Definitive Phage Type Research Articles

Comparative Genomic Analysis of Salmonella enterica Serovar Typhimurium Isolates from Passerines Reveals Two Lineages Circulating in Europe, New Zealand, and the United States.

Salmonella enterica serovar Typhimurium strains from passerines have caused wild bird deaths and human salmonellosis outbreaks in Europe, Oceania, and North America. Here, we performed comparative genomic analysis to explore the emergence, genetic relationship, and evolution of geographically dispersed passerine isolates. We found that passerine isolates from Europe and the United States clustered to form two lineages (EU and US passerine lineages), which were distinct from major S. Typhimurium lineages circulating in other diverse hosts (e.g., humans, cattle, pigs, chickens, and other avian hosts, such as pigeons and ducks). Further, passerine isolates from New Zealand clustered to form a sublineage (NZ passerine lineage) of the US passerine lineage. We inferred that the passerine isolates mutated at a rate of 3.2 × 10-7 substitutions/site/year, and the US, EU, and NZ passerine lineages emerged in approximately 1952, 1970, and 1996, respectively. Isolates from the three lineages presented genetic similarity, such as lack of antimicrobial resistance genes and accumulation of the same virulence pseudogenes. In addition, genetic diversity due to microevolution existed in the three passerine lineages. Specifically, pseudogenization in the type 1 fimbrial gene fimC (deletion of G at position 87) was detected only in the US and NZ passerine isolates, while single-base deletions in type 3 secretion system effector genes (i.e., gogB, sseJ, and sseK2) cooccurred solely in the EU passerine isolates. These findings provide insights into the evolution, host adaptation, and epidemiology of S. Typhimurium in passerines. IMPORTANCE Passerine-associated S. Typhimurium strains have been linked to human salmonellosis outbreaks in recent years. Here, we investigated the phylogenetic relationship of globally distributed passerine isolates and profiled their genomic similarity and diversity. Our study reveals two passerine-associated S. Typhimurium lineages circulating in Europe, Oceania, and North America. Isolates from the two lineages presented phylogenetic and genetic signatures that were distinct from those of isolates from other hosts. The findings shed light on the host adaptation of S. Typhimurium in passerines and are important for source attribution of S. Typhimurium strains to avian hosts. Further, we found that S. Typhimurium definitive phage type 160 (DT160) from passerines, which caused decades-long human salmonellosis outbreaks in New Zealand and Australia, formed a sublineage of the US passerine lineage, suggesting that DT160 might have originated from passerines outside Oceania. Our study demonstrates the importance of whole-genome sequencing and genomic analysis of historical microbial collections to modern epidemiologic surveillance.

Intra-macrophage expression of ArtAB toxin gene in Salmonella.

Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) definitive phage type 104 (DT104), S. Worthington, and S. bongori produce ArtAB toxin, which catalyses ADP-ribosylation of pertussis toxin-sensitive G protein. ArtAB gene (artAB) is encoded on a prophage in Salmonella, and prophage induction by SOS-inducing agents is associated with increases in ArtAB production in vitro. However, little is known about the expression of artAB in vivo. Here, we showed a significant increase in artAB transcription of DT104 within macrophage-like RAW264.7 cells. Intracellular expression of ArtAB was also observed by immunofluorescence staining. The induced expression of artAB in DT104 and S. bongori was enhanced by treatment of RAW264.7 cells with phorbol 12-myristate 13-acetate (PMA), which stimulates the production of reactive oxygen species (ROS); however, such induction was not observed in S. Worthington. Upregulation of oxyR, a major regulator of oxidative stress, and cI, a repressor of prophage induction, was observed in S. Worthington within RAW264.7 cells treated with PMA but not in the DT104 strain. Although the expression of oxyR was increased, artAB was upregulated in S. bongori, which lacks the cI gene in the incomplete artAB-encoded prophage. Taken together, oxidative stress plays a role in the production of artAB toxins in macrophages, and high expression levels of oxyR and cI are responsible for the low expression of artAB. Therefore, strain variation in the level of artAB expression within macrophages could be explained by differences in the oxidative stress response of bacteria and might be reflected in its virulence.

