Diversity of phage types among archived cultures of the Demerec collection of Salmonella enterica serovar Typhimurium strains

The existence of several thousand Salmonella enterica serovar Typhimurium LT2 and LT7 cultures originally collected by M. Demerec and sealed in agar stab vials for 33 to 46 years is a resource for evolutionary and mutational studies. Cultures from 74 of these vials, descendants of cells sealed and stored in nutrient agar stabs several decades ago, were phage typed by the Callow and Felix, Lilleengen, and Anderson systems. Among 53 LT2 archived strains, 16 had the same phage type as the nonarchival sequenced LT2 strain. The other 37 archived cultures differed in phage typing pattern from the sequenced strain. These 37 strains were divided into 10 different phage types. Among the 19 LT7 strains, only one was similar to the parent by phage typing, while 18 were different. These 18 strains fell into eight different phage types. The typing systems were developed to track epidemics from source to consumer, as well as geographic spread. The value of phage typing is dependent upon the stability of the phage type of any given strain throughout the course of the investigation. Thus, the variation over time observed in these archived cultures is particularly surprising. Possible mechanisms for such striking diversity may include loss of prophages, prophage mosaics as a result of recombination events, changes in phage receptor sites on the bacterial cell surface, or mutations in restriction-modification systems.     

Diversity of Phage Types among Archived Cultures of the Demerec Collection of Salmonella enterica serovar Typhimurium Strains

The existence of several thousand Salmonella enterica serovar Typhimurium LT2 and LT7 cultures originally collected by M. Demerec and sealed in agar stab vials for 33 to 46 years is a resource for evolutionary and mutational studies. Cultures from 74 of these vials, descendants of cells sealed and stored in nutrient agar stabs several decades ago, were phage typed by the Callow and Felix, Lilleengen, and Anderson systems. Among 53 LT2 archived strains, 16 had the same phage type as the nonarchival sequenced LT2 strain. The other 37 archived cultures differed in phage typing pattern from the sequenced strain. These 37 strains were divided into 10 different phage types. Among the 19 LT7 strains, only one was similar to the parent by phage typing, while 18 were different. These 18 strains fell into eight different phage types. The typing systems were developed to track epidemics from source to consumer, as well as geographic spread. The value of phage typing is dependent upon the stability of the phage type of any given strain throughout the course of the investigation. Thus, the variation over time observed in these archived cultures is particularly surprising. Possible mechanisms for such striking diversity may include loss of prophages, prophage mosaics as a result of recombination events, changes in phage receptor sites on the bacterial cell surface, or mutations in restriction-modification systems.     

Genomic structure of the Salmonella enterica serovar Typhimurium DT 64 bacteriophage ST64T: evidence for modular genetic architecture

The complete sequence of the double-stranded DNA genome of a serotype-converting temperate bacteriophage, ST64T, was determined. The 40,679-bp genomic sequence of ST64T has an overall GC content of 47.5% and was reminiscent of a number of lambdoid phages, in particular, P22. Inferred proteins of ST64T which exhibited a high degree of sequence similarity to P22 proteins (>90%) included the functional serotype conversion cassette, integrase, excisionase, Abc1, Abc2, early antitermination (gp24), NinD, NinH, NinZ, packaging (gp3 and gp2), head (with the exception of gp26, gp7, gp20, and gp16), and tail proteins. The putative immunity genes were highly related to those of Salmonella enterica serotype Typhimurium phage L, whereas the lysis genes were almost identical to those of S. enterica serovar Typhimurium PS3.     

