scholarly journals Development of a Tiered Multilocus Sequence Typing Scheme for Members of the Lactobacillus acidophilus Complex

2013 ◽  
Vol 79 (23) ◽  
pp. 7220-7228 ◽  
Author(s):  
Padmini Ramachandran ◽  
David W. Lacher ◽  
Erika A. Pfeiler ◽  
Christopher A. Elkins
Keyword(s):  
Lactobacillus Acidophilus ◽  
Multilocus Sequence Typing ◽  
Random Amplified Polymorphic Dna ◽  
Rflp Analysis ◽  
Housekeeping Genes ◽  
Nucleotide Substitutions ◽  
Content Type ◽  
Mlst Scheme ◽  
Pcr Rflp ◽  
Pcr Targeting

ABSTRACTMembers of theLactobacillus acidophiluscomplex are associated with functional foods and dietary supplements because of purported health benefits they impart to the consumer. Many characteristics of these microorganisms are reported to be strain specific. Therefore, proper strain typing is essential for safety assessment and product labeling, and also for monitoring strain integrity for industrial production purposes. Fifty-two strains of theL. acidophiluscomplex (L. acidophilus,L. amylovorus,L. crispatus,L. gallinarum,L. gasseri, andL. johnsonii) were genotyped using two established methods and compared to a novel multilocus sequence typing (MLST) scheme. PCR restriction fragment length polymorphism (PCR-RFLP) analysis of thehsp60gene with AluI and TaqI successfully clustered 51 of the 52 strains into the six species examined, but it lacked strain-level discrimination. Random amplified polymorphic DNA PCR (RAPD-PCR) targeting the M13 sequence resulted in highly discriminatory profiles but lacked reproducibility. In this study, an MLST scheme was developed using the conserved housekeeping genesfusA,gpmA,gyrA,gyrB,lepA,pyrG, andrecA, which identified 40 sequence types that successfully clustered all of the strains into the six species. Analysis of the observed alleles suggests that nucleotide substitutions within five of the seven MLST loci have reached saturation, a finding that emphasizes the highly diverse nature of theL. acidophiluscomplex and our unconventional application of a typically intraspecies molecular typing tool. Our MLST results indicate that this method could be useful for characterization and strain discrimination of a multispecies complex, with the potential for taxonomic expansion to a broader collection ofLactobacillusspecies.

scholarly journals Multilocus Sequence Typing of Leuconostoc gelidum subsp. gasicomitatum, a Psychrotrophic Lactic Acid Bacterium Causing Spoilage of Packaged Perishable Foods

2015 ◽  
Vol 81 (7) ◽  
pp. 2474-2480 ◽  
Author(s):  
Riitta Rahkila ◽  
Per Johansson ◽  
Elina Säde ◽  
Lars Paulin ◽  
Petri Auvinen ◽  
...  
Keyword(s):  
Population Structure ◽  
Lactic Acid ◽  
Lactic Acid Bacterium ◽  
Multilocus Sequence Typing ◽  
Genetic Material ◽  
Housekeeping Genes ◽  
Content Type ◽  
Mlst Scheme ◽  
Global Comparison ◽  
Leuconostoc Gelidum

ABSTRACTLeuconostoc gelidumsubsp.gasicomitatumis a psychrotrophic lactic acid bacterium (LAB) that causes spoilage of a variety of modified-atmosphere-packaged (MAP) cold-stored food products. During the past 10 years, this spoilage organism has been increasingly reported in MAP meat and vegetable products in northern Europe. In the present study, the population structure within 252L. gelidumsubsp.gasicomitatumstrains was determined based on a novel multilocus sequence-typing (MLST) scheme employing seven housekeeping genes. These strains had been isolated from meat and vegetable sources over a time span of 15 years, and all 68 previously detected pulsed-field gel electrophoresis (PFGE) genotypes were represented. A total of 46 sequence types (STs) were identified, with a majority of the strains (>60%) belonging to three major STs, which were grouped into three clonal complexes (CCs) and 17 singletons by Global Optimal eBURST (goeBURST). The results by Bayesian analysis of population structure (BAPS) mostly correlated with the grouping by goeBURST. Admixture analysis by BAPS indicated a very low level of exchange of genetic material between the subpopulations. Niche specificity was observed within the subpopulations: CC1 and BAPS cluster 1 consisted mostly of strains from a variety of MAP meats, whereas vegetable strains grouped together with strains from MAP poultry within CC2 and BAPS cluster 2. The MLST scheme presented in this study provides a shareable and continuously growing sequence database enabling global comparison of strains associated with spoilage cases. This will further advance our understanding of the microbial ecology of this industrially important LAB.

