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1.  Multilocus Sequence Typing Scheme for Bacteria of the Bacillus cereus Group 
In this study we developed a multilocus sequence typing (MLST) scheme for bacteria of the Bacillus cereus group. This group, which includes the species B. cereus, B. thuringiensis, B. weihenstephanensis, and B. anthracis, is known to be genetically very diverse. It is also very important because it comprises pathogenic organisms as well as bacteria with industrial applications. The MLST system was established by using 77 strains having various origins, including humans, animals, food, and soil. A total of 67 of these strains had been analyzed previously by multilocus enzyme electrophoresis, and they were selected to represent the genetic diversity of this group of bacteria. Primers were designed for conserved regions of housekeeping genes, and 330- to 504-bp internal fragments of seven such genes, adk, ccpA, ftsA, glpT, pyrE, recF, and sucC, were sequenced for all strains. The number of alleles at individual loci ranged from 25 to 40, and a total of 53 allelic profiles or sequence types (STs) were distinguished. Analysis of the sequence data showed that the population structure of the B. cereus group is weakly clonal. In particular, all five B. anthracis isolates analyzed had the same ST. The MLST scheme which we developed has a high level of resolution and should be an excellent tool for studying the population structure and epidemiology of the B. cereus group.
doi:10.1128/AEM.70.1.191-201.2004
PMCID: PMC321270  PMID: 14711642
2.  L-alanine-induced germination in Bacillus licheniformis -the impact of native gerA sequences 
BMC Microbiology  2014;14:101.
Background
L-alanine, acting through the GerA receptor, was recently found to be an efficient germinant in Bacillus licheniformis ATCC14580/DSM13.
Results
In this study, we show that several of 46 examined B. licheniformis strains germinate remarkably slower than the type strain when exposed to L-alanine. These strains are not necessarily closely related, as determined by MLST (multi-locus sequence typing). Three of the slow-germinating strains were further examined in order to see whether nucleotide substitutions in the gerA sequences were responsible for the slow L-alanine germination. This was performed by complementing the transformable type strain derivate MW3ΔgerAA with gerA variants from the three slow-germinating strains; NVH1032, NVH1112 and NVH800.
Conclusions
A wide selection of B. licheniformis strains was evaluated for L-alanine-induced germination efficiency. Our results show that gerA substitutions could only partially explain why spores of some B. licheniformis strains responded slower than others in the presence of L-alanine.
doi:10.1186/1471-2180-14-101
PMCID: PMC4021175  PMID: 24755193
Bacillus licheniformis; Germination; L-alanine; gerA; Genotype; Germinant receptor
3.  Multi-Locus Sequence Analysis Reveals Profound Genetic Diversity among Isolates of the Human Pathogen Bartonella bacilliformis 
Bartonella bacilliformis is the aetiological agent of human bartonellosis, a potentially life threatening infection of significant public health concern in the Andean region of South America. Human bartonellosis has long been recognised in the region but a recent upsurge in the number of cases of the disease and an apparent expansion of its geographical distribution have re-emphasized its contemporary medical importance. Here, we describe the development of a multi-locus sequence typing (MLST) scheme for B. bacilliformis and its application to an archive of 43 isolates collected from patients across Peru. MLST identified eight sequence types among these isolates and the delineation of these was generally congruent with those of the previously described typing scheme. Phylogenetic analysis based on concatenated sequence data derived from MLST loci revealed that seven of the eight sequence types were closely related to one another; however, one sequence type, ST8, exhibited profound evolutionary divergence from the others. The extent of this divergence was akin to that observed between other members of the Bartonella genus, suggesting that ST8 strains may be better considered as members of a novel Bartonella genospecies.
Author Summary
Bartonellosis (Carrion's disease) is caused by the bacterium Bartonella bacilliformis and is encountered across the Andean cordillera, particularly in Peru. B. bacilliformis is transmitted by arthropods, probably sandflies, and exploits humans as a reservoir host, establishing chronic infections characterized by intra-erythrocytic bacteremia. It this thought that asymptomatic infections are common among the residents of bartonellosis-endemic regions. However, several thousand cases of B. bacilliformis-induced severe haemolytic anaemia are reported each year, frequently among visitors to endemic regions but also among inhabitants of an increasing number of new foci of disease. To better understand the epidemiology of bartonellosis, a clearer understanding of how B. bacilliformis strains circulating in endemic regions and in new foci are related to one another is required. Here, we describe the development and application of a new genotyping tool for addressing this shortfall, namely multilocus sequence typing (MLST). We applied MLST to 43 B. bacilliformis isolates of varying provenance, delineating eight distinct genotypes, some of which had obvious epidemiological correlates. Surprisingly, one genotype exhibited profound divergence from the other seven, and the extent of this divergence suggested that some bartonellosis may be caused by a Bartonella species specifically related to, yet distinct from, B. bacilliformis.
doi:10.1371/journal.pntd.0001248
PMCID: PMC3139668  PMID: 21811647
4.  Multilocus Sequence Typing of Mycoplasma hyorhinis Strains Identified by a Real-Time TaqMan PCR Assay 
Journal of Clinical Microbiology  2014;52(5):1664-1671.
A real-time TaqMan PCR assay based on the gene encoding the protein p37 was developed to detect Mycoplasma hyorhinis. Its specificity was validated with 29 epidemiologically unrelated M. hyorhinis strains (28 field strains and one reference strain) and other mycoplasma species or with other microorganisms commonly found in pigs. The estimated detection limit of this qPCR assay was 125 microorganism equivalents/μl. The same 29 epidemiologically unrelated M. hyorhinis strains and four previously fully sequenced strains were typed by two portable typing methods, the sequencing of the p37 gene and a multilocus sequence typing (MLST) scheme. The first method revealed 18 distinct nucleotide sequences and insufficient discriminatory power (0.934). The MLST scheme was developed with the sequenced genomes of the M. hyorhinis strains HUB-1, GDL-1, MCLD, and SK76 and based on the genes dnaA, rpoB, gyrB, gltX, adk, and gmk. In total, 2,304 bp of sequence was analyzed for each strain. MLST was capable of subdividing the 33 strains into 29 distinct sequence types. The discriminatory power of the method was >0.95, which is the threshold value for interpreting typing results with confidence (D = 0.989). Population analysis showed that recombination in M. hyorhinis occurs and that strains are diverse but with a certain clonality (one unique clonal complex was identified). The new qPCR assay and the robust MLST scheme are available for the acquisition of new knowledge on M. hyorhinis epidemiology. A web-accessible database has been set up for the M. hyorhinis MLST scheme at http://pubmlst.org/mhyorhinis/.
doi:10.1128/JCM.03437-13
PMCID: PMC3993664  PMID: 24622092
5.  Short read sequence typing (SRST): multi-locus sequence types from short reads 
BMC Genomics  2012;13:338.
