[Show abstract][Hide abstract] ABSTRACT: Retrospectively, we investigated the epidemiology of a massive Salmonella enterica serovar Typhi outbreak in Zambia during 2010 to 2012. Ninety-four isolates were susceptibility tested by MIC determinations. Whole genome sequence typing (WGST) of 33 isolates and bioinformatic analysis identified the MLST, haplotype, plasmid replicon, antimicrobial resistance genes, and the genetic relatedness by Single Nucleotide Polymorphism (SNP) analysis and genomic deletions. The outbreak affected 2,040 patients with a fatality rate of 0.5%. Most isolates (83.0%) were multi-drug resistant (MDR). The isolates belonged to MLST ST1 and a new variant of the haplotype; H58B. Most isolates contained a chromosomally translocated region containing seven antimicrobial resistance genes; catA1, blaTEM-1, dfrA7, sul1, sul 2, strA, and strB, fragments of incQ1plasmid replicon, class 1 integron, and the mer operon. The genomic analysis revealed an overall 415 SNPs difference and 35 deletions among 33 of the isolates whole genome sequenced. In comparison with other genomes of H58, the Zambian isolates separated from genomes from Central Africa and India with 34 and 52 SNPs, respectively. The phylogenetic analysis indicates that 32 isolates of the 33 sequenced belonged to a tight clonal group, distinct from other H58 genomes included in the study. The small numbers of SNPs identified within this group are consistent with short-term transmission that can be expected over a period of 2 years. The phylogenetic analysis and deletions suggest that a single MDR clone was responsible for the outbreak during which occasional other S. Typhi lineages including sensitive ones continued to co-circulate. The common view is that the emerging global S. Typhi haplotype; H58B, containing the MDR incHI1 plasmid is responsible for the majority of typhoid infections in Asia and sub-Saharan Africa; we found that a new variant of the haplotype harbouring a chromosomally translocated region containing the MDR islands of incHI1 plasmid emerged in Zambia. This could chance the perception of the term "classical MDR typhoid" currently being solely associated with the incHI1 plasmid. It might be more common than anticipated that S. Typhi haplotype; H58B harbour either the incHI1 plasmid and /or a chromosomally translocated MDR region.
[Show abstract][Hide abstract] ABSTRACT: The rapid evolution of bacteria is crucial to their survival, and is among other things caused by exchange, transfer and uptake of DNA. Conjugation is one of the main mechanisms by which bacteria share their DNA, and is thought to be controlled by varied bacterial immune systems. Contradictory results, based on phenotypical studies, have been presented on Restriction-Modification systems as a barrier for conjugation and other means of uptake of exogenous DNA. In this study, we show that inactivation of the R.EcoKI restriction enzyme in strain Escherichia coli K-12 strain MG1655 increases the conjugational transfer of plasmid pOLA52, which carriers two EcoKI recognition sites. Interestingly, the results were not absolute, and uptake of un-methylated pOLA52 was still observed in the wild-type strain (with intact hsdR gene), but with a reduction of 85% compared to the mutant recipient having a disrupted hsdR gene. This leads to the conclusion that EcoKI Restriction-Modification affects the uptake of DNA by conjugation, but is not a major barrier for plasmid transfer.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Antimicrobial Chemotherapy 06/2014; · 5.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Emergence of antimicrobial resistance (AMR) in the animal reservoir forms a risk for human health. The use of antimicrobials in animals is the major cause of development of AMR in bacteria in animals. In the 1990s, the use of antimicrobials in animals, particularly as a growth promoter, led to alarming levels of AMR in many countries. This paper analyses the emergence of AMR in Denmark in terms of contributing factors that formed fertile ground from which AMR could develop. New technologies in combination with scientific unknowns led to the unexpected development of cross-resistance and an uncertainty about transmission to and risk for humans. Conflict of interests and varying susceptibility to risk between agriculture, health and commercial stakeholders complicated intervention. In addition, unintended economic incentives from old legislation resulted in a situation where the use of antimicrobials in general was stimulated. Complications of alarming high levels of AMR in animals, and a general discontent about this situation (including with farmers and vets) demanded a solution. National surveillance in DANMAP involving all stakeholders from the farm-to-fork food chain was setup to counteract scientific unknowns and conflicts of interest; new legislation was developed; and unintended economic incentives reduced. The current analysis may help to better understand the AMR problem in general and what may be done to counteract it.
