Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella

Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy-1 and Gifsy-2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy-2 prophage, codes for the [Cu, Zn] superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III-translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy-1 and Gifsy-2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels-1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy-3, which contains the phoP/phoQ-activated pagJ gene and the gene for the secreted leucine-rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEPhi. Most phage-associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria.     

Transposition of the heat-stable toxin astA gene into a gifsy-2-related prophage of Salmonella enterica serovar Abortusovis

The horizontal transfer and acquisition of virulence genes via mobile genetic elements have been a major driving force in the evolution of Salmonella pathogenicity. Serovars of Salmonella enterica carry variable assortments of phage-encoded virulence genes, suggesting that temperate phages play a pivotal role in this process. Epidemic isolates of S. enterica serovar Typhimurium are consistently lysogenic for two lambdoid phages, Gifsy-1 and Gifsy-2, carrying known virulence genes. Other serovars of S. enterica, including serovars Dublin, Gallinarum, Enteritidis, and Hadar, carry distinct prophages with similarity to the Gifsy phages. In this study, we analyzed Gifsy-related loci from S. enterica serovar Abortusovis, a pathogen associated exclusively with ovine infection. A cryptic prophage, closely related to serovar Typhimurium phage Gifsy-2, was identified. This element, named Gifsy-2AO, was shown to contribute to serovar Abortusovis systemic infection in lambs. Sequence analysis of the prophage b region showed a large deletion which covers genes encoding phage tail fiber proteins and putative virulence factors, including type III secreted effector protein SseI (GtgB, SrfH). This deletion was identified in most of the serovar Abortusovis isolates tested and might be dependent on the replicative transposition of an adjacent insertion sequence, IS1414, previously identified in pathogenic Escherichia coli strains. IS1414 encodes heat-stable toxin EAST1 (astA) and showed multiple genomic copies in isolates of serovar Abortusovis. To our knowledge, this is the first evidence of intergeneric transfer of virulence genes via insertion sequence elements in Salmonella. The acquisition of IS1414 (EAST1) and its frequent transposition within the chromosome might improve the fitness of serovar Abortusovis within its narrow ecological niche.     

Distribution of Gifsy-3 and of Variants of ST64B and Gifsy-1 Prophages amongst Salmonella enterica Serovar Typhimurium Isolates: Evidence that Combinations of Prophages Promote Clonality

Salmonella isolates harbour a range of resident prophages which can influence their virulence and ability to compete and survive in their environment. Phage gene profiling of a range of phage types of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) indicates a significant level of correlation of phage gene profile with phage type as well as correlation with genotypes determined by a combination of multi-locus variable-number tandem repeat (VNTR) typing and clustered regularly interspaced short palindromic repeats (CRISPR) typing. Variation in phage gene profiles appears to be partly linked to differences in composition of variants of known prophages. We therefore conducted a study of the distribution of variants of ST64B and Gifsy-1 prophages and coincidently the presence of Gifsy-3 prophage in a range of S. Typhimurium phage types and genotypes. We have discovered two variants of the DT104 variant of ST64B and at least two new variants of Gifsy-1 as well as variants of related phage genes. While there is definite correlation between phage type and the prophage profile based on ST64B and Gifsy-1 variants we find stronger correlation between the VNTR/CRISPR genotype and prophage profile. Further differentiation of some genotypes is obtained by addition of the distribution of Gifsy-3 and a sequence variant of the substituted SB26 gene from the DT104 variant of ST64B. To explain the correlation between genotype and prophage profile we propose that suites of resident prophages promote clonality possibly through superinfection exclusion systems.     

