Eighty-five Enterococcus faecalis isolates collected from animals (40 isolates), meju (a Korean fermented soybean product; 27 isolates), humans (10 isolates), and various environmental samples (8 isolates) were subjected to multilocus sequence typing (MLST) to identify genetic differences between sa...
Eighty-five Enterococcus faecalis isolates collected from animals (40 isolates), meju (a Korean fermented soybean product; 27 isolates), humans (10 isolates), and various environmental samples (8 isolates) were subjected to multilocus sequence typing (MLST) to identify genetic differences between samples of different origins. MLST analysis resulted in 44 sequence types (STs), and the eBURST algorithm clustered the STs into 21 clonal complexes (CCs) and 17 singletons. The predominant STs, ST695 (21.1%, 18/85) and ST694 (9.4%, 8/85), were singletons, and only contained isolates originating from meju. None of the STs in the current study belonged to CC2 or CC9, which comprise clinical isolates with high levels of antibiotic resistance. The E. faecalis isolates showed the highest rates of resistance to tetracycline (32.9%), followed by erythromycin (9.4%) and vancomycin (2.4%). All isolates from meju were sensitive to these three antibiotics. Hence, MLST uncovered genetic diversity within E. faecalis, and clustering of the STs using eBURST revealed a correlation between the genotypes and origins of the isolates.
Eighty-five Enterococcus faecalis isolates collected from animals (40 isolates), meju (a Korean fermented soybean product; 27 isolates), humans (10 isolates), and various environmental samples (8 isolates) were subjected to multilocus sequence typing (MLST) to identify genetic differences between samples of different origins. MLST analysis resulted in 44 sequence types (STs), and the eBURST algorithm clustered the STs into 21 clonal complexes (CCs) and 17 singletons. The predominant STs, ST695 (21.1%, 18/85) and ST694 (9.4%, 8/85), were singletons, and only contained isolates originating from meju. None of the STs in the current study belonged to CC2 or CC9, which comprise clinical isolates with high levels of antibiotic resistance. The E. faecalis isolates showed the highest rates of resistance to tetracycline (32.9%), followed by erythromycin (9.4%) and vancomycin (2.4%). All isolates from meju were sensitive to these three antibiotics. Hence, MLST uncovered genetic diversity within E. faecalis, and clustering of the STs using eBURST revealed a correlation between the genotypes and origins of the isolates.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
문제 정의
In addition, MLST has been used to study the long-term epidemiology of bacterial species, and has provided insights into population structure and patterns of evolutionary descent [20]. Therefore, our goal was to investigate genetic differences among E. faecalis isolates from fermented foods, animals, and the environment using MLST, and to evaluate their potential for use as starter cultures. Additionally, antibiotic resistance profiles of each of the isolates was examined to understand the relationship with genetic diversity.
가설 설정
According to the public MLST database, ST403 single-locus variants (designated ST29, ST244, ST292, and ST416) also tend to be associated with poultry. Thus, we hypothesized that CC403 clones might have become accustomed to poultry hosts. Similarly, ST4 in CC4 appears to be undergoing host expansion through both single- and double-locus variants, which have been isolated from poultry.
제안 방법
We could not identify a correlation between ST and the origin of the isolates, but clustering of the STs by eBURST analysis revealed a correlation between the genetic backgrounds and origins of the isolates, as well as antibiotic resistance patterns. Although the isolates used in this study could not be perfectly classified according to their origin, the applicability of this MLST scheme was proven by the clustering of isolates from meju. In addition, we confirmed that the meju isolates are likely to be safe for use as starter cultures in the food industry because of their antibiotic sensitivity and genetic clustering away from the clonal groups usually associated with pathogenicity.
Table 2. Sequence types of the Enterococcus faecalis strains used in this study, as well as their corresponding eBURST clonal group and allele numbers.
The eBURST algorithm clustered the 44 STs into 21 CCs and 17 singletons using the default stringent definition for groups, which assigned STs with shared alleles at six of the seven loci to the same group (Fig. 2). The dominant clonal group CC16 contained seven isolates from two STs, whereas CC4, CC21, CC165, CC648, and CC698 each contained two STs and comprised three, two, two, three, and three isolates, respectively.
The sequences of the seven amplified genes from each isolate were proofread using SeqMan (DNASTAR, USA), and high-quality double-stranded sequences were used for further analyses. The rate of occurrence of genetic diversity and the ratio of nonsynonymous (dN) to synonymous (dS) substitutions per nucleotide site were calculated using MEGA ver.
This study is the first to use MLST to analyze the genetic diversity within E. faecalis isolated from the Korean fermented food meju and from several other sources. We could not identify a correlation between ST and the origin of the isolates, but clustering of the STs by eBURST analysis revealed a correlation between the genetic backgrounds and origins of the isolates, as well as antibiotic resistance patterns.
대상 데이터
Fourteen of the STs (excluding the two major STs) comprised at least two isolates. Eight STs (ST277, ST314, ST387, ST403, ST694, ST695, ST699, and STK9) contained isolates originating from only one origin, whereas the remaining six STs contained isolates from two or three origins (Table 2).
