Importance of the Exopolysaccharide Matrix in Antimicrobial Tolerance of Pseudomonas aeruginosa Aggregates

Goltermann, Lise; Tolker‐Nielsen, Tim

doi:10.1128/aac.02696-16

Cited by 81 publications

(70 citation statements)

References 23 publications

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“…Indeed, the increased survival rates seen for P. aeruginosa cultures inoculated directly from colonies on plates are in accordance with the findings that these cultures had more aggregated biomass and that tobramycin, an antibiotic known for killing actively growing cells (35), failed to kill parts of nonattached aggregates (25). Therefore, it is likely that the increased tobramycin tolerance of cultures inoculated using method 2 is a result of cells being protected either due to reduced metabolic activity in parts of the larger aggregated population or as a result of Psl providing a shielding effect against tobramycin (36,37). This hidden difference in antibiotic tolerance among macroscopically identical cultures could have major effects on the outcomes of various studies; for example, this could be the case in susceptibility testing of antimicrobial agents, where aggregation in cultures may induce various fractions of tolerant cells.…”

Section: Discussionsupporting

confidence: 81%

The Inoculation Method Could Impact the Outcome of Microbiological Experiments

Kragh

Alhede

Rybtke

et al. 2018

Appl Environ Microbiol

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111

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For the last 150 years, bacteria have primarily been investigated in liquid bacth cultures (LBC). Contrary to most expectations, these cultures are not a homogeneous mixture of single-celled bacteria as free-floating bacterial aggregates eventually develop in most LBC. These aggregates share characteristics with biofilms such as increased antibiotic tolerance. We have investigated how aggregates develop and what influences this development in LBC of We focused on how the method of inoculation impacted aggregation by assessing aggregate frequency and size using confocal laser scanning microscopy. Four traditional methods of initiating an overnight bacterial culture were investigated comprising inoculation directly from frozen culture, using agar grown cells, or using cells grown in liquid. We discovered a direct link between the inoculation method and the size and frequency of biofilm aggregates in a LBC, 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 towards tobramycin indicating that the inoculation method has a profound impact on antibiotic tolerance. We also observed a mechanism where pre-formed 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 and also produce aggregates in LBC. Our results stress the importance of inoculation consistency throughout experiments and the substantial impact aggregate development in LBC has on the output of microbiological experiments. Liquid pure cultures are fundamental to the field of microbiological research. These cultures are normally thought of as a homogeneous mix of single cell bacteria. The present study shows how this is not always true. Bacteria may aggregate in these liquid cultures. The aggregation can be induced by the method chosen for inoculation. The presence of aggregates can significantly change the outcome of experiments by altering the phenotype of the cultures. The study found a mechanism where pre-formed aggregates are able to recruit surrounding single cells in a form of snowball effect creating more and larger aggregates in the culture. Once formed, these aggregates are hard to remove. Aggregates in liquid cultures may be an immense unseen challenge for microbiologists.

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Section: Discussionsupporting

confidence: 81%

The Inoculation Method Could Impact the Outcome of Microbiological Experiments

Kragh

Alhede

Rybtke

et al. 2018

Appl Environ Microbiol

Self Cite

111

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“…Previous studies have also observed nonattached aggregates in otitis media, nonhealing wounds, and soft tissue fillers (9,10). These aggregates share characteristics with surface-attached biofilms, such that they are also highly resilient to both antimicrobial treatment and host immune responses (15)(16)(17)(18)(19)(20). The aggregate phenotype is not unique to P. aeruginosa and has been described for Achromobacter, Borrelia spp., Mycobacterium abscessus, Staphylococcus aureus, Staphylococcus epidermidis, and Vibrio cholerae in a multitude of in vitro and in vivo systems (21)(22)(23)(24)(25)(26).…”

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confidence: 83%

Motility-Independent Formation of Antibiotic-Tolerant Pseudomonas aeruginosa Aggregates

