• Journal of Microbiology and Biotechnology
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• 제33권3호
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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.

• 한국식품과학회지
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• 제54권2호
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• pp.241-246
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• 2022

본 연구는 S. aureus에 대한 tuberostemonine의 항균 및 항생물막 효과에 대하여 확인하였다. S. aureus에 대한 tuberostemonine의 생장 저해 효과가 없음을 확인하였으나, crystal violet 염색법과 CLSM 이미지 측정을 통해 tuberostemonine이 유의한 S. aureus 항생물막 효과 가지는 것을 알 수 있었다. S. aureus의 생물막 형성과 분해 관련 유전자인 icaA와 agrA의 발현은 tuberostemonine를 처리하였을 때 유의미하게 각각 감소 또는 증가하는 것으로 나타났다. 따라서 본 연구에서 생물막 형성 저해 및 분해 효과가 확인된 천연화합물인 tuberostemonine은 S. aureus의 내성 발생 위험이 적은 새로운 항생물막제제로서 사용가능 할 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제14권6호
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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.

• 대한임상검사과학회지
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• 제50권2호
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• pp.100-109
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• 2018

다제내성(multidrug-resistant, MDR) Acinetobacter baumannii 균주의 출현 및 확산이 전 세계적으로 보고되어 왔는데, 이들 대부분은 생물막 형성능력을 가지고 있다. 생물막 형성은 소독 및 건조에 대해 세균이 저항할 수 있도록 해주는 중요한 병독성 인자이다. 본 연구에서는 생물막을 형성하는 A. baumannii 임상 분리주를 대상으로 항균제 내성 기전에 대한 유전적 기초를 조사하였다. 크리스탈 바이올렛 분석법을 통해 생물막 형성능력을 확인한 결과 46균주가 생물막을 형성하는 것으로 나타났다. 이어서 REP-PCR을 수행하여 서로 다른 클론에 속하는 16 균주를 선택한 후 이 균주들을 대상으로 항균제 내성 인자를 검출하였다. 본 연구에서 분리된 9개 non-MDR (0.96) 균주와 7개의 MDR (1.05) 균주의 평균 생물량은 서로 달랐지만, 이 차이는 유의하지 않았다. 대부분의 생물막 형성 MDR 균주는 다양한 항균제 내성인자 ($bla_{OXA-23}$, armA 및 gyrA 및 parC의 돌연변이)를 가지고 있는 것으로 나타났다. 그러나 대부분의 non-MDR 균주는 항균제 내성인자를 포함하지 않는 것으로 나타났다. 우리의 결과는 A. baumannii 균주의 생물막 형성능력이 항균제 감수성과 상관관계가 적음을 시사한다. 또한 MDR A. baumannii 임상 분리주의 출현은 대개 항균제 내성과 관련된 유전자의 돌연변이나 또는 다양한 항균제 내성인자의 획득에 의한 것으로 사료된다.

• Journal of Microbiology and Biotechnology
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• 제26권8호
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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.

• 한국환경과학회지
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• 제23권6호
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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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• 제26권3호
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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.

• 미생물학회지
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• 제42권4호
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• pp.277-285
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• 2006

본 연구에서는 신경인성 방광으로 요도 카테터를 유치하고 있는 환자의 카테터로부터 카테터 내 요로감염(Catheter-Associate Urinary Tract Infection; CA-UTI)에 관여하는 박테리아인 Escherichia coli, Pseudomonas aeruginosa 그리고 Staphylococcus aureus를 순수 분리, 동정하였다. 이 균주들을 대상으로 하여 quorum sensing mechanism을 규명하는 기초 연구로 각 균주의 quorum sensing 신호물질인 autoinducer (AIs)를 합성하는 유전자의 mRNA 발현을 확인하고, 정략분석 하였다. 각 세 균주를 단일과 세 균주의 혼합으로 24시간, 30일 동안 배양하며 일정 시간 간격으로 sample을 얻었다. 이 중 24시간 배양한 sample을 가지고 reverse transcription polymerase chain reaction (RT-PCR)을 수행하여 각 AIs 합성 유전자가 발현되는 최초 박테리아 밀도를 확인하였다. 단일배양에서 E. coli와 S. aureus의 AIs 합성 유전자(ygaG와 luxS)의 mRNA가 발현되는 최초 박테리아 밀도는 $2.4{\times}20^5$ CFU/ml, $5.4{\times}10^6$ CFU/ml 이었으며 P. aeruginosa의 rhlI와 lasI의 경우 $6.9{\times}10^4$ CFU/ml로 나타났다. 세 균주의 혼합배양에서 ygaG와 luxS의 mRNA가 발현되는 최초 박테리아 밀도는$7.3{\times}10^5$ CFU/ml, $1.6{\times}10^7$ CFU/ml이었으며 rhlI와 lasI의 경우 $2.1{\times}10^5$ CFU/ml로 나타났다. 또한 30일 배양한 sample의 RT-PCR 결과, 배양초기부터 각 AIs 합성 유전자들의 mRNA가 30일 동안 일정한 양만큼 지속적으로 발현됨을 확인하였다. Real-time RT-PCR을 이용한 AIs 합성 유전자의 mRNA 발현을 정량 분석한 결과 각 균주에서 단일배양보다 혼합배양시 AIs 합성 유전자의 발현이 더 많았다. 가장 많은 발현량의 차이를 보인 경우 E. coli ygaG의 mRNA 발현량은 단일배양보다 세 균주의 혼합배양시 최고 약 30배 이상이 증가하였고, P. aeruginosa rhlI의 경우 단일배양보다 혼합배양시 최고 약 40배, P. aeruginosa lasI의 경우 최고 약 250배 그리고 S. aureus luxS의 경우는 단일배양보다 혼합배양시 최고 약 5배 이상 mRNA 발현량이 증가하였다. 또한 세 균주의 4가지 유전자 중 P. aeruginosa의 rhlI와 lasI의 mRNA가 가장 많은 양으로 발현됨을 확인하였다.