Eva Litrup

Virulotyping and antimicrobial resistance typing of Salmonella enterica serovars relevant to human health in Europe

The combination of virulence gene and antimicrobial resistance gene typing using DNA arrays is a recently developed genomics-based approach to bacterial molecular epidemiology. We have now applied this technology to 523 Salmonella enterica subsp. enterica strains collected from various host sources and public health and veterinary institutes across nine European countries. The strain set included the five predominant Salmonella serovars isolated in Europe (Enteritidis, Typhimurium, Infantis, Virchow, and Hadar). Initially, these strains were screened for 10 potential virulence factors (avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) by polymerase chain reaction. The results indicated that only 14 profiles comprising these genes (virulotypes) were observed throughout Europe. Moreover, most of these virulotypes were restricted to only one (n = 9) or two (n = 4) serovars. The data also indicated that the virulotype did not vary significantly with host source or geographical location. Subsequently, a representative subset of 77 strains was investigated using a microarray designed to detect 102 virulence and 49 resistance determinants. The results confirmed and extended the previous observations using the virulo-polymerase chain reaction screen. Strains belonging to the same serovar grouped together, indicating that the broader virulence-associated gene complement corresponded with the serovar. There were, however, some differences in the virulence gene profiles between strains belonging to an individual serovar. This variation occurred primarily within those virulence genes that were prophage encoded, in fimbrial clusters or in the virulence plasmid. It seems likely that such changes enable Salmonella to adapt to different environmental conditions, which might be reflected in serovar-specific ecology. In this strain subset a number of resistance genes were detected and were serovar restricted to a varying degree. Once again the profiles of those genes encoding resistance were similar or the same for each serovar in all hosts and countries investigated.

Progressive genome-wide introgression in agricultural Campylobacter coli

Hybridization between distantly related organisms can facilitate rapid adaptation to novel environments, but is potentially constrained by epistatic fitness interactions among cell components. The zoonotic pathogens Campylobacter coli and C. jejuni differ from each other by around 15% at the nucleotide level, corresponding to an average of nearly 40 amino acids per protein‐coding gene. Using whole genome sequencing, we show that a single C. coli lineage, which has successfully colonized an agricultural niche, has been progressively accumulating C. jejuni DNA. Members of this lineage belong to two groups, the ST‐828 and ST‐1150 clonal complexes. The ST‐1150 complex is less frequently isolated and has undergone a substantially greater amount of introgression leading to replacement of up to 23% of the C. coli core genome as well as import of novel DNA. By contrast, the more commonly isolated ST‐828 complex bacteria have 10–11% introgressed DNA, and C. jejuni and nonagricultural C. coli lineages each have <2%. Thus, the C. coli that colonize agriculture, and consequently cause most human disease, have hybrid origin, but this cross‐species exchange has so far not had a substantial impact on the gene pools of either C. jejuni or nonagricultural C. coli. These findings also indicate remarkable interchangeability of basic cellular machinery after a prolonged period of independent evolution.

Multilocus sequence typing performed on Campylobacter coli isolates from humans, broilers, pigs and cattle originating in Denmark

Aims:  To assess whether Campylobacter coli isolated from different sources in Denmark constitute separate populations.

A phylogenetic group of Escherichia coli associated with active left-sided Inflammatory Bowel Disease

BackgroundEscherichia coli have been found in increased numbers in tissues from patients with Inflammatory Bowel Disease (IBD) and adherent-invasive E. coli have been found in resected ileum from patients with Crohns disesae. This study aimed to characterize possible differences in phylogenetic group (triplex PCR), extraintestinal pathogenic E. coli (ExPEC) genes and multilocus sequence type (MLST) between E. coli strains isolated from IBD patients with past or present involvement of the left side of the colon and from controls.ResultsFecal samples were collected from 18 patients and from 10 healthy controls. Disease activity was evaluated by sigmoidoscopy. Interestingly, E. coli strains of the phylogenetic group B2 were cultured from 60% of patients with IBD compared to 11% of healthy controls (p < 0.05). Furthermore, when comparing the number of E. coli B2 strains with at least one positive ExPEC gene among different groups, 86% were found positive among active IBD patients, significantly more than 13% among inactive IBD patients (p < 0.05), and 11% among healthy controls (p < 0.05). The B2 phylogenetic group was found in a specific cluster based on MLST, but no further separation between E. coli strains associated with active compared to inactive IBD was achieved.ConclusionIn conclusion, E. coli of the phylogenetic group B2 were isolated more frequently from IBD patients with past or present involvement of the left side of the colon compared to healthy controls, and B2 strains with ExPEC genes were found more frequently among IBD patients with active disease compared to patients with inactive disease.

