Genotypic and Phenotypic Trends in Antibiotic Resistant Pathogenic <i>Escherichia coli</i> Isolated from Humans and Farm Animals in South Korea

Unno, Tatsuya,Han, Dukki,Jang, Jeonghwan,Widmer, Kenneth,Ko, GwangPyo,Sadowsky, Michael J.,Hur, Hor-Gil Japanese Society of Microbial Ecology · The Japane 2011 Microbes and environments Vol.26 No.3

<P>South Korea uses more antibiotics for animal production than any other member of the OECD. The excessive use could potentially lead to a greater incidence of antibiotic resistant microorganisms, some of which may be pathogenic. In this study, we examined potential diarrheagenic and extraintestinal pathogenic <I>E. coli</I> (ExPEC) isolates using virulence gene profiling (<I>eaeA</I>, <I>est</I>, <I>elt</I>, <I>ipaH</I>, <I>stx1</I> and <I>stx2</I> as diarrheagenic and <I>afa/dra</I>, <I>iutA</I>, <I>kpsMT II</I>, <I>papA/C</I>, and <I>sfa/foc</I> as extraintestinal pathogenic virulence genes). A cluster analysis of DNA fingerprint data indicated that certain genotypes of chicken <I>E. coli</I> isolates are likely ExPEC. Moreover, our results showed 38 of the 60 potential diarrheagenic and ExPEC isolates obtained from animals and humans were also resistant to antibiotics, mostly to tetracycline groups, penicillin groups, and folate pathway inhibitors. Our results suggest that the use of antibiotics in agriculture should be controlled due to potential impacts on human health.</P>

Effects of Antibiotic Growth Promoter and Characterization of Ecological Succession in Swine Gut Microbiota

( Tatsuya Unno ),( Jung Man Kim ),( Robin B Guevarra ),( Son G Nguyen ) 한국미생물 · 생명공학회 2015 Journal of microbiology and biotechnology Vol.25 No.4

Ever since the ban on antibiotic growth promoters (AGPs), the livestock death rate has increased owing to pathogenic bacterial infections. There is a need of developing AGP alternatives; however, the mechanisms by which AGP enhances livestock growth performance are not clearly understood. In this study, we fed 3-week-old swine for 9 weeks with and without AGPs containing chlortetracycline, sulfathiazole, and penicillin to investigate the effects of AGPs on swine gut microbiota. Microbial community analysis was done based on bacterial 16S rRNA genes using MiSeq. The use of AGP showed no growth promoting effect, but inhibited the growth of potential pathogens during the early growth stage. Our results showed the significant increase in species richness after the stabilization of gut microbiota during the post-weaning period (4-week-old). Moreover, the swine gut microbiota was divided into four clusters based on the distribution of operational taxonomic units, which was significantly correlated to the swine weight regardless of AGP treatments. Taxonomic abundance analysis indicated a negative correlation between host weight and the abundance of the family Prevotellaceae species, but showed positive correlation to the abundance of the family Spirochaetaceae, Clostridiaceae_1, and Peptostreptococcaeae species. Although no growth performance enhancement was observed, the use of AGP inhibited the potential pathogens in the early growth stage of swine. In addition, our results indicated the ecological succession of swine gut microbiota according to swine weight. Here, we present a characterization of swine gut microbiota with respect to the effects of AGPs on growth performance.

Fecal pollution: new trends and challenges in microbial source tracking using next‐generation sequencing

Unno, Tatsuya,Staley, Christopher,Brown, Clairessa M.,Han, Dukki,Sadowsky, Michael J.,Hur, Hor‐,Gil BLACKWELL SCIENCE 2018 ENVIRONMENTAL MICROBIOLOGY Vol.20 No.9

