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Results from the Oxford nanopore minION sequencer “early-release” program enable identification of the insertion site of an antibiotic resistance island in Salmonella Typhi. Short-read, high-throughput sequencing technology cannot identify the chromosomal position of repetitive insertion sequences that typically flank horizontally acquired genes such as bacterial virulence genes and antibiotic resistance genes. The MinION nanopore sequencer can produce long sequencing reads on a device similar in size to a USB memory stick. Here we apply a MinION sequencer to resolve the structure and chromosomal insertion site of a composite antibiotic resistance island in Salmonella Typhi Haplotype 58. Nanopore sequencing data from a single 18-h run was used to create a scaffold for an assembly generated from short-read Illumina data. Our results demonstrate the potential of the MinION device in clinical laboratories to fully characterize the epidemic spread of bacterial pathogens.

Background Non-typhoid Salmonella Typhimurium ( S. Typhimurium) accounts for a high number of registered salmonellosis cases, and O-serotyping is one important tool for monitoring epidemiology and spread of the disease. Moreover, variations in glucosylated O-antigens are related to immunogenicity and spread in the host. However, classical autoagglutination tests combined with the analysis of specific genetic markers cannot always reliably register phase variable glucose modifications expressed on Salmonella O-antigens and additional tools to monitor O-antigen glucosylation phenotypes of S. Typhimurium would be desirable. Results We developed a test for the phase variable O-antigen glucosylation state of S. Typhimurium using the tailspike proteins (TSP) of Salmonella phages 9NA and P22. We used this ELISA like tailspike adsorption (ELITA) assay to analyze a library of 44 Salmonella strains. ELITA was successful in discriminating strains that carried glucose 1-6 linked to the galactose of O-polysaccharide backbone (serotype O1) from non-glucosylated strains. This was shown by O-antigen compositional analyses of the respective strains with mass spectrometry and capillary electrophoresis. The ELITA test worked rapidly in a microtiter plate format and was highly O-antigen specific. Moreover, TSP as probes could also detect glucosylated strains in flow cytometry and distinguish multiphasic cultures differing in their glucosylation state. Conclusions Tailspike proteins contain large binding sites with precisely defined specificities and are therefore promising tools to be included in serotyping procedures as rapid serotyping agents in addition to antibodies. In this study, 9NA and P22TSP as probes could specifically distinguish glucosylation phenotypes of Salmonella on microtiter plate assays and in flow cytometry. This opens the possibility for flow sorting of cell populations for subsequent genetic analyses or for monitoring phase variations during large scale O-antigen preparations necessary for vaccine production.

Systemic infections caused by Salmonella enterica are an ongoing public health problem especially in Sub-Saharan Africa. Essentially typhoid fever is associated with high mortality particularly because of the increasing prevalence of multidrug-resistant strains. Thus, a rapid blood-culture based bacterial species diagnosis including an immediate sub-differentiation of the various serovars is mandatory. At present, MALDI-TOF based intact cell mass spectrometry (ICMS) advances to a widely used routine identification tool for bacteria and fungi. In this study, we investigated the appropriateness of ICMS to identify pathogenic bacteria derived from Sub-Saharan Africa and tested the potential of this technology to discriminate S. enterica subsp. enterica serovar Typhi (S. Typhi) from other serovars. Among blood culture isolates obtained from a study population suffering from febrile illness in Ghana, no major misidentifications were observed for the species identification process, but serovars of Salmonella enterica could not be distinguished using the commercially available Biotyper database. However, a detailed analysis of the mass spectra revealed several serovar-specific biomarker ions, allowing the discrimination of S. Typhi from others. In conclusion, ICMS is able to identify isolates from a sub-Saharan context and may facilitate the rapid discrimination of the clinically and epidemiologically important serovar S. Typhi and other non-S. Typhi serovars in future implementations.