Correlation of phenotype with the genotype of egg-contaminating Salmonella enterica serovar Enteritidis

The genotype of Salmonella enterica serovar Enteritidis was correlated with the phenotype using DNA-DNA microarray hybridization, ribotyping, and Phenotype MicroArray analysis to compare three strains that differed in colony morphology and phage type. No DNA hybridization differences were found between two phage type 13A (PT13A) strains that varied in biofilm formation; however, the ribotype patterns were different. Both PT13A strains had DNA sequences similar to that of bacteriophage Fels2, whereas the PT4 genome to which they were compared, as well as a PT4 field isolate, had a DNA sequence with some similarity to the bacteriophage ST64b sequence. Phenotype MicroArray analysis indicated that the two PT13A strains and the PT4 field isolate had similar respiratory activity profiles at 37 degrees C. However, the wild-type S. enterica serovar Enteritidis PT13A strain grew significantly better in 20% more of the 1,920 conditions tested when it was assayed at 25 degrees C than the biofilm-forming PT13A strain grew. Statistical analysis of the respiratory activity suggested that S. enterica serovar Enteritidis PT4 had a temperature-influenced dimorphic metabolism which at 25 degrees C somewhat resembled the profile of the biofilm-forming PT13A strain and that at 37 degrees C the metabolism was nearly identical to that of the wild-type PT13A strain. Although it is possible that lysogenic bacteriophage alter the balance of phage types on a farm either by lytic competition or by altering the metabolic processes of the host cell in subtle ways, the different physiologies of the S. enterica serovar Enteritidis strains correlated most closely with minor, rather than major, genomic changes. These results strongly suggest that the pandemic of egg-associated human salmonellosis that came into prominence in the 1980s is primarily an example of bacterial adaptive radiation that affects the safety of the food supply.     

Host restriction of Salmonella enterica serotype Typhimurium pigeon isolates does not correlate with loss of discrete genes

The definitive phage types (DT) 2 and 99 of Salmonella enterica serotype Typhimurium are epidemiologically correlated with a host range restricted to pigeons, in contrast to phage types with broader host ranges such as epidemic cattle isolates (DT104 and DT204). To determine whether phage types with broad host range possess genetic islands absent from host-restricted phage types, we compared the genomes of four pigeon isolates to serotype Typhimurium strain LT2 using a DNA microarray. Three of the four isolates tested caused fluid accumulation in bovine ligated ileal loops, but they had reduced colonization of liver and spleen in susceptible BALB/c mice and were defective for intestinal persistence in Salmonella-resistant CBA mice. The genomes of the DT99 and DT2 isolates were extremely similar to the LT2 genome, with few notable differences on the level of complete individual genes. Two large groups of genes representing the Fels-1 and Fels-2 prophages were missing from the DT2 and DT99 phage types we analyzed. One of the DT99 isolates examined was lacking a third cluster of five chromosomal genes (STM1555 to -1559). Results of the microarray analysis were extended using Southern analysis to a collection of 75 serotype Typhimurium clinical isolates of 24 different phage types. This analysis revealed no correlation between the presence of Fels-1, Fels-2, or STM1555 to -1559 and the association of phage types with different host reservoirs. We conclude that serotype Typhimurium phage types with broad host range do not possess genetic islands influencing host restriction, which are absent from the host-restricted pigeon isolates.     

Salmonella phage ST64B encodes a member of the SseK/NleB effector family

Salmonella enterica is a species of bacteria that is a major cause of enteritis across the globe, while certain serovars cause typhoid, a more serious disease associated with a significant mortality rate. Type III secreted effectors are major contributors to the pathogenesis of Salmonella infections. Genes encoding effectors are acquired via horizontal gene transfer, and a subset are encoded within active phage lysogens. Because the acquisition of effectors is in flux, the complement of effectors possessed by various Salmonella strains frequently differs. By comparing the genome sequences of S. enterica serovar Typhimurium strain SL1344 with LT2, we identified a gene with significant similarity to SseK/NleB type III secreted effector proteins within a phage ST64B lysogen that is absent from LT2. We have named this gene sseK3. SseK3 was co-regulated with the SPI-2 type III secretion system in vitro and inside host cells, and was also injected into infected host cells. While no role for SseK3 in virulence could be identified, a role for the other family members in murine typhoid was found. SseK3 and other phage-encoded effectors were found to have a significant but sparse distribution in the available Salmonella genome sequences, indicating the potential for more uncharacterised effectors to be present in less studied serovars. These phage-encoded effectors may be principle subjects of contemporary selective processes shaping Salmonella-host interactions.     