scholarly journals Development of a Multilocus Sequence Typing Scheme for Molecular Typing of Mycoplasma pneumoniae

2015 ◽  
Vol 53 (10) ◽  
pp. 3195-3203 ◽  
Author(s):  
Rebecca J. Brown ◽  
Matthew T. G. Holden ◽  
O. Brad Spiller ◽  
Victoria J. Chalker
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Multilocus Sequence Typing ◽  
Genomic Sequence ◽  
Variable Number Tandem Repeat ◽  
Housekeeping Genes ◽  
Variable Number ◽  
Respiratory Pathogen ◽  
Content Type ◽  
Mlst Scheme ◽  
Lower Respiratory Disease

Mycoplasma pneumoniaeis a major human respiratory pathogen causing both upper and lower respiratory disease in humans of all ages, and it can also result in other serious extrapulmonary sequelae. A multilocus sequence typing (MLST) scheme forM. pneumoniaewas developed based on the sequences of eight housekeeping genes (ppa,pgm,gyrB,gmk,glyA,atpA,arcC, andadk) and applied to 55M. pneumoniaeclinical isolates and the two type strains M129 and FH. A total of 12 sequence types (STs) resulted for 57M. pneumoniaeisolates tested, with a discriminatory index of 0.21 STs per isolate. The MLST loci used in this scheme were shown to be stable in 10 strains following 10 sequential subculture passages. Phylogenetic analysis of concatenated sequences of the eight loci indicated two distinct genetic clusters that were directly linked to multilocus variable-number tandem repeat analysis (MLVA) type. Genetic MLST clustering was confirmed by genomic sequence analysis, indicating that the MLST scheme developed in this study is representative of the genome. Furthermore, this MLST scheme was shown to be more discriminatory than both MLVA and P1 typing for theM. pneumoniaeisolates examined, providing a method for further and more detailed analysis of observed epidemic peaks ofM. pneumoniaeinfection. This scheme is supported by a public Web-based database (http://pubmlst.org/mpneumoniae).

scholarly journals Genetic diversity in Italian Lactobacillus sanfranciscensis strains assessed by multilocus sequence typing and pulsed-field gel electrophoresis analyses

Microbiology ◽  
2010 ◽  
Vol 156 (7) ◽  
pp. 2035-2045 ◽  
Author(s):  
Claudia Picozzi ◽  
Gaia Bonacina ◽  
Ileana Vigentini ◽  
Roberto Foschino
Keyword(s):  
Multilocus Sequence Typing ◽  
Geographical Area ◽  
Housekeeping Genes ◽  
Processing Conditions ◽  
Clonal Population ◽  
Factors Affecting ◽  
Lactobacillus Sanfranciscensis ◽  
Mlst Scheme ◽  
Sequence Types

Lactobacillus sanfranciscensis is a lactic acid bacterium that characterizes the sourdough environment. The genetic differences of 24 strains isolated in different years from sourdoughs, mostly collected in Italy, were examined and compared by PFGE and multilocus sequence typing (MLST). The MLST scheme, based on the analysis of six housekeeping genes (gdh, gyrA, mapA, nox, pgmA and pta) was developed for this study. PFGE with the restriction enzyme ApaI proved to have higher discriminatory power, since it revealed 22 different pulsotypes, while 19 sequence types were recognized through MLST analysis. Notably, restriction profiles generated from three isolates collected from the same firm but in three consecutive years clustered in a single pulsotype and showed the same sequence type, emphasizing the fact that the main factors affecting the dominance of a strain are correlated with processing conditions and the manufacturing environment rather than the geographical area. All results indicated a limited recombination among genes and the presence of a clonal population in L. sanfranciscensis. The MLST scheme proposed in this work can be considered a useful tool for characterization of isolates and for in-depth examination of the strain diversity and evolution of this species.

