Infection of chickens with antimicrobial-resistant Salmonella enterica Typhimurium DT193 and monophasic Salmonella Typhimurium-like variants: an emerging risk to the poultry industry?

Antimicrobial-resistant Salmonella enterica poses a particular risk to public health, and in particular isolates belonging to clonal lineages such as Salmonella Typhimurium DT104 cause epidemics across species including poultry. In recent years, antimicrobial-resistant S. Typhimurium DT193 and specifically the monophasic S. Typhimurium-like variants of this phage type, serotypes 4,12:i:- and 4,5,12:i:-, have become an increasing risk to public health in Europe and the USA and now account for nearly one-half of human S. Typhimurium infections in the UK. Unlike S. Typhimurium that possesses two forms of flagella which can vary between phase 1 and phase 2 during infection, monophasic variants possess only phase 1 flagella. These monophasic antimicrobial-resistant variants have become a major problem in pig production but human cases have also been associated with poultry consumption and have been found in UK flocks through surveillance schemes since 2010. In this study we determined the ability of antimicrobial-resistant DT193 serotype 4,12:i:- and 4,5,12:i:- isolates from pigs to infect chickens. All isolates were found to colonize the caeca and liver. All but one isolate of serotype 4,5,12:i:- also infected the spleen. Levels of infection and pathology were comparable with those found with the virulent S. Typhimurium isolate 4/74. These findings indicate that both S. Typhimurium DT193 and monophasic variants of this phage type usually associated with pigs are capable of colonizing the chicken. This shows that both S. Typhimurium DT193 and monophasic variants represent a significant and potential emerging threat to poultry production from “spill-over” of these isolates from the pig industry or other sources.     

Characterization of Salmonella enterica serovar Typhimurium and monophasic Salmonella serovar 1,4,[5],12:i:- isolates in Thailand

Duplex PCR was developed to screen Salmonella enterica serovar Typhimurium phage type DT104 and related strains in Thailand because a phage typing laboratory of serovar Typhimurium is not available. Of 46 isolates of serovar Typhimurium and 32 isolates of S. enterica serovar 1,4,[5],12:i:-, 15 (33%) and 30 (94%) were duplex PCR positive, respectively. All isolates were submitted for phage typing to analyze the specificity of the PCR assay. Among serovar Typhimurium isolates that yielded positive duplex PCRs, only seven isolates were phage types DT104 or U302, and eight isolates were undefined types, whereas the negative PCR isolates were either other phage types, including DT7, DT12, DT66, DT79, DT166, DT170, DT193, and DT208 or an undefined type. The serovar Typhimurium and serovar 1,4,[5],12:i:- isolates that were duplex PCR positive were further subtyped by using XbaI PFGE to reveal their genetic relatedness. All serovar Typhimurium phage type DT104 strains had indistinguishable chromosomal patterns. The isolates of phage type U302 and most of the serovar 1,4,[5],12:i:- isolates that were duplex PCR positive yielded similar pulsed-field gel electrophoresis patterns. The patterns of PCR-negative isolates distinctly differed from the patterns of PCR-positive isolates. A total of 26% of all isolates had a dominant R-type ACSSuTG that was not found in the isolates of phage type DT104.     

Evidence for a Second Genomic Island Conferring Multidrug Resistance in a Clonal Group of Strains of Salmonella enterica Serovar Typhimurium and its Monophasic Variant Circulating in Italy,Denmark, and the United Kingdom