Inducible prophages contribute to Salmonella virulence in mice

We show that Salmonella typhimurium harbours two fully functional prophages, Gifsy-1 and Gifsy-2, that can be induced by standard treatments or, more effectively, by exposing bacteria to hydrogen peroxide. Curing bacteria for the Gifsy-2 prophage significantly reduces Salmonella's ability to establish a systemic infection in mice. Cured strains recover their virulence properties upon relysogenization. Phage Gifsy-2 carries the sodC gene for a periplasmic [Cu,Zn]-superoxide dismutase previously implicated in the bacterial defences against killing by macrophages. The contribution of the Gifsy-1 prophage to virulence - undetectable in the presence of Gifsy-2 as prophage - becomes significant in cells that lack Gifsy-2 but carry the sodC gene integrated in the chromosome. This confirms the involvement of Gifsy-2-encoded SodC protein in Salmonella pathogenicity and suggests that the Gifsy-1 prophage carries one or more additional virulence genes that have a functional equivalent on the Gifsy-2 genome.     

Pathogenic potential and horizontal gene transfer in ovine gastrointestinal Escherichia coli

Aims: To demonstrate that a thorough characterization and virulotyping of Escherichia coli strains isolated from sheep over time leads to new insights into ovine E. coli potentially becoming human pathogens through horizontal gene transfer. Methods and Results: One hundred and fifty E. coli isolates from two sheep, sampled over 3 weeks, were characterized by serotyping, virulotyping, genotyping using multiple locus variable number tandem repeats analysis (MLVA) and susceptibility to phage infection in vitro. The 35 MLVA profiles and the serotype and virulotypes of the strains were closely associated. Many MLVA profiles differed in one locus independent of serotypes. Escherichia coli isolates of the same serotype or virulotype had identical or very similar MLVA profiles. No transductants that incorporated the bacteriophages were found in vivo, but six E. coli isolates were susceptible to the phage infection in vitro. Changes in MLVA profiles were seen after acquisition of Stx phages in vitro only. Conclusions: The sheep carried Stx phage susceptible E. coli that possessed virulence markers associated with human pathogenicity. Changes in bacterial genomes by phage transfer may complicate outbreak source investigations. Serotype has to be taken into account when evaluating strain relationships by MLVA. Significance and Impact of the Study: Sheep carry E. coli that encode for virulence markers and belong to serogroups known to be human pathogens. In addition, a selection of isolates was found to be susceptible to horizontal transfer of Shiga toxin genes by means of bacteriophages in vitro, and the transfer resulted in a discernible change of the MLVA patterns of E. coli.     

OmpC is the receptor for Gifsy-1 and Gifsy-2 bacteriophages of Salmonella

Mutations in the Salmonella enterica serovar Typhimurium ompC gene conferred resistance to Gifsy-1 and Gifsy-2 bacteriophages. Selection for complementing plasmids yielded clones of ompC. Introduction of an ompC clone into Escherichia coli conferred the ability to adsorb Gifsy phage. These data show that OmpC is the receptor for Gifsy-1 and Gifsy-2 phages.     

Bacteriophage crosstalk: coordination of prophage induction by trans-acting antirepressors

Many species of bacteria harbor multiple prophages in their genomes. Prophages often carry genes that confer a selective advantage to the bacterium, typically during host colonization. Prophages can convert to infectious viruses through a process known as induction, which is relevant to the spread of bacterial virulence genes. The paradigm of prophage induction, as set by the phage Lambda model, sees the process initiated by the RecA-stimulated self-proteolysis of the phage repressor. Here we show that a large family of lambdoid prophages found in Salmonella genomes employs an alternative induction strategy. The repressors of these phages are not cleaved upon induction; rather, they are inactivated by the binding of small antirepressor proteins. Formation of the complex causes the repressor to dissociate from DNA. The antirepressor genes lie outside the immunity region and are under direct control of the LexA repressor, thus plugging prophage induction directly into the SOS response. GfoA and GfhA, the antirepressors of Salmonella prophages Gifsy-1 and Gifsy-3, each target both of these phages' repressors, GfoR and GfhR, even though the latter proteins recognize different operator sites and the two phages are heteroimmune. In contrast, the Gifsy-2 phage repressor, GtgR, is insensitive to GfoA and GfhA, but is inactivated by an antirepressor from the unrelated Fels-1 prophage (FsoA). This response is all the more surprising as FsoA is under the control of the Fels-1 repressor, not LexA, and plays no apparent role in Fels-1 induction, which occurs via a Lambda CI-like repressor cleavage mechanism. The ability of antirepressors to recognize non-cognate repressors allows coordination of induction of multiple prophages in polylysogenic strains. Identification of non-cleavable gfoR/gtgR homologues in a large variety of bacterial genomes (including most Escherichia coli genomes in the DNA database) suggests that antirepression-mediated induction is far more common than previously recognized.     