scholarly journals Determination of Molecular Phylogenetics of Vibrio parahaemolyticus Strains by Multilocus Sequence Typing

2008 ◽  
Vol 190 (8) ◽  
pp. 2831-2840 ◽  
Author(s):  
Narjol González-Escalona ◽  
Jaime Martinez-Urtaza ◽  
Jaime Romero ◽  
Romilio T. Espejo ◽  
Lee-Ann Jaykus ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Population Structure ◽  
Multilocus Sequence Typing ◽  
Vibrio Parahaemolyticus ◽  
Molecular Phylogenetics ◽  
Clonal Complex ◽  
Housekeeping Genes ◽  
The United States ◽  
Health Concern ◽  
Mlst Scheme

ABSTRACT Vibrio parahaemolyticus is an important human pathogen whose transmission is associated with the consumption of contaminated seafood. There is a growing public health concern due to the emergence of a pandemic strain causing severe outbreaks worldwide. Many questions remain unanswered regarding the evolution and population structure of V. parahaemolyticus. In this work, we describe a multilocus sequence typing (MLST) scheme for V. parahaemolyticus based on the internal fragment sequences of seven housekeeping genes. This MLST scheme was applied to 100 V. parahaemolyticus strains isolated from geographically diverse clinical (n = 37) and environmental (n = 63) sources. The sequences obtained from this work were deposited and are available in a public database (http://pubmlst.org/vparahaemolyticus ). Sixty-two unique sequence types were identified, and most (50) were represented by a single isolate, suggesting a high level of genetic diversity. Three major clonal complexes were identified by eBURST analysis. Separate clonal complexes were observed for V. parahaemolyticus isolates originating from the Pacific and Gulf coasts of the United States, while a third clonal complex consisted of strains belonging to the pandemic clonal complex with worldwide distribution. The data reported in this study indicate that V. parahaemolyticus is genetically diverse with a semiclonal population structure and an epidemic structure similar to that of Vibrio cholerae. Genetic diversity in V. parahaemolyticus appears to be driven primarily by frequent recombination rather than mutation, with recombination ratios estimated at 2.5:1 and 8.8:1 by allele and site, respectively. Application of this MLST scheme to more V. parahaemolyticus strains and by different laboratories will facilitate production of a global picture of the epidemiology and evolution of this pathogen.

scholarly journals Multilocus Sequence Typing for Characterization of Methicillin-Resistant and Methicillin-Susceptible Clones ofStaphylococcus aureus

2000 ◽  
Vol 38 (3) ◽  
pp. 1008-1015 ◽  
Author(s):  
Mark C. Enright ◽  
Nicholas P. J. Day ◽  
Catrin E. Davies ◽  
Sharon J. Peacock ◽  
Brian G. Spratt
Keyword(s):  
Multilocus Sequence Typing ◽  
Reference Strain ◽  
Housekeeping Genes ◽  
Invasive Disease ◽  
Healthy Individuals ◽  
Allelic Profile ◽  
Methicillin Resistant ◽  
Mlst Scheme ◽  
Hospital Acquired

A multilocus sequence typing (MLST) scheme has been developed forStaphylococcus aureus. The sequences of internal fragments of seven housekeeping genes were obtained for 155 S. aureusisolates from patients with community-acquired and hospital-acquired invasive disease in the Oxford, United Kingdom, area. Fifty-three different allelic profiles were identified, and 17 of these were represented by at least two isolates. The MLST scheme was highly discriminatory and was validated by showing that pairs of isolates with the same allelic profile produced very similar SmaI restriction fragment patterns by pulsed-field gel electrophoresis. All 22 isolates with the most prevalent allelic profile were methicillin-resistant S. aureus (MRSA) isolates and had allelic profiles identical to that of a reference strain of the epidemic MRSA clone 16 (EMRSA-16). Four MRSA isolates that were identical in allelic profile to the other major epidemic MRSA clone prevalent in British hospitals (clone EMRSA-15) were also identified. The majority of isolates (81%) were methicillin-susceptible S. aureus (MSSA) isolates, and seven MSSA clones included five or more isolates. Three of the MSSA clones included at least five isolates from patients with community-acquired invasive disease and may represent virulent clones with an increased ability to cause disease in otherwise healthy individuals. The most prevalent MSSA clone (17 isolates) was very closely related to EMRSA-16, and the success of the latter clone at causing disease in hospitals may be due to its emergence from a virulent MSSA clone that was already a major cause of invasive disease in both the community and hospital settings. MLST provides an unambiguous method for assigning MRSA and MSSA isolates to known clones or assigning them as novel clones via the Internet.