During the 2000s, a new clonal group with resistances to ampicillin, streptomycin, sulfonamides, and tetracycline (ASSuT) emerged in Italy among strains of Salmonella enterica serovar Typhimurium and its monophasic variant, Salmonella enterica subspecies enterica serovar 4,[5],12:i:−. The PulseNet Europe database allowed us to identify ASSuT strains of both S. Typhimurium and its monophasic variant, isolated in Denmark and the United Kingdom, with the same or very closely related pulsed-field gel electrophoresis (PFGE) patterns as the Italian strains, suggesting that the ASSuT clonal group is circulating in different European countries. With the aim of analyzing the molecular basis of antibiotic resistance, resistance genes were identified and their localization was investigated in 66 ASSuT strains and, as controls, in 11 strains with different resistance patterns and PFGE profiles, belonging both to S. Typhimurium and to its monophasic variant, isolated from humans in Italy, Denmark, and the United Kingdom. All the ASSuT strains were positive for the following resistance genes: bla_TEM-1, strA-strB, sul2, and tet(B). A localization experiment demonstrated that the ASSuT resistance genes are chromosomally located. This study confirms that a multidrug-resistant clonal group, ASSuT, of S. Typhimurium and its monophasic variant has emerged and is circulating in Italy, Denmark, and the United Kingdom. Moreover, the results of this work demonstrate that the multidrug resistance in this clonal group of Salmonella strains is conferred by a new genomic island.Salmonella enterica serovar Typhimurium is one of the most important food-borne zoonotic pathogens across the world, and the increasing occurrence and spread of multidrug-resistant (MDR) strains is a matter of concern. MDR S. Typhimurium definitive phage type 104 (DT104), resistant to ampicillin, chloramphenicol, streptomycin and spectinomycin, sulfonamides, and tetracyclines (resistance type [R type] ACSSpSuT), is a clear example of a clone that emerged in one country (the United Kingdom in the 1980s) and subsequently became widely distributed in at least four continents (41).Recently strains of S. Typhimurium and its monophasic variant, Salmonella enterica serovar 4,[5],12:i:−, characterized by the four-drug resistance (tetraresistance) pattern ASSuT (with or without additional resistances but lacking resistance to chloramphenicol), has emerged in Italy (5). These strains accounted for more than 30% of isolates from cases of human infection during the last 5 years and were also observed among isolates from different farm animal species (20). Pulsed-field gel electrophoresis (PFGE) analysis showed that these tetraresistant strains belonged to a unique clonal group, different from the DT104 ACSSpSuT clone (13). Using the PulseNet Europe database (27), it has been possible to verify that ASSuT strains of S. Typhimurium and its monophasic variant isolated in Denmark and the United Kingdom exhibited PFGE patterns that were the same as or very closely related to those of the Italian strains, suggesting that an ASSuT clonal group was circulating in different European countries.In Denmark, strains with the ASSuT R type accounted for 4.4% of S. Typhimurium strains from humans over the period from 1997 to 2002 (16); during the period from 2003 to 2008, the frequency of ASSuT strains from humans increased, varying from 10 to 22%. Moreover, a study of strains from pigs highlighted that from 2002 to 2006, the main phage type became DT120, and 50% of strains of this phage type belonged to R type ASSuT. Over the same period, the frequency of isolation of DT104 strains decreased (15).In England and Wales, the ASSuT resistance pattern, with additional resistance to furazolidone (Fu), was first detected in 1964 in S. Typhimurium DT29. Such strains, which possessed resistance determinants on both conjugative and nonconjugative plasmids, caused substantive outbreaks in both cattle and humans until 1971 (41). During the 1980s there was an increase, from 1% in 1981 to 8% in 1990, of strains with the ASSuT R type, mainly belonging to DT193 (39). Since 1996, DT193 has been second only to DT104 in cases of human infection; of these strains, 56% were multiresistant, with strains of R type ASSuT predominating. Previous studies have shown that strains of DT193 of R type ASSuT have often been associated with pigs, and outbreaks associated with pigs have been reported (40). In addition, in 2006, strains of DT120 of R type ASSuT, with a unique PFGE profile (STYMXB.0083, according to PulseNet nomenclature), caused an outbreak in northeast England (3). From January 2004 to December 2006, there were 489 strains of R type ASSuT, with or without additional resistances, from a total of 4,426 isolates from human infections (11%) reported to the Health Protection Agency (HPA Salmonella data set 2009; www.HPA.org.uk).In DT104 strains of R type ACSSuSpT, the resistance genes for ampicillin (antibiogram designation A) (bla_PSE-1), chloramphenicol (antibiogram designation C) (floR), streptomycin-spectinomycin (antibiogram designation SSp) (aadA2), sulfonamides (antibiogram designation Su) (sul1), and tetracyclines (antibiogram designation T) [tet(G)] are chromosomally integrated within the 43-kb Salmonella genomic island 1 (SGI-1) (4). In contrast, the genes responsible for the tetraresistance in the ASSuT clonal group have not been identified so far.The aim of this study was to analyze the molecular basis and location of ASSuT tetraresistance in S. Typhimurium and its monophasic variant strains from cases of human infection in Italy, Denmark, and the United Kingdom over the period from 2003 to 2006.     