Demirdjian

Sanchez

Hopkins

et al. 2019

Appl Environ Microbiol

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Pseudomonas aeruginosa is a bacterial pathogen that causes severe chronic infections in immunocompromised individuals. This bacterium is highly adaptable to its environments, which frequently select for traits that promote bacterial persistence. A clinically significant temporal adaptation is the formation of surface- or cell-adhered bacterial biofilms that are associated with increased resistance to immune and antibiotic clearance. Extensive research has shown that bacterial flagellar motility promotes formation of such biofilms, whereupon the bacteria subsequently become nonmotile. However, recent evidence shows that antibiotic-tolerant nonattached bacterial aggregates, distinct from surface-adhered biofilms, can form, and these have been reported in the context of lung infections, otitis media, nonhealing wounds, and soft tissue fillers. It is unclear whether the same bacterial traits are required for aggregate formation as for biofilm formation. In this report, using isogenic mutants, we demonstrate that P. aeruginosa aggregates in liquid cultures are spontaneously formed independent of bacterial flagellar motility and independent of an exogenous scaffold. This contrasts with the role of the flagellum to initiate surface-adhered biofilms. Similarly to surface-attached biofilms, these aggregates exhibit increased antibiotic tolerance compared to planktonic cultures. These findings provide key insights into the requirements for aggregate formation that contrast with those for biofilm formation and that may have relevance for the persistence and dissemination of nonmotile bacteria found within chronic clinical infections. IMPORTANCE In this work, we have investigated the role of bacterial motility with regard to antibiotic-tolerant bacterial aggregate formation. Previous work has convincingly demonstrated that P. aeruginosa flagellar motility promotes the formation of surface-adhered biofilms in many systems. In contrast, aggregate formation by P. aeruginosa was observed for nonmotile but not for motile cells in the presence of an exogenous scaffold. Here, we demonstrate that both wild-type P. aeruginosa and mutants that genetically lack motility spontaneously form antibiotic-tolerant aggregates in the absence of an exogenously added scaffold. Additionally, we also demonstrate that wild-type (WT) and nonmotile P. aeruginosa bacteria can coaggregate, shedding light on potential physiological interactions and heterogeneity of aggregates.

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“…Although P. aeruginosa isolates from cystic fibrosis patients produce alginate constitutively [18], loss of alginate production does not affect biofilm growth [19]. However, Pel, Psl and alginate do increase the survival of bacteria during infection [20][21][22].…”

Section: Introductionmentioning

confidence: 99%

Pseudomonas aeruginosa biofilm formation on endotracheal tubes requires multiple two-component systems

Badal

Jayarani

Kollaran

et al. 2020

Journal of Medical Microbiology

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Introduction. Indwelling medical devices such as endotracheal tubes (ETTs), urinary catheters, vascular access devices, tracheostomies and feeding tubes are often associated with hospital-acquired infections. Bacterial biofilm formed on the ETTs in intubated patients is a significant risk factor associated with ventilator-associated pneumonia. Pseudomonas aeruginosa is one of the four frequently encountered bacteria responsible for causing pneumonia, and the biofilm formation on ETTs. However, understanding of biofilm formation on ETT and interventions to prevent biofilm remains lagging. The ability to sense and adapt to external cues contributes to their success. Thus, the biofilm formation is likely to be influenced by the two-component systems (TCSs) that are composed of a membrane-associated sensor kinase and an intracellular response regulator. Aim. This study aims to establish an in vitro method to analyse the P. aeruginosa biofilm formation on ETTs, and identify the TCSs that contribute to this process. Methodology. In total, 112 P. aeruginosa PA14 TCS mutants were tested for their ability to form biofilm on ETTs, their effect on quorum sensing (QS) and motility. Results. Out of 112 TCS mutants studied, 56 had altered biofilm biomass on ETTs. Although the biofilm formation on ETTs is QS-dependent, none of the 56 loci controlled quorum signal. Of these, 18 novel TCSs specific to ETT biofilm were identified, namely, AauS, AgtS, ColR, CopS, CprR, NasT, KdpD, ParS, PmrB, PprA, PvrS, RcsC, PA14_11120, PA14_32580, PA14_45880, PA14_49420, PA14_52240, PA14_70790. The set of 56 included the GacS network, TCS proteins involved in fimbriae synthesis, TCS proteins involved in antimicrobial peptide resistance, and surface-sensing. Additionally, several of the TCS-encoding genes involved in biofilm formation on ETTs were found to be linked to flagellum-dependent swimming motility. Conclusions. Our study established an in vitro method for studying P. aeruginosa biofilm formation on the ETT surfaces. We also identified novel ETT-specific TCSs that could serve as targets to prevent biofilm formation on indwelling devices frequently used in clinical settings.

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Importance of the Exopolysaccharide Matrix in Antimicrobial Tolerance of Pseudomonas aeruginosa Aggregates

Cited by 81 publications

References 23 publications

The Inoculation Method Could Impact the Outcome of Microbiological Experiments

The Inoculation Method Could Impact the Outcome of Microbiological Experiments

Motility-Independent Formation of Antibiotic-Tolerant Pseudomonas aeruginosa Aggregates

Pseudomonas aeruginosa biofilm formation on endotracheal tubes requires multiple two-component systems

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