Characterisation of Phage Susceptibility Variation in Salmonellaenterica Serovar Typhimurium DT104 and DT104b.

The surge in mortality and morbidity rates caused by multidrug-resistant (MDR) bacteria prompted a renewal of interest in bacteriophages (phages) as clinical therapeutics and natural biocontrol agents. Nevertheless, bacteria and phages are continually under the pressure of the evolutionary phage–host arms race for survival, which is mediated by co-evolving resistance mechanisms. In Anderson phage typing scheme of Salmonella Typhimurium, the epidemiologically related definitive phage types, DT104 and DT104b, display significantly different phage susceptibility profiles. This study aimed to characterise phage resistance mechanisms and genomic differences that may be responsible for the divergent phage reaction patterns in S. Typhimurium DT104 and DT104b using whole genome sequencing (WGS). The analysis of intact prophages, restriction–modification systems (RMS), plasmids and clustered regularly interspaced short palindromic repeats (CRISPRs), as well as CRISPR-associated proteins, revealed no unique genetic determinants that might explain the variation in phage susceptibility among the two phage types. Moreover, analysis of genes coding for potential phage receptors revealed no differences among DT104 and DT104b strains. However, the findings propose the need for experimental assessment of phage-specific receptors on the bacterial cell surface and analysis of bacterial transcriptome using RNA sequencing which will explain the differences in bacterial susceptibility to phages. Using Anderson phage typing scheme of Salmonella Typhimurium for the study of bacteria-phage interaction will help improving our understanding of host–phage interactions which will ultimately lead to the development of phage-based technologies, enabling effective infection control.

Influence of SOS-inducing agents on the expression of ArtAB toxin gene in Salmonella enterica and Salmonella bongori.

Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) definitive phage type 104 (DT104), S. enterica subspecies enterica serovar Worthington (S. Worthington) and S. bongori produce ArtA and ArtB (ArtAB) toxin homologues, which catalyse ADP-ribosylation of pertussis toxin-sensitive G protein. ArtAB gene (artAB) is encoded on prophage in DT104 and its expression is induced by mitomycin C (MTC) and hydrogen peroxide (H2O2) that trigger the bacterial SOS response. Although the genetic regulatory mechanism associated with artAB expression is not characterized, it is thought to be associated with prophage induction, which occurs when the RecA-mediated SOS response is triggered. Here we show that subinhibitory concentration of quinolone antibiotics that are SOS-inducing agents, also induce ArtAB production in these Salmonella strains. Both MTC and fluoroquinolone antibiotics such as enrofloxacin-induced artA and recA transcription and artAB-encoding prophage (ArtAB-prophage) in DT104 and S. Worthington. However, in S. bongori, which harbours artAB genes on incomplete prophage, artA transcription was induced by MTC and enrofloxacin, but prophage induction was not observed. Taken together, these results suggest that SOS response followed by induction of artAB transcription is essential for ArtAB production. H2O2-mediated induction of ArtAB prophage and efficient production of ArtAB was observed in DT104 but not in S. Worthington and S. bongori. Therefore, induction of artAB expression with H2O2 is strain-specific, and the mode of action of H2O2 as an SOS-inducing agent might be different from those of MTC and quinolone antibiotics.