Redundancy in the requirement for the glycolytic enzymes phosphofructokinase (Pfk) 1 and 2 in the in vivo fitness of Salmonella enterica serovar Typhimurium

To assess the role of the glycolytic enzyme phosphofructokinase (Pfk) in the in vivo fitness of the pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) we have generated single and double gene deletion mutants of the two known isoforms of this enzyme, pfkA and pfkB. In a mouse model of typhoid fever, bacterial counts in the spleen and liver were similar between wild type and single pfkA and pfkB mutant-infected mice. However, a double pfkAB mutant was significantly attenuated for growth in vivo. This defect was complemented by provision of either pfkA or pfkB on pBR322. Together these data show that Pfk activity is required for the full in vivo fitness of S. Typhimurium with functional redundancy between pfkA and pfkB. The level of attenuation of the pfkAB double mutant was not sufficient for its consideration as a live attenuated vaccine strain.     

Prevalence and characterization of multidrug-resistant (type ACSSuT) Salmonella enterica serovar Typhimurium strains in isolates from four gosling farms and a hatchery farm

Salmonella enterica serovar Typhimurium strains of phage types DT104 and U302 are often resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (the ACSSuT resistance type) and are major zoonotic pathogens. Increased consumption of goose meat may enhance the risk of transferring S. enterica serovar Typhimurium and other enteric pathogens from geese to human due to the consumption of meats from infected geese or improper preparation of meats. Therefore, we characterized S. enterica serovar Typhimurium strains isolated from four goose farms (farms A, B, C, and D) and one hatchery farm (farm E) to determine the epidemic and genetic differences among them. Antibiotic susceptibility tests and multiplex PCR confirmed that 77.6% (52/67) of strains were ACSSuT strains isolated from farms A, C, and E. Antibiotic-susceptible strains were isolated mostly from farm B, and no strain was observed in farm D. All ACSSuT strains harbored a 94.7-kb virulence plasmid and contained one 1.1-kb conserved segment identical to that of Salmonella genomic island 1. Four genotypes were determined among these S. enterica serovar Typhimurium isolates by pulsed-field gel electrophoresis analysis of XbaI-digested DNA fragments. Most isolates (85.29%; 29/34) of major genotype Ib were ACSSuT strains isolated mainly from goslings of farm C and egg membranes of farm E, a hatchery farm, suggesting that S. enterica serovar Typhimurium strains in isolates from goslings might originate from its hatchery, from the egg membranes to the gosling fluff after hatching. Multiple phage types, types 8, 12, U283, DT104, and U302, were identified. In conclusion, geese were a reservoir of diverse multidrug-resistant (type ACSSuT) S. enterica serovar Typhimurium strains, and each farm was colonized with genetically closely related S. enterica serovar Typhimurium strains.     