In January 2013, the National Reference Centre for Salmonella (NRC) detected a salmonellosis cluster in Saxony-Anhalt, Germany, caused by uncommon O4 non-agglutinating, monophasic Salmonella (S.) Typhimurium DT193. Circulating predominant monophasic S. Typhimurium DT193 clones typically display resistance phenotype ASSuT. We investigated common exposures to control the outbreak, and conducted microbiological investigations to assess the strains' phenotype. We conducted a case-control study defining cases as persons living or working in Saxony-Anhalt diagnosed with the O4 non-agglutinating strain between January and March 2013. We selected two controls contemporarily reported with norovirus infection, frequency-matched on residence and age group, per case. We interviewed regarding food consumption, especially pork and its place of purchase. We calculated odds ratios (ORs) with 95% confidence intervals (95% CI) using logistic regression. The NRC investigated human and food isolates by PCR, SDS-PAGE, MLST, PFGE, MLVA and susceptibility testing. Altogether, 68 O4 non-agglutinating human isolates were confirmed between January and April 2013. Of those, 61 were assigned to the outbreak (median age 57 years, 44% female); 83% cases ≥ 60 years were hospitalized. Eating raw minced pork from butcheries within 3 days was associated with disease (31 cases, 28 controls; OR adjusted for sex: 3.6; 95% CI: 1.0-13). Phage type DT193 and MLST ST34 were assigned, and isolates' lipopolysaccharide (LPS) matched control strains. Isolates linked to Saxony-Anhalt exhibited PFGE type 5. ASSuT- and ACSSuT phenotype proportions were 34 and 39% respectively; 54% were resistant to chloramphenicol. Three pork isolates matched the outbreak strain. Raw minced pork was the most likely infection vehicle in this first reported outbreak caused by O4 non-agglutinating, mostly chloramphenicol-resistant S. Typhimurium DT193. High hospitalization proportions demand awareness on the risk of consumption of raw pork among elderly. LPS analysis indicated O4 expression; therefore, testing with antisera from different lots is recommendable in unexpected agglutination reactions.

Salmonella enterica serovar Typhimurium definitive type 2 (DT2) is host restricted to Columba livia (rock or feral pigeon) but is also closely related to S . Typhimurium isolates that circulate in livestock and cause a zoonosis characterized by gastroenteritis in humans. DT2 isolates formed a distinct phylogenetic cluster within S . Typhimurium based on whole-genome-sequence polymorphisms. Comparative genome analysis of DT2 94-213 and S . Typhimurium SL1344, DT104, and D23580 identified few differences in gene content with the exception of variations within prophages. However, DT2 94-213 harbored 22 pseudogenes that were intact in other closely related S . Typhimurium strains. We report a novel in silico approach to identify single amino acid substitutions in proteins that have a high probability of a functional impact. One polymorphism identified using this method, a single-residue deletion in the Tar protein, abrogated chemotaxis to aspartate in vitro . DT2 94-213 also exhibited an altered transcriptional profile in response to culture at 42°C compared to that of SL1344. Such differentially regulated genes included a number involved in flagellum biosynthesis and motility. IMPORTANCE Whereas Salmonella enterica serovar Typhimurium can infect a wide range of animal species, some variants within this serovar exhibit a more limited host range and altered disease potential. Phylogenetic analysis based on whole-genome sequences can identify lineages associated with specific virulence traits, including host adaptation. This study represents one of the first to link pathogen-specific genetic signatures, including coding capacity, genome degradation, and transcriptional responses to host adaptation within a Salmonella serovar. We performed comparative genome analysis of reference and pigeon-adapted definitive type 2 (DT2) S . Typhimurium isolates alongside phenotypic and transcriptome analyses, to identify genetic signatures linked to host adaptation within the DT2 lineage. Whereas Salmonella enterica serovar Typhimurium can infect a wide range of animal species, some variants within this serovar exhibit a more limited host range and altered disease potential. Phylogenetic analysis based on whole-genome sequences can identify lineages associated with specific virulence traits, including host adaptation. This study represents one of the first to link pathogen-specific genetic signatures, including coding capacity, genome degradation, and transcriptional responses to host adaptation within a Salmonella serovar. We performed comparative genome analysis of reference and pigeon-adapted definitive type 2 (DT2) S . Typhimurium isolates alongside phenotypic and transcriptome analyses, to identify genetic signatures linked to host adaptation within the DT2 lineage.