scholarly journals Global Multilocus Sequence Typing Analysis of Mycoplasma bovis Isolates Reveals Two Main Population Clusters

2014 ◽  
Vol 53 (3) ◽  
pp. 789-794 ◽  
Author(s):  
R. S. Rosales ◽  
C. P. Churchward ◽  
C. Schnee ◽  
K. Sachse ◽  
I. Lysnyansky ◽  
...  
Keyword(s):  
Multilocus Sequence Typing ◽  
Housekeeping Genes ◽  
Population Based ◽  
Economic Losses ◽  
Mycoplasma Bovis ◽  
Content Type ◽  
Clinical Presentations ◽  
High Discriminatory Power ◽  
Clonal Nature

Mycoplasma bovisis a major bovine pathogen associated with bovine respiratory disease complex and is responsible for substantial economic losses worldwide.M. bovisis also associated with other clinical presentations in cattle, including mastitis, otitis, arthritis, and reproductive disorders. To gain a better understanding of the genetic diversity of this pathogen, a multilocus sequence typing (MLST) scheme was developed and applied to the characterization of 137M. bovisisolates from diverse geographical origins, obtained from healthy or clinically infected cattle. Afterin silicoanalysis, a final set of 7 housekeeping genes was selected (dnaA,metS,recA,tufA,atpA,rpoD, andtkt). MLST analysis demonstrated the presence of 35 different sequence types (STs) distributed in two main clonal complexes (CCs), defined at the double-locus variant level, namely, CC1, which included most of the British and German isolates, and CC2, which was a more heterogeneous and geographically distant group of isolates, including European, Asian, and Australian samples. Index of association analysis confirmed the clonal nature of the investigatedM. bovispopulation, based on MLST data. This scheme has demonstrated high discriminatory power, with the analysis showing the presence of genetically distant and divergent clusters of isolates predominantly associated with geographical origins.

scholarly journals Genetic Characterization of Didymella bryoniae Isolates Infecting Watermelon and Other Cucurbits in Florida and Georgia

Plant Disease ◽  
2015 ◽  
Vol 99 (11) ◽  
pp. 1488-1499 ◽  
Author(s):  
Binoy Babu ◽  
Yonas W. Kefialew ◽  
Ping-Fang Li ◽  
Xing-Ping Yang ◽  
Sheeja George ◽  
...  
Keyword(s):  
Citrullus Lanatus ◽  
Random Amplified Polymorphic Dna ◽  
Rflp Analysis ◽  
Genetic Profile ◽  
Its Sequence ◽  
Didymella Bryoniae ◽  
Stem Blight ◽  
Gummy Stem Blight ◽  
Its Sequence Analysis ◽  
Pcr Rflp

Gummy stem blight caused by Didymella bryoniae (anamorph Phoma cucurbitacearum) is a major fungal disease of watermelon (Citrullus lanatus) and other cucurbits. Thirty-five isolates of Didymella and Phoma spp. associated with symptoms of gummy stem blight on watermelon, Canary melon (Cucumis melo), muskmelon (C. melo), and winter squash (Cucurbita maxima) from Florida and Georgia were characterized based on morphology on agar media, pathogenicity on ‘Melody’ watermelon, the internal transcribed spacer (ITS) sequence of ribosomal DNA (rDNA), random amplified polymorphic DNA (RAPD) analysis, and polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) analysis. All of the isolates were pathogenic on watermelon but differed in virulence. RAPD and ITS sequence analysis indicated genetic variability among the isolates but PCR-RFLP analysis did not show any variability. ITS sequence phylogenetic analysis identified two isolates, DB-05 and DB-33, which had a greater identity to that of D. bryoniae isolates from China (98 to 100% sequence homology) than other isolates from Florida and Georgia (95 to 98%). These two isolates possessed a single nucleotide substitution of A to G at position 131 of the ITS1 region. The study characterized the genetic profile of a collection of D. bryoniae isolates from Florida and Georgia in relation to isolates from other U.S. states and countries.