Background
Multi-locus sequence typing (MLST) has become the gold standard for population analyses of bacterial pathogens. This method focuses on the sequences of a small number of loci (usually seven) to divide the population and is simple, robust and facilitates comparison of results between laboratories and over time. Over the last decade, researchers and population health specialists have invested substantial effort in building up public MLST databases for nearly 100 different bacterial species, and these databases contain a wealth of important information linked to MLST sequence types such as time and place of isolation, host or niche, serotype and even clinical or drug resistance profiles. Recent advances in sequencing technology mean it is increasingly feasible to perform bacterial population analysis at the whole genome level. This offers massive gains in resolving power and genetic profiling compared to MLST, and will eventually replace MLST for bacterial typing and population analysis. However given the wealth of data currently available in MLST databases, it is crucial to maintain backwards compatibility with MLST schemes so that new genome analyses can be understood in their proper historical context.
Results
We present a software tool, SRST, for quick and accurate retrieval of sequence types from short read sets, using inputs easily downloaded from public databases. SRST uses read mapping and an allele assignment score incorporating sequence coverage and variability, to determine the most likely allele at each MLST locus. Analysis of over 3,500 loci in more than 500 publicly accessible Illumina read sets showed SRST to be highly accurate at allele assignment. SRST output is compatible with common analysis tools such as eBURST, Clonal Frame or PhyloViz, allowing easy comparison between novel genome data and MLST data. Alignment, fastq and pileup files can also be generated for novel alleles.
Conclusions
SRST is a novel software tool for accurate assignment of sequence types using short read data. Several uses for the tool are demonstrated, including quality control for high-throughput sequencing projects, plasmid MLST and analysis of genomic data during outbreak investigation. SRST is open-source, requires Python, BWA and SamTools, and is available from http://srst.sourceforge.net.
doi:10.1186/1471-2164-13-338
PMCID: PMC3460743  PMID: 22827703
MLST; Short read; Illumina; Sequence analysis; Plasmid; Chromosome; Microbiology; Bacteria; Population analysis; Outbreak
6.  A Single Multilocus Sequence Typing (MLST) Scheme for Seven Pathogenic Leptospira Species 
Background
The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species.
Methodology and Findings
We modified the existing scheme by replacing one of the seven MLST loci (fadD was changed to caiB), as the former gene did not appear to be present in some pathogenic species. Comparison of the original and modified schemes using data for L. interrogans and L. kirschneri demonstrated that the discriminatory power of the two schemes was not significantly different. The modified scheme was used to further characterize 325 isolates (L. alexanderi [n = 5], L. borgpetersenii [n = 34], L. interrogans [n = 222], L. kirschneri [n = 29], L. noguchii [n = 9], L. santarosai [n = 10], and L. weilii [n = 16]). Phylogenetic analysis using concatenated sequences of the 7 loci demonstrated that each species corresponded to a discrete clade, and that no strains were misclassified at the species level. Comparison between genotype and serovar was possible for 254 isolates. Of the 31 sequence types (STs) represented by at least two isolates, 18 STs included isolates assigned to two or three different serovars. Conversely, 14 serovars were identified that contained between 2 to 10 different STs. New observations were made on the global phylogeography of Leptospira spp., and the utility of MLST in making associations between human disease and specific maintenance hosts was demonstrated.
Conclusion
The new MLST scheme, supported by an updated MLST website, allows the characterization and species assignment of isolates of the seven major pathogenic species associated with leptospirosis.
Author Summary
Leptospirosis is a common zoonotic disease worldwide. Genotyping of the causative organisms provides important insights into disease transmission and informs preventive strategies and vaccine development. Multilocus sequence typing (MLST) is the most widespread genotyping methodology for bacterial pathogens, but the Leptospira scheme supported by a public MLST database is currently only applicable to L. interrogans and L. kirschneri. The purpose of this study was to extend the scheme to a total of seven pathogenic Leptospira species. This was achieved through the development of a modified scheme in which one of the seven MLST loci was replaced, together with newly designed primers for the remaining 6 loci. Comparison of the original and modified scheme demonstrated that they were very similar, hence sequence type (ST) assignments were largely carried over to the modified scheme. Phylogenetic trees reconstructed from concatenated sequences of the seven loci of the modified scheme demonstrated perfect classification of isolates into seven pathogenic species, which resided in clearly distinct phylogenetic clusters. Congruence was low between STs and serovars. The MLST scheme was used to gain new insights into the population genetic structure of Leptospira species associated with clinical disease and maintenance hosts in Asia.
doi:10.1371/journal.pntd.0001954
PMCID: PMC3554523  PMID: 23359622
7.  Development of a Tiered Multilocus Sequence Typing Scheme for Members of the Lactobacillus acidophilus Complex 
Applied and Environmental Microbiology  2013;79(23):7220-7228.
Members of the Lactobacillus acidophilus complex 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 the L. acidophilus complex (L. acidophilus, L. amylovorus, L. crispatus, L. gallinarum, L. gasseri, and L. 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 the hsp60 gene 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 genes fusA, gpmA, gyrA, gyrB, lepA, pyrG, and recA, 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 the L. acidophilus complex 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 of Lactobacillus species.
doi:10.1128/AEM.02257-13
PMCID: PMC3837765  PMID: 24038697
8.  High-Resolution Two-Locus Clonal Typing of Extraintestinal Pathogenic Escherichia coli 
Multilocus sequence typing (MLST) is usually based on the sequencing of 5 to 8 housekeeping loci in the bacterial chromosome and has provided detailed descriptions of the population structure of bacterial species important to human health. However, even strains with identical MLST profiles (known as sequence types or STs) may possess distinct genotypes, which enable different eco- or pathotypic lifestyles. Here we describe a two-locus, sequence-based typing scheme for Escherichia coli that utilizes a 489-nucleotide (nt) internal fragment of fimH (encoding the type 1 fimbrial adhesin) and the 469-nt internal fumC fragment used in standard MLST. Based on sequence typing of 191 model commensal and pathogenic isolates plus 853 freshly isolated clinical E. coli strains, this 2-locus approach—which we call CH (fumC/fimH) typing—consistently yielded more haplotypes than standard 7-locus MLST, splitting large STs into multiple clonal subgroups and often distinguishing different within-ST eco- and pathotypes. Furthermore, specific CH profiles corresponded to specific STs, or ST complexes, with 95% accuracy, allowing excellent prediction of MLST-based profiles. Thus, 2-locus CH typing provides a genotyping tool for molecular epidemiology analysis that is more economical than standard 7-locus MLST but has superior clonal discrimination power and, at the same time, corresponds closely to MLST-based clonal groupings.