Genetic Types,Gene Repertoire,and Evolution of Isolates of the Salmonella enterica Serovar 4,5,12:i:? Spanish Clone Assigned to Different Phage Types

Salmonella enterica subsp. enterica 4,[5],12:i:− is one of the most prevalent serovars associated with human infections worldwide. Two multidrug-resistant clones, designated Spanish and European clones, are recognized as having importance for public health and are subject to control measures in the European Union. In this study, 23 clinical isolates belonging to the Spanish clone were characterized by multilocus sequence typing, multiple-locus variable number tandem repeat analysis (MLVA), PCR amplification and sequencing, and a DNA microarray targeting 263 genes, in order to provide new insights into their origins and further evolution. The derived data were compared with information available from other studies for S. 4,[5],12:i:− isolates of both the Spanish and the European clones, to identify differential molecular markers which could be potentially used as surveillance tools in the control of dissemination of this serovar. The isolates analyzed were assigned to sequence type 19 and to 17 MLVA patterns, with 3-13-16-NA-311 being the most prevalent. Highly similar virulence, metabolic, and prophage-associated gene profiles were identified, but DNA mobility markers distinguished five genotypes. Two types of deletions, caused by insertion of IS26, presumably donated by pUO-STmR/RV1-like plasmids typically found in the Spanish clone, affected the fljAB operon and surrounding DNA. The Spanish and European clones differ in sequence type, MLVA patterns, gene repertoire, and fljAB deletion type. The observed variability supports an independent evolution of the two successful monophasic clones from different Salmonella enterica serovar Typhimurium ancestors and can be taken into consideration for epidemiological surveillance.     

The Salmonella enterica serovar Typhimurium QseB response regulator negatively regulates bacterial motility and swine colonization in the absence of the QseC sensor kinase

Salmonella enterica serovar Typhimurium (S. Typhimurium) responds to the catecholamine, norepinephrine by increasing bacterial growth and enhancing motility. In this study, iron with or without the siderophore, ferrioxamine E also enhanced bacterial motility. Iron-enhanced motility was growth-rate dependent, while norepinephrine-enhanced motility was growth-rate independent. The outer membrane catecholate receptors, IroN, FepA and CirA (required for norepinephrine-enhanced growth) were not required for norepinephrine-enhanced motility, nor was ExbD of the energy-transducing TonB-ExbB-ExbD ferri-siderophore uptake system. Examination of the QseBC two-component system revealed that qseB and qseBC mutants have motility phenotypes similar to wild-type S. Typhimurium, while motility of the qseC mutant was significantly decreased (P < 0.01). Each mutant of the QseBC system, as well as mutants of qseE and pmrA, responded to norepinephrine with increased motility, suggesting that other genes are involved in norepinephrine-enhanced motility of S. Typhimurium. In the swine host, fecal shedding of the qseBC mutant was similar to wild-type S. Typhimurium, whereas fecal shedding of the qseC mutant was significantly decreased (P < 0.01). Our data indicate that, in a qseC mutant, the QseB response regulator decreases motility and swine colonization; inactivation of the qseBC operon restores these bacterial phenotypes, classifying QseB as a negative regulator of bacterial motility and swine colonization.     

Fluorescence in situ hybridization (FISH) for rapid identification of Salmonella spp. from agar and blood culture broth—An option for the tropics?

Background

Salmonella enterica is an important cause of diarrhea with the potential to cause systemic infection including sepsis, particularly in the tropics. Sepsis in particular requires quick and reliable identification to allow a rapid optimization of antibiotic therapy. We describe the establishment and evaluation of fluorescence in situ hybridization (FISH) as a rapid and easy-to-perform molecular identification procedure from agar and blood culture broths.

Methods

Two newly developed FISH probes with specificity for Salmonella spp. were evaluated with 10 reference strains, 448 clinical isolates of Gram-negative bacteria from Germany and Ghana including 316 Salmonella spp. strains, and 39 environmental Salmonella spp. isolates from rivers and streams in Ghana. One FISH probe was further tested with 207 pre-incubated blood culture broths from Germany with Gram-negative rod-shaped bacteria in Gram stain.