Molecular characterization of Irish Salmonella enterica serotype typhimurium: detection of class I integrons and assessment of genetic relationships by DNA amplification fingerprinting

Salmonella enterica is among the principal etiological agents of food-borne illness in humans. Increasing antimicrobial resistance in S. enterica is a cause for worldwide concern. There is concern at present in relation to the increasing incidence of human infection with antimicrobial agent-resistant strains of S. enterica serotype Typhimurium, in particular of phage type DT104. Integrons appear to play an important role in the dissemination of antimicrobial resistance genes in many Enterobacteriaceae including S. enterica. In this study the antimicrobial susceptibilities and phage types of 74 randomly collected strains of S. enterica serotype Typhimurium from the Cork region of southern Ireland, obtained from human, animal (clinical), and food sources, were determined. Each strain was examined for integrons and typed by DNA amplification fingerprinting (DAF). Phage type DT104 predominated (n = 48). Phage types DT104b (n = 3), -193 (n = 9), -195 (n = 6), -208 (n = 3), -204a (n = 2), PT U302 (n = 1), and two nontypeable strains accounted for the remainder. All S. enterica serotype Typhimurium DT104 strains were resistant to ampicillin, chloramphenicol, streptomycin, Sulfonamide Duplex, and tetracycline, and one strain was additionally resistant to trimethoprim. All DT104 strains but one were of a uniform DAF type (designated DAF-I) and showed a uniform pattern of integrons (designated IP-I). The DT104b and PT U302 strains also exhibited the same resistance phenotype, and both had the DAF-I and IP-I patterns. The DAF-I pattern was also observed in a single DT193 strain in which no integrons were detectable. Greater diversity of antibiograms and DAF and IP patterns among non-DT104 phage types was observed. These data indicate a remarkable degree of homogeneity at a molecular level among contemporary isolates of S. enterica serotype Typhimurium DT104 from animal, human, and food sources in this region.     

Bacteriophage ST64B, a genetic mosaic of genes from diverse sources isolated from Salmonella enterica serovar typhimurium DT 64

The complete sequence of the double-stranded DNA (dsDNA) genome of the Salmonella enterica serovar Typhimurium ST64B bacteriophage was determined. The 40,149-bp genomic sequence of ST64B has an overall G+C content of 51.3% and is distinct from that of P22. The genome architecture is similar to that of the lambdoid phages, particularly that of coliphage lambda. Most of the putative tail genes showed sequence similarity to tail genes of Mu, a nonlambdoid phage. In addition, it is likely that these tail genes are not expressed due to insertions of fragments of genes related to virulence within some of the open reading frames. This, together with the inability of ST64B to produce plaques on a wide range of isolates, suggests that ST64B is a defective phage. In contrast to the tail genes, most of the head genes showed similarity to those of the lambdoid phages HK97 and HK022, but these head genes also have significant sequence similarities to those of several other dsDNA phages infecting diverse bacterial hosts, including Escherichia, Pseudomonas, Agrobacterium, Caulobacter, Mesorhizobium, and Streptomyces: This suggests that ST64B is a genetic mosaic that has acquired significant portions of its genome from sources outside the genus Salmonella.     

Resuscitation of a defective prophage in Salmonella cocultures

Widely studied Salmonella enterica serovar Typhimurium strains ATCC 14028s and SL1344 harbor a cryptic ST64B prophage unable to produce infectious virions. We found that coculturing either strain with an isogenic sibling lacking the prophage leads to the appearance of active forms of the virus. Active phage originates from reversion of a +1 frameshift mutation at a monotonous G:C run in a presumptive tail assembly pseudogene.     