doi:10.1128/AEM.06663-11
PMCID: PMC3294456  PMID: 22226951
9.  Comparison of Two Multilocus Sequence Based Genotyping Schemes for Leptospira Species 
Background
Several sequence based genotyping schemes have been developed for Leptospira spp. The objective of this study was to genotype a collection of clinical and reference isolates using the two most commonly used schemes and compare and contrast the results.
Methods and Findings
A total of 48 isolates consisting of L. interrogans (n = 40) and L. kirschneri (n = 8) were typed by the 7 locus MLST scheme described by Thaipadungpanit et al., and the 6 locus genotyping scheme described by Ahmed et al., (termed 7L and 6L, respectively). Two L. interrogans isolates were not typed using 6L because of a deletion of three nucleotides in lipL32. The remaining 46 isolates were resolved into 21 sequence types (STs) by 7L, and 30 genotypes by 6L. Overall nucleotide diversity (based on concatenated sequence) was 3.6% and 2.3% for 7L and 6L, respectively. The D value (discriminatory ability) of 7L and 6L were comparable, i.e. 92.0 (95% CI 87.5–96.5) vs. 93.5 (95% CI 88.6–98.4). The dN/dS ratios calculated for each locus indicated that none were under positive selection. Neighbor joining trees were reconstructed based on the concatenated sequences for each scheme. Both trees showed two distinct groups corresponding to L. interrogans and L. kirschneri, and both identified two clones containing 10 and 7 clinical isolates, respectively. There were six instances in which 6L split single STs as defined by 7L into closely related clusters. We noted two discrepancies between the trees in which the genetic relatedness between two pairs of strains were more closely related by 7L than by 6L.
Conclusions
This genetic analysis indicates that the two schemes are comparable. We discuss their practical advantages and disadvantages.
Author Summary
Two independent multilocus sequence based genotyping schemes (denoted here as 7L and 6L for schemes with 7 and 6 loci, respectively) are in use for Leptospira spp., which has led to uncertainty as to which should be adopted by the scientific community. The purpose of this study was to apply the two schemes to a single collection of pathogenic Leptospira, evaluate their performance, and describe the practical advantages and disadvantages of each scheme. We used a variety of phylogenetic approaches to compare the output data and found that the two schemes gave very similar results. 7L has the advantage that it is a conventional multi-locus sequencing typing (MLST) scheme based on housekeeping genes and is supported by a publically accessible database by which genotypes can be readily assigned as known or new sequence types by any investigator, but is currently only applicable to L. interrogans and L. kirschneri. Conversely, 6L can be applied to all pathogenic Leptospira spp., but is not a conventional MLST scheme by design and is not available online. 6L sequences from 271 strains have been released into the public domain, and phylogenetic analysis of new sequences using this scheme requires their download and offline analysis.
doi:10.1371/journal.pntd.0001374
PMCID: PMC3210738  PMID: 22087342
10.  Four Genotyping Schemes for Phylogenetic Analysis of Pseudomonas aeruginosa: Comparison of Their Congruence with Multi-Locus Sequence Typing 
PLoS ONE  2013;8(12):e82069.
Several molecular typing schemes have been proposed to differentiate among isolates and clonal groups, and hence establish epidemiological or phylogenetic links. It has been widely accepted that multi-locus sequence typing (MLST) is the gold standard for phylogenetic typing/long-term epidemiological surveillance, but other recently described methods may be easier to carry out, especially in settings with limited access to DNA sequencing. Comparing the performance of such techniques to MLST is therefore of relevance. A study was therefore carried out with a collection of P. aeruginosa strains (n = 133) typed by four typing schemes: MLST, multiple-locus variable number tandem repeat analysis (MLVA), pulsed-field gel electrophoresis (PFGE) and the commercial DiversiLab microbial typing system (DL). The aim of this study was to compare the results of each typing method with MLST. The Simpson's indices of diversity were 0.989, 0.980, 0.961 and 0.906 respectively for PFGE, MLVA, DL and MLST. The congruence between techniques was measured by the adjusted Wallace index (W): this coefficient indicates the probability that a pair of isolates which is assigned to the same type by one typing method is also typed as identical by the other. In this context, the congruence between techniques was recorded as follow: MLVA-type to predict MLST-type (93%), PFGE to MLST (92%), DL to MLST (64.2%), PFGE to MLVA (63.5%) and PFGE to DL (61.7%). Conversely, for all above combinations, prediction was very poor. The congruence was increased at the clonal complex (CC) level. MLST is regarded the gold standard for phylogenetic classification of bacteria, but is rather laborious to carry out in many settings. Our data suggest that MLVA can predict the MLST-type with high accuracy, and even higher when studying the clonal complex level. Of the studied three techniques MLVA was therefore the best surrogate method to predict MLST.
doi:10.1371/journal.pone.0082069
PMCID: PMC3859543  PMID: 24349186
11.  Isolation, Characterization, and Identification of Bacterial Contaminants in Semifinal Gelatin Extracts 
Bacterial contamination of gelatin is of great concern. Indeed, this animal colloid has many industrial applications, mainly in food and pharmaceutical products. In a previous study (E. De Clerck and P. De Vos, Syst. Appl. Microbiol. 25:611-618), contamination of a gelatin production process with a variety of gram-positive and gram-negative bacteria was demonstrated. In this study, bacterial contamination of semifinal gelatin extracts from several production plants was examined. Since these extracts are subjected to harsh conditions during production and a final ultrahigh-temperature treatment, the bacterial load at this stage is expected to be greatly reduced. In total, 1,129 isolates were obtained from a total of 73 gelatin batches originating from six different production plants. Each of these batches was suspected of having bacterial contamination based on quality control testing at the production plant from which it originated. For characterization and identification of the 1,129 bacterial isolates, repetitive-element PCR was used to obtain manageable groups. Representative strains were identified by means of 16S rRNA genesequencing, species-specific gyrB PCR, and gyrA and rpoB sequencing and were tested for gelatinase activity. The majority of isolates belonged to members of Bacillus or related endospore-forming genera. Representative strains were identified as Bacillus cereus, Bacillus coagulans, Bacillus fumarioli, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus sonorensis, Bacillus subtilis, Bacillus gelatini, Bacillus thermoamylovorans, Anoxybacillus contaminans, Anoxybacillus flavithermus, Brevibacillus agri, Brevibacillus borstelensis, and Geobacillus stearothermophilus. The majority of these species include strains exhibiting gelatinase activity. Moreover, some of these species have known pathogenic properties. These findings are of great concern with regard to the safety and quality of gelatin and its applications.
