Short read sequence typing (SRST): multi-locus sequence types from short reads

ABSTRACT: 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 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 assigns alleles using read mapping and an allele assignment score incorporating sequence coverage and variability, to determine the most likely allele. 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.     

Molecular phylogenetic analysis of Candida tropicalis isolates by multi-locus sequence typing

Multi-locus sequencing data for 242 different isolates of Candida tropicalis generated a dendrogram showing 235 strains assigned to a single large recently evolved group which contained several small clonal clusters. Haplotype analysis of a representative strain subset revealed a high level of recombination events in an otherwise clonal population. Pairs of isolates from single sources showed non-identity attributable to loss of heterozygosity in some genes in a manner similar to that established for C. albicans.     

Delineation of Stenotrophomonas spp. by multi-locus sequence analysis and MALDI-TOF mass spectrometry

The genus Stenotrophomonas is genetically and phenotypically heterogeneous. Of the nine species now accepted, only S. maltophilia is of clinical importance. Based on DNA-sequences of seven house keeping genes, it encompasses genogroups of DNA-similarity below 97% that predominantly comprise strains of environmental origin. Therefore, in order to unravel the uneven distribution of environmental isolates within genogroups and reveal genetic relationships within the genus, there is need for an easy and reliable approach for the identification and delineation of Stenotrophomonas spp. In this first study, a multi-locus sequence analysis (MLSA) with seven housekeeping genes (atpD, gapA, guaA, mutM, nuoD, ppsA and recA) was applied for analysis of 21 S. maltophilia of environmental origin, Stenotrophomonas spp. and related genera. The genotypic findings were compared with the results of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analyses. Our MLSA provided reliable inter- and intra-species discrimination of all tested isolates that correlated with the MALDI-TOF mass spectrometry data. One distantly related genogroup of environmental S. maltophilia strains needs to be reclassified as S. rhizophila. However, there are still remaining delineated S. maltophilia genogroups of predominantly environmental origin. Our data provide further evidence that ‘Pseudomonas’ beteli is a heterotypic synonym of S. maltophilia. Based on MLSA and MALDI-TOF data, Stenotrophomonas sp. (DSM 2408) belongs to S. koreensis.     

Six monophyletic lineages identified within Cryptococcus neoformans and Cryptococcus gattii by multi-locus sequence typing.

Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic basidiomycetous yeasts in which six haploid genotypic groups have been distinguished. The two haploid genotypic groups of C. neoformans have been described as variety grubii and variety neoformans. The four C. gattii genotypic groups have, however, not been described as separate taxa. One hundred and seventeen isolates representing all six haploid genotypic groups were selected for multi-locus sequence typing using six loci to investigate if the isolates consistently formed monophyletic lineages. Two monophyletic lineages, corresponding to varieties grubii and neoformans, were consistently present within C. neoformans, supporting the current classification. In addition, four monophyletic lineages corresponding to the previously described genotypic groups were consistently found within C. gattii, indicating that these lineages should be considered different taxa as well.     

Six monophyletic lineages identified within Cryptococcus neoformans and Cryptococcus gattii by multi-locus sequence typing

Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic basidiomycetous yeasts in which six haploid genotypic groups have been distinguished. The two haploid genotypic groups of C. neoformans have been described as variety grubii and variety neoformans. The four C. gattii genotypic groups have, however, not been described as separate taxa. One hundred and seventeen isolates representing all six haploid genotypic groups were selected for multi-locus sequence typing using six loci to investigate if the isolates consistently formed monophyletic lineages. Two monophyletic lineages, corresponding to varieties grubii and neoformans, were consistently present within C. neoformans, supporting the current classification. In addition, four monophyletic lineages corresponding to the previously described genotypic groups were consistently found within C. gattii, indicating that these lineages should be considered different taxa as well.     

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.     

Biodegradation of cyanide by Trichoderma spp. and Fusarium spp.

The rate of degradation of cyanide by certain strains of the Trichoderma spp. was evaluated. For comparison two Fusarium spp., which had previously been demonstrated to degrade metallocyanides were also studied. Studies were carried out to assess the rate of degradation using cyanide as the sole source of carbon or in the presence of glucose. Biodegradation was observed in flask cultures using cyanide as the sole carbon source. Strong evidence of cyanide biodegradation and co-metabolism emerged from studies with flask cultures where glucose was provided as a co-substrate. The rate of degradation of 2000 ppm CN⁻ was enhanced almost three times in the presence of glucose. A concomitant increase in fungal biomass was also observed in all the strains over the experimental period. Growth yield calculations performed provided values that were comparable to those reported in literature for one-carbon substrates.     

