Epidemiological tracking and population assignment of the non-clonal bacterium, Burkholderia pseudomallei

Rapid assignment of bacterial pathogens into predefined populations is an important first step for epidemiological tracking. For clonal species, a single allele can theoretically define a population. For non-clonal species such as Burkholderia pseudomallei, however, shared allelic states between distantly related isolates make it more difficult to identify population defining characteristics. Two distinct B. pseudomallei populations have been previously identified using multilocus sequence typing (MLST). These populations correlate with the major foci of endemicity (Australia and Southeast Asia). Here, we use multiple Bayesian approaches to evaluate the compositional robustness of these populations, and provide assignment results for MLST sequence types (STs). Our goal was to provide a reference for assigning STs to an established population without the need for further computational analyses. We also provide allele frequency results for each population to enable estimation of population assignment even when novel STs are discovered. The ability for humans and potentially contaminated goods to move rapidly across the globe complicates the task of identifying the source of an infection or outbreak. Population genetic dynamics of B. pseudomallei are particularly complicated relative to other bacterial pathogens, but the work here provides the ability for broad scale population assignment. As there is currently no independent empirical measure of successful population assignment, we provide comprehensive analytical details of our comparisons to enable the reader to evaluate the robustness of population designations and assignments as they pertain to individual research questions. Finer scale subdivision and verification of current population compositions will likely be possible with genotyping data that more comprehensively samples the genome. The approach used here may be valuable for other non-clonal pathogens that lack simple group-defining genetic characteristics and provides a rapid reference for epidemiologists wishing to track the origin of infection without the need to compile population data and learn population assignment algorithms.     

Core genome conservation of Staphylococcus haemolyticus limits sequence based population structure analysis

The notoriously multi-resistant Staphylococcus haemolyticus is an emerging pathogen causing serious infections in immunocompromised patients. Defining the population structure is important to detect outbreaks and spread of antimicrobial resistant clones. Currently, the standard typing technique is pulsed-field gel electrophoresis (PFGE). In this study we describe novel molecular typing schemes for S. haemolyticus using multi locus sequence typing (MLST) and multi locus variable number of tandem repeats (VNTR) analysis. Seven housekeeping genes (MLST) and five VNTR loci (MLVF) were selected for the novel typing schemes. A panel of 45 human and veterinary S. haemolyticus isolates was investigated. The collection had diverse PFGE patterns (38 PFGE types) and was sampled over a 20 year-period from eight countries. MLST resolved 17 sequence types (Simpsons index of diversity [SID]=0.877) and MLVF resolved 14 repeat types (SID=0.831). We found a low sequence diversity. Phylogenetic analysis clustered the isolates in three (MLST) and one (MLVF) clonal complexes, respectively. Taken together, neither the MLST nor the MLVF scheme was suitable to resolve the population structure of this S. haemolyticus collection. Future MLVF and MLST schemes will benefit from addition of more variable core genome sequences identified by comparing different fully sequenced S. haemolyticus genomes.     

Multilocus sequence typing

Multilocus sequence typing (MLST) provides a new approach to molecular epidemiology that can identify and track the global spread of virulent or antibiotic-resistant isolates of bacterial pathogens using the Internet. MLST databases, together with interrogation software, are available for Neisseria meningitidis and Streptococcus pneumoniae and databases for Streptococcus pyogenes and Staphylococcus aureus will be released shortly.     