Food Control 06/2014; 40:185–192. · 2.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The emergence of resistance in food animals has been associated to the consumption of antimicrobials in veterinary medicine. Consequently, monitoring programs have been designed to monitor the occurrence of antimicrobial resistant bacteria. This study analyses the amount of antimicrobial agents used in nine European countries from 2005 to 2011, and compares by univariate analysis the correlations between consumptions of each of the following antimicrobial classes; tetracycline, penicillins, cephalosporins, quinolones and macrolides. An overview of resistance in zoonotic and commensal bacteria in Europe focusing on Salmonella, Escherichia coli, Campylobacter sp and Enterococcus sp, during the same period of time based on monitoring programs is also assessed. With the exception of cephalosporins, linear regressions showed strong positive associations between the consumption of the four different antimicrobial classes. Substantial differences between countries were observed in the amount of antimicrobials used to produce 1 kg of meat. Moreover, large variations in proportions of resistant bacteria were reported by the different countries, suggesting differences in veterinary practice. Despite the withdrawn of a specific antimicrobial from “on farm” use, persistence over the years of bacteria resistant to this particular antimicrobial agent, was still observed. There were also differences in trends of resistance associated to specific animal species. In order to correlate the use of antimicrobial agents to the presence of resistance, surveillance of antimicrobial consumption by animal species should be established. Subsequently, intervention strategies could be designed to minimize the occurrence of resistance.
[Show abstract][Hide abstract] ABSTRACT: Here we design and develop two easy-to-use web-tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from Enterobacteriaceae. These tools will facilitate bacterial typing based on draft genomes of multi-drug resistant Enterobacteriaceae by a rapid detection of known plasmid types.Replicon sequences were collected from 559 fully sequenced plasmids present at the NCBI nucleotide database associated with the family Enterobacteriaceae, to build a consensus database for integration into a web-tool called PlasmidFinder usable for replicon sequence analysis of raw as well as group of contigs or completely assembled and closed plasmids sequencing data. The PlasmidFinder database currently consists of 116 replicon sequences that match at least at 80% nucleotide identity with all replicon sequences identified in the 559 fully sequenced plasmids. For pMLST analysis, a weekly updated database 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 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 sub-type genomic sequencing data of plasmids, revealing both known plasmid ST's as well as new alleles and ST variants.In conclusion, testing 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 often associated with antimicrobial resistance in clinically relevant bacterial pathogens.
Antimicrobial Agents and Chemotherapy 04/2014; · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fast and accurate identification and typing of pathogens is 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 E.coli (VTEC).In Denmark, Statens Serum Institut (SSI) routinely receives all suspected VTEC isolates. During a 7-week period in the fall 2012, all incoming isolates were concurrently subjected to WGS using IonTorrent PGM. Real-time bioinformatic analysis was performed using web-tools (www.genomicepidemiology.org) for species determination, MLST typing, 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 characterised 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, and is thus a superior alternative to conventional typing strategies. This approach may also be applied for typing and surveillance of other pathogens.
Journal of clinical microbiology 02/2014; · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: One of the first questions that emerge when encountering a prokaryotic organism of interest is 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 dataset of complete genomes; 1) SpeciesFinder, which is based on the complete 16S rRNA gene, 2) Reads2Type that searches for species-specific 50-mers in either the 16S rRNA gene or the GyrB gene (for the Enterobacteraceae family), 3) The rMLST method that samples up to 53 ribosomal genes, 4) TaxonomyFinder, which is based on species-specific functional protein domain profiles, and finally 5) KmerFinder, which examines the number of co-occurring k-mers. The performances of the methods were subsequently evaluated on three datasets 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 only sample 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%-97% of the isolates in the evaluations sets.
Journal of clinical microbiology 02/2014; · 4.16 Impact Factor
[Show abstract][Hide abstract] 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.
PLoS ONE 01/2014; 9(1):e84566. · 3.53 Impact Factor
[Show abstract][Hide abstract] 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.
PLoS ONE 01/2014; 9(2):e87991. · 3.53 Impact Factor
[Show abstract][Hide abstract] 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.
PLoS ONE 01/2014; 9(2):e89018. · 3.53 Impact Factor
[Show abstract][Hide abstract] 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.