scholarly journals Multilocus Sequence Typing Scheme for the Characterization of 936-Like Phages Infecting Lactococcus lactis

2012 ◽  
Vol 78 (13) ◽  
pp. 4646-4653 ◽  
Author(s):  
Maxim Moisan ◽  
Sylvain Moineau
Keyword(s):  
Lactococcus Lactis ◽  
Multilocus Sequence Typing ◽  
Phylogenetic Analyses ◽  
Clonal Evolution ◽  
Phage Group ◽  
Content Type ◽  
Mlst Scheme ◽  
Routine Identification ◽  
Set Up

ABSTRACTLactococcus lactisphage infections are costly for the dairy industry because they can slow down the fermentation process and adversely impact product safety and quality. Although many strategies have been developed to better control phage populations, new virulent phages continue to emerge. Thus, it is beneficial to develop an efficient method for the routine identification of new phages within a dairy plant to rapidly adapt antiphage tactics. Here, we present a multilocus sequence typing (MLST) scheme for the characterization of the 936-like phages, the most prevalent phage group infectingL. lactisstrains worldwide. The proposed MLST system targets the internal portion of five highly conserved genomic sequences belonging to the packaging, morphogenesis, and lysis modules. Our MLST scheme was used to analyze 100 phages with different restriction fragment length polymorphism (RFLP) patterns isolated from 11 different countries between 1971 and 2010. PCR products were obtained for all the phages analyzed, and sequence analysis highlighted the high discriminatory power of the MLST system, detecting 93 different sequence types. A conserved locus within thelysgene (coding for endolysin) was the most discriminative, with 65 distinct alleles. The locus within themcpgene (major capsid protein) was the most conserved (54 distinct alleles). Phylogenetic analyses of the concatenated sequences exhibited a strong concordance of the clusters with the phage host range, indicating the clonal evolution of these phages. A public database has been set up for the proposed MLST system, and it can be accessed athttp://pubmlst.org/bacteriophages/.

scholarly journals Development of a Multilocus Sequence Typing Tool for High-Resolution Genotyping of Enterocytozoon bieneusi

2011 ◽  
Vol 77 (14) ◽  
pp. 4822-4828 ◽  
Author(s):  
Yaoyu Feng ◽  
Na Li ◽  
Theresa Dearen ◽  
Maria L. Lobo ◽  
Olga Matos ◽  
...  
Keyword(s):  
High Resolution ◽  
Multilocus Sequence Typing ◽  
Enterocytozoon Bieneusi ◽  
Whole Genome Sequence ◽  
Rrna Gene ◽  
Content Type ◽  
Health Significance ◽  
The One ◽  
Pcr Targeting ◽  
Typing Tool

ABSTRACTThus far, genotyping ofEnterocytozoon bieneusihas been based solely on DNA sequence analysis of the internal transcribed spacer (ITS) of the rRNA gene. Both host-adapted and zoonotic (human-pathogenic) genotypes ofE. bieneusihave been identified. In this study, we searched for microsatellite and minisatellite sequences in the whole-genome sequence database ofE. bieneusiisolate H348. Seven potential targets (MS1 to MS7) were identified. Testing of the seven targets by PCR using two human-pathogenicE. bieneusigenotypes (A and Peru10) led to the selection of four targets (MS1, MS3, MS4, and MS7). Further analysis of the four loci with an additional 24 specimens of both host-adapted and zoonoticE. bieneusigenotypes indicated that most host-adapted genotypes were not amplified by PCR targeting these loci. In contrast, 10 or 11 of the 13 specimens of the zoonotic genotypes were amplified by PCR at each locus. Altogether, 12, 8, 7, and 11 genotypes of were identified at MS1, MS3, MS4, and MS7, respectively. Phylogenetic analysis of the nucleotide sequences obtained produced a genetic relationship that was similar to the one at the ITS locus, with the formation of a large group of zoonotic genotypes that included mostE. bieneusigenotypes in humans. Thus, a multilocus sequence typing tool was developed for high-resolution genotyping ofE. bieneusi.Data obtained in the study should also have implications for understanding the taxonomy ofEnterocytozoonspp., the public health significance ofE. bieneusiin animals, and the sources of humanE. bieneusiinfections.

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