TY - JOUR
T1 - Inoculation method could impact the outcome of microbiological experiments
AU - Kragh, Kasper Nørskov
AU - Alhede, Maria
AU - Rybtke, Morten
AU - Stavnsberg, Camilla
AU - Tolker-Nielsen, Tim
AU - Whiteley, Marvin
AU - Bjarnsholt, Thomas
N1 - Copyright © 2017 American Society for Microbiology.
PY - 2018/3/1
Y1 - 2018/3/1
N2 - For the past 150 years, bacteria have been investigated primarily in liquid batch cultures. Contrary to most expectations, these cultures are not homogeneous mixtures of single-cell bacteria, because free-floating bacterial aggregates eventually develop in most liquid batch cultures. These aggregates share characteristics with biofilms, such as increased antibiotic tolerance. We investigated how aggregates develop and what influences this development in liquid batch cultures of Pseudomonas aeruginosa. We focused on how the method of inoculation affected aggregation by assessing aggregate frequency and size using confocal laser scanning microscopy. Several traditional methods of initiating an overnight bacterial culture, i.e., inoculation directly from frozen cultures, inoculation using agar-grown cells, or inoculation using cells grown in liquid cultures, were investigated. We discovered a direct link between the inoculation method and the size and frequency of biofilm aggregates in liquid batch cultures, with inoculation directly from a plate resulting in the most numerous and largest aggregates. These large aggregates had an overall impact on the cultures' subsequent tolerance toward tobramycin, indicating that the inoculation method has a profound impact on antibiotic tolerance. We also observed a mechanism whereby preformed aggregates recruited single cells from the surrounding culture in a "snowball effect," building up aggregated biomass in the culture. This recruitment was found to rely heavily on the exopolysaccharide Psl. Additionally, we found that both Escherichia coli and Staphylococcus aureus produced aggregates in liquid batch cultures. Our results stress the importance of inoculation consistency throughout experiments and the substantial impact aggregate development in liquid batch cultures may have on the outcomes of microbiological experiments.
AB - For the past 150 years, bacteria have been investigated primarily in liquid batch cultures. Contrary to most expectations, these cultures are not homogeneous mixtures of single-cell bacteria, because free-floating bacterial aggregates eventually develop in most liquid batch cultures. These aggregates share characteristics with biofilms, such as increased antibiotic tolerance. We investigated how aggregates develop and what influences this development in liquid batch cultures of Pseudomonas aeruginosa. We focused on how the method of inoculation affected aggregation by assessing aggregate frequency and size using confocal laser scanning microscopy. Several traditional methods of initiating an overnight bacterial culture, i.e., inoculation directly from frozen cultures, inoculation using agar-grown cells, or inoculation using cells grown in liquid cultures, were investigated. We discovered a direct link between the inoculation method and the size and frequency of biofilm aggregates in liquid batch cultures, with inoculation directly from a plate resulting in the most numerous and largest aggregates. These large aggregates had an overall impact on the cultures' subsequent tolerance toward tobramycin, indicating that the inoculation method has a profound impact on antibiotic tolerance. We also observed a mechanism whereby preformed aggregates recruited single cells from the surrounding culture in a "snowball effect," building up aggregated biomass in the culture. This recruitment was found to rely heavily on the exopolysaccharide Psl. Additionally, we found that both Escherichia coli and Staphylococcus aureus produced aggregates in liquid batch cultures. Our results stress the importance of inoculation consistency throughout experiments and the substantial impact aggregate development in liquid batch cultures may have on the outcomes of microbiological experiments.
U2 - 10.1128/aem.02264-17
DO - 10.1128/aem.02264-17
M3 - Journal article
C2 - 29269495
SN - 0099-2240
VL - 84
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
IS - 5
M1 - e02264-17
ER -