doi:10.1128/AEM.70.6.3664-3672.2004
PMCID: PMC427776  PMID: 15184171
12.  Optimized Multilocus Sequence Typing (MLST) Scheme for Trypanosoma cruzi 
Trypanosoma cruzi, the aetiological agent of Chagas disease possess extensive genetic diversity. This has led to the development of a plethora of molecular typing methods for the identification of both the known major genetic lineages and for more fine scale characterization of different multilocus genotypes within these major lineages. Whole genome sequencing applied to large sample sizes is not currently viable and multilocus enzyme electrophoresis, the previous gold standard for T. cruzi typing, is laborious and time consuming. In the present work, we present an optimized Multilocus Sequence Typing (MLST) scheme, based on the combined analysis of two recently proposed MLST approaches. Here, thirteen concatenated gene fragments were applied to a panel of T. cruzi reference strains encompassing all known genetic lineages. Concatenation of 13 fragments allowed assignment of all strains to the predicted Discrete Typing Units (DTUs), or near-clades, with the exception of one strain that was an outlier for TcV, due to apparent loss of heterozygosity in one fragment. Monophyly for all DTUs, along with robust bootstrap support, was restored when this fragment was subsequently excluded from the analysis. All possible combinations of loci were assessed against predefined criteria with the objective of selecting the most appropriate combination of between two and twelve fragments, for an optimized MLST scheme. The optimum combination consisted of 7 loci and discriminated between all reference strains in the panel, with the majority supported by robust bootstrap values. Additionally, a reduced panel of just 4 gene fragments displayed high bootstrap values for DTU assignment and discriminated 21 out of 25 genotypes. We propose that the seven-fragment MLST scheme could be used as a gold standard for T. cruzi typing, against which other typing approaches, particularly single locus approaches or systematic PCR assays based on amplicon size, could be compared.
Author Summary
The single-celled parasite Trypanosoma cruzi occurs in mammals and insect vectors in the Americas. When transmitted to humans it causes Chagas disease (American trypanosomiasis) a major public health problem. T. cruzi is genetically diverse and currently split into six groups, known as TcI to TcVI. Multilocus sequence typing (MLST) is a method used for studying the population structure and diversity of pathogens and involves sequencing DNA of several different genes and comparing the sequences between isolates. Here, we assess 13 T. cruzi genes and select the best combination for diversity studies. Outputs reveal that a combination of 7 genes can be used for both lineage assignment and high resolution studies of genetic diversity, and a reduced combination of four loci for lineage assignment. Application of MLST for assigning field isolates of T. cruzi to genetic groups and for detailed investigation of diversity provides a valuable approach to understanding the taxonomy, population structure, genetics, ecology and epidemiology of this important human pathogen.
doi:10.1371/journal.pntd.0003117
PMCID: PMC4148231  PMID: 25167160
13.  Classification of Bartonella Strains Associated with Straw-Colored Fruit Bats (Eidolon helvum) across Africa Using a Multi-locus Sequence Typing Platform 
PLoS Neglected Tropical Diseases  2015;9(1):e0003478.
Bartonellae are facultative intracellular bacteria and are highly adapted to their mammalian host cell niches. Straw-colored fruit bats (Eidolon helvum) are commonly infected with several bartonella strains. To elucidate the genetic diversity of these bartonella strains, we analyzed 79 bartonella isolates from straw-colored fruit bats in seven countries across Africa (Cameroon, Annobon island of Equatorial Guinea, Ghana, Kenya, Nigeria, Tanzania, and Uganda) using a multi-locus sequencing typing (MLST) approach based on nucleotide sequences of eight loci (ftsZ, gltA, nuoG, ribC, rpoB, ssrA, ITS, and 16S rRNA). The analysis of each locus but ribC demonstrated clustering of the isolates into six genogroups (E1 – E5 and Ew), while ribC was absent in the isolates belonging to the genogroup Ew. In general, grouping of all isolates by each locus was mutually supportive; however, nuoG, gltA, and rpoB showed some incongruity with other loci in several strains, suggesting a possibility of recombination events, which were confirmed by network analyses and recombination/mutation rate ratio (r/m) estimations. The MLST scheme revealed 45 unique sequence types (ST1 – 45) among the analyzed bartonella isolates. Phylogenetic analysis of concatenated sequences supported the discrimination of six phylogenetic lineages (E1 – E5 and Ew) corresponding to separate and unique Bartonella species. One of the defined lineages, Ew, consisted of only two STs (ST1 and ST2), and comprised more than one-quarter of the analyzed isolates, while other lineages contained higher numbers of STs with a smaller number of isolates belonging to each lineage. The low number of allelic polymorphisms of isolates belonging to Ew suggests a more recent origin for this species. Our findings suggest that at least six Bartonella species are associated with straw-colored fruit bats, and that distinct STs can be found across the distribution of this bat species, including in populations of bats which are genetically distinct.
Author Summary
Bats, with over 1000 recognized species, represent about 20% of all classified mammalian species worldwide. These mammals have a wide range of ecologies and life-history traits, and are now widely recognized as important reservoirs of many pathogens. Bartonella species have been found distributed in a wide range of mammalian species, including bats. About half of recognized Bartonella species, including one bat-associated species, have been associated with human illness. Previous studies have shown that Bartonella species are extremely diverse, with or without evident specificity to their mammalian hosts. Possessing many unique aspects, bartonellae can serve as a useful biological marker to study how microorganisms have evolved and diversified along with their animal hosts in evolutionary history. In this study, we applied multi-locus sequence typing, or MLST, to study the genetic differences of straw-colored fruit bat (Eidolon helvum)-associated Bartonella species. Our studies suggest Bartonella species have both exchanged genetic materials among species through recombination events and lost genes that are perhaps superfluous to their life cycles, which includes an intracellular stage in mammals.
doi:10.1371/journal.pntd.0003478
PMCID: PMC4311972  PMID: 25635826
14.  Bayesian semi-supervised classification of bacterial samples using MLST databases 
BMC Bioinformatics  2011;12:302.