This study determined the passage time and phage propagation time of salmonella specific phages, Felix O1 and S16, in 10 bearded dragons, based on re‐isolation from cloacal swabs and faecal samples following oral administration, as a possible tool for reducing salmonella shedding. In Study 1, Felix O1 was administered orally for 12 consecutive days. Over 60 days, swabs were taken from the oral cavity and cloaca and qualitative Salmonella detection as well as salmonella quantification from faecal samples were performed. In Study 2, a phage cocktail (Felix O1 and S16) was administered to half of the tested animals. Salmonella (S.) Eastbourne was also given orally to all animals. Oral and cloacal swabs were tested as in Study 1, and faecal samples were collected for phage quantification. Various Salmonella serovars were detectable at the beginning of the study. The numbers of serovars detected declined over the course of the study. S. Kisarawe was most commonly detected. Salmonella titres ranged from 102 to 107 cfu/g faeces. The phages (Felix O1 and S16) were detectable for up to 20 days after the last administration. The initial phage titres ranged from 103 to 107 pfu/ml. The study shows that the phages were able to replicate in the intestine, and were shed for a prolonged period and therefore could contribute to a reduction of Salmonella shedding. Bearded dragons are common exotic pets which shed Salmonella spp. regularly. This study was performed to gain knowledge how Salmonella specific phages affect the shedding and colonization off this zoonotic bacteria.

Host-specific serovars of Salmonella enterica often have large-scale chromosomal rearrangements that occur by recombination between rrn operons. Two hypotheses have been proposed to explain these rearrangements: (i) replichore imbalance from horizontal gene transfer drives the rearrangements to restore balance, or (ii) the rearrangements are a consequence of the host-specific lifestyle. Although recent evidence has refuted the replichore balance hypothesis, there has been no direct evidence for the lifestyle hypothesis. To test this hypothesis, we determined the rrn arrangement type for 20 Salmonella enterica serovar Typhi strains obtained from human carriers at periodic intervals over multiple years. These strains were also phage typed and analyzed for rearrangements that occurred over long-term storage versus routine culturing. Strains isolated from the same carrier at different time points often exhibited different arrangement types. Furthermore, colonies isolated directly from the Dorset egg slants used to store the strains also had different arrangement types. In contrast, colonies that were repeatedly cultured always had the same arrangement type. Estimated replichore balance of isolated strains did not improve over time, and some of the rearrangements resulted in decreased replicore balance. Our results support the hypothesis that the restricted lifestyle of host-specific Salmonella is responsible for the frequent chromosomal rearrangements in these serovars. IMPORTANCE Although it was previously thought that bacterial chromosomes were stable, comparative genomics has demonstrated that bacterial chromosomes are dynamic, undergoing rearrangements that change the order and expression of genes. While most Salmonella strains have a conserved chromosomal arrangement type, rearrangements are very common in host-specific Salmonella strains. This study suggests that chromosome rearrangements in the host-specific Salmonella enterica serovar Typhi, the causal agent of typhoid fever, occur within the human host over time. The results also indicate that rearrangements can occur during long-term maintenance on laboratory medium. Although these genetic changes do not limit survival under slow-growth conditions, they may limit the survival of Salmonella Typhi in other environments, as predicted for the role of pseudogenes and genome reduction in niche-restricted bacteria. Although it was previously thought that bacterial chromosomes were stable, comparative genomics has demonstrated that bacterial chromosomes are dynamic, undergoing rearrangements that change the order and expression of genes. While most Salmonella strains have a conserved chromosomal arrangement type, rearrangements are very common in host-specific Salmonella strains. This study suggests that chromosome rearrangements in the host-specific Salmonella enterica serovar Typhi, the causal agent of typhoid fever, occur within the human host over time. The results also indicate that rearrangements can occur during long-term maintenance on laboratory medium. Although these genetic changes do not limit survival under slow-growth conditions, they may limit the survival of Salmonella Typhi in other environments, as predicted for the role of pseudogenes and genome reduction in niche-restricted bacteria.