PLoS ONE 01/2014; 9(8):e104984. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although the majority of bacteria are harmless or even beneficial to their host, others are highly virulent and can cause serious diseases, and even death. Due to the constantly decreasing cost of high-throughput sequencing there are now many completely sequenced genomes available from both human pathogenic and innocuous strains. The data can be used to identify gene families that correlate with pathogenicity and to develop tools to predict the pathogenicity of newly sequenced strains, investigations that previously were mainly done by means of more expensive and time consuming experimental approaches. We describe PathogenFinder (http://cge.cbs.dtu.dk/services/PathogenFinder/), a web-server for the prediction of bacterial pathogenicity by analysing the input proteome, genome, or raw reads provided by the user. The method relies on groups of proteins, created without regard to their annotated function or known involvement in pathogenicity. The method has been built to work with all taxonomic groups of bacteria and using the entire training-set, achieved an accuracy of 88.6% on an independent test-set, by correctly classifying 398 out of 449 completely sequenced bacteria. The approach here proposed is not biased on sets of genes known to be associated with pathogenicity, thus the approach could aid the discovery of novel pathogenicity factors. Furthermore the pathogenicity prediction web-server could be used to isolate the potential pathogenic features of both known and unknown strains.
PLoS ONE 12/2013; 8(10):e77302. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The comparison of the publicly available genomes of the virulent Salmonella serovar Typhimurium strains SL1344, 14028s, 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 strains collections; 1) 310 isolates of S. Typhimurium from human blood or stool samples and from food, animal and environmental reservoirs, and 2) 90 isolates belonging to other serovars. The region sb49-sb50 was found to be unique to S. Typhimurium and was strongly associated to 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 for systemic infections.
Journal of Medical Microbiology 12/2013; · 2.30 Impact Factor
[Show abstract][Hide abstract] 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 time and thereby improve control and treatment. A major bottle-neck is the availability of fast and reliable bioinformatics tools. This study was conducted to evaluate the applicability of WGS directly on clinical samples and to develop easy-to-use bioinformatics tools for analysis of the sequencing data. Thirty-five random urine samples from patients with suspected urinary tract infections were examined using conventional microbiology, WGS of isolated bacteria and by directly sequencing on pellets from the urine. A rapid method for analyzing the sequence data was developed. Bacteria were cultivated from 19 samples, but only in pure culture from 17. WGS improved the identification of the cultivated bacteria and almost complete agreement was observed between phenotypic and predicted antimicrobial susceptibility. Complete agreement was observed between species identification, multi-locus-sequence typing and phylogenetic relationship for the Escherichia coli and Enterococcus faecalis isolates when comparing the results of WGS of cultured isolates and directly from the urine samples. Sequencing directly from the urine enabled bacterial identification in polymicrobic 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 time. 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 bioinformatics tool was developed in this study.
Journal of clinical microbiology 10/2013; · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Salmonella enterica serovar Typhimurium ST313 is an invasive and phylogenetically distinct lineage present in sub-Saharan Africa. We report the presence of S. Typhimurium ST313 from patients in the Democratic Republic of Congo and Nigeria.
Eighteen S. Typhimurium ST313 isolates were characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). Additionally, six of the isolates were characterized by whole genome sequence typing (WGST). The presence of a putative virulence determinant was examined in 177 Salmonella isolates belonging to 57 different serovars.
All S. Typhimurium ST313 isolates harbored resistant genes encoded by blaTEM1b, catA1, strA/B, sul1, and dfrA1. Additionally, aac(6')1aa gene was detected. Phylogenetic analyses revealed close genetic relationships among Congolese and Nigerian isolates from both blood and stool. Comparative genomic analyses identified a putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and S. Dublin.
We showed in a limited number of isolates that S. Typhimurium ST313 is a prevalent sequence-type causing gastrointestinal diseases and septicemia in patients from Nigeria and DRC. We found three distinct phylogenetic clusters based on the origin of isolation suggesting some spatial evolution. Comparative genomics showed an interesting putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and invasive S. Dublin.
The Journal of Infection in Developing Countries 10/2013; 7(10):696-706. · 1.00 Impact Factor
[Show abstract][Hide abstract] 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 29 586 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.
Epidemiology and Infection 10/2013; · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Conjugation assays were carried out between Bacillus subtilis strains. The donor containing Tn916 was pre-exposed 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 of tet(M) using β-glucuronidase reporter assays.
Ethanol significantly (P = 0.01) increased the transfer of Tn916 by 5-fold, whereas hydrogen peroxide, chlorhexidine digluconate and sodium hypochlorite did not significantly affect the transfer frequency.
These results suggest that exposure to subinhibitory concentrations of ethanol may induce the transfer of Tn916-like elements and any resistance genes they contain.
Journal of Antimicrobial Chemotherapy 10/2013; · 5.34 Impact Factor