A rapid multiplex DNA suspension array method for Salmonella typhimurium subtyping using prophage-related markers

In this study we developed a preliminary proof of concept of method for Salmonella typhimurium subtyping using multiplex PCR-based phage locus typing and a multiplex Luminex DNA suspension array for product detection. Thirty markers were selected from prophages ST64B, ST64T, ST104, P22, Gifsy-1, sopEΦ and mostly phage-related AFLP fragments, and organised into two multiplex PCRs of 15 markers each. A two-group DNA suspension array was developed using a combination of flow cytometry and Luminex xMAP® technology. To assess its subtyping capability the method was applied to 438 non-epidemiological related S. typhimurium isolates of 56 phage types. Eighty-one profiles were generated. Isolates were divided into sixteen main prophage marker profile types. There was a strong tendency for isolates with the same phage type to have the same or closely related profiles and for groups of phage types to share the same profile. The discriminatory power of this method expressed as the Simpson's Index of Diversity (D) was 0.954. A panel of 12 selected markers achieved almost the same D value (0.952) as the 30 markers. This new method provides an alternative typing scheme for S. typhimurium epidemiological investigations. The developed array is in a high-throughput format which could easily be semi-automated, making the test fast and economical.     

Identification of GtgE,a novel virulence factor encoded on the Gifsy-2 bacteriophage of Salmonella enterica serovar Typhimurium

The Gifsy-2 temperate bacteriophage of Salmonella enterica serovar Typhimurium contributes significantly to the pathogenicity of strains that carry it as a prophage. Previous studies have shown that Gifsy-2 encodes SodCI, a periplasmic Cu/Zn superoxide dismutase, and at least one additional virulence factor. Gifsy-2 encodes a Salmonella pathogenicity island 2 type III secreted effector protein. Sequence analysis of the Gifsy-2 genome also identifies several open reading frames with homology to those of known virulence genes. However, we found that null mutations in these genes did not individually have a significant effect on the ability of S. enterica serovar Typhimurium to establish a systemic infection in mice. Using deletion analysis, we have identified a gene, gtgE, which is necessary for the full virulence of S. enterica serovar Typhimurium Gifsy-2 lysogens. Together, GtgE and SodCI account for the contribution of Gifsy-2 to S. enterica serovar Typhimurium virulence in the murine model.     

Phenotypes of lexA mutations in Salmonella enterica: evidence for a lethal lexA null phenotype due to the Fels-2 prophage

The LexA protein of Escherichia coli represses the damage-inducible SOS regulon, which includes genes for repair of DNA. Surprisingly, lexA null mutations in Salmonella enterica are lethal even with a sulA mutation, which corrects lexA lethality in E. coli. Nine suppressors of lethality isolated in a sulA mutant of S. enterica had lost the Fels-2 prophage, and seven of these (which grew better) had also lost the Gifsy-1 and Gifsy-2 prophages. All three phage genomes included a homologue of the tum gene of coliphage 186, which encodes a LexA-repressed cI antirepressor. The tum homologue of Fels-2 was responsible for lexA lethality and had a LexA-repressed promoter. This basis of lexA lethality was unexpected because the four prophages of S. enterica LT2 are not strongly UV inducible and do not sensitize strains to UV killing. In S. enterica, lexA(Ind(-)) mutants have the same phenotypes as their E. coli counterparts. Although lexA null mutants express their error-prone DinB polymerase constitutively, they are not mutators in either S. enterica or E. coli.     