Background
Worldwide effort on sampling and characterization of molecular variation within a large number of human and animal pathogens has lead to the emergence of multi-locus sequence typing (MLST) databases as an important tool for studying the epidemiology and evolution of pathogens. Many of these databases are currently harboring several thousands of multi-locus DNA sequence types (STs) enriched with metadata over traits such as serotype, antibiotic resistance, host organism etc of the isolates. Curators of the databases have thus the possibility of dividing the pathogen populations into subsets representing different evolutionary lineages, geographically associated groups, or other subpopulations, which are defined in terms of molecular similarities and dissimilarities residing within a database. When combined with the existing metadata, such subsets may provide invaluable information for assessing the position of a new set of isolates in relation to the whole pathogen population.
Results
To enable users of MLST schemes to query the databases with sets of new bacterial isolates and to automatically analyze their relation to existing curated sequences, we introduce here a Bayesian model-based method for semi-supervised classification of MLST data. Our method can use an MLST database as a training set and assign simultaneously any set of query sequences into the earlier discovered lineages/populations, while also allowing some or all of these sequences to form previously undiscovered genetically distinct groups. This tool provides probabilistic quantification of the classification uncertainty and is highly efficient computationally, thus enabling rapid analyses of large databases and sets of query sequences. The latter feature is a necessary prerequisite for an automated access through the MLST web interface. We demonstrate the versatility of our approach by anayzing both real and synthesized data from MLST databases. The introduced method for semi-supervised classification of sets of query STs is freely available for Windows, Mac OS X and Linux operative systems in BAPS 5.4 software which is downloadable at http://web.abo.fi/fak/mnf/mate/jc/software/baps.html. The query functionality is also directly available for the Staphylococcus aureus database at http://www.mlst.net and shortly will be available for other species databases hosted at this web portal.
Conclusions
We have introduced a model-based tool for automated semi-supervised classification of new pathogen samples that can be integrated into the web interface of the MLST databases. In particular, when combined with the existing metadata, the semi-supervised labeling may provide invaluable information for assessing the position of a new set of query strains in relation to the particular pathogen population represented by the curated database.
Such information will be useful both for clinical and basic research purposes.
doi:10.1186/1471-2105-12-302
PMCID: PMC3155509  PMID: 21791094
15.  Comparison of Multilocus Sequence Typing, Pulsed-Field Gel Electrophoresis, and Antimicrobial Susceptibility Typing for Characterization of Salmonella enterica Serotype Newport Isolates 
Journal of Clinical Microbiology  2006;44(7):2449-2457.
In the United States, multidrug-resistant phenotypes of Salmonella enterica serotype Newport (commonly referred to as MDR-AmpC) have emerged in animals and humans and have become a major public health problem. Although pulsed-field gel electrophoresis (PFGE) is the current “gold standard” typing method for Salmonella, multilocus sequence typing (MLST) may be more relevant to investigations exploring evolutionary and population biology relationships. In this study, 81 Salmonella enterica serotype Newport isolates from humans, food animals, and retail foods were examined for antimicrobial susceptibility and characterized using PFGE and MLST of seven genes, aroC, dnaN, hemD, hisD, purE, sucA, and thrA. Forty-nine percent of the isolates were resistant to nine or more of the tested antimicrobials. Salmonella isolates displayed resistance most often to sulfamethoxazole (57%), streptomycin (56%), tetracycline (56%), ampicillin (52%), and ceftiofur (49%) and, to a lesser extent, to kanamycin (19%), trimethoprim-sulfamethoxazole (17%), and gentamicin (11%). A total of 43 PFGE patterns were generated using XbaI, indicating a genetically diverse population. The largest PFGE cluster contained isolates from clinically ill swine, cattle, and humans. MLST resulted in 12 sequence types (STs), with one type encompassing 62% of the strains. Ten new sequence types and one novel allele type were identified. Furthermore, MLST typing showed that strains closely related by PFGE clustered in major STs, whereas more distantly related strains were separated into two clusters by PFGE. The results of this study demonstrated that the MLST scheme employed here clustered S. enterica serovar Newport isolates in distinct molecular populations, and strain discrimination was enhanced by combining PFGE, antimicrobial susceptibility, and MLST results.
doi:10.1128/JCM.00019-06
PMCID: PMC1489510  PMID: 16825363
16.  Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species 
Genome Biology  2004;5(10):r77.
The complete sequence of the Bacillus licheniformis ATCC 14580 genome was determined, revealing 4,208 predicted protein-coding genes, 7 rRNA operons and 72 tRNA genes.
Background
Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products. This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature.
Results
We determined the complete nucleotide sequence of the B. licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes. The B. licheniformis chromosome contains large regions that are colinear with the genomes of B. subtilis and Bacillus halodurans, and approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologs.
Conclusions
Despite the unmistakable organizational similarities between the B. licheniformis and B. subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species. Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B. licheniformis genome. The availability of a completed genome sequence for B. licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.
doi:10.1186/gb-2004-5-10-r77
PMCID: PMC545597  PMID: 15461803
17.  Multilocus Sequence Typing System for the Endosymbiont Wolbachia pipientis▿  
Applied and Environmental Microbiology  2006;72(11):7098-7110.
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/.
doi:10.1128/AEM.00731-06
PMCID: PMC1636189  PMID: 16936055
18.  Multilocus sequence typing (and phylogenetic analysis) of Campylobacter jejuni and Campylobacter coli strains isolated from clinical cases in Greece 
BMC Research Notes  2013;6:359.
Background
The molecular epidemiology of C. jejuni and C. coli clinical strains isolated from children with gastroenteritis, was investigated using the multilocus sequence typing method (MLST). This analysis establishes for the first time in Greece and constitutes an important tool for the epidemiological surveillance and control of Campylobacter infection in our country.
Methods
The MLST genotypes were compared with those gained by other typing methods (HS-typing, PFGE and FlaA typing) and were also phylogenetically analyzed, in order to uncover genetic relationships.