Multilocus sequence typing of Salmonella strains by high-throughput sequencing of selectively amplified target genes

Rapid development of next generation sequencing (NGS) technologies in recent years has made whole genome sequencing of bacterial genomes widely accessible. However, it is often unnecessary or not feasible to sequence the whole genome for most applications of genetic analyses in bacteria. Selectively capturing defined genomic regions followed by NGS analysis could be a promising approach for high-resolution molecular typing of a large set of strains. In this study, we describe a novel and straightforward PCR-based target-capturing method, hairpin-primed multiplex amplification (HPMA), which allows for simultaneous amplification of numerous target genes. To test the feasibility of NGS-based strain typing using HPMA, 20 target gene sequences were simultaneously amplified with barcode tagging in each of 41 Salmonella strains. The amplicons were then pooled and analyzed by 454 pyrosequencing. Analysis of the sequence data, as an extension of multilocus sequence typing (MLST), demonstrated the utility and potential of this novel typing method, MLST-seq, as a high-resolution strain typing method. With the rapidly increasing sequencing capacity of NGS, MLST-seq or its variations using different target enrichment methods can be expected to become a high-resolution typing method in the near future for high-throughput analysis of a large collection of bacterial strains.     

两株炭疽芽胞杆菌MLST 新序列型(ST)

【目的】2株炭疽芽胞杆菌(Bacillus anthracis)17003-14和17003-32的多位点序列分型(Multilocussequence typing,MLST)研究。【方法】选取B.anthracis基因组7个常见管家基因位点glpF、gmk、ilvD、pta、pur、pycA和tpi进行PCR扩增、测序,与MLST数据库中的等位基因序列进行比对,确定菌株的序列型(sequence type,ST)。【结果】B.anthracis 17003-14和17003-32的等位基因编号分别为113、31、1、43、1、53、7和113、31、1、43、1、53、37,比对结果显示这2株细菌的等位基因编号组合未见报道。【结论】17003-14和17003-32为新ST菌株,已被MLST数据库确认,注册号(pubMLST id)分别为id-1053和id-1054。     

Comparison of a semi-automated rep-PCR system and multilocus sequence typing for differentiation of Salmonella enterica isolates

The accurate sub-typing of Salmonella enterica isolates is essential for epidemiological investigations and surveillance of Salmonella infections. Salmonella isolates are currently identified using the Kauffman-White serotyping scheme. Multilocus sequence typing (MLST) schemes have been developed for the major bacterial pathogens including Salmonella and have assisted in understanding the molecular epidemiology and population biology of these organisms. Recently, the DiversiLab rep-PCR system has been developed using micro-fluidic chips to provide standardized, semi-automated fingerprinting for pathogens including S. enterica. In the current study, 71 isolates of S. enterica, representing 21 serovars, were analyzed using MLST and the DiversiLab rep-PCR system. MLST was able to identify 31 sequence types (STs), while the DiversiLab system revealed 38 DiversiLab types (DTs). The rep-PCR distinguished isolates of different serovars and showed greater discriminatory power (0.95) than MLST typing (0.89). Rep-PCR exhibited 92% concordance with MLST and 90% with serotyping, while the concordance level of MLST typing with serotyping was 96%, representing a strong association. Comparison of rep-PCR profiles with those held in an online library database led to the accurate prediction of serovar in 63% of cases and resulted in inaccurate predictions for 10% of profiles. MLST and the rep-PCR system may provide useful additional informative techniques for the molecular identification of S. enterica. We conclude that the DiversiLab rep-PCR system may provide a rapid (less than 4 h) and standardized method for sub-typing isolates of S. enterica.     

Multi-locus sequence typing: a tool for global epidemiology

The characterization of pathogenic isolates plays a pivotal role in the epidemiology of infectious diseases, generating the information necessary for identifying, tracking, and intervening against disease outbreaks. In 1998 multi-locus sequence typing (MLST) was proposed as a nucleotide sequence-based approach that could be applied to many bacterial pathogens. It combined developments in high-throughput sequencing and bioinformatics with established population genetics techniques to provide a portable, reproducible, and scalable typing system that reflected the population and evolutionary biology of bacterial pathogens. MLST schemes have been developed for a variety of procaryotic and eucaryotic pathogens and the data generated have contributed to both epidemiological surveillance and fundamental studies of pathogen biology.     