Genetic diversity of clinical Salmonella enterica serovar Typhimurium in a university hospital of south Tunisia, 2000–2013

Typhimurium is one of the main Salmonella serovar responsible for non-typhoidal gastro-enteritis in Tunisia. Here, we aimed to assess the genetic diversity of 88 clinical Salmonella Typhimurium strains recovered during 14 years from 2000 to 2013. Phage typing, CRISPR polymorphisms (CRISPOL), pulsed-field gel electrophoresis (PFGE), multi-locus variable-number tandem repeat analysis (MLVA) and Whole genome sequencing (WGS) were used to study the relatedness and spatio-temporal evolution of Salmonella Typhimurium populations (Typhimurium (n = 81), monophasic (n = 3) and nonmotile (n = 4) variants).Seven-locus MLST from whole genome assemblies showed that all isolates, except one, belonged to ST19. The isolates were divided into 10 definitive phage (DT) types, dominated by DT104-L (39.8%), DT41 (14.8%), DT116 (11.4%) and DT120 (5.7%). Fifty-seven MLVA patterns (DI, 0.978) were obtained compared to 11 different CRISPOL types and 15 PFGE types (DI,0.845). For cgMLST analysis, 20 profiles were found. A total of 3056 SNPs were identified from the whole genome of the 88 Salmonella Typhimurium isolates. These SNPs resolved these isolates into 86 SNP haplotypes. The phylogeny result allocated most Salmonella Typhimurium isolates into four distinct clades and seven subclades. Genetic diversity between the four clades ranged in the order of 249 to 720 nucleotide changes. The prevalent phage type DT104L formed a major clade on the phylogenetic tree. Pairwise SNP differences between the strains of this clade ranged between 0 and 59.SNP-based WGS typing seems to be the most valuable molecular markers for studying the evolutionary relationships of homogeneous serovar Typhimurium isolates.

Salmonella Typhimurium DT193 and DT99 are present in great and blue tits in Flanders, Belgium.

Endemic infections with the common avian pathogen Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium) may incur a significant cost on the host population. In this study, we determined the potential of endemic Salmonella infections to reduce the reproductive success of blue (Cyanistes caeruleus) and great (Parus major) tits by correlating eggshell infection with reproductive parameters. The fifth egg of each clutch was collected from nest boxes in 19 deciduous forest fragments. Out of the 101 sampled eggs, 7 Salmonella Typhimurium isolates were recovered. The low bacterial prevalence was reflected by a similarly low serological prevalence in the fledglings. In this study with a relatively small sample size, presence of Salmonella did not affect reproductive parameters (egg volume, clutch size, number of nestlings and number of fledglings), nor the health status of the fledglings. However, in order to clarify the impact on health and reproduction a larger number of samples have to be analyzed. Phage typing showed that the isolates belonged to the definitive phage types (DT) 193 and 99, and multi-locus variable number tandem repeat analysis (MLVA) demonstrated a high similarity among the tit isolates, but distinction to human isolates. These findings suggest the presence of passerine-adapted Salmonella strains in free-ranging tit populations with host pathogen co-existence.

Characterization of pertussis-like toxin from Salmonella spp. that catalyzes ADP-ribosylation of G proteins

Salmonella Typhimurium definitive phage type (DT) 104 produces a pertussis-like toxin (ArtAB-DT104), which catalyzes ADP-ribosylation of pertussis toxin sensitive G proteins. However, the prevalence of ArtAB and its toxicity have not been established. We report here that, in addition to DT104, S. Worthington, and S. bongori, produce ArtAB homologs, designated ArtAB-SW and ArtAB-Sb, respectively. We purified and characterized these ArtAB toxins, which comprise a 27-kDa A subunit (ArtA) and 13.8-kDa pentameric B subunits (ArtB). While the sequence of the A subunit, which is ADP-ribosyltransferase, is similar to the A subunit sequences of other ArtABs, the B subunit of ArtAB-Sb is divergent compared to the B subunit sequences of other ArtABs. Intraperitoneal injection of purified ArtABs was fatal in mice; the 50% lethal doses of ArtAB-DT104 and ArtAB-SW were lower than that of ArtAB-Sb, suggesting that ArtB plays an influential role in the toxicity of ArtABs. ArtABs catalyzed ADP-ribosylation of G proteins in RAW 264.7 murine macrophage-like cells, and increased intracellular cyclic AMP levels. ArtAB-DT104 and ArtAB-SW, but not ArtAB-Sb, stimulated insulin secretion in mice; however, unlike Ptx, ArtABs did not induce leukocytosis. This disparity in biological activity may be explained by differences in ADP-ribosylation of target G proteins.

