Showing papers by "Frank Møller Aarestrup published in 2014"

In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing

Abstract: In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration into a Web tool called PlasmidFinder that can be used for replicon sequence analysis of raw, contig group, or completely assembled and closed plasmid sequencing data. The PlasmidFinder database currently consists of 116 replicon sequences that match with at least at 80% nucleotide identity all replicon sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated from www.pubmlst.org and integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection of Salmonella enterica serovar Typhimurium isolates. PlasmidFinder identified a total of 103 replicons and between zero and five different plasmid replicons within each of 49 S . Typhimurium draft genomes tested. The pMLST Web tool was able to subtype genomic sequencing data of plasmids, revealing both known plasmid sequence types (STs) and new alleles and ST variants. In conclusion, testing of the two Web tools using both fully assembled plasmid sequences and WGS-generated draft genomes showed them to be able to detect a broad variety of plasmids that are often associated with antimicrobial resistance in clinically relevant bacterial pathogens.

Real-Time Whole-Genome Sequencing for Routine Typing, Surveillance, and Outbreak Detection of Verotoxigenic Escherichia coli

Abstract: Fast and accurate identification and typing of pathogens are essential for effective surveillance and outbreak detection. The current routine procedure is based on a variety of techniques, making the procedure laborious, time-consuming, and expensive. With whole-genome sequencing (WGS) becoming cheaper, it has huge potential in both diagnostics and routine surveillance. The aim of this study was to perform a real-time evaluation of WGS for routine typing and surveillance of verocytotoxin-producing Escherichia coli (VTEC). In Denmark, the Statens Serum Institut (SSI) routinely receives all suspected VTEC isolates. During a 7-week period in the fall of 2012, all incoming isolates were concurrently subjected to WGS using IonTorrent PGM. Real-time bioinformatics analysis was performed using web-tools (www.genomicepidemiology.org) for species determination, multilocus sequence type (MLST) typing, and determination of phylogenetic relationship, and a specific VirulenceFinder for detection of E. coli virulence genes was developed as part of this study. In total, 46 suspected VTEC isolates were characterized in parallel during the study. VirulenceFinder proved successful in detecting virulence genes included in routine typing, explicitly verocytotoxin 1 (vtx1), verocytotoxin 2 (vtx2), and intimin (eae), and also detected additional virulence genes. VirulenceFinder is also a robust method for assigning verocytotoxin (vtx) subtypes. A real-time clustering of isolates in agreement with the epidemiology was established from WGS, enabling discrimination between sporadic and outbreak isolates. Overall, WGS typing produced results faster and at a lower cost than the current routine. Therefore, WGS typing is a superior alternative to conventional typing strategies. This approach may also be applied to typing and surveillance of other pathogens.

Solving the problem of comparing whole bacterial genomes across different sequencing platforms.

Abstract: Whole genome sequencing (WGS) shows great potential for real-time monitoring and identification of infectious disease outbreaks. However, rapid and reliable comparison of data generated in multiple laboratories and using multiple technologies is essential. So far studies have focused on using one technology because each technology has a systematic bias making integration of data generated from different platforms difficult. We developed two different procedures for identifying variable sites and inferring phylogenies in WGS data across multiple platforms. The methods were evaluated on three bacterial data sets and sequenced on three different platforms (Illumina, 454, Ion Torrent). We show that the methods are able to overcome the systematic biases caused by the sequencers and infer the expected phylogenies. It is concluded that the cause of the success of these new procedures is due to a validation of all informative sites that are included in the analysis. The procedures are available as web tools.

Rapid Whole-Genome Sequencing for Detection and Characterization of Microorganisms Directly from Clinical Samples

Abstract: Whole-genome sequencing (WGS) is becoming available as a routine tool for clinical microbiology. If applied directly on clinical samples, this could further reduce diagnostic times and thereby improve control and treatment. A major bottleneck is the availability of fast and reliable bioinformatic tools. This study was conducted to evaluate the applicability of WGS directly on clinical samples and to develop easy-to-use bioinformatic tools for the analysis of sequencing data. Thirty-five random urine samples from patients with suspected urinary tract infections were examined using conventional microbiology, WGS of isolated bacteria, and direct sequencing on pellets from the urine samples. A rapid method for analyzing the sequence data was developed. Bacteria were cultivated from 19 samples but in pure cultures from only 17 samples. WGS improved the identification of the cultivated bacteria, and almost complete agreement was observed between phenotypic and predicted antimicrobial susceptibilities. Complete agreement was observed between species identification, multilocus sequence typing, and phylogenetic relationships for Escherichia coli and Enterococcus faecalis isolates when the results of WGS of cultured isolates and urine samples were directly compared. Sequencing directly from the urine enabled bacterial identification in polymicrobial samples. Additional putative pathogenic strains were observed in some culture-negative samples. WGS directly on clinical samples can provide clinically relevant information and drastically reduce diagnostic times. This may prove very useful, but the need for data analysis is still a hurdle to clinical implementation. To overcome this problem, a publicly available bioinformatic tool was developed in this study.

