• Journal of Microbiology and Biotechnology
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• v.33 no.3
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• pp.348-355
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• 2023

Epifluorescence microscopy with image analysis was evaluated as a biofilm quantification method (i.e., quantification of surface area colonized by biofilms), in comparison with crystal violet (CV) staining. We performed different experiments to generate multispecies biofilms with natural and artificial bacterial assemblages. First, four species were inoculated daily in 16 different sequences to form biofilms (surface colonization, 0.1%-56.6%). Second, a 9-species assemblage was allowed to form biofilms under 10 acylase treatment episodes (33.8%-55.6%). The two methods comparably measured the quantitative variation in biofilms, exhibiting a strong positive relationship (R² ≥ 0.7). Moreover, the two methods exhibited similar levels of variation coefficients. Finally, six synthetic and two natural consortia were allowed to form biofilms for 14 days, and their temporal dynamics were monitored. The two methods were comparable in quantifying four biofilms colonizing ≥18.7% (R² ≥ 0.64), but not for the other biofilms colonizing ≤ 3.7% (R² ≤ 0.25). In addition, the two methods exhibited comparable coefficients of variation in the four biofilms. Microscopy and CV staining comparably measured the quantitative variation of biofilms, exhibiting a strongly positive relationship, although microscopy cannot appropriately quantify the biofilms below the threshold colonization. Microscopy with image analysis is a promising approach for easily and rapidly estimating absolute quantity of multispecies biofilms.

• Korean Journal of Food Science and Technology
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• v.54 no.2
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• pp.241-246
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• 2022

Antibiotic resistance is a serious problem to food safety as well as human healthcare. To avoid this, there are several approaches for a new class of antibiotic agents that target only production of virulence factors such as biofilm without bacterial growth defect. The objective of this study was to investigate the antibiofilm activity of tuberostemonine in Staphylococcus aureus. Tuberostemonine significantly reduced the biofilm formation (26.07-47.02%) in the crystal violet assay whereas there were no effect on S. aureus growth. The dispersion in preformed biofilm was also observed by confocal laser scanning microscopy (CLSM). Quantification real-time PCR revealed that the icaA and agrA expression having an important role in biofilm production of S. aureus were strongly affected with tuberostemonine. These results suggest that tuberostemonine has potential for controlling biofilm formation and dispersion by effect on virulence regulation of S. aureus.

• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1114-1119
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• 2004

The biofilms on pipe walls in water distribution systems are of interest since they can lead to chlorine demand, coliform growth, pipe corrosion, and water taste and odor problems. As such, the study described in this paper is part of an AWWARF and Tampa Bay Water tailored collaboration project to determine the effect of blending different source waters on the water quality in various distribution systems. The project was based on 18 independent pilot distribution systems (PDS), each being fed by a different water blend (7 finished waters blended in different proportions). The source waters compared were groundwater, surface water, and brackish water, which were treated in a variety of pilot distribution systems, including reverse osmosis (RO) (desalination), both membrane and chemical softening, and ozonation-biological activated carbon (BAC), resulting in a total of 7 different finished waters. The observations from this study consistently demonstrated that unlined ductile iron was more heavily colonized by a biomass than galvanized steel, lined ductile iron, and PVC (in that order) and that the fixed biomass accumulation was more influenced by the nature of the supporting material than by the water quality (including the secondary residual levels). However, although the bulk liquid water cultivable bacterial counts (i.e. heterotrophic plate counts or HPCs) did not increase with a greater biofilm accumulation, the results also suggested that high HPCs corresponded to a low disinfectant residual more than a high biofilm inventory. Furthermore, temperature was found to affect the biofilms, plus the AOC was important when the residual was between 0.6 and 2.0 mg $Cl_2/l$. An additional aspect of the current study was that the potential of the exoproteolytic activity (PEPA) technique was used along with a traditional so-called destructive technique in which the biofilm was scrapped off the coupon surface, resuspended, and cultivated on an R2A agar. Both techniques indicated similar trends and relative comparisons among the PDSs, yet the culturable biofilm values for the traditional method were several orders of magnitude lower than the PEPA values.