High-resolution melting genotyping of Enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms

We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure.     

eBURST: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data

The introduction of multilocus sequence typing (MLST) for the precise characterization of isolates of bacterial pathogens has had a marked impact on both routine epidemiological surveillance and microbial population biology. In both fields, a key prerequisite for exploiting this resource is the ability to discern the relatedness and patterns of evolutionary descent among isolates with similar genotypes. Traditional clustering techniques, such as dendrograms, provide a very poor representation of recent evolutionary events, as they attempt to reconstruct relationships in the absence of a realistic model of the way in which bacterial clones emerge and diversify to form clonal complexes. An increasingly popular approach, called BURST, has been used as an alternative, but present implementations are unable to cope with very large data sets and offer crude graphical outputs. Here we present a new implementation of this algorithm, eBURST, which divides an MLST data set of any size into groups of related isolates and clonal complexes, predicts the founding (ancestral) genotype of each clonal complex, and computes the bootstrap support for the assignment. The most parsimonious patterns of descent of all isolates in each clonal complex from the predicted founder(s) are then displayed. The advantages of eBURST for exploring patterns of evolutionary descent are demonstrated with a number of examples, including the simple Spain(23F)-1 clonal complex of Streptococcus pneumoniae, "population snapshots" of the entire S. pneumoniae and Staphylococcus aureus MLST databases, and the more complicated clonal complexes observed for Campylobacter jejuni and Neisseria meningitidis.     

Multilocus sequence typing for global surveillance of meningococcal disease

The global surveillance of bacterial pathogens is particularly important for bacteria with diverse and dynamic populations that cause periodic epidemics or pandemics. The isolate characterization methods employed for surveillance should: (1) generate unambiguous data; (2) be readily implemented in a variety of scenarios and be reproducible among laboratories; (3) be scalable and preferably available in a high throughput format; and (4) be cost effective. Multilocus sequence typing (MLST) was designed to meet these criteria and has been implemented effectively for a wide range of microorganisms. The 'Impact of meningococcal epidemiology and population biology on public health in Europe (EU-MenNet)' project had amongst its objectives: (1) to disseminate meningococcal MLST and sequence-based typing throughout Europe by establishing a centre for training and data generation, and (2) to produce a comprehensive Europe-wide picture of meningococcal disease epidemiology for the first time. Data produced from the project have shown the distribution of a relatively small number of STs, clonal complexes and PorA types that account for a large proportion of the disease-associated isolates in Europe. The project demonstrates how molecular typing can be combined with epidemiological data via the Internet for global disease surveillance.     

High-resolution melting system to perform multilocus sequence typing of Campylobacter jejuni

Multi-locus sequence typing (MLST) has emerged as the state-of-the-art method for resolving bacterial population genetics but it is expensive and time consuming. We evaluated the potential of high resolution melting (HRM) to identify known MLST alleles of Campylobacter jejuni at reduced cost and time. Each MLST locus was amplified in two or three sub fragments, which were analyzed by HRM. The approach was investigated using 47 C. jejuni isolates, previously characterized by classical MLST, representing isolates from diverse environmental, animal and clinical sources and including the six most prevalent sequence types (ST) and the most frequent alleles. HRM was then applied to a validation set of 84 additional C. jejuni isolates from chickens; 92% of the alleles were resolved in 35 hours of laboratory time and the cost of reagents per isolate was $20 compared with $100 for sequence-based typing. HRM has the potential to complement sequence-based methods for resolving SNPs and to facilitate a wide range of genotyping studies.     

The relative contributions of recombination and mutation to the divergence of clones of Neisseria meningitidis.

