Towards the development of a DNA-sequence based approach to serotyping of Salmonella enterica

Summary of fliC sequence variation within the g-complex

All polymorphisms within the g-complex sequences analysed are displayed in Figure 2 The target region (highlighted) was selected because it conferred multiple sero-specific amino acid substitutions and was variable at the DNA level. In the 17 bp nucleotide sequence assayed, 15 sequence types were identified (Figure 3). This region was assayed against the test panel of 17 Salmonella strains belonging to the g-complex and was able to exclusively identify sequence motifs corresponding to phase 1 flagellar serotypes. The serotypes not differentiated by this assay ([f],g,m, [p], g,m, g,m,s and g,m, [p],s or non-motile Gallinarum) were known from full sequencing to be identical at the target region.

Amino acid differences between g-complex strains identified by full sequencing

The following polymorphisms located in fliC of the g-complex are likely to be involved in specific epitope formation: two amino acid sequence types were observed in 25 fliC-[f],g,m, [p] sequences obtained from Salmonella enterica serovar Enteritidis strains. Twenty-three S. Enteritidis strains demonstrated complete conservation in their DNA sequence (B16, B18, JTCM02 and 20 phage type 4 strains (Enteritidis_b)). The sequence of B17 was congruent with published S. Enteritidis (M84980) (Enteritidis_a), and exhibited a single amino acid (Ser>Gly at 302) substitution compared to sequence type Enteritidis_b. Published S. Othmarschen (U06455) fliC-g,m, [t] sequence inferred the same amino acid sequence as Enteritidis_a but exhibited a silent mutation at the DNA level. As the fliC sequence for these two serotypes was identical it was apparent that the sequence included here represented an S. Othmarschen strain in which the t factor was absent.

Published S. Gallinarum sequences demonstrated 100% DNA homology to Enteritidis_b except for a SNP encoding a stop codon in M84975. S. Pullorum and S. Gallinarum are non-motile as they do not express flagella. Antisera to the g factor antigen react strongly with induced-motility S. Pullorum culture, indicating that g epitopes are expressed in these cells [19]. This correlates with our sequence data as S. Pullorum clusters with g,m sequences (Figure 1). Biotype-specific polymorphisms for S. Pullorum were observed at amino acid position 91 and 323. Molecular identification of S. Pullorum and S. Gallinarum would be of considerable benefit as standard serotyping cannot differentiate these two serotypes.

FliC-g,q was differentiated from all other g-complex sequences by an Asp>Gly serotype-specific polymorphism observed at position 284 for S. Moscow. A Thr>Ala substitution at residue 304 conferred by a single nucleotide polymorphism (SNP) was identified between sequences of g,m and g,p, congruent with a previous report [20], and forms the basis for differentiation of these two serotypes. DNA polymorphisms, but no inferred amino acid substitutions, were observed between strains exhibiting g,m,s and g,m, [p],s. The p factor was not coded for by the fliC sequences of these strains.

S. Essen fliC-g,m was distinct from other g and m coding sequences by an Asp>Asn substitution at 283. fliC-g,p,s could be differentiated from fliC-g,p by a Thr>Ala substitution at 254. A motif of two amino acids at positions 302 and 307 was common to S. Derby, S. Agona, S. Adelaide, and S. Berta which exhibit phase 1 flagellar antigenic factors "f" and "g". This motif was exclusive to these serotypes. DNA sequence variation at corresponding positions allowed S. Derby and S. Agona to be distinguished from S. Adelaide and S. Berta. FliC-g,z₅₁; and fliC-m,t with fliC-g,m,t each form distinct clusters (Figure 1).

Summary of fliC sequence variation within the non-g complex

Sequence conservation within alleles that did not encode g or m,t antigenic factors was demonstrated by 97.8 – 99.1% homology and 80.35% homology was measured in the complex. The high level of variability between alleles in this group did not allow association of specific amino acids to epitope formation that was possible with the g-complex sequences. The quantity and distribution of polymorphic bases observed in this group (specified below) meant that there was a choice of regions that could be used for differentiation. Following testing of four possible regions, the region encompassing amino acids 248–250 was selected for use in the final non-g assay. Each serotype had a unique motif at the target region except fliC-l,v and fliC-l,z₁₃ which shared a sequence type (Figure 4).