The colanic acid gene cluster of Salmonella enterica has a complex history

The colanic acid gene cluster of Salmonella enterica LT2 was sequenced and compared with that of Escherichia coli K-12. The two clusters are similar with divergence slightly higher than average for genes of the two species. The cluster was divided into four blocks by GC content and seems likely to have transferred from a higher GC content species to the ancestor of E. coli and S. enterica. All 19 genes of K-12 and 13 genes of LT2 appear to have undergone random genetic drift with amelioration of the GC content. However, in the case of S. enterica, we believe that the six genes of the GDP-fucose pathway group were replaced relatively recently by genes closely related to those of the original donor species. Two repetitive elements were observed: a bacterial interspersed mosaic element in the intergenic region between wzx and wcaK in K-12 only and a RSA (repetitive sequence element) sequence between wcaJ and wzx in LT2 only.     

Cloning and characterization of the gene encoding the z66 antigen of Salmonella enterica serovar Typhi

Z66 antigen-positive strains of Salmonella enterica serovar Typhi change flagellin expression in only one direction from the z66 antigen to the d or j antigen, which is different from the phase variation of S. enterica serovar Typhimurium. In the present study, we identified a new flagellin gene in z66 antigen-positive strains of S. enterica serovar Typhi. The genomic structure of the region containing this new flagellin gene was similar to that of fljBA operon of biphasic S. enterica serovars. A fljA-like gene was present downstream of the new flagellin gene. A rho-independent terminator was located between the new flagellin gene and the fljA-like gene. Hin-like gene was not present upstream of the new flagellin gene. We generated a mutant strain of S. enterica serovar Typhi, which carries a deletion of the new flagellin gene. Western blotting revealed that the 51-kDa z66 antigen protein was absent from the population of proteins secreted by the mutant strain. Southern hybridization demonstrated that the z66 antigen-positive strains of S. enterica serovar Typhi carried the new flagellin gene and fliC on two different genomic EcoRI fragments. When z66 antigen-positive strains were incubated with anti-z66 antiserum, the flagellin expression by S. enterica serovar Typhi changed from z66 antigen to j antigen. The new flagellin gene and the fljA-like gene were absent in the strain with altered flagellin expression. These results suggested that the new flagellin gene is a fljB-like gene, which encodes the z66 antigen of S. enterica serovar Typhi, and that deletion of fljBA-like operon may explain why S. enterica serovar Typhi alters the flagellin expression in only one direction from the z66 antigen to the d or j antigen.     

The genome of Salmonella enterica serovar gallinarum: distinct insertions/deletions and rare rearrangements

Salmonella enterica serovar Gallinarum is a fowl-adapted pathogen, causing typhoid fever in chickens. It has the same antigenic formula (1,9,12:--:--) as S. enterica serovar Pullorum, which is also adapted to fowl but causes pullorum disease (diarrhea). The close relatedness but distinct pathogeneses make this pair of fowl pathogens good models for studies of bacterial genomic evolution and the way these organisms acquired pathogenicity. To locate and characterize the genomic differences between serovar Gallinarum and other salmonellae, we constructed a physical map of serovar Gallinarum strain SARB21 by using I-CeuI, XbaI, and AvrII with pulsed-field gel electrophoresis techniques. In the 4,740-kb genome, we located two insertions and six deletions relative to the genome of S. enterica serovar Typhimurium LT2, which we used as a reference Salmonella genome. Four of the genomic regions with reduced lengths corresponded to the four prophages in the genome of serovar Typhimurium LT2, and the others contained several smaller deletions relative to serovar Typhimurium LT2, including regions containing srfJ, std, and stj and gene clusters encoding a type I restriction system in serovar Typhimurium LT2. The map also revealed some rare rearrangements, including two inversions and several translocations. Further characterization of these insertions, deletions, and rearrangements will provide new insights into the molecular basis for the specific host-pathogen interactions and mechanisms of genomic evolution to create a new pathogen.     