Multilocus sequence typing (MLST) is a recently developed nucleotide sequence-based method for the definitive assignment of isolates within bacterial populations to specific clones. MLST uses the same principles as multilocus enzyme electrophoresis and provides data that can be used to investigate aspects of the population genetics and evolution of bacterial species. We used an MLST data set consisting of the sequences of approximately 450-bp fragments from seven housekeeping loci from a large strain collection of Neisseria meningitidis to estimate the relative impact of recombination compared with point mutation in the diversification of N. meningitidis clonal complexes. 126 meningococcal isolates were assigned to 10 clonal complexes, 9 of which contained minor clonal variants. The allelic variation within each complex was classified as a recombinational exchange or a putative point mutation through a comparison of the sequences of each variant allele with that of the allele typically found in the clonal complex. The nine clonal complexes contained a total of 23 allelic variants, and analysis of the sequences of these variant alleles revealed that a single nucleotide site in a meningococcal housekeeping gene is at least 80-fold more likely to change as a result of recombination than as a result of mutation. This value is estimated to be 10-50-fold for Escherichia coli and approximately 50-fold for Streptococcus pneumoniae.     

Investigating Burkholderia cepacia complex populations recovered from Italian maize rhizosphere by multilocus sequence typing

The Burkholderia cepacia complex (BCC) comprises at least nine closely related species of abundant environmental microorganisms. Some of these species are highly spread in the rhizosphere of several crop plants, particularly of maize; additionally, as opportunistic pathogens, strains of the BCC are capable of colonizing humans. We have developed and validated a multilocus sequence typing (MLST) scheme for the BCC. Although widely applied to understand the epidemiology of bacterial pathogens, MLST has seen limited application to the population analysis of species residing in the natural environment; we describe its novel application to BCC populations within maize rhizospheres. 115 BCC isolates were recovered from the roots of different maize cultivars from three different Italian regions over a 9-year period (1994-2002). A total of 44 sequence types (STs) were found of which 41 were novel when compared with existing MLST data which encompassed a global database of 1000 clinical and environmental strains representing nearly 400 STs. In this study of rhizosphere isolates approximately 2.5 isolates per ST was found, comparable to that found for the whole BCC population. Multilocus sequence typing also resolved inaccuracies associated with previous identification of the maize isolates based on recA gene restriction fragment length polymorphims and species-specific polymerase chain reaction. The 115 maize isolates comprised the following BCC species groups, B. ambifaria (39%), BCC6 (29%), BCC5 (10%), B. pyrrocinia (8%), B. cenocepacia IIIB (7%) and B. cepacia (6%), with BCC5 and BCC6 potentially constituting novel species groups within the complex. Closely related clonal complexes of strains were identified within B. cepacia, B. cenocepacia IIIB, BCC5 and BCC6, with one of the BCC5 clonal complexes being distributed across all three sampling sites. Overall, our analysis demonstrates that the maize rhizosphere harbours a massive diversity of novel BCC STs, so that their addition to our global MLST database increased the ST diversity by 10%.     

Multilocus sequence typing

Numerous computer-based statistical packages have been developed in recent years and it has become easier to analyze nucleotide sequence data and gather subsequent information that would not normally be available. Multilocus sequence typing (MLST) is used for characterizing isolates of bacterial and fungal species and uses nucleotide sequences of internal fragments of housekeeping genes. This method is finding a place in clinical microbiology and public health by providing data for epidemiological surveillance and development of vaccine policy. It adds greatly to our knowledge of the genetic variation that can occur within a species and has therefore been used for studies of population biology. Analysis requires the detailed interpretation of nucleotide sequence data obtained from housekeeping and nonhousekeeping genes. This is due to the amount of data generated from nucleotide sequencing and the information generated from an array of analytical tools improves our understanding of bacterial pathogens. This can benefit public health interventions and the development of enhanced therapies and vaccines. This review concentrates on the analytical tools used in MLST and their use in the clinical microbiology and public health fields.     

