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 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 Molecular Characterization of Mycoplasma pneumoniae Infections in Two Rural Populations of Thailand from 2009 to 2012

2017 ◽  
Vol 55 (7) ◽  
pp. 2222-2233 ◽  
Author(s):  
Toni Whistler ◽  
Pongpun Sawatwong ◽  
Maureen H. Diaz ◽  
Alvaro J. Benitez ◽  
Bernard J. Wolff ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Mycoplasma Pneumoniae ◽  
Variable Number Tandem Repeat ◽  
Population Based ◽  
Variable Number ◽  
23S Rrna ◽  
Nasopharyngeal Swab ◽  
Urban Centers ◽  
Content Type ◽  
Adhesion Protein

ABSTRACTStudies onMycoplasma pneumoniaein Thailand have focused on urban centers and have not included molecular characterization. In an attempt to provide a more comprehensive understanding of this organism, we conducted a systematic random sampling to identify 3,000 nasopharyngeal swab specimens collected from January 2009 through July 2012 during population-based surveillance for influenza-like illness in two rural provinces.M. pneumoniaewas detected by real-time PCR in 175 (5.8%) specimens. Genotyping was performed using the major adhesion protein (P1) and multilocus variable-number tandem-repeat analysis (MLVA). Of the 157 specimens typed, 97 were P1 type 1 and 60 were P1 type 2. Six different MLVA profiles were identified in 149 specimens, with 4/5/7/2 (40%) and 3/5/6/2 (26%) predominating. There was no discrete seasonality toM. pneumoniaeinfections. Examination of the 23S rRNA sequence for known polymorphisms conferring macrolide resistance revealed that all 141 tested to possess the genotype associated with macrolide susceptibility.

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 Study of Two Separate Types of Macrolide-Resistant Mycoplasma pneumoniae Outbreaks

2016 ◽  
Vol 60 (7) ◽  
pp. 4310-4314 ◽  
Author(s):  
Yingshuo Wang ◽  
Qian Ye ◽  
Dehua Yang ◽  
Zhimin Ni ◽  
Zhimin Chen
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Primary Schools ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Upper Respiratory Tract ◽  
Rt Pcr ◽  
Content Type ◽  
Upper Respiratory ◽  
Longitudinal Sampling

ABSTRACTTo study the complete natural process of aMycoplasma pneumoniaeoutbreak in a semiclosed room such as a primary school room, we investigated two separateM. pneumoniaeoutbreaks involving 81 students in total in two primary schools in Hangzhou, China.M. pneumoniaeisolates from pharyngeal swabs were detected by fluorescence quantitative real-time PCR (RT-PCR) and culture. The class in school M had 39 students, with 12 (30.8%) with positiveM. pneumoniaedetection results. The class from school J had 42 students, with 13 (31.0%) positive. The strains from two classes were confirmed to represent two clones (3/4/5/7/2 and 5/4/5/7/2) and to be macrolide resistant (A2063G) according to P1 and multilocus variable-number tandem-repeat analysis (MLVA) genotyping, determination of MIC of antibiotics, and sequencing. Students withM. pneumoniaeisolates detected were divided into three groups: those carrying the isolates, those with upper respiratory tract infection (URI), and those with pneumonia. Longitudinal sampling performed using pharyngeal swabs showed that the persistence ofM. pneumoniaewas longest in the group of students with pneumonia.M. pneumoniaecauses pneumonia outbreaks in schools, and the incidence of pneumonia has a higher rate than that of URI. The persistence ofM. pneumoniae, with a median duration of 79.50 days in the group of students with pneumonia, differs from that of the infection state.

scholarly journals Emergence of Macrolide-Resistant Mycoplasma pneumoniae in Hong Kong Is Linked to Increasing Macrolide Resistance in Multilocus Variable-Number Tandem-Repeat Analysis Type 4-5-7-2

2015 ◽  
Vol 53 (11) ◽  
pp. 3560-3564 ◽  
Author(s):  
Pak-Leung Ho ◽  
Pierra Y. Law ◽  
Betsy W. K. Chan ◽  
Chun-Wai Wong ◽  
Kelvin K. W. To ◽  
...  
Keyword(s):  
Hong Kong ◽  
Mycoplasma Pneumoniae ◽  
Tandem Repeat ◽  
Variable Number Tandem Repeat ◽  
Routine Care ◽  
Variable Number ◽  
Macrolide Resistance ◽  
Content Type ◽  
Repeat Analysis ◽  
Mlva Type