Background The Enterobacteriaceae comprise a large number of clinically relevant species with several individual subspecies. Overlapping virulence-associated gene pools and the high overall genome plasticity often interferes with correct enterobacterial strain typing and risk assessment. Array technology offers a fast, reproducible and standardisable means for bacterial typing and thus provides many advantages for bacterial diagnostics, risk assessment and surveillance. The development of highly discriminative broad-range microbial diagnostic microarrays remains a challenge, because of marked genome plasticity of many bacterial pathogens. Results We developed a DNA microarray for strain typing and detection of major antimicrobial resistance genes of clinically relevant enterobacteria. For this purpose, we applied a global genome-wide probe selection strategy on 32 available complete enterobacterial genomes combined with a regression model for pathogen classification. The discriminative power of the probe set was further tested in silico on 15 additional complete enterobacterial genome sequences. DNA microarrays based on the selected probes were used to type 92 clinical enterobacterial isolates. Phenotypic tests confirmed the array-based typing results and corroborate that the selected probes allowed correct typing and prediction of major antibiotic resistances of clinically relevant Enterobacteriaceae , including the subspecies level, e.g. the reliable distinction of different E. coli pathotypes. Conclusions Our results demonstrate that the global probe selection approach based on longest common factor statistics as well as the design of a DNA microarray with a restricted set of discriminative probes enables robust discrimination of different enterobacterial variants and represents a proof of concept that can be adopted for diagnostics of a wide range of microbial pathogens. Our approach circumvents misclassifications arising from the application of virulence markers, which are highly affected by horizontal gene transfer. Moreover, a broad range of pathogens have been covered by an efficient probe set size enabling the design of high-throughput diagnostics.

Multicellular behavior in Salmonella Typhimurium ATCC14028 called the rdar morphotype is characterized by the expression of the extracellular matrix components cellulose and curli fimbriae. Over 90% of S. Typhimurium and S. Enteritidis strains from human disease, food and animals expressed the rdar morphotype at 28 °C. Regulation of the rdar morphotype occurred via the response regulator ompR which activated transcription of csgD required for production of cellulose and curli fimbriae. Serovar-specific regulation of csgD required rpoS in S. Typhimurium, but was partially independent of rpoS in S. Enteritidis. Rarely, strain-specific temperature-deregulated expression of the rdar morphotype was observed. The host-restricted serovars S. Typhimurium var. Copenhagen phage type DT2 and DT99, Salmonella Typhi and Salmonella Choleraesuis did not express the rdar morphotype, while in Salmonella Gallinarum cellulose expression at 37 °C was seen in some strains. Therefore, the expression pattern of the rdar morphotype is serovar specific and correlates with a disease phenotype breaching the intestinal epithelial cell lining.

Salmonella spp. are recognized as some of the most common causes of enteritis worldwide. This study aimed to identify clinically isolated S. Typhimurium in western Kenya and to assess antimicrobial resistance profiles and strain inter-relatedness. The study was performed in rural Maseno, Nyanza province in Kenya, between February 2004 and June 2005. Sixty-three patients with diarrhoea and fever were recruited. S. Typhimurium isolates were confirmed using serotyping, biochemical testing, and 16S rRNA sequencing. Susceptibility to 20 antimicrobials was determined and specific resistance genes were identified by polymerase chain reaction (PCR). Strain diversity was further analyzed using pulsed-field gel electrophoresis (PFGE), fluorescence amplified fragment length polymorphism (fAFLP), and multi-locus-variable-number-tandem regions (MLVNTR). Twenty S. Typhimurium strains were isolated in the course of the study and their identity was confirmed by 16S rRNA gene sequencing. All 20 S. Typhimurium strains were resistant to ampicillin, streptomycin and sulfamethoxazole; ciprofloxacin resistance and phage DT104 were not detected. PFGE, plasmid profiling, and analysis of selected VNTR loci revealed further heterogeneity among the strains in the study. S. Typhimurium was commonly isolated from patients with diarrhoea and fever in Maseno. Considerable phenotypic and genotypic diversity was observed among isolates, suggesting that strains belonging to multiple lineages are responsible for disease in the study region. Multiple resistance was common and mediated by a variety of resistance genes but not by phage DT104.