Results
Among 68 C. jejuni strains, 41 different MLST-Sequence Types (MLST-STs) were found. Fifty six strains or 34 MLST-STs could be sorted into 15 different MLST-Sequence Type Complexes (MLST-STCs), while twelve strains or seven MLST-STs did not match any of the MLST-STCs of the database. Twenty C. coli strains belonged to 14 different MLST-STs. Eleven MLST-STs were classified in the same MLST-STC (828), and three were unclassifiable. There was no significant association between the MLST-STs and the results of the other typing methods.
Phylogenetic analysis revealed that some strains, classified to the species of C. jejuni, formed a separate, phylogenetically distinct group. In eight strains some alleles belonging to the taxonomic cluster of C. jejuni, were also detected in C. coli and vice versa, a phenomenon caused by the genetic mosaic encountered inside the genus Campylobacter.
Conclusions
The MLST-ST determination proved to be a very useful tool for the typing as well as the identification of Campylobacter on the species level.
doi:10.1186/1756-0500-6-359
PMCID: PMC3844450  PMID: 24010733
Campylobacter jejuni; Campylobacter coli; Serotyping; FlaA typing; PFGE; MLST; Phylogenetic analysis
19.  Allele Intersection Analysis: A Novel Tool for Multi Locus Sequence Assignment in Multiply Infected Hosts 
PLoS ONE  2011;6(7):e22198.
Wolbachia are wide-spread, endogenous α-Proteobacteria of arthropods and filarial nematodes. 15–75% of all insect species are infected with these endosymbionts that alter their host's reproduction to facilitate their spread. In recent years, many insect species infected with multiple Wolbachia strains have been identified. As the endosymbionts are not cultivable outside living cells, strain typing relies on molecular methods. A Multi Locus Sequence Typing (MLST) system was established for standardizing Wolbachia strain identification. However, MLST requires hosts to harbour individual and not multiple strains of supergroups without recombination. This study revisits the applicability of the current MLST protocols and introduces Allele Intersection Analysis (AIA) as a novel approach. AIA utilizes natural variations in infection patterns and allows correct strain assignment of MLST alleles in multiply infected host species without the need of artificial strain segregation. AIA identifies pairs of multiply infected individuals that share Wolbachia and differ in only one strain. In such pairs, the shared MLST sequences can be used to assign alleles to distinct strains. Furthermore, AIA is a powerful tool to detect recombination events. The underlying principle of AIA may easily be adopted for MLST approaches in other uncultivable bacterial genera that occur as multiple strain infections and the concept may find application in metagenomic high-throughput parallel sequencing projects.
doi:10.1371/journal.pone.0022198
PMCID: PMC3137623  PMID: 21789233
20.  Efficient recombinant expression and secretion of a thermostable GH26 mannan endo-1,4-β-mannosidase from Bacillus licheniformis in Escherichia coli 
Background
Mannans are one of the key polymers in hemicellulose, a major component of lignocellulose. The Mannan endo-1,4-β-mannosidase or 1,4-β-D-mannanase (EC 3.2.1.78), commonly named β-mannanase, is an enzyme that can catalyze random hydrolysis of β-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans. The enzyme has found a number of applications in different industries, including food, feed, pharmaceutical, pulp/paper industries, as well as gas well stimulation and pretreatment of lignocellulosic biomass for the production of second generation biofuel. Bacillus licheniformis is a Gram-positive endospore-forming microorganism that is generally non-pathogenic and has been used extensively for large-scale industrial production of various enzymes; however, there has been no previous report on the cloning and expression of mannan endo-1,4-β-mannosidase gene (manB) from B. licheniformis.
Results
The mannan endo-1,4-β-mannosidase gene (manB), commonly known as β-mannanase, from Bacillus licheniformis strain DSM13 was cloned and overexpressed in Escherichia coli. The enzyme can be harvested from the cell lysate, periplasmic extract, or culture supernatant when using the pFLAG expression system. A total activity of approximately 50,000 units could be obtained from 1-l shake flask cultures. The recombinant enzyme was 6 × His-tagged at its C-terminus, and could be purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The specific activity of the purified enzyme when using locust bean gum as substrate was 1672 ± 96 units/mg. The optimal pH of the enzyme was between pH 6.0 - 7.0; whereas the optimal temperature was at 50 - 60°C. The recombinant β-mannanase was stable within pH 5 - 12 after incubation for 30 min at 50°C, and within pH 6 - 9 after incubation at 50°C for 24 h. The enzyme was stable at temperatures up to 50°C with a half-life time of activity (τ1/2) of approximately 80 h at 50°C and pH 6.0. Analysis of hydrolytic products by thin layer chromatography revealed that the main products from the bioconversion of locus bean gum and mannan were various manno-oligosaccharide products (M2 - M6) and mannose.
Conclusion
Our study demonstrates an efficient expression and secretion system for the production of a relatively thermo- and alkali-stable recombinant β-mannanase from B. licheniformis strain DSM13, suitable for various biotechnological applications.
doi:10.1186/1475-2859-9-20
PMCID: PMC2868798  PMID: 20380743
21.  Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira species 
Background
Leptospira are the parasitic bacterial organisms associated with a broad range of mammalian hosts and are responsible for severe cases of human Leptospirosis. The epidemiology of leptospirosis is complex and dynamic. Multiple serovars have been identified, each adapted to one or more animal hosts. Adaptation is a dynamic process that changes the spatial and temporal distribution of serovars and clinical manifestations in different hosts. Serotyping based on repertoire of surface antigens is an ambiguous and artificial system of classification of leptospiral agents. Molecular typing methods for the identification of pathogenic leptospires up to individual genome species level have been highly sought after since the decipherment of whole genome sequences. Only a few resources exist for microbial genotypic data based on individual techniques such as Multiple Locus Sequence Typing (MLST), but unfortunately no such databases are existent for leptospires.
Results
We for the first time report development of a robust MLST method for genotyping of Leptospira. Genotyping based on DNA sequence identity of 4 housekeeping genes and 2 candidate genes was analyzed in a set of 120 strains including 41 reference strains representing different geographical areas and from different sources. Of the six selected genes, adk, icdA and secY were significantly more variable whereas the LipL32 and LipL41 coding genes and the rrs2 gene were moderately variable. The phylogenetic tree clustered the isolates according to the genome-based species.