Multilocus sequence typing system for the endosymbiont Wolbachia pipientis

The eubacterial genus Wolbachia comprises one of the most abundant groups of obligate intracellular bacteria, and it has a host range that spans the phyla Arthropoda and Nematoda. Here we developed a multilocus sequence typing (MLST) scheme as a universal genotyping tool for Wolbachia. Internal fragments of five ubiquitous genes (gatB, coxA, hcpA, fbpA, and ftsZ) were chosen, and primers that amplified across the major Wolbachia supergroups found in arthropods, as well as other divergent lineages, were designed. A supplemental typing system using the hypervariable regions of the Wolbachia surface protein (WSP) was also developed. Thirty-seven strains belonging to supergroups A, B, D, and F obtained from singly infected hosts were characterized by using MLST and WSP. The number of alleles per MLST locus ranged from 25 to 31, and the average levels of genetic diversity among alleles were 6.5% to 9.2%. A total of 35 unique allelic profiles were found. The results confirmed that there is a high level of recombination in chromosomal genes. MLST was shown to be effective for detecting diversity among strains within a single host species, as well as for identifying closely related strains found in different arthropod hosts. Identical or similar allelic profiles were obtained for strains harbored by different insect species and causing distinct reproductive phenotypes. Strains with similar WSP sequences can have very different MLST allelic profiles and vice versa, indicating the importance of the MLST approach for strain identification. The MLST system provides a universal and unambiguous tool for strain typing, population genetics, and molecular evolutionary studies. The central database for storing and organizing Wolbachia bacterial and host information can be accessed at http://pubmlst.org/wolbachia/.     

Multilocus sequence typing--what is resolved?

Nucleotide sequence-based methods for bacterial typing (multilocus sequence typing; MLST) allow rapid and global comparisons between results from different laboratories. Combining this advantage with the reduced cost of high throughput sequencing, increasing automation and the amenability of sequence data for evolutionary analysis, it seems inevitable that sequence-based typing will eventually predominate over gel-based methods such as pulsed-field gel electrophoresis (PFGE) for most bacterial species. The increasing availability of multiple genome sequences for single pathogenic species, and the recent development of many new MLST schemes, means that a re-examination of the utility of multilocus sequencing, and in particular the choice of gene loci, is now appropriate.     

High-throughput multilocus sequence typing: bringing molecular typing to the next level

Multilocus sequence typing (MLST) is a widely used system for typing microorganisms by sequence analysis of housekeeping genes. The main advantage of MLST in comparison to other typing techniques is the unambiguity and transferability of sequence data. However, a main disadvantage is the high cost of DNA sequencing. Here we introduce a high-throughput MLST (HiMLST) method that employs next-generation sequencing (NGS) technology (Roche 454), to generate large quantities of high-quality MLST data at low costs. The HiMLST protocol consists of two steps. In the first step MLST target genes are amplified by PCR in multi-well plates. During this PCR the amplicons of each bacterial isolate are provided with a unique DNA barcode, the multiplex identifier (MID). In the second step all amplicons are pooled and sequenced in a single NGS-run. The MLST profile of each individual isolate can be retrieved easily using its unique MID. With HiMLST we have profiled 575 isolates of Legionella pneumophila, Staphylococcus aureus, Pseudomonas aeruginosa and Streptococcus pneumoniae in mixed species HiMLST experiments. In conclusion, the introduction of HiMLST paves the way for a broad employment of the MLST as a high-quality and cost-effective method for typing microbial species.     

Comparison of direct genome restriction enzyme analysis and pulsed-field gel electrophoresis for typing of Vibrio vulnificus and their correspondence with multilocus sequence typing data

We compared the potential of direct genome restriction enzyme analysis (DGREA) and pulsed-field gel electrophoresis (PFGE) for discriminating Vibrio vulnificus isolates from clinical (23) and environmental (17) sources. The genotypes generated by both methodologies were compared to previous multilocus sequence typing (MLST) data. DGREA established clearer relationships among V. vulnificus strains and was more consistent with MLST than with PFGE. DGREA is a very promising tool for epidemiological and ecological studies of V. vulnificus.