Forty-four different STs were identified for the 85 isolates, and 17 STs consisted of a single isolate (Table 2). Eleven new STs (ST695, ST696, ST697, ST698, ST699, ST700, ST703, ST704, ST705, ST706, and ST707) were identified as a result of new alleles for six of the seven genes (all except gyd): gdh (2 alleles), pstS (2), gki (3), aroE (5), xpt (1), and yiqL (5), whereas 12 new STs (STK1–STK12) were identified on the basis of a new combination of the existing alleles (Table 2).
Lee of the Korea Environmental Microorganisms Bank, Suwon, Korea. The 10 E. faecalis isolates from humans were kindly provided by Dr. Yoon from Kangwon National University, Chuncheon, Korea. The 40 E.
The phylogenetic tree revealed three different clusters; however, isolates were not clustered on the basis of origin, although a few branches contained isolates from the same origin. The 27 isolates from meju were allocated into three STs (ST648, ST694, and ST695), which were located on distinct branches, and were clustered with isolates from other origins. Therefore, we assumed that the current MLST data for E.
org/efaecalis/). The ST was determined through a combination of the seven alleles. New STs identified in the present study were deposited in the MLST database.
The two ST403 isolates identified in this study, belonging to CC403, originated from poultry. According to the public MLST database, ST403 single-locus variants (designated ST29, ST244, ST292, and ST416) also tend to be associated with poultry.
이론/모형
Antibiotic minimum inhibitory concentrations (MICs) were determined using the broth microdilution method according to the guidelines of the Clinical and Laboratory Standards Institute [24].
New STs identified in the present study were deposited in the MLST database. Phylogenetic analysis based on the STs was performed using the maximum likelihood method. STs were analyzed to determine CCs using the eBURST algorithm to identify single-locus variants, double-locus variants, and singletons [19].
Phylogenetic analysis based on sequence typing data, strain origins, and antibiotic susceptibility profiles. The data were compared using simple matching coefficients, and were clustered using the maximum likelihood method. Branches with bootstrap values <50% have been collapsed.
The sequences of the seven amplified genes from each isolate were proofread using SeqMan (DNASTAR, USA), and high-quality double-stranded sequences were used for further analyses. The rate of occurrence of genetic diversity and the ratio of nonsynonymous (dN) to synonymous (dS) substitutions per nucleotide site were calculated using MEGA ver. 6.06 [22], according to the Nei and Gojobori method [23]. Allele numbers were assigned by comparing the sequence at each locus to known alleles in the E.
성능/효과
Eleven new STs (ST695, ST696, ST697, ST698, ST699, ST700, ST703, ST704, ST705, ST706, and ST707) were identified as a result of new alleles for six of the seven genes (all except gyd): gdh (2 alleles), pstS (2), gki (3), aroE (5), xpt (1), and yiqL (5), whereas 12 new STs (STK1–STK12) were identified on the basis of a new combination of the existing alleles (Table 2).
Seven housekeeping gene fragments from all 85 E. faecalis isolates were sequenced, and the allelic variation of each gene sequence was determined. The number of polymorphic sites within each gene ranged from 8 (gyd) to 27 (gki and aroE) (Table 1).
The taxonomic identities of all isolates were confirmed by sequence analysis of near-complete 16S rRNA gene regions [10], and all showed >99.9% identity to the type strain ATCC 19433.
faecalis isolates from French cheese were associated with CC25 and CC55, which are rarely associated with clinical isolates [35]. These results suggested that the clonal groups of E. faecalis from fermented foods might be distinguished from those of pathogenic strains, as CCs appear to depend on strain origin.
참고문헌 (36)
Santos MM, Piccirillo C, Castro PM, Kalogerakis N, Pintado ME. 2012. Bioconversion of oleuropein to hydroxytyrosol by lactic acid bacteria. World J. Microbiol. Biotechnol. 28: 2435- 2440.
Foulquie Moreno MR, Sarantinopoulos P, Tsakalidou E, De Vuyst L. 2006. The role and application of enterococci in food and health. Int. J. Food Microbiol. 106: 1-24.
Franz CM, van Belkum MJ, Holzapfel WH, Abriouel H, Galvez A. 2007. Diversity of enterococcal bacteriocins and their grouping in a new classification scheme. FEMS Microbiol. Rev. 31: 293-310.
Hidron AI, Edwards JR, Patel J, Horan TC, Sievert DM, Pollock DA, et al. 2008. NHSN annual update: antimicrobialresistant pathogens associated with healthcare-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006-2007. Infect. Control Hosp. Epidemiol. 29: 996-1011.
Perez-Pulido R, Abriouel H, Ben Omar N, Lucas R, Martinez-Canamero M, Galvez A. 2006. Safety and potential risks of enterococci isolated from traditional fermented capers. Food Chem. Toxicol. 44: 2070-2077.
DiazGranados CA, Zimmer SM, Klein M, Jernigan JA. 2005. Comparison of mortality associated with vancomycinresistant and vancomycin-susceptible enterococcal bloodstream infections: a meta-analysis. Clin. Infect. Dis. 41: 327-333.
