacteriophages activity against planktonic and biofilm bacteria. Thus, the wide applicability of this ultra-sensitive and automated technique provides a further advance in the field of clinical microbiology and biomedical sciences.
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Abbreviations
- CAMHB+2.5% LHB:
-
Cation Adjusted Müller Hinton Broth supplemented with 2.5% lysate horse blood
- CFUs:
-
Colony-forming units
- GC:
-
Growth control
- HA:
-
Hyaluronic acid
- HA/Levo:
-
Levofloxacin-loaded hyaluronic acid hydrogel
- HA/PBS:
-
Phosphate buffered saline/hyaluronic acid hydrogel
- Htot :
-
Total heat produced
- IMC:
-
Isothermal microcalorimetry
- k:
-
Growth rate constant
- λ:
-
Lag phase
- MBBC:
-
Minimum biofilm bactericidal concentration
- MBEC:
-
Minimum biofilm eradicating concentration
- MBPC:
-
Minimum biofilm preventing concentration
- MHIC:
-
Minimum heat inhibiting concentration
- MHICb :
-
Minimum heat inhibiting concentration for biofilm
- MIC:
-
Minimum inhibiting concentration
- Pmax :
-
Maximum heat flow peak
- PBS:
-
Phosphate buffered saline
- S. pyogenes :
-
Streptococcus pyogenes
- Tmax :
-
Time of the maximum heat flow peak
- TTD:
-
Time to detection
- TSA:
-
Trypticase soy agar
- TSA+2.5% LHB:
-
Trypticase soy agar supplemented with 2.5% lysate horse blood
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Acknowledgments
An educational grant was provided by the PRO-IMPLANT Foundation (Berlin, Germany). Authors acknowledge Dr. Gerardo Abbandonato for his valuable contribution to microcalorimetric data analysis.
Authors’ Contribution
MEB, MGM, and MDL conceived and designed the work. MEB and MGM performed the experiments. MGM analyzed the data with the contribution of MEB, AT, and MDL. MDL and MEB drafted the manuscript, with the contribution of MGM, MC, AK, and TT. All authors reviewed and revised the first and final drafts of this manuscript.
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Butini, M.E. et al. (2018). Real-Time Antimicrobial Susceptibility Assay of Planktonic and Biofilm Bacteria by Isothermal Microcalorimetry. In: Donelli, G. (eds) Advances in Microbiology, Infectious Diseases and Public Health. Advances in Experimental Medicine and Biology(), vol 1214. Springer, Cham. https://doi.org/10.1007/5584_2018_291
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