Conclusion
The main advantages of MLST over other typing methods for leptospires include reproducibility, robustness, consistency and portability. The genetic relatedness of the leptospires can be better studied by the MLST approach and can be used for molecular epidemiological and evolutionary studies and population genetics.
doi:10.1186/1476-0711-5-28
PMCID: PMC1664579  PMID: 17121682
22.  Microdiversity of Deep-Sea Bacillales Isolated from Tyrrhenian Sea Sediments as Revealed by ARISA, 16S rRNA Gene Sequencing and BOX-PCR Fingerprinting 
Microbes and Environments  2013;28(3):361-369.
With respect to their terrestrial relatives, marine Bacillales have not been sufficiently investigated. In this report, the diversity of deep-sea Bacillales, isolated from seamount and non-seamount stations at 3,425 to 3,580 m depth in the Tyrrhenian Sea, was investigated using PCR fingerprinting and 16S rRNA sequence analysis. The isolate collection (n=120) was de-replicated by automated ribosomal intergenic spacer analysis (ARISA), and phylogenetic diversity was analyzed by 16S rRNA gene sequencing of representatives of each ARISA haplotype (n=37). Phylogenetic analysis of isolates showed their affiliation to six different genera of low G+C% content Gram-positive Bacillales: Bacillus, Staphylococcus, Exiguobacterium, Paenibacillus, Lysinibacillus and Terribacillus. Bacillus was the dominant genus represented by the species B. licheniformis, B. pumilus, B. subtilis, B. amyloliquefaciens and B. firmus, typically isolated from marine sediments. The most abundant species in the collection was B. licheniformis (n=85), which showed seven distinct ARISA haplotypes with haplotype H8 being the most dominant since it was identified by 63 isolates. The application of BOX-PCR fingerprinting to the B. licheniformis sub-collection allowed their separation into five distinct BOX genotypes, suggesting a high level of intraspecies diversity among marine B. licheniformis strains. This species also exhibited distinct strain distribution between seamount and non-seamount stations and was shown to be highly prevalent in non-seamount stations. This study revealed the great microdiversity of marine Bacillales and contributes to understanding the biogeographic distribution of marine bacteria in deep-sea sediments.
doi:10.1264/jsme2.ME13013
PMCID: PMC4070960  PMID: 24005887
Deep-sea sediments; Bacillales; ARISA fingerprinting; Microdiversity; BOX-PCR
23.  Cronobacter, the emergent bacterial pathogen Enterobacter sakazakii comes of age; MLST and whole genome sequence analysis 
BMC Genomics  2014;15(1):1121.
Background
Following the association of Cronobacter spp. to several publicized fatal outbreaks in neonatal intensive care units of meningitis and necrotising enterocolitis, the World Health Organization (WHO) in 2004 requested the establishment of a molecular typing scheme to enable the international control of the organism. This paper presents the application of Next Generation Sequencing (NGS) to Cronobacter which has led to the establishment of the Cronobacter PubMLST genome and sequence definition database (http://pubmlst.org/cronobacter/) containing over 1000 isolates with metadata along with the recognition of specific clonal lineages linked to neonatal meningitis and adult infections
Results
Whole genome sequencing and multilocus sequence typing (MLST) has supports the formal recognition of the genus Cronobacter composed of seven species to replace the former single species Enterobacter sakazakii. Applying the 7-loci MLST scheme to 1007 strains revealed 298 definable sequence types, yet only C. sakazakii clonal complex 4 (CC4) was principally associated with neonatal meningitis. This clonal lineage has been confirmed using ribosomal-MLST (51-loci) and whole genome-MLST (1865 loci) to analyse 107 whole genomes via the Cronobacter PubMLST database. This database has enabled the retrospective analysis of historic cases and outbreaks following re-identification of those strains.
Conclusions
The Cronobacter PubMLST database offers a central, open access, reliable sequence-based repository for researchers. It has the capacity to create new analysis schemes ‘on the fly’, and to integrate metadata (source, geographic distribution, clinical presentation). It is also expandable and adaptable to changes in taxonomy, and able to support the development of reliable detection methods of use to industry and regulatory authorities. Therefore it meets the WHO (2004) request for the establishment of a typing scheme for this emergent bacterial pathogen. Whole genome sequencing has additionally shown a range of potential virulence and environmental fitness traits which may account for the association of C. sakazakii CC4 pathogenicity, and propensity for neonatal CNS.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-1121) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-1121
PMCID: PMC4377842  PMID: 25515150
Emergent bacterial pathogen; Cronobacter; MLST; Genomic analysis
24.  Characterisation of Clostridium difficile Hospital Ward–Based Transmission Using Extensive Epidemiological Data and Molecular Typing 
PLoS Medicine  2012;9(2):e1001172.
A population-based study in Oxfordshire (UK) hospitals by Sarah Walker and colleagues finds that in an endemic setting with good infection control, ward-based contact cannot account for most new cases of Clostridium difficile infection.
Background
Clostridium difficile infection (CDI) is a leading cause of antibiotic-associated diarrhoea and is endemic in hospitals, hindering the identification of sources and routes of transmission based on shared time and space alone. This may compromise rational control despite costly prevention strategies. This study aimed to investigate ward-based transmission of C. difficile, by subdividing outbreaks into distinct lineages defined by multi-locus sequence typing (MLST).
Methods and Findings
All C. difficile toxin enzyme-immunoassay-positive and culture-positive samples over 2.5 y from a geographically defined population of ∼600,000 persons underwent MLST. Sequence types (STs) were combined with admission and ward movement data from an integrated comprehensive healthcare system incorporating three hospitals (1,700 beds) providing all acute care for the defined geographical population. Networks of cases and potential transmission events were constructed for each ST. Potential infection sources for each case and transmission timescales were defined by prior ward-based contact with other cases sharing the same ST. From 1 September 2007 to 31 March 2010, there were means of 102 tests and 9.4 CDIs per 10,000 overnight stays in inpatients, and 238 tests and 15.7 CDIs per month in outpatients/primary care. In total, 1,276 C. difficile isolates of 69 STs were studied. From MLST, no more than 25% of cases could be linked to a potential ward-based inpatient source, ranging from 37% in renal/transplant, 29% in haematology/oncology, and 28% in acute/elderly medicine to 6% in specialist surgery. Most of the putative transmissions identified occurred shortly (≤1 wk) after the onset of symptoms (141/218, 65%), with few >8 wk (21/218, 10%). Most incubation periods were ≤4 wk (132/218, 61%), with few >12 wk (28/218, 13%). Allowing for persistent ward contamination following ward discharge of a CDI case did not increase the proportion of linked cases after allowing for random meeting of matched controls.