Assigning Escherichia coli strains to phylogenetic groups: multi-locus sequence typing versus the PCR triplex method

It is well recognized that Escherichia coli consists of a number of distinct phylo-groups and that strains of the different phylo-groups vary in their ecological niches, life-history characteristics and propensity to cause disease. Consequently, much can be learnt by assigning a strain of E. coli to one of the recognized phylo-groups. A triplex PCR-based method that enables strains of E. coli to be assigned to a phylo-group using a dichotomous key approach based on the presence or absence of two genes (chuA and yjaA) and an anonymous DNA fragment (TSPE4.C2) has been developed. However, the accuracy with which this method assigns strains to their correct phylo-group has not been adequately evaluated. Consequently, 662 strains of E. coli were characterized using a multi-locus sequence typing approach. Unsupervised population assignment algorithms were used to assign strains to phylo-groups based on the multi-locus sequence typing data. The analyses revealed that 85-90% of E. coli strains can be assigned to a phylo-group and that 80-85% of the phylo-group memberships assigned using the Clermont method are correct. However, the accuracy with which strains are assigned to the correct phylo-group depends on their Clermont genotype. For example, strains yielding a Clermont genotype consistent with phylo-groups B1 and B2 are assigned correctly 95% of the time. Strains failing to yield any PCR products using the Clermont method are seldom members of phylo-group A and strains with such a genotype should not be assigned to a phylo-group.     

Development of a sequence typing scheme for differentiation of Salmonella Enteritidis strains

A DNA sequence typing scheme based on the caiC and SEN0629 loci was developed for differentiation of Salmonella Enteritidis strains and validated using a diverse collection of 102 isolates representing 38 phage types from different sources, year of isolation, geographical locations and epidemiological backgrounds. caiC encodes a probable crotonobetaine/carnitine-CoA ligase, and SEN0629 is a pseudogene. Our system allowed for discrimination of 16 sequence types (STs) among the 102 isolates analysed and intraphage type differentiation. Our findings also suggested that the stability of phage typing may be adversely affected by the occurrence of phage type conversion events. During a confirmatory phage typing analysis performed by a reference laboratory, 13 of 31 S. Enteritidis strains representing nine phage types were assigned phage types that differed from the ones originally determined by the same reference laboratory. It is possible that this phenomenon passes largely unrecognized in reference laboratories performing routine phage typing analyses. Our results demonstrate that phage typing is an unstable system displaying limited reproducibility and that the two-loci sequence typing scheme is highly discriminatory, stable, truly portable and has the potential to become the new gold standard for epidemiological typing of S .?Enteritidis strains.     

驴源兽疫链球菌的分离鉴定及多位点序列分型分析

【目的】本研究采集了新疆地区驴腺疫病料,从样品中分离鉴定可疑病原菌并对其进行基因水平的分型与进化分析。【方法】对采集的新疆地区某驴场驴腺疫病驴颌下淋巴结拭子进行细菌分离培养,对2个分离株(HT111、HT321)进行染色、培养、生化试验、药敏试验,对其16S rRNA及SeM基因进行测序和进化分析,并通过PCR扩增7个管家基因,利用多位点序列分型(MLST)方法鉴定其分子分型。【结果】2个分离株均属马链球菌兽疫亚种,其中HT111对测试的13种抗生素中的3种(青霉素、克拉霉素和四环素)耐药,HT321对8种抗生素(阿莫西林、头孢呋辛、头孢噻呋、青霉素、克拉霉素、克林霉素、土霉素和四环素)耐药。序列分型发现了一种新的ST型为ST420 (HT321),分离株HT111为ST179型。【结论】本研究在新疆地区首次分离了驴源马链球菌兽疫亚种,并鉴定出一个新的ST型(ST420),为驴腺疫的流行病学提供了参考数据。     

Single-nucleotide primer extension assay for detection and sequence typing of "Dehalococcoides" spp

A single-nucleotide primer extension (SNuPE) assay in combination with taxon-specific 16S rRNA gene PCR analysis was developed for the detection and typing of populations of the genus "Dehalococcoides". The specificity of the assay was evaluated with 16S rRNA gene sequences obtained from an isolate and an environmental sample representing two Dehalococcoides subgroups, i.e., the Cornell and the Pinellas subgroups. Only one sequence type, belonging to the Pinellas subgroup, was detected in a Bitterfeld-Wolfen region aquifer containing chlorinated ethenes as the main contaminants. The three-primer hybridization assay thus provided a fast and easy-to-implement method for confirming the specificity of taxon-specific PCR and allowed rapid additional taxonomic classification into subgroups. This study demonstrates the great potential of SNuPE as a novel approach for rapid parallel detection of microorganisms and typing of different nucleic acid signature sequences from environmental samples.     

