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

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.

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

Microbiology ◽

10.1099/mic.0.037341-0 ◽

2010 ◽

Vol 156 (7) ◽

pp. 2035-2045 ◽

Cited By ~ 54

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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Identification of Three Major Clones of Multiply Antibiotic-Resistant Streptococcus pneumoniae in Taiwanese Hospitals by Multilocus Sequence Typing

Journal of Clinical Microbiology ◽

10.1128/jcm.36.12.3514-3519.1998 ◽

1998 ◽

Vol 36 (12) ◽

pp. 3514-3519 ◽

Cited By ~ 70

Author(s):

Zhi-Yuan Shi ◽

Mark C. Enright ◽

Paul Wilkinson ◽

David Griffiths ◽

Brian G. Spratt

Keyword(s):

Streptococcus Pneumoniae ◽

Molecular Typing ◽

Multilocus Sequence Typing ◽

Housekeeping Genes ◽

Multidrug Resistant ◽

Single Locus ◽

Allelic Profile ◽

Antibiotic Resistant ◽

Typing Procedure

In this paper we demonstrate the advantages of a new molecular typing procedure, multilocus sequence typing, for the unambiguous characterization of penicillin-resistant pneumococci. The sequences of ∼450-bp fragments of seven housekeeping genes were determined for 74 penicillin-resistant Taiwanese isolates of Streptococcus pneumoniae (MIC of penicillin > 0.5 μg/ml). The combination of alleles at the seven loci defined an allelic profile for each strain, and a dendrogram, based on the pairwise mismatches in allelic profiles, grouped 86% of the isolates into one of three penicillin-resistant clones for which the MICs of penicillin were 1 to 2 μg/ml. Isolates within each clone had identical alleles at all seven loci or differed at only a single locus, and the fingerprints of their pbp1A, pbp2B, and pbp2X genes were uniform. Isolates of the Taiwan-19F clone and the Taiwan-23F clone were resistant to penicillin, tetracycline, and erythromycin but were susceptible to chloramphenicol. A second serotype 23F clone and serotype 19F variants of this clone were resistant to penicillin, tetracycline, chloramphenicol, and, in some cases, erythromycin. Comparisons of the allelic profiles of the three major clones with those of reference isolates of the known penicillin-resistant clones showed that the Taiwan-19F and Taiwan-23F clones were previously undescribed, whereas the second serotype 23F clone was indistinguishable from the Spanish multidrug-resistant serotype 23F clone. Single isolates of the Spanish penicillin-resistant serotype 9V clone and the Spanish multidrug-resistant serotype 6B clone were also identified in the collection.

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

Journal of Bacteriology ◽

10.1128/jb.01808-07 ◽

2008 ◽

Vol 190 (8) ◽

pp. 2831-2840 ◽

Cited By ~ 131

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.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.01910-14 ◽

2014 ◽

Vol 53 (3) ◽

pp. 789-794 ◽

Cited By ~ 30

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.

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Characterization of Staphylococcus aureus Isolates from Retail Chicken Carcasses and Pet Workers in Northwest Arkansas

Journal of Food Protection ◽

10.4315/0362-028x.jfp-11-251 ◽

2012 ◽

Vol 75 (1) ◽

pp. 174-178 ◽

Cited By ~ 14

Author(s):

IRENE HANNING ◽

DAVID GILMORE ◽

SEAN PENDLETON ◽

SCOTT FLECK ◽

ASHLEY CLEMENT ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Multilocus Sequence Typing ◽