Macrolide-resistantMycoplasma pneumoniae(MRMP) is rapidly emerging in Asia, but information on the temporal relationship between the increase in macrolide resistance and changes in strain types is scarce. Between 2011 and 2014,M. pneumoniaeinfection was diagnosed by PCR as part of routine care in a health care region in Hong Kong. Testing was initiated by clinicians, mainly in patients with suspectedM. pneumoniaepneumonia. Specimens positive forM. pneumoniaewere retrospectively investigated by macrolide resistance genotyping and a four-locus (Mpn13 to -16) multilocus variable-number tandem-repeat analysis (MLVA) scheme. The overall percentage ofM. pneumoniae-positive specimens was 17.9%, with annual rates ranging from 9.8% to 27.2%. The prevalence of MRMP had rapidly increased from 13.6% in 2011 to 30.7% in 2012, 36.6% in 2013, and 47.1% in 2014 (P= 0.038). Two major MLVA types, 4-5-7-2 and 3-5-6-2, accounted for 75% to 85% of the infections each year. MLVA types 4-5-7-2 and 3-5-6-2 predominated among macrolide-resistant and macrolide-sensitive groups, respectively. The increase in MRMP was mainly caused by increasing macrolide resistance in the prevalent MLVA type 4-5-7-2, changing from 25.0% in 2011 to 59.1% in 2012, to 89.7% in 2013, and to 100% in 2014 (P< 0.001). In conclusion, increasing MRMP in Hong Kong was linked to a single MLVA type, which was both prevalent and increasingly resistant to macrolides.

scholarly journals Potentially Human-Pathogenic Escherichia coli O26 in Norwegian Sheep Flocks

2011 ◽  
Vol 77 (14) ◽  
pp. 4949-4958 ◽  
Author(s):  
C. Sekse ◽  
M. Sunde ◽  
B.-A. Lindstedt ◽  
P. Hopp ◽  
T. Bruheim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Human Pathogens ◽  
Content Type ◽  
Representative Sampling ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
Close Relationship ◽  
Large Survey

ABSTRACTA national survey ofEscherichia coliO26 in Norwegian sheep flocks was conducted, using fecal samples to determine the prevalence. In total, 491 flocks were tested, andE. coliO26 was detected in 17.9% of the flocks. One hundred forty-twoE. coliO26 isolates were examined for flagellar antigens (H typing) and four virulence genes, includingstxandeae, to identify possible Shiga toxin-producingE. coli(STEC) and enteropathogenicE. coli(EPEC). Most isolates (129 out of 142) were identified asE. coliO26:H11. They possessedeaeand may have potential as human pathogens, although only a small fraction were identified as STEC O26:H11, giving a prevalence in sheep flocks of only 0.8%. Correspondingly, the sheep flock prevalence of atypical EPEC (aEPEC) O26:H11 was surprisingly high (15.9%). The genetic relationship between theE. coliO26:H11 isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA), identifying 63 distinct PFGE profiles and 22 MLVA profiles. Although the MLVA protocol was less discriminatory than PFGE and a few cases of disagreement were observed, comparison by partition mapping showed an overall good accordance between the two methods. A close relationship between a few isolates of aEPEC O26:H11 and STEC O26:H11 was identified, but all theE. coliO26:H11 isolates should be considered potentially pathogenic to humans. The present study consisted of a representative sampling of sheep flocks from all parts of Norway. This is the first large survey of sheep flocks focusing onE. coliO26 in general, including results of STEC, aEPEC, and nonpathogenic isolates.

scholarly journals Prediction of Local Transmission of Mycobacterium tuberculosis Isolates of a Predominantly Beijing Lineage by Use of a Variable-Number Tandem-Repeat Typing Method Incorporating a Consensus Set of Hypervariable Loci

2017 ◽  
Vol 56 (1) ◽  
Author(s):  
Yoshiro Murase ◽  
Kiyohiko Izumi ◽  
Akihiro Ohkado ◽  
Akio Aono ◽  
Kinuyo Chikamatsu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Tandem Repeat ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Trend Test ◽  
Typing Method ◽  
Content Type ◽  
Tuberculosis Transmission ◽  
High Prevalence ◽  
Hypervariable Loci