Prevalence and characterization of Salmonella among humans in Ghana

BackgroundNon-typhoidal Salmonella (NTS) is a public health problem worldwide and particularly in Africa with high disease burden. This study characterized Salmonella isolates from humans in Ghana to determine serovar distribution, phage types, and antimicrobial resistance. Further, the clonal relatedness among isolates was determined.MethodsOne hundred and thirty-seven Salmonella isolates (111 clinical and 26 public toilet) were characterized using standard serotyping, phage typing, and antimicrobial susceptibility testing methods. The molecular epidemiology of common serovars (Salmonella Typhimurium and Salmonella Enteritidis) was established by pulsed field gel electrophoresis (PFGE).ResultsTwenty-two serovars were identified with S. Enteritidis, S. Typhimurium, and Salmonella Derby as the most dominant. One hundred and twelve isolates showed resistance to more than one antimicrobial. Fifty-eight (n = 58/112; 54.5%) strains were multi-resistant with low resistance to cephalosporins ceftazidime (8.0%), cefotaxime (4.5%), and cefoxitin (2.7%) with synergy to clavulanic acid indicating possible ESBLs. Isolates showed high resistance to trimethoprim (66.1%), tetracycline (61.6%), ampicillin (57.1%), sulfamethoxazole (46.4%), chloramphenicol (33.9%), and ciprofloxacin (25.0%). The most common resistance pattern of multi-resistant serovars was to ampicillin, chloramphenicol, sulphonamide, and trimethoprim. S. Enteritidis (18/43) strains reacted with typing phages but did not conform to any phage type with PT14B and PT4 as predominant definitive phage types. Six S. Typhimurium strains reacted but did not conform to any recognized phage type while seven were non-typable. The predominant definitive phage types were DT1 and DT22. PFGE patterns of human S. Enteritidis were closely related to patterns of poultry isolates obtained in a previous study in Ghana.ConclusionsCephalosporin resistance is uncommon among Salmonella from humans in Ghana. Poultry may be an important source of human salmonellosis. There is an urgent need for the implementation of routine surveillance of antimicrobial use and bacterial resistance among humans in Ghana.

The occurrence ofSalmonellaspp. in duck eggs on sale at retail or from catering in England

Human salmonellosis outbreaks in England and Northern Ireland due to Salmonella enterica serovar Typhimurium definitive phage type (DT) 8 have been identified as associated with the consumption of duck eggs since 2010. This study has shown that Salmonella spp. was detected in 1·4% of ducks egg samples providing baseline data for risk assessors, regulators and the food industry. This may be helpful in communicating risks associated with the consumption of this product as well as evaluating risk management options to control food safety including vaccination of ducks.

Genomic Analysis of Salmonella enterica Serovar Typhimurium from Wild Passerines in England and Wales.

Passerine salmonellosis, caused by certain definitive phage types (DTs) of Salmonella Typhimurium, has been documented as a cause of wild passerine mortality since the 1950s in many countries, often in the vicinity of garden bird feeding stations. To gain better insight into its epidemiology and host-pathogen interactions, we sequenced the genomes of a collection of 11 isolates from wild passerine salmonellosis in England and Wales. Phylogenetic analysis showed these passerine isolates to be closely related to each other and to form a clade that is distinct from other strains of S Typhimurium, which included a multidrug-resistant isolate from invasive nontyphoidal Salmonella disease that shares the same phage type as several of the passerine isolates. Closely related to wild passerine isolates and within the same clade were four S Typhimurium isolates from humans as well as isolates from horses, poultry, cattle, an unspecified wild bird, and a domestic cat and dog with similar DTs and/or multilocus sequence types. This suggests the potential for cross-species transmission, and the genome sequences provide a valuable resource to investigate passerine salmonellosis further.