<P>In this minireview, we expand upon traditional microbial source tracking (MST) methods by discussing two recently developed, next-generation-sequencing (NGS)-based MST approaches to identify sources of fecal pollution in recreational waters. One method defines operational taxonomic units (OTUs) that are specific to a fecal source, e.g., humans and animals or shared among multiple fecal sources to determine the magnitude and likely source association of fecal pollution. The other method uses SourceTracker, a program using a Bayesian algorithm, to determine which OTUs have contributed to an environmental community based on the composition of microbial communities in multiple fecal sources. Contemporary NGS-based MST tools offer a promising avenue to rapidly characterize fecal source contributions for water monitoring and remediation efforts at a broader and more efficient scale than previous molecular MST methods. However, both NGS methods require optimized sequence processing methodologies (e.g. quality filtering and clustering algorithms) and are influenced by primer selection for amplicon sequencing. Therefore, care must be taken when extrapolating data or combining datasets. Furthermore, traditional limitations of library-dependent MST methods, including differential decay of source material in environmental waters and spatiotemporal variation in source communities, remain to be fully understood. Nevertheless, increasing use of these methods, as well as expanding fecal taxon libraries representative of source communities, will help improve the accuracy of these methods and provide promising tools for future MST investigations.</P>

Bioinformatic Suggestions on MiSeq-Based Microbial Community Analysis

Unno, Tatsuya The Korean Society for Microbiology and Biotechnol 2015 Journal of microbiology and biotechnology Vol.25 No.6

Recent sequencing technology development has revolutionized fields of microbial ecology. MiSeq-based microbial community analysis allows us to sequence more than a few hundred samples at a time, which is far more cost-effective than pyrosequencing. The approach, however, has not been preferably used owing to computational difficulties of processing huge amounts of data as well as known Illumina-derived artefact problems with amplicon sequencing. The choice of assembly software to take advantage of paired-end sequencing and methods to remove Illumina artefacts sequences are discussed. The protocol we suggest not only removed erroneous reads, but also dramatically reduced computational workload, which allows even a typical desktop computer to process a huge amount of sequence data generated with Illumina sequencers. We also developed a Web interface (<uri>http://biotech.jejunu.ac.kr/ ~abl/16s/</uri>) that allows users to conduct fastq-merging and mothur batch creation. The study presented here should provide technical advantages and supports in applying MiSeq-based microbial community analysis.

Evaluation of Intestinal Function Improvement Based on Gut Microbiota

Tatsuya Unno 한국식품영양과학회 2016 한국식품영양과학회 학술대회발표집 Vol.2016 No.10

Integrated online system for a pyrosequencing-based microbial source tracking method that targets Bacteroidetes 16S rDNA.

Unno, Tatsuya,Di, Doris Y W,Jang, Jeonghwan,Suh, Yae Seul,Sadowsky, Michael J,Hur, Hor-Gil American Chemical Society 2012 Environmental science & technology Vol.46 No.1

<P>Genotypic microbial source tracking (MST) methods are now routinely used to determine sources of fecal contamination impacting waterways. We previously reported the development of a pyrosequencing-based MST method that assigns contamination sources based on shared operational taxonomic units (OTUs) between fecal and environmental bacterial communities. Despite decreasing sequencing costs, pyrosequencing-based MST approaches are not used in routine water quality monitoring studies due in large part to difficulties in handling massive data sets and difficulties in determining sources of fecal contamination. In the studies presented here we describe the development of an online MST tool, PyroMiST ( http://env1.gist.ac.kr/aeml/MST.html) that uses total bacterial or Bacteroidetes 16S rDNA pyrosequencing reads to determine fecal contamination of waterways. The program cd-hit was used for OTU assignment and a Perl script was used to calculate the number of shared OTUs. The analyses require only a small number of pyrosequencing reads from environmental samples. Our results indicate that PyroMiST provides a user-friendly web interface for pyrosequence data that significantly reduces analysis time required to determine potential sources of fecal contamination in the environment.</P>

Absence of Escherichia coli Phylogenetic Group B2 Strains in Humans and Domesticated Animals from Jeonnam Province, Republic of Korea

Unno, Tatsuya,Han, Dukki,Jang, Jeonghwan,Lee, Sun-Nim,Ko, GwangPyo,Choi, Ha Young,Kim, Joon Ha,Sadowsky, Michael J.,Hur, Hor-Gil American Society for Microbiology 2009 Applied and environmental microbiology Vol.75 No.17