Kara A, Devrim I, Bayram N, Katipoglu N, Kiran E, Oruc Y, et al. 2015. Risk of vancomycin-resistant enterococci bloodstream infection among patients colonized with vancomycin-resistant enterococci. Braz. J. Infect. Dis. 19: 58-61.
Jeong DW, Kim HR, Jung G, Han S, Kim CT, Lee JH. 2014. Bacterial community migration in the ripening of doenjang, a traditional Korean fermented soybean food. J. Microbiol. Biotechnol. 24: 648-660.
Nallapareddy SR, Wenxiang H, Weinstock GM, Murray BE. 2005. Molecular characterization of a widespread, pathogenic, and antibiotic resistance-receptive Enterococcus faecalis lineage and dissemination of its putative pathogenicity island. J. Bacteriol. 187: 5709-5718.
Kawalec M, Pietras Z, Danilowicz E, Jakubczak A, Gniadkowski M, Hryniewicz W, et al. 2007. Clonal structure of Enterococcus faecalis isolated from Polish hospitals: characterization of epidemic clones. J. Clin. Microbiol. 45: 147-153.
Kuch A, Willems RJ, Werner G, Coque TM, Hammerum AM, Sundsfjord A, et al. 2012. Insight into antimicrobial susceptibility and population structure of contemporary human Enterococcus faecalis isolates from Europe. J. Antimicrob. Chemother. 67: 551-558.
Campanile F, Bartoloni A, Bartalesi F, Borbone S, Mangani V, Mantella A, et al. 2003. Molecular alterations of VanA element in vancomycin-resistant enterococci isolated during a survey of colonized patients in an Italian intensive care unit. Microb. Drug Resist. 9: 191-199.
Ruiz-Garbajosa P, Bonten MJ, Robinson DA, Top J, Nallapareddy SR, Torres C, et al. 2006. Multilocus sequence typing scheme for Enterococcus faecalis reveals hospitaladapted genetic complexes in a background of high rates of recombination. J. Clin. Microbiol. 44: 2220-2228.
Kuhn I, Burman LG, Haeggman S, Tullus K, Murray BE. 1995. Biochemical fingerprinting compared with ribotyping and pulsed-field gel electrophoresis of DNA for epidemiological typing of enterococci. J. Clin. Microbiol. 33: 2812-2817.
Feil EJ, Li BC, Aanensen DM, Hanage WP, Spratt BG. 2004. eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J. Bacteriol. 186: 1518-1530.
Chung YS, Kwon KH, Shin S, Kim JH, Park YH, Yoon JW. 2014. Characterization of veterinary hospital-associated isolates of Enterococcus species in Korea. J. Microbiol. Biotechnol. 24: 386-393.
Nei M, Gojobori T. 1986. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol. Biol. Evol. 3: 418-426.
Clinical and Laboratory Standards Institute. 2007. Performance standards for antimicrobial susceptibility testing; seventeenth informational supplement. CLSI domument M100-S17. CLSI, Wayne, PA. USA.
Guerrero-Ramos E, Cordero J, Molina-Gonzalez D, Poeta P, Igrejas G, Alonso-Calleja C, et al. 2016. Antimicrobial resistance and virulence genes in enterococci from wild game meat in Spain. Food Microbiol. 53: 156-164.
Furlaneto-Maia L, Rocha KR, Siqueira VL, Furlaneto MC. 2014. Comparison between automated system and PCR-based method for identification and antimicrobial susceptibility profile of clinical Enterococcus spp. Rev. Instit. Med. Trop. Sao Paulo 56: 97-103.
Quinones D, Kobayashi N, Nagashima S. 2009. Molecular epidemiologic analysis of Enterococcus faecalis isolates in Cuba by multilocus sequence typing. Microb. Drug Resist. 15: 287-293.
Poulsen LL, Bisgaard M, Son NT, Trung NV, An HM, Dalsgaard A. 2012. Enterococcus faecalis clones in poultry and in humans with urinary tract infections, Vietnam. Emerg. Infect. Dis. 18: 1096-1100.
Lopez M, Rezusta A, Seral C, Aspiroz C, Marne C, Aldea MJ, et al. 2012. Detection and characterization of a ST6 clone of vanB2-Enterococcus faecalis from three different hospitals in Spain. Eur. J. Clin. Microbiol. Infect. Dis. 31: 257-260.
Rathnayake IU, Hargreaves M, Huygens F. 2011. Genotyping of Enterococcus faecalis and Enterococcus faecium isolates by use of a set of eight single nucleotide polymorphisms. J. Clin. Microbiol. 49: 367-372.
Solheim M, Brekke MC, Snipen LG, Willems RJ, Nes IF, Brede DA. 2011. Comparative genomic analysis reveals significant enrichment of mobile genetic elements and genes encoding surface structure-proteins in hospital-associated clonal complex 2 Enterococcus faecalis. BMC Microbiol. 11: 3.
Jamet E, Akary E, Poisson MA, Chamba JF, Bertrand X, Serror P. 2012. Prevalence and characterization of antibiotic resistant Enterococcus faecalis in French cheeses. Food Microbiol. 31: 191-198.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.