Results

Evaluation of the newly designed FISH probes demonstrated sensitivity of 99.2% and specificity of 98.4% for clinical isolates, sensitivity of 97.4% for environmental Salmonella spp. isolates, and sensitivity of 100% and specificity of 99.5% for blood culture materials.

Conclusions

FISH proved to be highly reliable for a rapid identification of Salmonella spp. directly from pre-incubated blood culture broths as well as after growth on agar. The inexpensive and easy-to-perform procedure is particularly suitable for resource-limited areas where more sophisticated procedures are not available.     

P087, a lactococcal phage with a morphogenesis module similar to an Enterococcus faecalis prophage

The virulent lactococcal phage P087 was isolated from a dairy environment in 1978. This phage was then recognized as the reference member for one of the ten phage groups currently known to infect Lactococcus lactis strains. The double-stranded DNA genome of this Siphoviridae phage is composed of 60,074 bp and is circularly permuted. Five tRNA and 88 orfs were found within an uncommon genome architecture. Eleven structural proteins were also identified through SDS-PAGE and LC-MS/MS analyses. Of note, 11 translated orfs from the structural module of phage P087 have identities to gene products found in a prophage located in the genome of Enterococcus faecalis V583. The alignment of both genomic sequences suggests that DNA exchanges could occur between these two phages which are infecting low G+C bacteria found in similar ecological niches.     

Genomic and phenotypic comparison of two Salmonella Typhimurium strains responsible for consecutive salmonellosis outbreaks in New Zealand

Salmonella enterica serovar Typhimurium DT160 was the predominant cause of notified human salmonellosis cases in New Zealand from 2000 to 2010, before it was superseded by another S. Typhimurium strain, DT56 variant (DT56v). Whole genome sequencing and phenotypic testing were used to compare 109 DT160 isolates with eight DT56v isolates from New Zealand animal and human sources. Phylogenetic analysis provided evidence that DT160 and DT56v strains were distantly related with an estimated date of common ancestor between 1769 and 1821. The strains replicated at different rates but had similar antimicrobial susceptibility profiles. Both strains were resistant to the phage expressed from the chromosome of the other strain, which may have contributed to the emergence of DT56v. DT160 contained the pSLT virulence plasmid, and the sseJ and sseK2 genes that may have contributed to the higher reported prevalence compared to DT56v. A linear pBSSB1-family plasmid was also found in one of the DT56v isolates, but there was no evidence that this plasmid affected bacterial replication or antimicrobial susceptibility. One of the DT56v isolates was also sequenced using long-read technology and found to contain an uncommon chromosome arrangement for a Typhimurium isolate. This study demonstrates how comparative genomics and phenotypic testing can help identify strain-specific elements and factors that may have influenced the emergence and supersession of bacterial strains of public health importance.     

Phenotype MicroArray™ in the metabolic characterisation of Salmonella serotypes Agona, Enteritidis, Give, Hvittingfoss, Infantis, Newport and Typhimurium

The Phenotype MicroArray™ (PM) technology was used to study the metabolic characteristics of 29 Salmonella strains belonging to seven serotypes of S. enterica spp. enterica. Strains of serotypes Typhimurium (six strains among definite phage types DTs 1, 40 and 104) and Agona (two strains) were tested for 949 substrates, Enteritidis (six strains of phage type PT1), Give, Hvittingfoss, Infantis and Newport strains (two of each) were tested for 190 substrates and seven other Agona strains for 95 substrates. The strains represented 18 genotypes in pulsed-field gel electrophoresis (PFGE). Among 949 substrates, 18 were identified that could be used to differentiate between the strains of those seven serotypes or within a single serotype. Unique metabolic differences between the Finnish endemic Typhimurium DT1 and Agona strains were detected, for example, in the metabolism of d-tagatose, d-galactonic acid γ-lactone and l-proline as a carbon source. Thus, the PM technique is a useful tool for identifying potential differential markers on a metabolic basis that could be used for epidemiological surveillance.     