Some amino acid sequences were not identical within non-g alleles, including i, r, d, e,h, a and z₄,z₂₃ (Additional file 1). A previous study of fliC-i sequences reported no variation in a 260 bp region among seven Typhimurium strains [17]. Six full S. Typhimurium fliC s and a fragment spanning nucleotides 434–1090, corresponding to amino acids 159 – 400, of a further 20 S. Typhimurium strains were sequenced. Three distinct DNA sequences which resulted in translated differences in the expressed peptides were observed within the serotype. Sequence type "Typhimurium_a" was detected in 18 strains, identical to the sequenced strain LT2. Sequence type "Typhimurium_b" was detected in four strains and was differentiated by a SNP at 768, conferring a 256 Glu>Lys substitution. Sequence type "Typhimurium_c" conferred a Glu>Lys substitution and an Ala>Thr substitution at 263 and was found in two strains: 571896 and 571913. Strains 571896 and 571913 were phage type DT104 however, other strains tested did not conform to recognised phage typing patterns so no assured correlation could be made with phage type or other phenotype. S. Choleraesuis sequence (fliC-c) differed from that published (AF159459) at one nucleotide, conferring amino acid substitution of Thr >Ser at codon 99.

FliC sequences of nine S. Heidelberg strains were identical, consistent with the results of a previous report [18]. The published sequence for fliC-r of S. Rubislaw (X04505) differed from S. Heidelberg at three amino acids. The S. Muenchen sequence determined in this study differs in twelve amino acids to the published S. Muenchen (X03395), and differed in 25 amino acids from the S. Duisberg sequence in this study. S. Anatum, S. Newport and S. Saintpaul exhibit factors e,h in their phase 1 flagellar. Amino acid sequence was conserved in two strains of S. Saintpaul but distinct for each serotype due to four amino acid substitutions at codons 192, 213, 238, 356. S. Brandenburg and S. Panama exhibit l,v in the phase 1 antigen, no inferred amino acid differences were detected. FliC-l,v sequences clustered with fliC-l,z₁₃(Figure 1).

FliC from three strains exhibiting the z₄ antigenic factor in phase 1 flagellar were sequenced. Cluster analysis grouped these sequences together in the non-g group although they contain regions of sequence similar to g-complex strains (amino acid positions 96 – 164). Z₄,z₂₄ is distinct from z₄,z₂₃ and z₄,z₂₃ sequences varied within the serotype at seven amino acid positions: 235, 237, 239, 242, 253, 351 and 369. The complex mosaic nature of fliC is evident from analysis of amino acid alignment of sequences in particular strains from subgroups in the SARC collection (see Materials and Methods).

Molecular serotyping assays

By comparison of amino acid sequences coding for antigens of the different serotypes, sero-specific motifs were identified. Individual regions of fliC were selected for the g-group and non-g group to provide unique sequence for as many serotypes as possible, while keeping the assay simple to perform and analyse. Two multiplex PCRs were developed for the production of fliC amplicon of g-complex strains and fliC amplicon of non-g strains. Sero-specific motifs in each amplicon were consequently identified by sequencing-by-synthesis.

G-complex assay

Fifteen sequence types were identified in the 17 bp of nucleotide sequence assayed (Figure 3). Twenty-seven strains were tested and each produced a recognised sequence motif which differentiated between serotypes. Serotypes would be fully resolved through the detection of further polymorphisms, for example g, [s],t and g,t can be separated through additional detection of a A>G change at nucleotide position 777 conferring amino acid Ser>Gly substitution specific to g,t.

Non-g assay

Fourteen sequence types were identified in the 9 bp of nucleotide sequence assayed (Figure 4). Thirty strains were tested, each producing a recognised sequence motif allowing separation of serotypes. Serotypes l,v and l,z₁₃ gave the same motif at the target region but could be separated by nucleotide substitution A>G at position 783 conferring a Thr>Ala change.

