Shiga Toxin-Producing Escherichia coli in Diarrheal Stool of Swedish Children: Evaluation of Polymerase Chain Reaction Screening and Duration of Shiga Toxin Shedding.

BACKGROUND: Shiga toxin (Stx)-producing Escherichia coli (STECs) are the most common cause of acute renal failure in children. The present study evaluated a 10-year STEC polymerase chain reaction screening regimen in children. METHODS: All routine stool culture specimens from patients below 10 years of age (n = 10 342) from May 2003 through April 2013 in the County of Jönköping, Sweden, were included. Patients were divided in 1 group where analyses of STEC were requested by the clinician (n = 2366) and 1 screening group (n = 7976). Patients who were positive for STEC were tested weekly until they were negative. Clinical data were collected through a questionnaire and by reviewing medical records. RESULTS: In specimens from 191 patients, stx was found (162 index cases). The prevalence was 1.8% in the requested group and 1.5% in the screening group (P = .5). Diarrhea was the most frequent symptom reported in 156 cases and of these 29 (19%) had hemorrhagic colitis (HC) and 7 children developed hemolytic uremic syndrome (HUS). No difference regarding severity of symptoms between the groups was found. Stx2 predominated in cases with HC (P < .0001) and HUS (P = .04). Median stx shedding duration was 20 days (1-256 days), and no difference in duration was seen between stx types (P = .106-1.00) and presence of eaeA (P = .72). CONCLUSIONS: Most STEC cases were found in the screening group with comparable prevalence and disease severity as in patients where analysis was requested. Furthermore, non-O157 serotypes caused severe disease when carrying stx2, and prolonged shedding of STEC may be a risk for transmission.

Insights to genetic characterization tools for epidemiological tracking of Francisella tularensis in Sweden.

Tularaemia, caused by the bacterium Francisella tularensis, is endemic in Sweden and is poorly understood. The aim of this study was to evaluate the effectiveness of three different genetic typing systems to link a genetic type to the source and place of tularemia infection in Sweden. Canonical single nucleotide polymorphisms (canSNPs), MLVA including five variable number of tandem repeat loci and PmeI-PFGE were tested on 127 F. tularensis positive specimens collected from Swedish case-patients. All three typing methods identified two major genetic groups with near-perfect agreement. Higher genetic resolution was obtained with canSNP and MLVA compared to PFGE; F. tularensis samples were first assigned into ten phylogroups based on canSNPs followed by 33 unique MLVA types. Phylogroups were geographically analysed to reveal complex phylogeographic patterns in Sweden. The extensive phylogenetic diversity found within individual counties posed a challenge to linking specific genetic types with specific geographic locations. Despite this, a single phylogroup (B.22), defined by a SNP marker specific to a lone Swedish sequenced strain, did link genetic type with a likely geographic place. This result suggests that SNP markers, highly specific to a particular reference genome, may be found most frequently among samples recovered from the same location where the reference genome originated. This insight compels us to consider whole-genome sequencing (WGS) as the appropriate tool for effectively linking specific genetic type to geography. Comparing the WGS of an unknown sample to WGS databases of archived Swedish strains maximizes the likelihood of revealing those rare geographically informative SNPs.

Rapid comparative genomic analysis for clinical microbiology: the Francisella tularensis paradigm.

It is critical to avoid delays in detecting strain manipulations, such as the addition/deletion of a gene or modification of genes for increased virulence or antibiotic resistance, using genome analysis during an epidemic outbreak or a bioterrorist attack. Our objective was to evaluate the efficiency of genome analysis in such an emergency context by using contigs produced by pyrosequencing without time-consuming finishing processes and comparing them to available genomes for the same species. For this purpose, we analyzed a clinical isolate of Francisella tularensis subspecies holarctica (strain URFT1), a potential biological weapon, and compared the data obtained with available genomic sequences of other strains. The technique provided 1,800,530 bp of assembled sequences, resulting in 480 contigs. We found by comparative analysis with other strains that all the gaps but one in the genome sequence were caused by repeats. No new genes were found, but a deletion was detected that included three putative genes and part of a fourth gene. The set of 35 candidate LVS virulence attenuation genes was identified, as well as a DNA gyrase mutation associated with quinolone resistance. Selection for variable sequences in URFT1 allowed the design of a strain-specific, highly effective typing system that was applied to 74 strains and six clinical specimens. The analysis presented herein may be completed within approximately 6 wk, a duration compatible with that required by an urgent context. In the bioterrorism context, it allows the rapid detection of strain manipulation, including intentionally added virulence genes and genes that support antibiotic resistance.

Outbreak of Salmonella Thompson infections linked to imported rucola lettuce.

On November 15, 2004, a cluster of three cases of Salmonella Thompson infection was registered by the Norwegian reference laboratory. In the following days further cases occurred, prompting a case-control study among the first 13 cases and 26 matched controls. By December 31, 21 cases had been reported, with the first onset on October 24. Consumption of rucola lettuce (Eruca sativa, also known as rocket salad or arugula) (OR 8,8 [1,2-infinity]) and mixed salad (OR 5,0 [1,0-infinity]) was associated with illness. On November 26, Swedish authorities notified the finding of Salmonella Thompson in rucola lettuce through the EU Rapid Alert System for Food and Feed. Later, several countries reported finding this and other Salmonella serovars and Campylobacter in rucola produced in Italy. In response to our alert through the international Enter-net surveillance network, Sweden and England also reported an increase of cases. Salmonella Thompson isolates from products and patients from several countries showed high similarity by pulsed-field gel electrophoresis, but some isolates showed significant differences. We think that the outbreak in Norway reflected a larger international outbreak caused by rucola imported from one Italian producer. Findings of other pathogens indicate a massive contamination, possibly caused by irrigation with nonpotable water. Rapid international information exchange is invaluable when investigating outbreaks caused by internationally marketed products.