Whole genome sequencing provides possible explanations for the difference in phage susceptibility among two Salmonella Typhimurium phage types (DT8 and DT30) associated with a single foodborne outbreak.

BackgroundPhage typing has been used for decades as a rapid, low cost approach for the epidemiological surveillance of Salmonella enterica subsp. enterica serovar Typhimurium. Although molecular methods are replacing phage typing the system is still in use and provides a valuable model for study of phage-host interaction. Phage typing depends on the pattern of bacterial resistance or sensitivity to a panel of specific bacteriophages. In the phage typing scheme, S. Typhimurium definitive phage types (DT) 8 and 30 differ greatly in their susceptibility to the 30 typing phages of S. Typhimurium; DT8 is susceptible to 11 phages whereas DT30 is resistant to all typing phages except one phage although both DT8 and DT30 were reported to be associated with a single foodborne salmonellosis outbreak in Ireland between 2009 and 2011. We wished to study the genomic correlates of the DT8 and DT30 difference in phage susceptibility using the whole genome sequence (WGS) of S. Typhimurium DT8 and DT30 representatives.ResultsComparative genome analysis revealed that both S. Typhimurium DT8 and DT30 are lysogenic for three prophages including two S. Typhimurium associated prophages (Gifsy-2 and ST64B) and one S. Enteritidis associated prophage (Enteritidis lysogenic phage S) which has not been detected previously in S. Typhimurium. Furthermore, DT8 and DT30 contain identical clustered regularly interspaced short palindromic repeats (CRISPRs). Interestingly, S. Typhimurium DT8 harbours an accessory genome represented by a virulence plasmid that is highly related to the pSLT plasmid of S. Typhimurium strain LT2 (phage typed as DT4) and codes a unique methyltransferase (MTase); M.EcoGIX related MTase. This plasmid is not detected in DT30. On the other hand, DT30 carries a unique genomic island similar to the integrative and conjugative element (ICE) of Enterotoxigenic Escherichia coli (ETEC) and encodes type IV secretion pathway system (T4SS) and several hypothetical proteins. This genomic island is not detected in DT8.ConclusionsWe suggest that differences in phage susceptibility between DT8 and DT30 may be related to acquisition of ICE in DT30 and loss of pSLT like plasmid that might be associated with DT30 resistance to almost all phages used in the typing scheme. Additional studies are required to determine the significance of the differences among DT8 and DT30 in relation to the difference in phage susceptibility. This study represents an initial step toward understanding the molecular basis of this host-phage relationship.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1687-6) contains supplementary material, which is available to authorized users.

Complete Proteome of a Quinolone-Resistant Salmonella Typhimurium Phage Type DT104B Clinical Strain

Salmonellosis is one of the most common and widely distributed foodborne diseases. The emergence of Salmonella strains that are resistant to a variety of antimicrobials is a serious global public health concern. Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) is one of these emerging epidemic multidrug resistant strains. Here we collate information from the diverse and comprehensive range of experiments on Salmonella proteomes that have been published. We then present a new study of the proteome of the quinolone-resistant Se20 strain (phage type DT104B), recovered after ciprofloxacin treatment and compared it to the proteome of reference strain SL1344. A total of 186 and 219 protein spots were recovered from Se20 and SL1344 protein extracts, respectively, after two-dimensional gel electrophoresis. The signatures of 94% of the protein spots were successfully identified through matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). Three antimicrobial resistance related proteins, whose genes were previously detected by polymerase chain reaction (PCR), were identified in the clinical strain. The presence of these proteins, dihydropteroate synthase type-2 (sul2 gene), aminoglycoside resistance protein A (strA gene) and aminoglycoside 6'-N-acetyltransferase type Ib-cr4 (aac(6')-Ib-cr4 gene), was confirmed in the DT104B clinical strain. The aac(6')-Ib-cr4 gene is responsible for plasmid-mediated aminoglycoside and quinolone resistance. This is a preliminary analysis of the proteome of these two S. Typhimurium strains and further work is being developed to better understand how antimicrobial resistance is developing in this pathogen.