Different sugar residues of the lipopolysaccharide outer core are required for early interactions of Salmonella enterica serovars Typhi and Typhimurium with epithelial cells

The role of lipopolysaccharide (LPS) in entry of Salmonella Typhimurium into epithelial cells remains unclear. In this study, we tested the ability of a series of mutants with deletions in genes for the synthesis and assembly of the O antigen and the outer core of LPS to adhere to and invade HeLa, BHK, and IB3 epithelial cells lines. Mutants devoid of O antigen, or that synthesized only one O antigen unit, or with altered O antigen chain lengths were as able as the wild type to enter epithelial cells, indicating that this polysaccharide is not required for invasion of epithelial cells in vitro. In contrast, the LPS core plays a role in the interaction of S. Typhimurium with epithelial cells. The minimal core structure required for adherence and invasion comprised the inner core and residues Glc I–Gal I of the outer core. A mutant of S. Typhimurium that produced a truncated LPS core lacking the terminal galactose residue had a significant lower level of adherence to and ingestion by the three epithelial cell lines than did strains with this characteristic. Complementation of the LPS production defect recovered invasion to parental levels. Heat-killed bacteria with a core composed of Glc I–Gal I, but not bacteria with a core composed of Glc I, inhibited uptake of the wild type by HeLa cells. A comparison of the chemical structure of the S. Typhi core with the published chemical structure of that of S. Typhimurium indicated that the Glc I–Gal I–Glc II backbone is conserved in both serovars. However, S. Typhi requires a terminal glucose for maximal invasion. Therefore, our data indicate that critical saccharide residues of the outer core play different roles in the early interactions of serovars Typhi and Typhimurium with epithelial cells.     

Immunological responses against Salmonella enterica serovar Typhimurium Braun lipoprotein and lipid A mutant strains in Swiss-Webster mice: potential use as live-attenuated vaccines

We generated and characterized Salmonella enterica serovar Typhimurium mutants that were deleted for the genes encoding Braun lipoprotein (lpp) alone or in conjunction with the msbB gene, which codes for an enzyme required for the acylation of the lipid A moiety of lipopolysaccharide. Two copies of the lpp gene, designated as lppA and lppB, exist on the chromosome of S. Typhimurium. These mutants were highly attenuated in a mouse infection model and induced minimal histopathological changes in mouse organs compared to those seen in infection with wild-type (WT) S. Typhimurium. The lppB/msbB and the lppAB/msbB mutants were maximally attenuated, and hence further examined in this study for their ability to induce humoral and cellular immune responses. Importantly, infection of out-bred Swiss-Webster mice with the mutant S. Typhimurium generated superior T helper cell type 2 (Th2) responses compared to WT S. Typhimurium, as determined by measuring IgG subclasses and cytokines. WT S. Typhimurium induced higher levels of IgG2a in sera of infected mice, while the lppB/msbB and lppAB/msbB mutants mounted higher levels of IgG1 as determined by an enzyme-linked immunosorbent assay. Mice immunized with lppB/msbB and lppAB/msbB mutants rapidly cleared WT S. Typhimurium upon subsequent rechallenge, and naïve mice passively immunized with sera from animals infected with S. Typhimurium mutants were protected against subsequent challenge with WT S. Typhimurium. Splenic T cells produced higher levels of interferon-gamma following ex vivo exposure to WT S. Typhimurium, while splenic T cells infected with the above-mentioned two mutants evoked higher levels of interleukin-6. Further, mice infected with lppB/msbB and lppAB/msbB mutants showed much higher levels of splenic T cell activation as measured by CD44⁺ expression on CD4⁺ T cells by flow cytometry and by incorporation of ³H-thymidine compared to mice that were infected with WT S. Typhimurium. We expect the lppB/msbB and lppAB/msbB mutants to be excellent live-attenuated vaccine candidates, because they induced minimal inflammatory responses and evoked stronger and specific antibody and cellular immune responses.     