• Korean Journal of Clinical Laboratory Science
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• v.50 no.2
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• pp.100-109
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• 2018

The emergence and dissemination of multidrug-resistant (MDR) Acinetobacter baumannii isolates have been reported worldwide, with most of these possessing the ability to form biofilms. Biofilm formation is an important virulence factor associated with the resistance to disinfection and desiccation. This study examined the genetic basis of antimicrobial resistance mechanisms of biofilm-forming A. baumannii clinical isolates. Imaging and quantification of biofilms were performed by a crystal violet assay and 46 biofilm-forming A. baumannii isolates were selected. Subsequently, 16 isolates belonging to different clones were identified using REP-PCR, and detection of the antimicrobial determinants in the isolates was carried out. The 16 isolates included 9 non-MDR and 7 MDR isolates. The mean biomass $OD_{560}$ values of the non-MDR (0.96) and MDR (1.05) isolates differed but this difference was not significant. In this study, most biofilm-forming MDR A. baumannii isolates contained various antimicrobial resistance determinants ($bla_{OXA-23}$, armA, and mutations of gyrA and parC). On the other hand, most biofilm-forming non-MDR A. baumannii isolates did not contain antimicrobial resistance determinants. These results suggest that there is little correlation between the biofilm-forming ability and antimicrobial susceptibility in A. baumannii isolates. In addition, the emergence of MDR A. baumannii clinical isolates is generally caused by mutations of the genes associated with antimicrobial resistance and/or the acquisition of various antimicrobial resistance determinants.

• Journal of Microbiology and Biotechnology
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• v.26 no.8
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• pp.1481-1489
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• 2016

The emergence and dissemination of carbapenemase-producing Acinetobacter baumannii isolates have been reported worldwide, and A. baumannii isolates harboring bla_OXA-23 are often resistant to various antimicrobial agents. Antimicrobial resistance can be particularly strong for biofilm-forming A. baumannii isolates. We investigated the genetic basis for carbapenem resistance and biofilm-forming ability of multidrug-resistant (MDR) clinical isolates. Ninety-two MDR A. baumannii isolates were collected from one university hospital located in the Chungcheong area of Korea over a 5-year period. Multiplex PCR and DNA sequencing were performed to characterize carbapenemase and bap genes. Clonal characteristics were analyzed using REP-PCR. In addition, imaging and quantification of biofilms were performed using a crystal violet assay. All 92 MDR A. baumannii isolates involved in our study contained the bla_OXA-23 and bap genes. The average absorbance of biomass in Bap-producing strains was much greater than that in non-Bap-producing strains. In our study, only three REP-PCR types were found, and the isolates showing type A or type B were found more than 60 times among unique patients during the 5 years of surveillance. These results suggest that the isolates have persisted and colonized for 5 years, and biofilm formation ability has been responsible for their persistence and colonization.

• Journal of Environmental Science International
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• v.23 no.6
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• pp.1101-1109
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• 2014

In this study we followed biofilm formation and development in a granular activated carbon (GAC) filter on pilot-scale during the 12 months of operation. GAC particles and water samples were sampled from four different depths (-5, -25, -50 and -90 cm from surface of GAC bed) and attached biomass were measured with adenosine tri-phosphate (ATP) analysis and heterotrophic plate count (HPC) method. The attached biomass accumulated rapidly on the GAC particles of top layer throughout all levels in the filter during the 160 days (BV 23,000) of operation and maintained a steady-state afterward. During steady-state, biomass (ATP and HPC) concentrations of top layer in the BAC filer were $2.1{\mu}g{\cdot}ATP/g{\cdot}GAC$ and $3.3{\times}10^8cells/g{\cdot}GAC$, and 85%, 83% and 99% of the influent total biodegradable dissolved organic carbon ($BDOC_{total}$), $BDOC_{slow}$ and $BDOC_{rapid}$ were removed, respectively. During steady-state process, biomass (ATP and HPC) concentrations of middle layer (-50 cm) and bottom layer (-90 cm) in the BAC filter were increased consistently. Biofilm development (growth rate) proceed highest rate in the top layer of filter (${\mu}_{ATP}=0.73day^{-1}$; ${\mu}_{HPC}=1,74day^{-1}$) and 78%~87% slower in the bottom layer (${\mu}_{ATP}=0.14day^{-1}$; ${\mu}_{HPC}=0.34day^{-1}$). This study shows that the combination of different analytical methods allows detailed quantification of the microbiological activity in drinking water biofilter.