Comparison of DeltarelA strains of Escherichia coli and Salmonella enterica serovar Typhimurium suggests a role for ppGpp in attenuation regulation of branched-chain amino acid biosynthesis

The growth recovery of Escherichia coli K-12 and Salmonella enterica serovar Typhimurium DeltarelA mutants were compared after nutritional downshifts requiring derepression of the branched-chain amino acid pathways. Because wild-type E. coli K-12 and S. enterica serovar Typhimurium LT2 strains are defective in the expression of the genes encoding the branch point acetohydroxy acid synthetase II (ilvGM) and III (ilvIH) isozymes, respectively, DeltarelA derivatives corrected for these mutations were also examined. Results indicate that reduced expression of the known global regulatory factors involved in branched-chain amino acid biosynthesis cannot completely explain the observed growth recovery defects of the DeltarelA strains. In the E. coli K-12 MG1655 DeltarelA background, correction of the preexisting rph-1 allele which causes pyrimidine limitations resulted in complete loss of growth recovery. S. enterica serovar Typhimurium LT2 DeltarelA strains were fully complemented by elevated basal ppGpp levels in an S. enterica serovar Typhimurium LT2 DeltarelA spoT1 mutant or in a strain harboring an RNA polymerase mutation conferring a reduced RNA chain elongation rate. The results are best explained by a dependence on the basal levels of ppGpp, which are determined by relA-dependent changes in tRNA synthesis resulting from amino acid starvations. Expression of the branched-chain amino acid operons is suggested to require changes in the RNA chain elongation rate of the RNA polymerase, which can be achieved either by elevation of the basal ppGpp levels or, in the case of the E. coli K-12 MG1655 strain, through pyrimidine limitations which partially compensate for reduced ppGpp levels. Roles for ppGpp in branched-chain amino acid biosynthesis are discussed in terms of effects on the synthesis of known global regulatory proteins and current models for the control of global RNA synthesis by ppGpp.     

Genomic diversification among archival strains of Salmonella enterica serovar typhimurium LT7

To document genomic changes during long periods of storage, we analyzed Salmonella enterica serovar Typhimurium LT7, a mutator strain that was previously reported to have higher rates of mutations compared to other serovar Typhimurium strains such as LT2. Upon plating directly from sealed agar stabs that had been stocked at room temperature for up to four decades, many auxotrophic mutants derived from LT7 gave rise to colonies of different sizes. Restreaking from single colonies consistently yielded colonies of diverse sizes even when we repeated single-colony isolation nine times. Colonies from the first plating had diverse genomic changes among and even within individual vials, including translocations, inversions, duplications, and point mutations, which were detected by rare-cutting endonuclease analysis with pulsed-field gel electrophoresis. Interestingly, even though the colony size kept diversifying, all descendents of the same single colonies from the first plating had the same sets of detected genomic changes. We did not detect any colony size or genome structure diversification in serovar Typhimurium LT7 stocked at -70 degrees C or in serovar Typhimurium LT2 stocked either at -70 degrees C or at room temperature. These results suggest that, although colony size diversification occurred during rapid growth, all detected genomic changes took place during the storage at room temperature and were carried over to their descendents without further changes during rapid growth in rich medium. We constructed a genomic cleavage map on the LT7 strain that had been stocked at -70 degrees C and located all of the detected genomic changes on the map. We speculated on the significance of mutators for survival and evolution under environmentally stressed conditions.     

Characterization of the genomes of a diverse collection of Salmonella enterica serovar Typhimurium definitive phage type 104

Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) has caused significant morbidity and mortality in humans and animals for almost three decades. We completed the full DNA sequence of one DT104 strain, NCTC13348, and showed that significant differences between the genome of this isolate and the genome of the previously sequenced strain Salmonella serovar Typhimurium LT2 are due to integrated prophage elements and Salmonella genomic island 1 encoding antibiotic resistance genes. Thirteen isolates of Salmonella serovar Typhimurium DT104 with different pulsed-field gel electrophoresis (PFGE) profiles were analyzed by using multilocus sequence typing (MLST), plasmid profiling, hybridization to a pan-Salmonella DNA microarray, and prophage-based multiplex PCR. All the isolates belonged to a single MLST type, sequence type ST19. Microarray data demonstrated that the gene contents of the 13 DT104 isolates were remarkably conserved. The PFGE DNA fragment size differences in these isolates could be explained to a great extent by differences in the prophage and plasmid contents. Thus, here the nature of variation in different Salmonella serovar Typhimurium DT104 isolates is further defined at the gene and whole-genome levels, illustrating how this phage type evolves over time.     