ABSTRACT Strain genotyping based on the variable-number tandem repeat (VNTR) is widely applied for identifying the transmission of Mycobacterium tuberculosis. A consensus set of four hypervariable loci (1982, 3232, 3820, and 4120) has been proposed to improve the discrimination of Beijing lineage strains. Herein, we evaluated the utility of these four hypervariable loci for tracing local tuberculosis transmission in 981 cases over a 14-month period in Japan (2010 to 2011). We used six different VNTR systems, with or without the four hypervariable loci. Patient ages and weighted standard distances (a measure of the dispersion of genotype-clustered cases) were used as proxies for estimating local tuberculosis transmission. The highest levels of isolate discrimination were achieved with VNTR systems that incorporated the four hypervariable loci (i.e., the Japan Anti-Tuberculosis Association [JATA]18-VNTR, mycobacterial interspersed repetitive unit [MIRU]28-VNTR, and 24Beijing-VNTR). The clustering rates by JATA12-VNTR, MIRU15-VNTR, JATA15-VNTR, JATA18-VNTR, MIRU28-VNTR, and 24Beijing-VNTR systems were 52.2%, 51.0%, 39.0%, 24.1%, 23.1%, and 22.0%, respectively. As the discriminative power increased, the median weighted standard distances of the clusters tended to decrease (from 311 to 80 km, P < 0.001, Jonckheere-Terpstra trend test). Concurrently, the median ages of patients in the clusters tended to decrease (from 68 to 60 years, P < 0.001, Jonckheere-Terpstra trend test). These findings suggest that strain typing using the four hypervariable loci improves the prediction of active local tuberculosis transmission. The four-locus set can therefore contribute to the targeted control of tuberculosis in settings with high prevalence of Beijing lineage strains.

scholarly journals Examination of Taxonomic Uncertainties Surrounding Brucella abortus bv. 7 by Phenotypic and Molecular Approaches

2013 ◽  
Vol 80 (5) ◽  
pp. 1570-1579 ◽  
Author(s):  
Bruno Garin-Bastuji ◽  
Virginie Mick ◽  
Gilles Le Carrou ◽  
Sebastien Allix ◽  
Lorraine L. Perrett ◽  
...  
Keyword(s):  
Brucella Abortus ◽  
Variable Number Tandem Repeat ◽  
Variable Number ◽  
Epidemiological Methods ◽  
Content Type ◽  
Phenotypic Profile ◽  
Molecular Approaches ◽  
Content Classification ◽  
Species Specific ◽  
Kenyan Strain

ABSTRACTBrucellataxonomy is perpetually being reshuffled, at both the species and intraspecies levels. Biovar 7 ofBrucella abortuswas suspended from theApproved Lists of Bacterial NamesBrucellaclassification in 1988, because of unpublished evidence that the reference strain 63/75 was a mixture ofB. abortusbiovars 3 and 5. To formally clarify the situation, all isolates previously identified asB. abortusbv. 7 in the AHVLA and ANSES strain collections were characterized by classical microbiological and multiple molecular approaches. Among the 14 investigated strains, including strain 63/75, only four strains, isolated in Kenya, Turkey, and Mongolia, were pure and showed a phenotypic profile in agreement with the former biovar 7, particularly agglutination with both anti-A/anti-M monospecific sera. These results were strengthened by molecular strategies. Indeed, genus- and species-specific methods allowed confirmation that the four pure strains belonged to theB. abortusspecies. The combination of most approaches excluded their affiliation with the recognized biovars (biovars 1 to 6 and 9), while some suggested that they were close to biovar 3.These assays were complemented by phylogenetic and/or epidemiological methods, such as multilocus sequence analysis (MLSA) and variable-number tandem repeat (VNTR) analysis. The results of this polyphasic investigation allow us to propose the reintroduction of biovar 7 into theBrucellaclassification, with at least three representative strains. Interestingly, the Kenyan strain, sharing the same biovar 7 phenotype, was genetically divergent from other three isolates. These discrepancies illustrate the complexity ofBrucellataxonomy. This study suggests that worldwide collections could include strains misidentified asB. abortusbv. 7, and it highlights the need to verify their real taxonomic position.

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.

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