Genetic characterization of the Guinea-Bissau family of Mycobacterium tuberculosis complex strains.

In a previous study of Mycobacterium tuberculosis complex isolates from Guinea-Bissau in West Africa, we identified a unique group of strains, designated here as the Guinea-Bissau family of strains, which, although genotypically closely related, phenotypically demonstrated a considerable heterogeneity. We conducted here a detailed genotypic analysis of a subset (n = 35) of these isolates. Based on the data obtained, and by comparison of known corresponding genes in mycobacteria outside the M. tuberculosis complex, we propose that the Guinea-Bissau strains belong to a unique branch of the M. tuberculosis complex tree in between classical M. tuberculosis and classical M. bovis. These observations are discussed in their significance in M. tuberculosis complex classification.

Prevalence of the new, SPI1-like, pathogenicity island ETT2 among Escherichia coli.

The new pathogenicity island ETT2 has been identified by PCR and gene probes among various intestinal serovars and pathovars of E. coli, in particular among EHEC/STEC. However, ETT2 was not detected among extra-intestinal and non-pathogenic E. coli strains or other enteric bacteria including various S. enterica serovars. A considerable molecular diversity of ETT2 among various E. coli serovars was found. The occurrence of ETT2 among E. coli is independent of the presence of other virulence properties, e.g. the pathogenicity islands LEE, LPA, or HPI.

The Salmonella enterica subspecies I specific centisome 7 genomic island encodes novel protein families present in bacteria living in close contact with eukaryotic cells.

We have determined the genetic structure of the Salmonella enterica centisome 7 genomic island (SCI) located at the aspV loci in S. enterica subspecies I strains. The 47-kb long genomic island encodes 37 putative proteins, including the previously described saf fimbrial operon and the sinR transcriptional regulator. Other open reading frames (designated sci A to Z) in the island encode putative proteins with homologies to virulence-associated proteins in a number of gram-negative bacteria such as Pseudomonas aeruginosa, Yersinia pestis and enterohemorrhagic Escherichia coli, bacteria that have the ability to interact with and manipulate eukaryotic cells. The Sci proteins have putative cytoplasmic, periplasmic and outer membrane localizations pointing to a role in extracellular processes such as secretion or organelle biosynthesis. The genes encoding Sci-like proteins are clustered in all sequenced bacterial genomes available in the databases and a core set can be defined by the presence of genes encoding proteins with similarity to the SciB, C, G, H, I, O proteins. The SCI genomic island DNA sequences are restricted to Salmonella strains belonging to S. enterica subspecies I and deletion of the entire island affects the ability of the organisms to enter eukaryotic cells.

Characterization of a pore-forming cytotoxin expressed by Salmonella enterica serovars typhi and paratyphi A.

Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the clyA gene that has been characterized so far only in Escherichia coli. Using DNA sequence analysis and PCR, we established that clyA is conserved in the human-specific typhoid Salmonella enterica serovars Typhi and Paratyphi A and that the entire clyA gene locus is absent in many other S. enterica serovars, including Typhimurium. The gene products, designated ClyA(STy) and ClyA(SPa), show >/=90% amino acid identity to E. coli cytolysin A, ClyA(EC), and they are immunogenically related. The Salmonella proteins showed a pore-forming activity and are hence functional homologues to ClyA(EC). The chromosomal clyA(STy) gene locus was expressed at detectable levels in the serovar Typhi strains S2369/96 and S1112/97. Furthermore, in the serovar Typhi vaccine strain Ty21a, expression of clyA(STy) reached phenotypic levels, as detected on blood agar plates. The hemolytic phenotype was abolished by the introduction of an in-frame deletion in the clyA(STy) chromosomal locus of Ty21a. Transcomplementation of the mutant with a cloned clyA(STy) gene restored the hemolytic phenotype. To our knowledge, Ty21a is the first reported phenotypically hemolytic Salmonella strain in which the genetic determinant has been identified.

A new approach to genome mapping and sequencing: slalom libraries.

We describe here an efficient strategy for simultaneous genome mapping and sequencing. The approach is based on physically oriented, overlapping restriction fragment libraries called slalom libraries. Slalom libraries combine features of general genomic, jumping and linking libraries. Slalom libraries can be adapted to different applications and two main types of slalom libraries are described in detail. This approach was used to map and sequence (with approximately 46% coverage) two human P1-derived artificial chromosome (PAC) clones, each of approximately 100 kb. This model experiment demonstrates the feasibility of the approach and shows that the efficiency (cost-effectiveness and speed) of existing mapping/sequencing methods could be improved at least 5-10-fold. Furthermore, since the efficiency of contig assembly in the slalom approach is virtually independent of length of sequence reads, even short sequences produced by rapid, high throughput sequencing techniques would suffice to complete a physical map and a sequence scan of a small genome.