Shiga toxin-producing Escherichia coli strains from cattle as a source of the Stx2a bacteriophages present in enteroaggregative Escherichia coli O104:H4 strains

Enteroaggregative, Shiga toxin-producing E. coli (EAEC-STEC) O104:H4 strains are emerging pathogens causing life threatening diseases in humans. EAEC-STEC O104:H4 strains isolated between 2001 and 2011 were found to harbor a distinct type of Shiga toxin 2a- (Stx2a) encoding prophage. This phage type shows only <65% genetic similarity to so far described viable Stx phages due to differences in the modules for DNA replication, metabolism, regulation and host specificity. Stx production in EAEC is rarely observed and the source of the Stx2a phage in the EAEC-STEC O104:H4 strains is not known. We identified two DNA segments derived from orf15 and the cI gene of the O104:H4 Stx2a phage P13374 that are characteristic for Stx2a prophages present in EAEC-STEC O104:H4 strains. By PCR, these sequences were detected in 14 (5.8%) of 241 Stx2-positive STEC from animals and food. Infectious Stx2a phages could be isolated from four bovine STEC strains. These were found highly similar to P13374 for orf15, cI and stx2a sequences, the chromosomal integration site (wrbA), for phage DNA restriction profiles, virion morphology and superinfection immunity. Stx2a phages of the four bovine STEC strains formed lysogens on the E. coli K-12 strain C600. Phage P13374 from an EAEC-STEC O104:H4 outbreak strain and one of the bovine STEC phages (P13803) lysogenized the Stx-negative EAEC O104:H4 strain CB14647 by integrating in the wrbA gene of CB14647 and converted it into a Stx2a producer. Our findings provide experimental evidence that EAEC-STEC O104:H4 strains have evolved by uptake of Stx2a phages from the bovine reservoir.     

Atypical, fljB-negative Salmonella enterica subsp. enterica strain of serovar 4,5,12:i:- appears to be a monophasic variant of serovar Typhimurium

An fljB-negative, multidrug-resistant Salmonella enterica serovar 4,5,12:i:- phage type DT U302 strain (resistant to ampicillin, chloramphenicol, sulfonamide, gentamicin, streptomycin, tetracycline, and sulfamethoxazole-trimethoprim) emerged and spread in Spain in 1997. Sequences specific for Salmonella serovar Typhimurium and phage type DT 104 and U302 were present in this atypical Salmonella strain, suggesting that it is a monophasic Salmonella serovar Typhimurium variant.     

Enhanced vaccine antigen delivery by Salmonella using antibiotic-free operator–repressor titration-based plasmid stabilisation compared to chromosomal integration

Live attenuated bacteria provide the potential to replace traditional needle-based vaccination with an orally administered vaccine. The heterologous antigen gene is usually transformed as a multi-copy plasmid into the bacterial cell, but plasmids in live bacterial vaccine strains are often unstable, so an alternative approach is to integrate the single-copy antigen gene into the bacterial chromosome. We report a comparison between the chromosomally integrated and the plasmid-borne Bacillus anthracis protective antigen gene in live Salmonella enterica serovar Typhimurium, using the Operator–Repressor Titration (ORT) system to ensure stable plasmid maintenance. These studies demonstrate that the stabilised plasmid approach of gene expression produced greater amounts of antigenic protein, which in turn resulted in higher antibody responses and levels of protection in mice.     

B cell precursors are targets for Salmonella infection

We previously reported that, in mice, B cells are a reservoir for bacteria during Salmonella infection. Here, we show that, within the bone marrow, B cells and their precursors are targeted for infection by Salmonella enterica serovar typhimurium. Our data suggest that B cells within the bone marrow may be a bacterial niche during chronic Salmonella infection.     