Conclusions
In an endemic setting with well-implemented infection control measures, ward-based contact with symptomatic enzyme-immunoassay-positive patients cannot account for most new CDI cases.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Hospital-acquired infections are common and occur when patients are infected with an organism found in the hospital or health-care environment. Hospital-acquired infections can sometimes cause no symptoms but often lead to illness or even death. A leading hospital-acquired infection is with the anaerobic bacterium Clostridium difficile, which causes gastrointestinal problems, including diarrhea, leading to severe illness and even death, especially in older patients or patients who are already seriously ill. Between 7% and 26% of elderly adult inpatients in hospitals may be asymptomatic carriers of C. difficile, and the spores that are formed by this organism can live outside of the human body for long periods of time and are notoriously resistant to most routine surface-cleaning methods. Following major hospital-associated outbreaks around the world, C. difficile infection has become a prime target for expensive prevention and infection control strategies.
Why Was This Study Done?
Prevention strategies and infection control measures have contributed to reducing the incidence of C. difficile infection, however, to date, there have not been any robust evaluations of the impact of such strategies in reducing the spread of infection at the individual level. In order to implement improved, cost-effective policies, and to work out how to reduce incidence even further, a better understanding of person-to-person spread is crucial, especially as infection with C. difficile depends on a combination of factors, such as antibiotic exposure and host susceptibility. Therefore, the researchers conducted this study to examine in detail the transmission of C. difficile in hospital wards in order to give more insight and information on the nature of person-to-person spread.
What Did the Researchers Do and Find?
The researchers used a population-based study in Oxfordshire, UK, to investigate hospital ward–based transmission of defined C. difficile strains from symptomatic patients by identifying C. difficile infection from routine clinical microbiological samples from 1 September 2007 to 31 March 2010. Throughout this period, Oxfordshire hospitals operated a rigorous infection control policy monitored by infection control staff, in which stool samples for C. difficile testing were taken from admitted patients with persistent diarrhea, and from patients with any diarrhea who were 65 years or older. The researchers tested all stool samples for C. difficile toxins by enzyme immunoassay, cultured positive samples, and genotyped C. difficile isolates by using multi-locus sequence typing (to identify strains, that is, sequence types), and finally, constructed networks of cases and potential transmissions (by tracing contacts for up to 26 weeks) for each sequence type identified.
In order to show which ward-based contacts potentially incorporated direct person-to-person spread and indirect transmission via the environment during shared ward exposure, the researchers analysed links (ward contacts) between the first case (the donor) and the second case (the recipient) for all pairs of cases with the same sequence type. The researchers then calculated the minimum infectious period by measuring the time between the first infected stool sample from the donor and ward contact with the recipient, and calculated the incubation period as the time between this ward contact and the first infected stool sample in the recipient. To reduce the possibility of shared ward contacts occurring by chance, the researchers used patients with negative enzyme immunoassay stool samples as controls to estimate how often such ward contacts reflected actual transmission rather than chance.
Over the study period, almost 30,000 stool samples from almost 15,000 patients were tested for C. difficile, with 4.4% (1,282) found positive for C. difficile in enzyme immunoassay and culture. With genotyping, the researchers identified 69 strains (sequence types) of C. difficile. The researchers found that the majority (66%) of cases of C. difficile infection were not linked to known cases, and only 23% had a credible ward-based donor sharing the same sequence type of C. difficile. Furthermore, the researchers found that most probable transmissions occurred less than one week after the onset of symptoms, with a minority (10%) occurring after eight weeks. Most incubation periods were less than four weeks, but a few (13%) were more than 12 weeks. Importantly, even after allowing for the random meeting of matched controls and for persistent ward contamination, the proportion of linked cases did not increase following ward discharge of a C. difficile infection case.
What Do These Findings Mean?
These findings show that in an endemic setting with well-implemented infection control measures, ward-based contact with symptomatic, enzyme-immunoassay-positive patients cannot account for most new cases of C. difficile infection. Crucially, these findings mean that C. difficile infection might not be effectively controlled by current strategies to prevent person-to-person spread. Although the researchers were able to distinguish different strains of C. difficile, there were insufficient numbers of these different strains to deduce whether the results they obtained might be different if there was a different combination of strain types, that is, if some strains were spreading more in hospitals than others. Finally, in order to determine what other types of control interventions are required to reduce the spread of C. difficile, a better understanding of other routes of transmission and reservoirs of infectivity is needed.
Additional Information
Please access these web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001172.
This study is further discussed in a PLoS Medicine Perspective by Stephan Harbarth and Matthew Samore
The US Centers for Disease Control and Prevention provides information about C. difficile infection, as does the UK Health Protection Agency
The World Health Organization has published a guide for preventing hospital-acquired infections
doi:10.1371/journal.pmed.1001172
PMCID: PMC3274560  PMID: 22346738
25.  Insights into the Emergent Bacterial Pathogen Cronobacter spp., Generated by Multilocus Sequence Typing and Analysis 
Cronobacter spp. (previously known as Enterobacter sakazakii) is a bacterial pathogen affecting all age groups, with particularly severe clinical complications in neonates and infants. One recognized route of infection being the consumption of contaminated infant formula. As a recently recognized bacterial pathogen of considerable importance and regulatory control, appropriate detection, and identification schemes are required. The application of multilocus sequence typing (MLST) and analysis (MLSA) of the seven alleles atpD, fusA, glnS, gltB, gyrB, infB, and ppsA (concatenated length 3036 base pairs) has led to considerable advances in our understanding of the genus. This approach is supported by both the reliability of DNA sequencing over subjective phenotyping and the establishment of a MLST database which has open access and is also curated; http://www.pubMLST.org/cronobacter. MLST has been used to describe the diversity of the newly recognized genus, instrumental in the formal recognition of new Cronobacter species (C. universalis and C. condimenti) and revealed the high clonality of strains and the association of clonal complex 4 with neonatal meningitis cases. Clearly the MLST approach has considerable benefits over the use of non-DNA sequence based methods of analysis for newly emergent bacterial pathogens. The application of MLST and MLSA has dramatically enabled us to better understand this opportunistic bacterium which can cause irreparable damage to a newborn baby’s brain, and has contributed to improved control measures to protect neonatal health.
doi:10.3389/fmicb.2012.00397
PMCID: PMC3504363  PMID: 23189075
Cronobacter; MLST; MLSA; sequence typing; ST4

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