The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage

Toxigenic strains of Pasteurella multocida produce a 146 kDa toxin (PMT) that acts as a potent mitogen. Sequence analysis of the structural gene for PMT, toxA, previously suggested it was horizontally acquired, because it had a low G + C content relative to the P. multocida genome. To address this, the sequence of DNA flanking toxA was determined. The sequence analysis showed the presence of homologues to bacteriophage tail protein genes and a bacteriophage antirepressor, suggesting that the toxin gene resides within a prophage. In addition to phage genes, the toxA flanking DNA contained a homologue of a restriction/modification system that was shown to be functional. The presence of a bacteriophage was demonstrated in spent medium from toxigenic P. multocida isolates. Its production was increased by mitomycin C addition, a treatment that is known to induce the lytic cycle of many temperate bacteriophages. The genomes of bacteriophages from three different toxigenic P. multocida strains had similar but not identical restriction profiles, and were approximately 45-50 kb in length. The prophages from two of these had integrated at the same site in the chromosome, in a tRNA gene. Southern blot analysis confirmed that these bacteriophages contained the toxA gene.     

Specialized Transducing Phages Derived from Salmonella Phage P22

Salmonella phage P22 has been used in the construction of three sorts of specialized transducing phage: P22 proAB, P22 proABlac and P22 argF. The bacterial genes carried are derived from E. coli K12. Since E. coli and Salmonella chromosomes recombine very poorly, E. coli genes cannot be transduced into Salmonella recipients by P22's generalized transduction mechanism. Therefore, stable inheritance of E. coli material provides a means of detecting specialized transduction. Formation of these phages was possible because the P22 prophage recognizes an attachment site in the E. coli F' prolac episome. Salmonella strains carrying the F' prolac episome can be lysogenized by P22 so as to leave the prophage inserted into the E. coli material of the F' factor. Improper prophage excision can then lead to formation of P22 specialized phages carrying E. coli genetic material.     

Evolutionary genetics of the isocitrate dehydrogenase gene (icd) in Escherichia coli and Salmonella enterica.

Sequences of the icd gene, encoding isocitrate dehydrogenase (IDH), were obtained for 33 strains representing the major phylogenetic lineages of Escherichia coli and Salmonella enterica. Evolutionary relationships of the strains based on variation in icd are generally similar to those previously obtained for several other housekeeping and for invasion genes, but the sequences of S. enterica subspecies V strains are unusual in being almost intermediate between those of the other S. enterica subspecies and E. coli. For S. enterica, the ratio of synonymous (silent) to nonsynonymous (replacement) nucleotide substitutions between pairs of strains was larger than comparable values for 12 other housekeeping and invasion genes, reflecting unusually strong purifying selection against amino acid replacement in the IDH enzyme. All amino acids involved in the catalytic activity and conformational changes of IDH are strictly conserved within and between species. In E. coli, the level of variation at the 3' end of the gene is elevated by the presence in some strains of a 165-bp replacement sequence supplied by the integration of either lambdoid phage 21 or defective prophage element e14. The 72 members of the E. coli Reference Collection (ECOR) and five additional E. coli strains were surveyed for the presence of phage 21 (as prophage) by PCR amplification of a phage 21-specific fragment in and adjacent to the host icd, and the sequence of the phage 21 segment extending from the 3' end of icd through the integrase gene (int) was determined in nine strains of E. coli. Phage 21 was found in 39% of E. coli strains, and its distribution among the ECOR strains is nonrandom. In two ECOR strains, the phage 21 int gene is interrupted by a 1,313-bp insertion element that has 99.3% nucleotide sequence identity with IS3411 of E. coli. The phylogenetic relationships of phage 21 strains derived from sequences of two different genomic regions were strongly incongruent, providing evidence of frequent recombination.     