The stability of the targeted polymorphisms in Salmonella phase 1 flagellar antigens was demonstrated through testing on a panel of 55 isolates. The SNP responsible for the antigenic difference between serotypes g,m and g,p was within the target region and so could be differentiated by the assay. The amino acid substitution that separated fliC-g,p,u was also encoded within the sequence assayed. Antigens i, r, c, d, b, e,h, k, a, z₄₁, z, z₁₀, z₄,z₂₃, z₄z₂₄, g,q, g,m,p, g,p,u, [f],g,t, g,z₅₁ and biotype S. Pullorum gave unique motifs, l,v and l,z₁₃ shared a motif. Some serotypes for which certain factors may be present or absent (denoted by square brackets in antigenic formulae) were not separated from similar serotypes: [f],g,m, [p], g,m and g,m, [p],s; [f],g,m, [p] and g,m, [t]; g, [s],t and g,t although these could be separated by other DNA polymorphisms as discussed. Two motifs were observed for k, each specific to S. Thompson and IIIb. Two motifs were observed for d, specific to S. Duisberg and S. Muenchen / S. Schwarzengrund. Published sequence data for fliC-m,t, from serotypes S. Banana, S. Oranienburg and S. Pensacola were included in assay design. The polymorphic region targeted by the assay is predicted to differentiate m,t sequences from other g-complex antigens, and also differentiate S. Pensacola from S. Banana and S. Oranienberg. Strains exhibiting factors m,t were not available for testing.

Future work

Alternative sero-specific polymorphisms identified in this study could be exploited by similar assays to allow further separation when antigens did not give unique pyrograms.

The alliance of the fliC assay to a fljB and rfb assay would allow the full antigenic formulae of Salmonella serotypes to be determined. Common phase 2 flagellar antigens will be selected for sequencing and together with published data will be analysed for sero-specific motifs and a short sequence assay designed with the approach described in this study.

In 1993, Luk et a l [8] outlined a simple length heterogeneity PCR for identification of Salmonella major serogroups A, B, C2, and D. They based their PCR on the presence/absence of genes or sequence polymorphisms within shared genes. Essentially, only serogroups A and D possess a gene to synthesise tyvelose but serogroup A genes carry an early stop codon and do not produce the sugar itself. Only groups B and C2 possess a gene to synthesise abequose but the sequences are distinct. We have also designed a preliminary pyrosequencing assay to distinguish these serogroups based on amplification and short sequences of these genes (data not shown).

In summary, epitopes are conformational and it is difficult to determine which amino acids would interact from a linear sequence. However, in the g-complex sequences some amino acid changes could be identified as responsible for differences in antigenic factors because variation was minimal. There is no common factor among the non-g antigens and the sequences are much more heterogenous; there are too many substitutions to draw conclusions about epitope specific sequences. Epitope mapping could be used to further investigate epitopes responsible for antigenic specificity.

Bacterial strains

Strains exhibiting the different phase 1 flagellar antigenic factors were selected from Salmonella Reference Collections A, B and C obtained from the University of Calgary. Multiple isolates of S. Enteritidis phage type 4, and S. Typhimurium phage type DT104 plus a panel of serotyped strains were gratefully received from the Salmonella Reference Laboratory, Health Protection Agency, Colindale (Additional file 2).

DNA preparation, PCR and sequencing

MagNA Pure instrument and Total Nucleic Acid Extraction Kit 1 (Roche, East Sussex). PCR reactions contained 1X PCR buffer, 20 pmoles of FL_START2, 20 pmoles rFSa1 [21], 1 U Taq polymerase, 0.25 mM of each dNTP, 4 mM MgCl₂ (Sigma-Aldrich, Dorset).