Association between phylogeny, virulence potential and serovars of Salmonella enterica

Salmonella enterica subsp. enterica is one of the leading causes of zoonotic food-borne disease worldwide. The consequence of these infections is a serious impact on economics of the society in the form of lost productivity and expenses for medical care. The objective of this study was to analyze the difference in genomic content between selected serovars, especially the content of pathogenicity genes and this was done with a DNA microarray. Furthermore, we investigated the phylogenetic relationship between serovars using multilocus sequence typing (MLST). We chose serovars Typhimurium and Enteritidis as they are responsible for 75% of human infections in Europe. Additionally, we included serovars Derby, Dublin, Saintpaul, 4,5,12:i:-, Java and 4,5,12:b:- which are suspected to have different degrees of virulence to humans. MLST analysis clustered strains according to serovar with the exception of Java and Derby. DNA microarray clustered strains according to serovar and serogroup except for serovar 4,5,12:b:-. Differences in content of pathogenicity related genes between serovars with various host preferences and virulence towards humans were not observed. However, our strains from the supposedly less virulent serovar Derby lacked a combination of genes important for virulence. It might be speculated that other serovars can sustain their pathogenicity lacking one or two of these genes, whereas lack of many virulence genes will result in reduced virulence. A partial lack of concordance between MLST and microarray was found and this can be explained by the underlying data. On one hand, microarray data include highly variable regions which are known to be involved in horizontal gene transfer. On the other hand, MLST data is restricted to seven sequences and disregards contribution of horizontally acquired genes when evaluating evolution. The DNA microarray and MLST analysis complement each other giving a clearer image of evolution of these serovars and, furthermore, a visualization of the horizontally acquired genes.

Molecular Characterization of Salmonella Typhimurium Highly Successful Outbreak Strains

Three large clusters of Salmonella Typhimurium infections in Denmark in 2008 and 2009 were defined by multilocus variable number of tandem repeat analysis (MLVA). One of these proved to be the hereto largest Danish cluster of salmonellosis with 1446 cases. Two smaller clusters with a total of 197 and 89 cases, respectively, were seen concurrently. These clusters shared epidemiological characteristics such as age distribution, geography, and time. To investigate the possible genetic relationship between the cluster strains, these were further characterized by phage typing, pulsed-field gel electrophoresis, and Optical Mapping. Although the MLVA method proved robust and well-performing in detecting and defining clusters, the employment of a second typing method detected an additional fourth cluster among the isolates. The cluster strains were stable throughout the almost 2-year period, even though we detected changes in three of five MLVA loci in a small fraction of isolates. These changes were mainly due to the gain or loss of single repeats. Optical Mapping of the large cluster strain indicated no increased content of virulence genes; however, Optical Mapping did reveal a large insert, a probable prophage, in the main cluster. This probable prophage may give the cluster strain a competitive advantage. The molecular methods employed suggested that the four clusters represented four distinct strains, although they seemed to be epidemiologically linked and shared genotypic characteristics.

Plasmid-borne colistin resistance gene mcr-3 in Salmonella isolates from human infections, Denmark, 2009–17

This report describes one Salmonella isolate harbouring both mcr-1 and mcr-3. We also found nine other Salmonella isolates positive for the plasmid-borne colistin resistance gene, mcr-3. The strains were isolated from patients in Denmark between 2009 and 2017 and five of the patients had travelled to Asia. In addition to mcr-3, all strains were found positive for blaTEM-1, strA, strB, sul2 and tet(A) or tet(B), and most strains were positive for blaCTX-M-55 and qnrS.

DNA microarray analysis of Salmonella serotype Typhimurium strains causing different symptoms of disease

BackgroundSalmonella enterica subsp. enterica is one of the leading food-borne pathogens in the USA and European countries. Outcome of human Salmonella serotype Typhimurium infections ranges from mild self-limiting diarrhoea to severe diarrhoea that requires hospitalization. Increased knowledge of the mechanisms that are responsible for causing infection and especially the severity of infection is of high interest.ResultsStrains were selected from patients with mild infections (n = 9) and patients with severe infections (n = 9) and clinical data allowed us to correct for known underlying diseases. Additionally, outbreak isolates (n = 3) were selected. Strains were analyzed on a DNA-DNA microarray for presence or absence of 281 genes covering marker groups of genes related to pathogenicity, phages, antimicrobial resistance, fimbriae, mobility, serotype and metabolism. Strains showed highly similar profiles when comparing virulence associated genes, but differences between strains were detected in the prophage marker group. The Salmonella virulence plasmid was present in 72% of the strains, but presence or absence of the virulence plasmid did not correspond to disease symptoms. A dendrogram clustered strains into four groups. Clustering confirmed DT104 as being a clonal phagetype. Clustering of the remaining strains was mainly correlated to presence or absence of the virulence plasmid and mobile elements such as transposons. Each of the four clusters in the tree represented an almost equal amount of strains causing severe or mild symptoms of infection.ConclusionsWe investigated clinical significance of known virulence factors of Salmonella serotype Typhimurium strains causing different disease symptoms, and conclude that the few detected differences in Salmonella serotype Typhimurium do not affect outcome of human disease.

Use of multiple-locus variable-number tandem-repeats analysis (MLVA) typing to characterize Salmonella Typhimurium DT41 broiler breeder infections.