A single step multiplex PCR for identification of six diarrheagenic E. coli pathotypes and Salmonella

E. coli is generally a commensal but includes some highly pathogenic strains carrying additional genes in plasmids and/or the chromosome. Based on these genes the pathogenic strains are divided into pathotypes including enteropathogenic (EPEC), enterohemorrhagic (EHEC), enterotoxigenic (ETEC), enteroaggregative (EAEC), enteroinvasive (EIEC) and diffusely adherent (DAEC) E. coli. Here, previously developed multiplex PCR strategies for these strains were integrated into one single step multiplex that differentiates all these E. coli pathotypes, usually based on multiple characteristic PCR products. This multiplex PCR works reliably for colony PCR. Two additional markers were added: one to detect most Enterobacteriacea, which acts as a positive control for successful PCR, and one to distinguish Salmonella. The multiplex correctly classified a set of 45 reference strains by colony PCR and 71 (45 + 26) strains by in silico PCR. It was then used to interrogate 44 clinical strains from bovine hosts resulting in detection of EAEC and DAEC determinants.     

Integrative Analysis of Salmonellosis in Israel Reveals Association of Salmonella enterica Serovar 9,12:l,v:? with Extraintestinal Infections,Dissemination of Endemic S. enterica Serovar Typhimurium DT104 Biotypes,and Severe Underreporting of Outbreaks

Salmonella enterica is the leading etiologic agent of bacterial food-borne outbreaks worldwide. This ubiquitous species contains more than 2,600 serovars that may differ in their host specificity, clinical manifestations, and epidemiology. To characterize salmonellosis epidemiology in Israel and to study the association of nontyphoidal Salmonella (NTS) serovars with invasive infections, 48,345 Salmonella cases reported and serotyped at the National Salmonella Reference Center between 1995 and 2012 were analyzed. A quasi-Poisson regression was used to identify irregular clusters of illness, and pulsed-field gel electrophoresis in conjunction with whole-genome sequencing was applied to molecularly characterize strains of interest. Three hundred twenty-nine human salmonellosis clusters were identified, representing an annual average of 23 (95% confidence interval [CI], 20 to 26) potential outbreaks. We show that the previously unsequenced S. enterica serovar 9,12:l,v:− belongs to the B clade of Salmonella enterica subspecies enterica, and we show its frequent association with extraintestinal infections, compared to other NTS serovars. Furthermore, we identified the dissemination of two prevalent Salmonella enterica serovar Typhimurium DT104 clones in Israel, which are genetically distinct from other global DT104 isolates. Accumulatively, these findings indicate a severe underreporting of Salmonella outbreaks in Israel and provide insights into the epidemiology and genomics of prevalent serovars, responsible for recurring illness.     

A comparison of two PCR-based typing methods with pulsed-field gel electrophoresis in Salmonella enterica serovar Enteritidis

Two novel molecular typing methods, multiple-locus variable-number tandem repeats (VNTR) analysis (MLVA) and multiple amplification of phage loci typing (MAPLT), were compared with pulsed-field gel electrophoresis (PFGE) for the discrimination of 128 Salmonella enterica serovar Enteritidis (S. Enteritidis) isolates. Selected epidemiologically unrelated isolates represented a cross-section of phage types routinely isolated in Australia and included 28 isolates that could not be assigned a phage type. Targeting 5 previously described loci, MLVA generated 61 different profiles with a Simpson index of diversity of DI=0.968. MLVA locus STTR-5 proved to be the most diverse with 11 different alleles detected with a Nei's diversity index value of D=0.769. Using 8 MAPLT primers previously developed for S. Typhimurium produced 36 different profiles with a DI value of 0.948. By contrast, PFGE only generated 13 different pulsed-field patterns, DI=0.873. Within each phage type there was variation in the extent to which either molecular method was able to discriminate the S. Enteritidis isolates. MAPLT provided more discrimination in terms of the number of profiles and DI value for phage type 1 and the untypable strains while MLVA was more discriminatory for phage types 14var and 26. There was a general lack of concordance of either molecular assay to phage type. These results suggest that both MAPLT and MLVA have excellent potential as tools for epidemiological studies of S. Enteritidis.