Epidemiological Evidence That Garden Birds Are a Source of Human Salmonellosis in England and Wales

The importance of wild bird populations as a reservoir of zoonotic pathogens is well established. Salmonellosis is a frequently diagnosed infectious cause of mortality of garden birds in England and Wales, predominantly caused by Salmonella enterica subspecies enterica serovar Typhimurium definitive phage types 40, 56(v) and 160. In Britain, these phage types are considered highly host-adapted with a high degree of genetic similarity amongst isolates, and in some instances are clonal. Pulsed field gel electrophoresis, however, demonstrated minimal variation amongst matched DT40 and DT56(v) isolates derived from passerine and human incidents of salmonellosis across England in 2000–2007. Also, during the period 1993–2012, similar temporal and spatial trends of infection with these S. Typhimurium phage types occurred in both the British garden bird and human populations; 1.6% of all S. Typhimurium (0.2% of all Salmonella) isolates from humans in England and Wales over the period 2000–2010. These findings support the hypothesis that garden birds act as the primary reservoir of infection for these zoonotic bacteria. Most passerine salmonellosis outbreaks identified occurred at and around feeding stations, which are likely sites of public exposure to sick or dead garden birds and their faeces. We, therefore, advise the public to practise routine personal hygiene measures when feeding wild birds and especially when handling sick wild birds.

Identification of Virulence Properties in Salmonella Typhimurium DT104 Using Caenorhabditis elegans

Salmonella enterica serover Typhimurium definitive phage type DT104, resistant to multiple antibiotics, is one of the most widespread Salmonella species in human infection worldwide. Although several cohort studies indicate that DT104 carrying the multidrug resistance (MDR) locus on salmonella genomic island 1 is a possible hyper-virulent strain compared to DT104 strains without MDR, or other Salmonella enterica serotypes, existing experimental evidence regarding virulence properties associated with the MDR region is controversial. To address this question, we constructed an isogenic MDR deletion (∆MDR) mutant strain of DT104, SNS12, by allelic exchange and used Caenorhabditis elegans as a host model to assess differences in virulence between these two strains. SNS12 exhibited decreased virulence in C. elegans, and we observed increased colonization and proliferation of the intestine of C. elegans by DT104. The immune response against MDR-carrying DT104 appears to function through a non-canonical Unfolded Protein Response (UPR) pathway, namely prion-like-(QN-rich)-domain-bearing protein pathway (PQN), in a ced-1 dependent manner in C. elegans. Further, we also demonstrate that genes of the PQN pathway and antimicrobial peptide gene abf-2, are expressed at higher transcriptional levels in worms immediately following exposure to DT104, in comparison with worms exposed to SNS12. Altogether, our results suggest that the MDR region of Salmonella Typhimurium DT104 has a direct role in virulence against Caenorhabditis elegans.

Genomic Analysis ofSalmonella entericaSerovar Typhimurium Definitive Phage Type 104

Genomic Analysis of<i>Salmonella enterica</i>Serovar Typhimurium Definitive Phage Type 104

Tetracycline accelerates the temporally-regulated invasion response in specific isolates of multidrug-resistant Salmonella enterica serovar Typhimurium