Hypervariable single and multi-locus DNA polymorphisms for genetic typing of non-human primates

The series of hypervariable, “minisatellite” loci characterized byJeffreys and coworkers in the human myoglobin gene have proved to be DNA sequences highly conserved throughout the eukaryotic genome, and hence the methodology developed for human DNA “fingerprinting” has found immediate application in an ever expanding number of species. Primatologists have not been slow to profit from a method which predicts individual recognition to a very high degree of probability, and initial studies have focused on paternity allocation (rather than paternity exclusion, as designated by the classical biochemical markers), adaptive aspects of socio-sexual behaviour patterns and mating systems. A number of probes with sequences corresponding to the common minisatellite core sequences have been used for probing genomic DNA, and synthetic, G-rich oligonucleotides (15 – 37 bases), corresponding to the core sequence of the minisatellite repeat unit, or simply di-, tri-, or tetranucleotide repeats, appear to be equally discriminatory. The multiple banding patterns produced on hybridization of these probes to restriction enzyme digests of DNA provide an advantage in that the probability of two unrelated individuals sharing the same banding pattern will be low. However, the uncertainty of linkage of the multiple loci identified precludes genotyping and population genetic analyses based on allele frequencies. In contrast, single locus analysis allows DNA typing using variable number tandem repeat (VNTR) or restriction fragment length (RFLP) DNA polymorphisms, and the merits and drawbacks relative to DNA fingerprinting are discussed. For the behavioural primatologists dealing with defined, accessible troops of primates, the value of multilocus DNA fingerprinting, in terms of established methodology and availability of probes applicable to species as phylogenetically wide-ranging as apes and prosimians, may well outweigh the loss of genotypic and population structure data.     

Multilocus sequence typing scheme for the characterization of 936-like phages infecting Lactococcus lactis

Lactococcus lactis phage 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 infecting L. lactis strains 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 the lys gene (coding for endolysin) was the most discriminative, with 65 distinct alleles. The locus within the mcp gene (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 at http://pubmlst.org/bacteriophages/.     

In vitro methods for antifungal susceptibility testing of Trichophyton spp.

In general, methods to test the susceptibility of fungi to antifungal drugs require standardized techniques, but so far there is no methodology that is widely applicable to dermatophytes. Here we introduced modifications to the protocols from documents of the National Committee for Clinical Laboratory Standards (CLSI) M38-A and the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) that are usually applied to moulds and fermentative yeasts, in order to adjust the conditions for the growth of dermatophytes. The modifications included: growth on potato dextrose agar supplemented with 2 % in-house rice flour to encourage sporulation, the addition of 2 % glucose to the culture media (RPMI-1640), and an incubation temperature of 28 °C. In addition, the incubation period was 7 d, the minimum inhibitory concentration (MIC) was defined as 80 % growth inhibition endpoints for azole agents, and the inocula only contained microconidia. Results obtained by both tested methodologies were very similar to the ones reported by other researchers. MIC₉₀ (MIC at which 90% of isolates tested were inhibited) values were identical for four out of five antifungal drugs tested and there was only a difference of one or two dilutions when MIC₅₀ values were compared. Although the modifications introduced did not interfere with the results, more studies are necessary to establish a standard technique to test susceptibility of dermatophytes to antifungal drugs.     

短梗霉产liamocins研究进展

短梗霉真菌(Aureobasidium spp.)是一种世界性的酵母样真菌,因其产生黑色素而被称为黑酵母.短梗霉的许多菌株都能分泌细胞外脂质liamocins.Liamocins具有表面活性、良好的抗癌和抗菌活性.本文综述了分泌liamocins的短梗霉的多样性及影响其产生liamocins的因素,总结了liamoci...     

希瓦氏菌生物被膜研究进展

希瓦氏菌（Shewanella spp.）是海产品中常见的优势腐败菌,易在食品加工设备表面形成生物被膜而难以清除。生物被膜的存在不仅会造成食品的持续污染和交叉污染,也会影响加工设备的使用,从而对国民健康和经济发展造成威胁。目前,针对希瓦氏菌生物被膜的研究主要集中在表型研究上,对其生物被膜形成分子机制的研究尚处于起步阶段。总结希瓦氏菌生物被膜的形成过程,重点论述希瓦氏菌生物被膜的形成机制并对希瓦氏菌生物被膜控制方法进行简要概括,展望未来的研究方向,以期为希瓦氏菌生物被膜的深入研究提供参考。     

The potential of Ulocladium botrytis for biological control of Orobanche spp.

A strain of Ulocladium botrytis isolated from diseased Orobanche crenata shoots caused disease on the parasitic weed in pathogenicity tests. The potential of the fungus to be developed as a mycoherbicide for Orobanche spp. was further investigated. Although the fungus significantly decreased O. crenata germination in vitro by 80%, it did not generally lead to a decreased number of O. crenata shoots or tubercles in inoculated root chambers or pots. However, the number of diseased or dead tubercles and underground shoots was significantly increased compared to the noninoculated treatments. Postemergence inoculation of O. crenata shoots with a conidial suspension resulted in the death of almost all inoculated plants 14 days after application under greenhouse conditions. In preliminary host-range studies, the pathogen caused disease on Orobanche cumana on sunflower whereas on Orobanche aegyptiaca shoots parasitizing tomato only minimal disease symptoms could be detected after postemergence inoculation. Based on the results of our investigations, we conclude that Ulocladium botrytis has only a limited potential to be used as a biocontrol agent against Orobanche spp.