Aims:  To characterize isolates of Salmonella Typhimurium DT41 obtained from infected flocks of broiler breeders by multiple‐locus variable‐number tandem‐repeats analysis (MLVA) and compare results with a diverse strain collection from Germany and United Kingdom and isolates from Danish patients.

Detection of mcr-1-encoding plasmid-mediated colistin-resistant Salmonella isolates from human infection in Denmark

In November 2015, Liu et al reported plasmid-mediated colistin resistance (encoded by mcr-1) in Escherichia coli and Klebsiella pneumoniae isolates from China [1]. Soon after, there were several reports of mcr-1 in E. coli or K. pneumoniae from other parts of the world, whereas only a few reports have been regarding the finding of mcr-1 in Salmonella spp. [2]. Statens Serum Institut (Copenhagen, Demark) receives isolates and patient information from all human clinical Salmonella infections in Denmark for surveillance. From 2008 to 2015, a total of 8397 Salmonella isolates (80% of all incoming isolates) were tested for colistin susceptibility using Sensititre Trek panels (Thermo Fisher Scientific,Waltham,MA). Of the 488 isolates with a colistinminimum inhibitory concentration (MIC) of >2 mg/L, 129 were tested for the presence of themcr-1 gene by PCR [1], excluding the serotypes Dublin and Enteritidis (359 isolates) due to increased MICs to colistin. Four isolates were found to be positive for themcr-1 gene, all beingmonophasic variants of Salmonella Typhimurium (4,5,12:i:and 4,12:i:-) (Table 1). One of the patients from 2014 had travelled to Thailand prior to the onset of disease, whereas the other patient from 2014 had an unknown travel history (Table 1). The two patients from 2015 had acquired their infection domestically and were from the same town in Denmark. Whole-genome sequencing (WGS) (MiSeq; Illumina Inc., SanDiego, CA) of the fourmcr-1-positive isolates and extraction of sequence types (STs) (https://github.com/tseemann/mlst) revealed two ST34 and two ST19 isolates (Table 1). The sequence datawere used to create a single nucleotide polymorphism (SNP) phylogeny (in-house pipeline), also including available WGS data of previously reportedmcr-1-positive S. Typhimurium isolates from Portugal (GenBank No. NZ_LFCC01000022), France (GenBank No. NZ_LKJD01000097) and local sequences representing the diversity within (mcr-1-negative) S. Typhimurium, and the monophasic variant isolated from patients in Denmark. The SNP analysis showed that the two ST19MCR1-producing strains were identical andwere located on a deep branch not related to any other of the selected strains. The two ST34 mcr1-positive strains were located in a group with all other ST34 strains included. The mcr-1-positive strains (including the Portuguese and French strains) were located on four different deep branches. Using ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/), various resistance genes were detected from theWGS data (Table 1). Of note, the mcr-1-positive isolate from the patient with a known travel history to Thailand harboured blaCTX-M-55 encoding an extendedspectrum β-lactamase (ESBL) enzyme and qnrS1 encoding ciprofloxacin resistance. Using PlasmidFinder (https://cge.cbs.dtu.dk/services/ PlasmidFinder/), an IncI2 replicon was detected in one isolate (1401R17698), and IncX4 replicons were detected in the three other isolates (Table 1). The initial finding of plasmid-bornemcr-1 by Liu et al identified the gene on a 64-kb IncI2 plasmid (pHNSHP45). Also, an IncI2 plasmid was detected in recent MCR-1-producing E. coli isolates from Denmark and China [3] (ESBL20150072 and A31-12, respectively) as well as an MCR-1-producing S. Typhimurium (S3) from a pig farm in Great Britain [4]. BLAST analysis using the GView Server (https://server.gview.ca/) of the sequencing data from 1401R17698, ESBL20150072, pA32-12 and S3 against pHNSHP45 suggested highly similar plasmids. Similarly, the full genome sequence of an IncX4 plasmid (pESTMCR) originating from an MCR1-producing E. coli isolated from pig slurry in Estonia is available (GenBank No. KU743383.1). BLAST atlas mapping using the GView Server shows very high similarity between this plasmid and the three IncX4-containing S. Typhimurium strains in the current study as well as to two MCR-1-producing Salmonella Paratyphi B isolates (12CEB4337SAL and 12CEB2196SAL) from France [5]. The Salmonella isolate from a patient with a known travel history to Thailand, in addition to beingmcr-1-positive, also carried blaCTX-M-55. Several other studies have detected isolates with both mcr-1 and blaCTX-M-55. Except for the Danish E. coli isolate with an unknown travel link as well as one Dutch E. coli isolate with a link to Peru, all isolates with both mcr-1 and blaCTX-M-55 had a link to Asia. BLAST atlas mapping of WGS data from the Salmonella isolates in the present study indicated that highly similar plasmids belonging to IncI2 and IncX4 are involved in the spread of mcr-1. Very high transfer rates