Into the well—A close look at the complex structures of a microtiter biofilm and the crystal violet assay

Kragh, Kasper Nørskov; Alhede, Morten; Kvich, Lasse; Bjarnsholt, Thomas

doi:10.1016/j.bioflm.2019.100006

Cited by 90 publications

(88 citation statements)

References 32 publications

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“…It must be noted, however, that adding liquid manipulation steps such as rinsing, fixation and gradual dehydration to the preparation of biofilms is likely to affect the appearance of biofilms and cause e.g., loss of biomass in each step. Kragh et al have demonstrated that media removal and rinsing can critically affect quantitative biofilm analysis compared to undisturbed biofilms [10]. In addition, we observed that while oligotrophic bacterial biofilms retained their overall microcolony architecture during fixing and dehydration steps, biofilms formed in growth medium lost most of their biomass and surface coverage during dehydration steps prior to SEM.…”

Section: Bacterial Biofilm Formation On Surfaces Coated With Nano-znosupporting

confidence: 48%

“…Even if biofilm formation on material of interest is studied, often-used methods of biofilm viability assessment are prone to critical failures. For example, viability staining with propidium iodide and suitable counterstains can dramatically underestimate viability in both oligotrophic [9] and growth medium biofilms [10] while colony counts, the gold standard of microbiology, depend on viable cell harvesting efficiency and dispersion of harvested aggregates prior to cultivation [11], [12].…”

Section: Introductionmentioning

confidence: 99%

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Selective antibiofilm properties and biocompatibility of nano-ZnO and nano-ZnO/Ag coated surfaces

Rosenberg

Visnapuu

Vija

et al. 2020

Preprint

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Spread of pathogenic microbes and antibiotic-resistant bacteria in health-care settings and public spaces is a serious public health challenge. Materials and surface-treatments that prevent solid surface colonization and biofilm formation or impede touch-transfer of viable microbes could provide means to decrease pathogen transfer from high-touch surfaces in critical applications. Both, ZnO and Ag nanoparticles have shown a great potential in antimicrobial applications. Although antimicrobial properties of such nanoparticle suspensions and their biocompatibility with human cells are well studied, less is known about nano-enabled solid surfaces. Here we demonstrate that surfaces coated with nano-ZnO or nano-ZnO/Ag composites possess species-selective medium-dependent antibiofilm activity against Escherichia coli, Staphylococcus aureus and Candida albicans. Colonization of nano-ZnO surfaces by E. coli and S. aureus was decreased in oligotrophic (nutrient-poor, no growth) conditions with E. coli showinghigher sensitivity to Ag and S. aureus to Zn, respectively. Minor to no effect was observed for bacteria in growth medium (nutrient-rich, exponential growth). Inversely, compared to uncoated surfaces, nano-ZnO surfaces enhanced biofilm formation by C. albicans in oligotrophic conditions and just a minor transient negative effect was seen in nutrient-rich medium. However, enhanced C. albicans biofilm formation on nano-ZnO surfaces was effectively counteracted by the addition of Ag. Our results not only showed that nano-ZnO/Ag coated solid surfaces have the potential to effectively decrease surface colonization by the bacteria E. coli and S. aureus but also indicated the importance of the use of application-appropriate test conditions and exposure medium in antimicrobial surface testing. Possible selective enhancement of biofilm formation by the yeast C. albicans on Zn-enabled surfaces should be taken into account in antimicrobial surface development.

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Section: Bacterial Biofilm Formation On Surfaces Coated With Nano-znosupporting

confidence: 48%

Section: Introductionmentioning

confidence: 99%

Selective antibiofilm properties and biocompatibility of nano-ZnO and nano-ZnO/Ag coated surfaces

Rosenberg

Visnapuu

Vija

et al. 2020

Preprint

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show abstract

“…It must be noted, however, that adding liquid manipulation steps such as rinsing, fixation and gradual dehydration to the preparation of biofilm samples is likely to affect the appearance of biofilms and cause e.g., loss of biomass in each step. Kragh et al have recently demonstrated that media removal and rinsing can critically affect quantitative biofilm analysis compared to undisturbed biofilms 10 . In addition, we observed that while oligotrophic bacterial biofilms retained their overall microcolony architecture during fixing and dehydration steps, biofilms formed in growth medium lost most of their biomass and surface coverage during dehydration steps prior to SEM.…”

Section: Discussionmentioning

confidence: 99%

Selective antibiofilm properties and biocompatibility of nano-ZnO and nano-ZnO/Ag coated surfaces