Benchmarking of Methods for Genomic Taxonomy

Abstract: One of the first issues that emerges when a prokaryotic organism of interest is encountered is the question of what it is--that is, which species it is The 16S rRNA gene formed the basis of the first method for sequence-based taxonomy and has had a tremendous impact on the field of microbiology Nevertheless, the method has been found to have a number of shortcomings In the current study, we trained and benchmarked five methods for whole-genome sequence-based prokaryotic species identification on a common data set of complete genomes: (i) SpeciesFinder, which is based on the complete 16S rRNA gene; (ii) Reads2Type that searches for species-specific 50-mers in either the 16S rRNA gene or the gyrB gene (for the Enterobacteraceae family); (iii) the ribosomal multilocus sequence typing (rMLST) method that samples up to 53 ribosomal genes; (iv) TaxonomyFinder, which is based on species-specific functional protein domain profiles; and finally (v) KmerFinder, which examines the number of cooccurring k-mers (substrings of k nucleotides in DNA sequence data) The performances of the methods were subsequently evaluated on three data sets of short sequence reads or draft genomes from public databases In total, the evaluation sets constituted sequence data from more than 11,000 isolates covering 159 genera and 243 species Our results indicate that methods that sample only chromosomal, core genes have difficulties in distinguishing closely related species which only recently diverged The KmerFinder method had the overall highest accuracy and correctly identified from 93% to 97% of the isolates in the evaluations sets

Evaluation of Whole Genome Sequencing for Outbreak Detection of Salmonella enterica

Abstract: Salmonella enterica is a common cause of minor and large food borne outbreaks. To achieve successful and nearly ‘real-time’ monitoring and identification of outbreaks, reliable sub-typing is essential. Whole genome sequencing (WGS) shows great promises for using as a routine epidemiological typing tool. Here we evaluate WGS for typing of S. Typhimurium including different approaches for analyzing and comparing the data. A collection of 34 S. Typhimurium isolates was sequenced. This consisted of 18 isolates from six outbreaks and 16 epidemiologically unrelated background strains. In addition, 8 S. Enteritidis and 5 S. Derby were also sequenced and used for comparison. A number of different bioinformatics approaches were applied on the data; including pan-genome tree, k-mer tree, nucleotide difference tree and SNP tree. The outcome of each approach was evaluated in relation to the association of the isolates to specific outbreaks. The pan-genome tree clustered 65% of the S. Typhimurium isolates according to the pre-defined epidemiology, the k-mer tree 88%, the nucleotide difference tree 100% and the SNP tree 100% of the strains within S. Typhimurium. The resulting outcome of the four phylogenetic analyses were also compared to PFGE reveling that WGS typing achieved the greater performance than the traditional method. In conclusion, for S. Typhimurium, SNP analysis and nucleotide difference approach of WGS data seem to be the superior methods for epidemiological typing compared to other phylogenetic analytic approaches that may be used on WGS. These approaches were also superior to the more classical typing method, PFGE. Our study also indicates that WGS alone is insufficient to determine whether strains are related or un-related to outbreaks. This still requires the combination of epidemiological data and whole genome sequencing results.

Characterization of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli obtained from Danish pigs, pig farmers and their families from farms with high or no consumption of third- or fourth-generation cephalosporins