Human Infection ◽

Methicillin Resistant ◽

Healthy Humans ◽

Northwest Arkansas ◽

Low Prevalence ◽

Raw Poultry ◽

Staphylococcus Spp

Staphylococcus aureus can be carried on the skin and nasal passages of humans and animals as a commensal. A case of human methicillin-resistant S. aureus infection resulting from contact with pork has been reported. Poultry carcasses are sold at retail with the skin intact, but pork and beef typically are not. Thus, the risk of methicillin-resistant S. aureus human infection from whole raw poultry carcasses may be greater than that of exposure from pork or beef. The objective of this study was to isolate and characterize S. aureus from whole retail poultry carcasses and compare the isolates to S. aureus isolates from humans. A total of 25 S. aureus isolates were collected from 222 whole poultry carcasses. The isolates were characterized phenotypically with antibiotic resistance disc diffusion assays and genotypically using multilocus sequence typing. A total of 17 S. aureus isolates obtained from healthy humans were included and characterized in the same way as the poultry isolates. Staphylococcus spp. were recovered from all poultry carcasses. Only 25 poultry carcasses (11.2%) were contaminated with S. aureus. Of these 25 isolates, 36% were resistant to at least one of the antibiotics tested and 20% were resistant to two or more antibiotics tested. However, 100% of the human isolates were resistant to at least one of the antibiotics and 94% were resistant to two or more antibiotics. The results of the multilocus sequence typing indicate that most of the isolates grouped according to source. These results indicate a low prevalence of S. aureus present in poultry, and the isolates were not phenotypically similar to human isolates. The low number of S. aureus isolates from this study indicates that chicken carcasses would appear to not be a significant source of this bacterium.

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

Applied and Environmental Microbiology ◽

10.1128/aem.02257-13 ◽

2013 ◽

Vol 79 (23) ◽

pp. 7220-7228 ◽

Cited By ~ 14

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.

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Staphylococcus aureus PBP4 Is Essential for β-Lactam Resistance in Community-Acquired Methicillin-Resistant Strains

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00049-08 ◽

2008 ◽

Vol 52 (11) ◽

pp. 3955-3966 ◽

Cited By ~ 105

Author(s):

Guido Memmi ◽

Sergio R. Filipe ◽

Mariana G. Pinho ◽

Zhibiao Fu ◽

Ambrose Cheung

Keyword(s):

Staphylococcus Aureus ◽

Methicillin Resistance ◽

Healthy Individuals ◽

Penicillin Binding Protein ◽

Methicillin Resistant ◽

Fold Reduction ◽

Resistant Strains ◽

Mrsa Strains ◽

Hospital Acquired ◽

Major Target

ABSTRACT Recent cases of infections caused by community-acquired methicillin-resistant Staphylococcus aureus (MRSA) (CA-MRSA) strains in healthy individuals have raised concerns worldwide. CA-MRSA strains differ from hospital-acquired MRSAs by virtue of their genomic background and increased virulence in animal models. Here, we show that in two common CA-MRSA isolates, USA300 and MW2 (USA400), a loss of penicillin binding protein 4 (PBP4) is sufficient to cause a 16-fold reduction in oxacillin and nafcillin resistance, thus demonstrating that mecA, encoding PBP2A, is not the sole determinant of methicillin resistance in CA-MRSA. The loss of PBP4 was also found to severely affect the transcription of PBP2 in cells after challenge with oxacillin, thus leading to a significant decrease in peptidoglycan cross-linking. Autolysis, which is commonly associated with the killing mechanism of penicillin and β-lactams, does not play a role in the reduced resistance phenotype associated with the loss of PBP4. We also showed that cefoxitin, a semisynthetic β-lactam that binds irreversibly to PBP4, is synergistic with oxacillin in killing CA-MRSA strains, including clinical CA-MRSA isolates. Thus, PBP4 represents a major target for drug rediscovery against CA-MRSA, and a combination of cefoxitin and synthetic penicillins may be an effective therapy for CA-MRSA infections.

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A novel multilocus sequence typing scheme for the opportunistic pathogen Propionibacterium acnes and characterization of type I cell surface-associated antigens

Microbiology ◽

10.1099/mic.0.049676-0 ◽

2011 ◽

Vol 157 (7) ◽

pp. 1990-2003 ◽

Cited By ~ 99

Author(s):

Andrew McDowell ◽

Anna Gao ◽

Emma Barnard ◽

Colin Fink ◽

Philip I. Murray ◽

...

Keyword(s):

Cell Surface ◽

Multilocus Sequence Typing ◽

Propionibacterium Acnes ◽

Antigenic Variation ◽

Dna Typing ◽

Single Locus ◽

Type I ◽

Mlst Scheme ◽

Type Ia

We have developed a novel multilocus sequence typing (MLST) scheme and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and random amplification of polymorphic DNA typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (STs). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB1), representing 64 % of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants, which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore support an association between acne and P. acnes strains from the type IA cluster and highlight the role of a widely disseminated clonal genotype in this condition. Characterization of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00931-12 ◽

2012 ◽

Vol 78 (13) ◽

pp. 4646-4653 ◽

Cited By ~ 12

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/.

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