PCR amplification of the fliC gene was performed with an 9700 GeneAmp PCR System (Applied Biosystems, Cheshire): 35 cycles of 95°C for 60 sec, 50°C for 60 sec, 72°C for 30 sec followed by a 7 min final extension at 72°C. PCR products were purified with Qiaquick spin columns (Qiagen Ltd, West Sussex) and quantitated by gel electrophoresis using Ready-to-Run pre-cast gels (Amersham Biosciences, Buckinghamshire). Fifty to one-hundred nanograms of the purified PCR product was used for cycle sequencing, with specific primers (Additional file 3) and the CEQ DTCS dye terminator kit (Beckman Coulter, Buckinghamshire). Excess dNTPs were removed from sequencing reactions using GenClean, a 96-well plate format gel filtration system (Genetix Ltd, Hampshire). Sequencing reactions were run on a CEQ 8000XL capillary sequencer (Beckman Coulter). Primers were designed on generated sequence aided by Eprimer3 [22] in a primer walking approach to complete sequencing of the full gene. Sequences generated have been submitted to GenBank (Accession numbers AY649696-AY6497242).

Sequence analysis

Data were analysed and assembled using SeqMan, a component of the DNA Star software package. Multiple alignments were created using BioEdit (Tom Hall, North Carolina State University).

Phylogeny inference package Phylip (Joe Felsenstein, University of Washington) was used to compute a distance matrix from protein sequences and build trees illustrating the relatedness of fliC sequences. Some previously published sequences were included (Additional file 3). Polymorphisms postulated to be serotype specific were identified from the alignments of full fliC sequences; in-house programme MOP-UPs [23] identified motifs and designed primers to user-specified groups of sequences in the alignment (Anthony Underwood, Health Protection Agency, London).

Assay design

Two multiplex PCRs were designed to amplify polymorphic regions of both g- and non-g complex fliC sequences. Amplicon sizes were approximately 316 bp for g-complex strains, 170 bp – 250 bp, (size varied according to serotype) from non-g strains. The order of nucleotide dispensation was tailored to enable the first two dispensations to act as negative and positive controls.

DNA preparation and Pyrosequencing

A 1 μl loop of cells was boiled in 100 μl of sterile distilled water for 10 min at 95°C. One microlitre of lysate was used for PCR. Two PCRs were run in parallel to amplify fragments of the fliC gene. Fifty microlitres of PCR product was prepared containing 1 U Taq polymerase, 0.25 mM of each dNTP, 4 mM MgCl₂. PCR for amplification of g-complex strains used three forward primers: 100 pmol GPYRO-A; 12.5 pmol GPYRO-B; and 12.5 pmol GPYRO-C; and 125 picomoles of reverse biotinylated primer G-REV. PCR for amplification of non-g strains used 14 forward primers (NON-G-PYRO-A, NON-G-PYRO-B etc.) in equal concentrations. Thirteen biotinylated reverse primers (NON-G-REV-A, NON-G-REV-B etc.) were used in equal concentrations. In each 50 μl reaction 125 pmol of mixed forward primer and 125 pmol mixed reverse primer was used. Primer sequences are detailed in Additional file 3.

Thermocycling was performed with an Applied Biosystems 9700 GeneAmp PCR System using a touch-down programme: initial denaturation step of 94°C for 2 min; followed by 17 cycles of 94°C for 20 sec, 66°C (-1°C per cycle) for 30 sec, 72°C for 30 sec; followed by 20 cycles of 94°C for 20 sec, 54°C for 30 sec, 72°C for 30 sec. The excess primers were removed using a filter plate and vacuum system (Genetix Ltd, Hampshire) before visualising the PCR products on the Ready-To-Run agarose system (as previously). Biotinylated single-stranded DNA was immobilized on streptavidin-coated sepharose beads (Amersham Biosciences, Buckinghamshire) with binding buffer. The mixture was agitated at 1400 rpm for 10 min at room temperature. Single stranded DNA bound to beads was isolated from the mixture using a series of wash steps for 5 seconds each in turn, 70% ethanol, 0.2M NaOH and washing buffer. Ninety-six samples were prepared in 2 minutes by automation of strand separation step using a vacuum preparation tool (Pyrosequencing AB, Uppsala, Sweden). A combination of pyrosequencing primers was used for each assay; 20 primers for the non-g complex assay, and three for the g-complex assay (Additional file 3) into which DNA was eluted. Pyrosequencing primers were annealed to single-stranded DNA on the beads by heating to 80°C for 2 minutes and allowed to cool slowly. Single stranded binding protein, enzyme mix, substrate mix and dNTPs (Pyrosequencing) were added sequentially by the instrument according to the programmed dispensation order.