The SopEPhi phage integrates into the ssrA gene of Salmonella enterica serovar Typhimurium A36 and is closely related to the Fels-2 prophage

Salmonella spp. are enteropathogenic gram-negative bacteria that use a large array of virulence factors to colonize the host, manipulate host cells, and resist the host's defense mechanisms. Even closely related Salmonella strains have different repertoires of virulence factors. Bacteriophages contribute substantially to this diversity. There is increasing evidence that the reassortment of virulence factor repertoires by converting phages like the GIFSY phages and SopEPhi may represent an important mechanism in the adaptation of Salmonella spp. to specific hosts and to the emergence of new epidemic strains. Here, we have analyzed in more detail SopEPhi, a P2-like phage from Salmonella enterica serovar Typhimurium DT204 that encodes the virulence factor SopE. We have cloned and characterized the attachment site (att) of SopEPhi and found that its 47-bp core sequence overlaps the 3' terminus of the ssrA gene of serovar Typhimurium. Furthermore, we have demonstrated integration of SopEPhi into the cloned attB site of serovar Typhimurium A36. Sequence analysis of the plasmid-borne prophage revealed that SopEPhi is closely related to (60 to 100% identity over 80% of the genome) but clearly distinct from the Fels-2 prophage of serovar Typhimurium LT2 and from P2-like phages in the serovar Typhi CT18 genome. Our results demonstrate that there is considerable variation among the P2-like phages present in closely related Salmonella spp.     

Inversions over the terminus region in Salmonella and Escherichia coli: IS200s as the sites of homologous recombination inverting the chromosome of Salmonella enterica serovar typhi

Genomic rearrangements (duplications and inversions) in enteric bacteria such as Salmonella enterica serovar Typhimurium LT2 and Escherichia coli K12 are frequent (10(-3) to 10(-5)) in culture, but in wild-type strains these genomic rearrangements seldom survive. However, inversions commonly survive in the terminus of replication (TER) region, where bidirectional DNA replication terminates; nucleotide sequences from S. enterica serovar Typhimurium LT2, S. enterica serovar Typhi CT18, E. coli K12, and E. coli O157:H7 revealed genomic inversions spanning the TER region. Assuming that S. enterica serovar Typhimurium LT2 represents the ancestral genome structure, we found an inversion of 556 kb in serovar Typhi CT18 between two of the 25 IS200 elements and an inversion of about 700 kb in E. coli K12 and E. coli O157:H7. In addition, there is another inversion of 500 kb in E. coli O157:H7 compared with E. coli K12. PCR analysis confirmed that all S. enterica serovar Typhi strains tested, but not strains of other Salmonella serovars, have an inversion at the exact site of the IS200 insertions. We conclude that inversions of the TER region survive because they do not significantly change replication balance or because they are part of the compensating mechanisms to regain chromosome balance after it is disrupted by insertions, deletions, or other inversions.     

Identification of specific gene sequences conserved in contemporary epidemic strains of Salmonella enterica

Genetic elements specific to recent and contemporary epidemic strains of Salmonella enterica were identified using comparative genomic analysis. Two epidemic multidrug-resistant (MDR) strains, MDR Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) and cephalosporin-resistant MDR Salmonella enterica serovar Newport, and an epidemic pansusceptible strain, Salmonella serovar Typhimurium DT160, were subjected to Salmonella gene microarray and suppression subtractive hybridization analyses. Their genome contents were compared with those of coexisting sporadic strains matched by serotype, geographic and temporal distribution, and host species origin. These paired comparisons revealed that epidemic strains of S. enterica had specific genes and gene regions that were shared by isolates of the same subtype. Most of these gene sequences are related to mobile genetic elements, including phages, plasmids, and plasmid-like and transposable elements, and some genes may encode proteins conferring growth or survival advantages. The emergence of epidemic MDR strains may therefore be associated with the presence of fitness-associated genetic factors in addition to their antimicrobial resistance genes.