• Journal of Microbiology and Biotechnology
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• v.26 no.3
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• pp.511-520
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• 2016

The most energy-demanding step of wastewater treatment is the aeration-dependent elimination of organic carbon. Microbial fuel cells (MFCs) offer an alternative strategy in which carbon elimination is conducted by anaerobic microorganisms that transport respiratory electrons originating from carbon oxidation to an anode. Hence, chemical energy is directly transformed into electrical energy. In this study, the use and stability of barcode-containing exoelectrogenic model biofilms under non-axenic wastewater treatment conditions are described. Genomic barcodes were integrated in Shewanella oneidensis, Geobacter sulfurreducens, and G. metallireducens. These barcodes are unique for each strain and allow distinction between those cells and naturally occurring wild types as well as quantification of the amount of cells in a biofilm via multiplex qPCR. MFCs were pre-incubated with these three strains, and after 6 days the anodes were transferred into MFCs containing synthetic wastewater with 1% wastewater sludge. Over time, the system stabilized and the coulomb efficiency was constant. Overall, the initial synthetic biofilm community represented half of the anodic population at the end of the experimental timeline. The part of the community that contained a barcode was dominated by G. sulfurreducens cells (61.5%), while S. oneidensis and G. metallireducens cells comprised 10.5% and 17.9%, respectively. To the best of our knowledge, this is the first study to describe the stability of a synthetic exoelectrogenic consortium under non-axenic conditions. The observed stability offers new possibilities for the application of synthetic biofilms and synthetically engineered organisms fed with non-sterile waste streams.

• Korean Journal of Microbiology
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• v.42 no.4
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• pp.277-285
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• 2006

The most biofilm forming bacteria in catheter, Esctherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus were isolated and identified from a patient's catheter occuring catheter-associated urinary tract infection (CA-UTI). We examined mRNA expression and its quantification of AIs synthetic genes encoding signal substance of quorum sensing from each bacterial species in order to elucidated quorum sensing mechanism. Both pure cultures for each bacterial strains and a mixed cultures with three were grown for 24 hr and 30 days. Initial densities to be able to detect mRNA expression oil single strains culture were shown at $2.4{\times}10^5$ CFU/ml, $5.4{\times}10^6$ CFU/ml of E. coli for ygaG and S. aureus for luxS, and at $6.9{\times}10^4$ CFU/ml of P. aeruginosa for rhlI and lasI. Also, in mixed culture of three, initial cell densities of mRNA expression were appear to at $7.3{\times}10^5$ CFU/ml, $1.6{\times}10^7$ CFU/ml of E. coli for ygaG and S. aureus for luxS, and at $2.1{\times}10^5$ CFU/ml of P. aeruginosa for rhlI and lasI. Each AIs synthetic gene was expressed in initial cell density and the mRNA expression of the genes were detected continously during 30 days. And then, the quantification of mRNA expression level of ygaG, rhlI, last, and luxS which were related AIs synthesis was done each time point by real-time RT-PCR. Interestingly, the mRNA levels of ygaG, rhlI, lasI, and luxS from the mixed culture was higher than those from each single strain culture. In the case of E. coli ygaG, the amount of transcript from the mixed culture was at least 30 times for that from single culture. In the case of P. aeruginosa rhlI and lasI, the amount of transcript from the mixed culture was at least 40 times and 250 times for that from single strain culture. In the case of S. aureus luxS, the amount of transcript from the mixed culture was at least 5 times for that from single strain culture. And specially, the mRNA expression of rhlI and lasI of P. aeruginosa showed the highest efficency among four AIs synthetic genes.