BackgroundMultidrug-resistant (MDR) Salmonella isolates are associated with increased morbidity compared to antibiotic-sensitive strains and are an important health and safety concern in both humans and animals. Salmonella enterica serovar Typhimurium is a prevalent cause of foodborne disease, and a considerable number of S. Typhimurium isolates from humans and livestock are resistant to three or more antibiotics. The majority of these MDR S. Typhimurium isolates are resistant to tetracycline, a commonly used and clinically and agriculturally relevant antibiotic. Because exposure of drug-resistant bacteria to antibiotics can affect cellular processes associated with virulence, such as invasion, we investigated the effect tetracycline had on the invasiveness of tetracycline-resistant MDR S. Typhimurium isolates.ResultsThe isolates selected and tested were from two common definitive phage types of S. Typhimurium, DT104 and DT193, and were resistant to tetracycline and at least three other antibiotics. Although Salmonella invasiveness is temporally regulated and normally occurs during late-log growth phase, tetracycline exposure induced the full invasive phenotype in a cell culture assay during early-log growth in several DT193 isolates. No changes in invasiveness due to tetracycline exposure occurred in the DT104 isolates during early-log growth or in any of the isolates during late-log growth. Real-time PCR was used to test expression of the virulence genes hilA, prgH, and invF, and these genes were significantly up-regulated during early-log growth in most isolates due to tetracycline exposure; however, increased virulence gene expression did not always correspond with increased invasion, and therefore was not an accurate indicator of elevated invasiveness. This is the first report to assess DT193 isolates, as well as the early-log growth phase, in response to tetracycline exposure, and it was the combination of both parameters that was necessary to observe the induced invasion phenotype.ConclusionsIn this report, we demonstrate that the invasiveness of MDR S. Typhimurium can be modulated in the presence of tetracycline, and this effect is dependent on growth phase, antibiotic concentration, and strain background. Identifying the conditions necessary to establish an invasive phenotype is important to elucidate the underlying factors associated with increased virulence of MDR Salmonella.

Prevalence of Salmonella enterica serovar 4,[5],12:i:- in England and Wales, 2010

Difficulties in accurately identifying serovar 4,[5],12:i:- as monophasic variants of Salmonella enterica serovar Typhimurium mean there is confusion in the reporting of serovars Typhimurium and 4,[5],12:i:-. To gain insight into the prevalence and diversity of these monophasic variants in England and Wales, screening for fljB, hin and the serovar 4,[5],12:i:- DT193-associated genomic island was conducted on 609 S. enterica isolates designated as definitive phage type (DT) 193, and 142 isolates serologically-defined as monophasic variants of serovar Typhimurium but belonging to phage types other than DT193. All latter 142 isolates were subtyped by multilocus variable-number tandem repeat analysis (MLVA). MLVA was also applied to 70 DT193 serologically-defined monophasic variant isolates. Results indicate that serovar 4,[5],12:i:- accounted for 108 of 209 (52%) of DT193 isolates with available serological data and 99 of 142 (70%) monophasic variant isolates belonging to other phage types. Of 609 DT193 isolates, 463 (76%) lacked fljB and hin. Moreover, genetically-related isolates of DTs 120, 191, 191a, 195, phage types U311 and U323, and reacts but does not conform (RDNC) and untypable (UT) strains were also lacking either hin and/or fljB. Of note, the serovar 4,[5],12:i:- DT193-associated genomic island was identified in not only 458 of 463 (99%) monophasic DT193 isolates, but also 25 of 139 (18%) biphasic DT193 isolates and 56 of 76 (74%) monophasic variants of other phage types. Accurate monitoring of the emergence of serovar 4,[5],12:i:- isolates is important to ascertain the public health impact of these strains; since 2012 the Health Protection Agency’s Salmonella Reference Unit has therefore begun determining full antigenic structures of all presumptive O:4 isolates in addition to routinely performing phage typing for identification of variants of serovar Typhimurium.