Rosenberg

Visnapuu

Vija

et al. 2020

Sci Rep

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Spread of pathogenic microbes and antibiotic-resistant bacteria in health-care settings and public spaces is a serious public health challenge. Materials that prevent solid surface colonization or impede touch-transfer of viable microbes could provide means to decrease pathogen transfer from high-touch surfaces in critical applications. ZnO and Ag nanoparticles have shown great potential in antimicrobial applications. Less is known about nano-enabled surfaces. Here we demonstrate that surfaces coated with nano-ZnO or nano-ZnO/Ag composites are not cytotoxic to human keratinocytes and possess species-selective medium-dependent antibiofilm activity against Escherichia coli, Staphylococcus aureus and Candida albicans. Colonization of nano-ZnO and nano-ZnO/Ag surfaces by E. coli and S. aureus was decreased in static oligotrophic conditions (no planktonic growth). Moderate to no effect was observed for bacterial biofilms in growth medium (supporting exponential growth). Inversely, nano-ZnO surfaces enhanced biofilm formation by C. albicans in oligotrophic conditions. However, enhanced C. albicans biofilm formation on nano-ZnO surfaces was effectively counteracted by the addition of Ag. Possible selective enhancement of biofilm formation by the yeast C. albicans on Zn-enabled surfaces should be taken into account in antimicrobial surface development. Our results also indicated the importance of the use of application-appropriate test conditions and exposure medium in antimicrobial surface testing. Biofilms are by far the preferred lifestyle of bacteria 1 , mostly in diverse nutrient-limited environmental niches. Biofilm communities cause biomass buildup on solid surfaces that results in major expenses in marine traffic, water systems maintenance and in the industrial sector. Biofilms can also harbor potential human pathogens in food industry, health-care facilities, drinking water systems and on high-touch surfaces in public spaces. It is estimated that hard to treat pathogenic biofilms account for over 80% of all human microbial infections 2 with antibiotic-resistant ESKAPE pathogens 3 including six nosocomial pathogens commonly associated with multidrug resistance and virulence (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) being the most problematic in the field. Biofilms pose a major medical challenge as they can be over 1,000-fold more tolerant to antibiotics than their planktonic counterparts 4. Spatially heterogeneous natural selection in biofilm milieu also contributes to antibiotic resistance development and transfer 5,6 as well as producing antibiotic-resistant bacteria that are more fit and not easily outcompeted in the absence of the drug 7. Controversially, most of the methods used to assess antimicrobial properties of surface materials, irrespective of their proposed application, either use planktonic cultures or study indirect effects such as release of antimicrobial compounds from the surfaces 8. Such methods might...

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“…While the microtiter crystal violet assay is one of the most widely used methods for assessing biofilm formation, because it is a fast, high-throughput screening tool with great value for microbiologists, this assay is known for its substantial deviation from experiment to experiment, and even from well to well. The microtiter biofilm assay involves several pitfalls and can result in scattered results due to heterogeneous structured growth, disruptive procedures, and shortcomings in quantifying biomass and viable cells [54,55]. These facts could also be responsible for the differences observed between research works.…”

Section: Biofilm Productionmentioning

confidence: 99%

Diversity, Antibiotic Resistance, and Biofilm-Forming Ability of Enterobacteria Isolated from Red Meat and Poultry Preparations

Capita¹,

Castaño-Arriba²,

Rodríguez-Melcón³

et al. 2020

Microorganisms

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A total of 44 samples of beef, pork, and poultry preparations were tested. Average counts (log cfu/g) of enterobacteria were 1.99 ± 0.99 (beef preparations), 1.96 ± 1.44 (pork), 2.09 ± 0.92 (chicken), and 2.17 ± 1.06 (turkey) (p > 0.05). Two hundred enterobacterial strains were identified and 13 genera (21 species) were distinguished, including species that are a significant cause of infection. The most common genera were Escherichia (32.5% of strains), Serratia (17.0%), Hafnia (12.5%), and Salmonella (12.0%). Isolates were screened by disc diffusion for susceptibility to 15 antibiotics. A total of 126 strains (63% of the isolates) were multirresistant (having resistance to two or more antibiotics), 46 (23%) were resistant to one antibiotic, and 28 (14%) were sensitive to all antibiotics. The average number of resistances per strain was 2.53 ± 2.05. A higher (p < 0.05) average number of resistances was observed in strains from turkey (3.14 ± 2.55) than in strains from beef (2.15 ± 1.22), pork (2.16 ± 1.39), or chicken (2.44 ± 2.22). At least 50% of strains showed resistance or reduced susceptibility to ampicillin, cefotaxime, ceftazidime, or streptomycin, considered to be “critically important” antimicrobial agents in human medicine. Seventy-nine strains (39.5%), 60 strains (30.0%), and 46 strains (23.0%) were weak, moderate, and strong biofilm producers (crystal violet assay), respectively. This investigation provides evidence that bacteria from red meat and poultry preparations pose major potential risk to consumers.

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Into the well—A close look at the complex structures of a microtiter biofilm and the crystal violet assay

Cited by 90 publications

References 32 publications

Selective antibiofilm properties and biocompatibility of nano-ZnO and nano-ZnO/Ag coated surfaces

Selective antibiofilm properties and biocompatibility of nano-ZnO and nano-ZnO/Ag coated surfaces

Selective antibiofilm properties and biocompatibility of nano-ZnO and nano-ZnO/Ag coated surfaces

Diversity, Antibiotic Resistance, and Biofilm-Forming Ability of Enterobacteria Isolated from Red Meat and Poultry Preparations

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