Characterization of closely related lactococcal starter strains which show differing patterns of bacteriophage sensitivity

AIMS: To characterize a group of closely related Lactococcus lactis subsp. lactis casein starter strains used commercially, which differ in their sensitivity to bacteriophages isolated from the same industrial environment. METHODS AND RESULTS: Nine strains of L. lactis, six of which had been used as starter cultures for lactic casein manufacture, were shown to be closely related by pulsed-field gel electrophoresis and total DNA profiles. Nineteen phages which propagated on one or more of these starter strains were isolated from industrial casein whey samples. The phages were all small isometric-headed and could be divided into five groups on the basis of host range on the nine strains. Most of the phages did not give a PCR product with primers designed to detect the two most common lactococcal small isometric phage species (936 and P335). The hosts could be divided into six groups depending on their phage sensitivity. Plasmids encoding genes for the cell envelope associated PI-type proteinase, lactose metabolism and specificity subunits of a type I restriction/modification system were identified. CONCLUSIONS: This work demonstrates how isolates of the same starter strain may come to be regarded as separate cultures because of their different origins, and how these closely related strains may differ in some of their industrially relevant characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: This situation may be very common among lactococci used as dairy starter cultures, and implies that the dairy industry worldwide depends on a small number of different strains.     

MLVA as a Tool for Public Health Surveillance of Human Salmonella Typhimurium: Prospective Study in Belgium and Evaluation of MLVA Loci Stability

Surveillance of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) is generally considered to benefit from molecular techniques like multiple-locus variable-number of tandem repeats analysis (MLVA), which allow earlier detection and confinement of outbreaks. Here, a surveillance study, including phage typing, antimicrobial susceptibility testing and the in Europe most commonly used 5-loci MLVA on 1,420 S. Typhimurium isolates collected between 2010 and 2012 in Belgium, was used to evaluate the added value of MLVA for public health surveillance. Phage types DT193, DT195, DT120, DT104, DT12 and U302 dominate the Belgian S. Typhimurium population. A combined resistance to ampicillin, streptomycin, sulphonamides and tetracycline (ASSuT) with or without additional resistances was observed for 42.5% of the isolates. 414 different MLVA profiles were detected, of which 14 frequent profiles included 44.4% of the S. Typhimurium population. During a serial passage experiment on selected isolates to investigate the in vitro stability of the 5 MLVA loci, variations over time were observed for loci STTR6, STTR10, STTR5 and STTR9. This study demonstrates that MLVA improves public health surveillance of S. Typhimurium. However, the 5-loci MLVA should be complemented with other subtyping methods for investigation of possible outbreaks with frequent MLVA profiles. Also, variability in these MLVA loci should be taken into account when investigating extended outbreaks and studying dynamics over longer periods.     

Characterization of grvA, an antivirulence gene on the gifsy-2 phage in Salmonella enterica serovar typhimurium

The lambdoid phage Gifsy-2 contributes significantly to Salmonella enterica serovar Typhimurium virulence. The phage carries the periplasmic superoxide dismutase gene, sodCI, and other unidentified virulence factors. We have characterized the gene grvA, a single open reading frame inserted in the opposite orientation in the tail operon of the Gifsy-2 phage. Contrary to what is observed with classic virulence genes, grvA null mutants were more virulent than wild type as measured by intraperitoneal competition assays in mice. We have termed this effect antivirulence. Wild-type grvA in single copy complemented this phenotype. However, grvA(+) on a multicopy plasmid also conferred the antivirulence phenotype. Neither a grvA null mutation nor the grvA(+) plasmid conferred a growth advantage or disadvantage in laboratory media. The antivirulence phenotype conferred by the grvA null mutation and the grvA(+) plasmid required wild-type sodCI but was independent of other virulence factors encoded on Gifsy-2. These results suggest that in a wild-type situation, GrvA decreases the pathogenicity of serovar Typhimurium in the host, most likely by affecting resistance to toxic oxygen species. These virulence phenotypes were independent of functional Gifsy-2 phage production. Our data suggest that the contribution of Gifsy-2 is a complicated sum of both positive virulence factors such as sodCI and antivirulence factors such as grvA.