Abstract: Objectives To compare and characterize extended-spectrum β-lactamase (ESBL)-producing Escherichia coli from pigsties, pig farmers and their families on farms with previous high or no use of third- or fourth-generation cephalosporins. Methods Twenty farms with no third- or fourth-generation cephalosporin use and 19 herds with previous frequent use were included. The ESBL-producing isolates detected in humans and pigs were characterized by ESBL genotype, PFGE, susceptibility to non-β-lactam antibiotics and phylotype, and selected isolates were characterized by multilocus sequence typing (MLST). Furthermore, transferability of bla(CTX-M-)1 from both human and pig isolates was studied and plasmid incompatibility groups were defined. The volunteers answered a questionnaire including epidemiological risk factors for carriage of ESBL-producing E. coli. Results ESBL-producing E. coli was detected in pigs on 79% of the farms with high consumption of cephalosporins compared with 20% of the pigs on farms with no consumption. ESBL-producing E. coli was detected in 19 of the 195 human participants and all but one had contact with pigs. The genes found in both humans and pigs at the same farms were blaCTX-M-1 (eight farms), bla(CTX-M-14) (one farm) and bla(SHV-12) (one farm). At four farms ESBL-producing E. coli isolates with the same CTX-M enzyme, phylotype, PFGE type and MLST type were detected in both pigs and farmers. The majority of the plasmids with bla(CTX-M-1) were transferable by conjugation and belonged to incompatibility group IncI1, IncF, or IncN. Conclusions The present study shows an increased frequency of ESBL-producing E. coli on farms with high consumption of third- or fourth-generation cephalosporins and indicates transfer of either ESBL-producing E. coli or plasmids between pigs and farmers.

Effect of subinhibitory concentrations of four commonly used biocides on the conjugative transfer of Tn916 in Bacillus subtilis

Abstract: Objectives: Large amounts of biocides are used to reduce and control bacterial growth in the healthcare sector, food production and agriculture. This work explores the effect of subinhibitory concentrations of four commonly used biocides (ethanol, hydrogen peroxide, chlorhexidine digluconate and sodium hypochlorite) on the conjugative transposition of the mobile genetic element Tn916. Methods: Conjugation assays were carried out betweenBacillussubtilis strains. The donor containing Tn916 was preexposed to subinhibitory concentrations of each biocide for a defined length of time, which was determined by an analysis of the transcriptional response of the promoter upstream oftet(M) using b-glucuronidase reporter assays. Results: Ethanol significantly (P¼0.01) increased the transfer of Tn916 by 5-fold, whereas hydrogen peroxide, chlorhexidine digluconate and sodium hypochlorite did not significantly affect the transfer frequency. Conclusions: These results suggest that exposure to subinhibitory concentrations of ethanol may induce the transfer of Tn916-like elements and any resistance genes they contain.

The Role of the st313-td Gene in Virulence of Salmonella Typhimurium ST313

Abstract: Multidrug-resistant Salmonella enterica serovar Typhimurium ST313 has emerged in sub-Saharan Africa causing severe infections in humans. Therefore, it has been speculated that this specific sequence type, ST313, carries factors associated with increased pathogenicity. We assessed the role in virulence of a gene with a yet unknown function, st313-td, detected in ST313 through comparative genomics. Additionally, the structure of the genomic island ST313-GI, harbouring the gene was determined. The gene st313-td was cloned into wild type S. Typhimurium 4/74 (4/74-C) as well as knocked out in S. Typhimurium ST313 02–03/002 (Δst313-td) followed by complementation (02-03/002-C). Δst313-td was less virulent in mice following i.p. challenge than the wild type and this phenotype could be partly complemented in trans, indicating that st313-td plays a role during systemic infection. The gene st313-td was shown not to affect invasion of cultured epithelial cells, while the absence of the gene significantly affects uptake and intracellular survival within macrophages. The gene st313-td was proven to be strongly associated to invasiveness, harboured by 92.5% of S. Typhimurium blood isolates (n = 82) and 100% of S. Dublin strains (n = 50) analysed. On the contrary, S. Typhimurium isolates of animal and food origin (n = 82) did not carry st313-td. Six human, non-blood isolates of S. Typhimurium from Belarus, China and Nepal harboured the gene and belonged to sequence types ST398 and ST19. Our data showed a global presence of the st313-td gene and in other sequence types than ST313. The gene st313-td was shown to be expressed during logarithmic phase of growth in 14 selected Salmonella strains carrying the gene. This study reveals that st313-td plays a role in S. Typhimurium ST313 pathogenesis and adds another chapter to understanding of the virulence of S. Typhimurium and in particular of the emerging sequence type ST313.