Molecular typing of Salmonella enterica serotype Typhimurium and serotype 4,5,12:i:- isolates from cattle by multiple-locus variable-number tandem-repeats analysis

To evaluate the usefulness of multiple-locus variable-number tandem-repeats analysis (MLVA) as a tool for the epidemiological analysis of bovine Salmonellosis, Salmonella enterica serotype Typhimurium and serotype 4,5,12:i:- isolates (544 and 18, respectively) obtained from cattle in Hokkaido, Japan, between 1977 and 2009, were characterised by MLVA. MLVA identified 184 profiles versus 121 profiles identified by pulsed-field gel electrophoresis (PFGE). Cluster analysis of the MLVA profiles demonstrated 3 major clusters (A, B, and C) and 3 minor clusters (D, E, and F). Cluster A was associated with PFGE cluster I, which included isolates of definitive phage type 104 (DT104), while cluster C was associated with PFGE cluster VII, which has been disseminating among cattle since 2002. An isolate of serotype Typhimurium belonging to MLVA cluster F, in which 10 serotype 4,5,12:i:- isolates were included, was found to have an MLVA profile closely related to those of serotype 4,5,12:i:- isolates, suggesting that such a strain may be an ancestral candidate for serotype 4,5,12:i:-. Overall, the discriminatory power of MLVA was higher than that of PFGE, and MLVA differentiated between the isolates of the DT104 family, which appeared to be clonal by PFGE. However, this depended on PFGE clusters because PFGE allowed greater discrimination between isolates within PFGE cluster IV and VI than MLVA. The combination of PFGE and MLVA data allowed for improved subtype discrimination and enabled the identification of recently disseminated clones. Hence, MLVA can be used in combination with PFGE to effectively accelerate the molecular epidemiologic investigation of Salmonella.

Phenotypic and Molecular Characterization of Human Salmonella enterica serovar 4,[5],12:i:- Isolates in Slovakia

Forty-three epidemiologically unrelated emerging Salmonella enterica subsp. enterica serovar 4,[5],12:i:- strains isolated during the period 2009-2010 in Slovakia were characterized by phenotypic and genotypic methods. Thirty-one isolates (72.1%) expressed resistance to ampicillin, streptomycin, sulfizoxazole, and tetracycline [R-type ASSuT]. The majority of the strains belonged to both definitive phage types DT193 (30.2%) and U311 (27.9%). Other phage types identified were U302 (6.9%), DT18 (4.7%), and DT194 (2.3%). Twelve strains (27.9%) were not typeable. Pulsed-field gel electrophoresis analysis identified three closely related major banding profiles (X1, X1a, and X2), suggesting the similarity and close epidemiological relationship between S. enterica serovar 4,[5],12:i:- strains. In two isolates with R-type ASSuT, phage type NT and in one isolate with R-type ACROSSuSxTTTMPNA, phage type DT193 class 1 integrons were found encoding bla(PSE-1) and dfrA, aadA1, respectively.

Revival of an old problem: an increase inSalmonella entericaserovar Typhimurium definitive phage type 8 infections in 2010 in England and Northern Ireland linked to duck eggs

Salmonella enterica serovar Typhimurium definitive phage type (DT) 8 is uncommon in humans in the UK. In July 2010, the Health Protection Agency reported an excess isolation rate of pan-susceptible S. Typhimurium DT8 in England and Northern Ireland. By the end of October, this amounted to 81 laboratory-confirmed human cases for all regions of England and Northern Ireland in 2010, an increase of 26% and 41% on 2009 and 2008, respectively. Descriptive epidemiological investigation found a strong association with infection and consumption of duck eggs. Duck eggs contaminated with S. Typhimurium DT8 were collected from a patient's home and also at farms in the duck-egg supply chain. Although duck eggs form a small part of total UK eggs sales, there has been significant growth in sales in recent years. This is the first known outbreak of salmonellosis linked to duck eggs in the UK since 1949 and highlighted the impact of a changing food source and market on the re-emergence of salmonellosis linked to duck eggs. Control measures by the duck-egg industry should be improved along with a continued need to remind the public and commercial caterers of the potential high risks of contracting salmonellosis from duck eggs.