<B>ABSTRACT</B><P>Multiplex PCR analyses of DNAs from genotypically unique <I>Escherichia coli</I> strains isolated from the feces of 138 humans and 376 domesticated animals from Jeonnam Province, South Korea, performed using primers specific for the <I>chuA</I> and <I>yjaA</I> genes and an unknown DNA fragment, TSPE4.C2, indicated that none of the strains belonged to <I>E. coli</I> phylogenetic group B2. In contrast, phylogenetic group B2 strains were detected in about 17% (8 of 48) of isolates from feces of 24 wild geese and in 3% (3 of 96) of isolates obtained from the Yeongsan River in Jeonnam Province, South Korea. The distribution of <I>E. coli</I> strains in phylogenetic groups A, B1, and D varied depending on the host examined, and there was no apparent seasonal variation in the distribution of strains in phylogenetic groups among the Yeongsan River isolates. The distribution of four virulence genes (<I>eaeA</I>, <I>hlyA</I>, <I>stx</I>1, and <I>stx</I>2) in isolates was also examined by using multiplex PCR. Virulence genes were detected in about 5% (38 of 707) of the total group of unique strains examined, with 24, 13, 13, and 9 strains containing <I>hlyA</I>, <I>eaeA</I>, <I>stx</I>2, and <I>stx</I>1, respectively. The virulence genes were most frequently present in phylogenetic group B1 strains isolated from beef cattle. Taken together, results of these studies indicate that <I>E. coli</I> strains in phylogenetic group B2 were rarely found in humans and domesticated animals in Jeonnam Province, South Korea, and that the majority of strains containing virulence genes belonged to phylogenetic group B1 and were isolated from beef cattle. Results of this study also suggest that the relationship between the presence and types of virulence genes and phylogenetic groupings may differ among geographically distinct <I>E. coli</I> populations.</P>

Introductory Review : Illumina-based Microbiome Analysis

Tatsuya Unno,Hyejun Jo,Jiwan Hong,Jiyon Kim 제주대학교 아열대농업생명과학연구소 2018 아열대농업생명과학연구지 Vol.34 No.1

Illumina를 이용한16S rRNA 기반 미생물생태분석에서 분변의 동결건조에 의한 인공적인 시퀀스 생성 감소효과

운노타쯔야 ( Tatsuya Unno ),김정만 ( Jungman Kim ) 한국응용생명화학회(구 한국농화학회) 2016 Journal of Applied Biological Chemistry (J. Appl. Vol.59 No.4

PCR 산물을 이용한 시퀀싱방법 중 Illumina 플랫폼으로 시퀀싱을 수행하면 100개 이상의 인위적인 시퀀스가 생겨나며, 그러한 인위적으로 형성되는 시퀀스에 의해 Operational taxonomic units를 기반으로 한 미생물생태 변화 및 네트워크 분석에 영향을 미친다. 이러한 문제점이 있음에도 불구하고 분변미생물생태를 분석하는데 Illumina에서 제공하고 있는 시퀀싱을 주된 방법으로 사용하고 있으며, 또한 그러한 시퀀스 기반의 분변미생물 생태분석 결과는 분변샘플상태(i.e., 분변 보관 기간, 분변양, 분변의 신선도)에 따라 상이하게 나타난다. 본 연구에서는 분변샘플의 동결건조가 시퀀스 데이터의 퀄리티를 향상시키는지 관해 조사하였으며, 이를 통해 분변샘플에 동결건조처리는 전체적인 미생물생태구조를 변화시키지는 않지만 인위적으로 형성되었을 가능성이 있는 시퀀스의 수를 감소시키는 것으로 확인되었다. 따라서, 분변으로부터 DNA를 추출하기 이전에 동결건조처리하는 방법을 Illumina 기반의 분변미생물생태분석에 사용하는 것을 권장한다. When used for amplicon sequencing, Illumina platforms produce more than hundreds of sequence artefacts, which affects operational taxonomic units based analyses such as differential abundance and network analyses. Nevertheless it has become a major tool for fecal microbial community analysis. In addition, results from sequence-based fecal microbial community analysis vary depending on conditions of samples (i.e., freshness, time of storage and quantity). We investigated if freeze-drying samples could improve quality of sequence data. Our results showed reduced number of possible artefacts while maintaining overall microbial community structure. Therefore, freeze-drying feces prior to DNA extraction is recommended for Illumina-based microbial community analysis.

NGS기반 민물품질 모니터링 목표

운노타쯔야 ( Tatsuya Unno ),박상정 ( Sangjung Park ),허호길 ( Hor Gil Hur ),( Michael J. Sadowsky ) 한국환경농학회 2012 한국환경농학회 학술대회집 Vol.2012 No.-