Genome-Wide High-Throughput Screening to Investigate Essential Genes Involved in Methicillin-Resistant Staphylococcus aureus Sequence Type 398 Survival

Abstract: Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) Sequence Type 398 (ST398) is an opportunistic pathogen that is able to colonize and cause disease in several animal species including humans. To better understand the adaptation, evolution, transmission and pathogenic capacity, further investigations into the importance of the different genes harboured by LA-MRSA ST398 are required. In this study we generated a genome-wide transposon mutant library in an LA-MRSA ST398 isolate to evaluate genes important for bacterial survival in laboratory and host-specific environments. The transposon mutant library consisted of approximately 1 million mutants with around 140,000 unique insertion sites and an average number of unique inserts per gene of 44.8. We identified LA-MRSA ST398 essential genes comparable to other high-throughput S. aureus essential gene studies. As ST398 is the most common MRSA isolated from pigs, the transposon mutant library was screened in whole porcine blood. Twenty-four genes were specifically identified as important for bacterial survival in porcine blood. Mutations in 23 of these genes resulted in attenuated bacterial fitness. Seven of the 23 genes were of unknown function, whereas 16 genes were annotated with functions predominantly related to carbon metabolism, pH shock and a variety of regulations and only indirectly to virulence factors. Mutations in one gene of unknown function resulted in a hypercompetitive mutant. Further evaluation of these genes is required to determine their specific relevance in blood survival.

Genome Sequences of Two Copper-Resistant Escherichia coli Strains Isolated from Copper-Fed Pigs.

Abstract: The draft genome sequences of two copper-resistant Escherichia coli strains were determined. These had been isolated from copper-fed pigs and contained additional putative operons conferring copper and other metal and metalloid resistances.

Genome Sequences of Three Highly Copper-Resistant Salmonella enterica subsp. I Serovar Typhimurium Strains Isolated from Pigs in Denmark.

Abstract: Salmonella typhimurium is the causative agent of typhoid fever, which causes nearly 21.7 million illnesses and 217,000 deaths around the world each year. Here, we describe the draft genome sequences of the Salmonella typhimurium strains S7, S15, and S23, isolated from copper-fed pigs in Denmark and containing additional putative determinants conferring resistances to copper and other metals and metalloids.

Analysis of the contribution of bacteriophage ST64B to in vitro virulence traits of Salmonella enterica serovar Typhimurium.

Abstract: Comparison of the publicly available genomes of the virulent Salmonella enterica serovar Typhimurium (S. Typhimurium) strains SL1344, 14028s and D23580 to that of the virulence-attenuated isolate LT2 revealed the absence of a full sequence of bacteriophage ST64B in the latter. Four selected ST64B regions of unknown function (sb7–sb11, sb46, sb49–sb50 and sb54) were mapped by PCR in two strain collections: (i) 310 isolates of S. Typhimurium from human blood or stool samples, and from food, animal and environmental reservoirs; and (ii) 90 isolates belonging to other serovars. The region sb49–sb50 was found to be unique to S. Typhimurium and was strongly associated with strains isolated from blood samples (100 and 28.4 % of the blood and non-blood isolates, respectively). The region was cloned into LT2 and knocked out in SL1344, and these strains were compared to wild-type isogenic strains in in vitro assays used to predict virulence association. No difference in invasion of the Int407 human cell line was observed between the wild-type and mutated strains, but the isolate carrying the whole ST64B prophage was found to have a slightly better survival in blood. The study showed a high prevalence and a strong association between the prophage ST64B and isolates of S. Typhimurium collected from blood, and may indicate that such strains constitute a selected subpopulation within this serovar. Further studies are indicated to determine whether the slight increase in blood survival observed in the strain carrying ST64B genes is of paramount importance for systemic infections.

Spatio-temporal analysis of Salmonella surveillance data in Thailand.

Abstract: SUMMARY This study evaluates the usefulness of spatio-temporal statistical tools to detect outbreaks using routine surveillance data where limited epidemiological information is available. A dataset from 2002 to 2007 containing information regarding date, origin, source and serotype of 29586 Salmonella isolates from Thailand was analysed. Data was grouped into human and non-human categories and the analysis was performed for the top five occurring serovars for each year of the study period. A total 91 human and 39 non-human significant spatio-temporal clusters were observed, accounting for 11% and 16% of the isolates, respectively. Serovar-specific associations between human and non-human clusters were also evaluated. Results show that these statistical tools can provide information for use in outbreak prevention and detection, in countries where only limited data is available. Moreover, it is suggested that monitoring non-human reservoirs can be relevant in predicting future Salmonella human cases.

Pasteurella multocida septicaemia and pneumonia after chemotherapy in a patient who had no known contact with animals

Abstract: Pasteurella multocida inhabits the upper respiratory tract of many animals. It can cause skin and soft tissue infections in humans, usually in association with animal bites. We present a case of a 66-year-old chemotherapy-induced immunocompromised patient with lung cancer, who was treated for pneumonia and septicaemia due to P. multocida. There was no anamnestic contact with animals, which underlines the fact that immunocompromised patients can suffer from serious systemic infections due to P. multocida - even with no known animal contact.