For over 70 years, the Kauffmann-White Salmonella
serotyping scheme has served as the primary method of subtyping Salmonella
and acted as an international language for microbiologists and public health scientists. Traditional methods for identifying serotype can be problematic because production and quality control of the hundreds of antisera required to identify the >2,500 serotypes are difficult and time-consuming, and many isolates can require 3 to 5 days or more for full determination of the serotype. New methods are being developed using DNA-based assays for serotype identification to replace traditional serotyping (4
). Ideally, these new methods should correlate with the Kauffmann-White serotyping scheme in order to preserve the continuity of the epidemiologic information based on serotype and the ability to communicate with laboratories still using traditional methods. Molecular methods based on other surrogate targets may not group the isolates similarly, making it difficult to compare results to historical surveillance data.
In order to preserve the link to serotype determined by traditional methods, we have developed a molecular serotyping assay for the H antigens by targeting the genes encoding the immunologically recognized antigen. In developing the assay, we focused on the 100 most common serotypes among isolates reported from humans in the United States (see Table S1 in the supplemental material). In the United States, more than 161,000 isolates, representing approximately 1,000 different serotypes, were reported to the CDC from 2002 to 2006; however, the 100 most common serotypes accounted for about 98% of the isolates.
The H-antigen assay described here used a 20-primer multiplex PCR to amplify the variable region of the flagellin genes of Salmonella
, followed by a hybridization of the PCR amplicons with 38 antigen-specific probes coupled to 36 different microspheres. The H-antigen PCR and hybridization conditions were optimized to match the conditions reported for the Salmonella
O group assay (6
), which allows the O- and H-antigen assays to be run simultaneously, with only the primer and probe combinations being different.
Signal strength, reported in MFI values, was quite variable between probes. Each probe generally has a characteristic MFI range as well as a typical MFI value for the background. It is unknown why some probes produced stronger MFI values than others (e.g., H:v mean MFI value of 15,042 and H:x mean MFI value of 1,596). Factors such as G+C content, length of oligonucleotide, location of probe target within the amplicon, balance of G+C across the probe sequence, and location of substitutions within the probe have been considered; however, none of these appeared to correlate directly with signal strength. Increased strength of hairpin formation predicted in simulation by probe design software correlated with decreased signal in preliminary tests; therefore, efforts were made to reduce the likelihood of hairpin formation in the design of the probes.
PCR product length has also been suggested to impact signal strength. Preliminary assays with longer PCR amplicons (1,100 to 1,300 bp) produced MFI values in 2,000-to-4,000 MFI value range. However, the levels for five probes (H:2, -m/g,m, -v, -y, and -z29) were unacceptably low (e.g., MFI values of <1,000). To increase the overall MFI values, we designed the 20-primer multiplex PCR described here, which resulted in a PCR amplicon length of 300 to 600 bp. Primers were designed to amplify conserved sequences within multiple antigen groups when possible (e.g., two forward primers and one reverse primer amplify all 1-complex, EN-complex, and L-complex antigen alleles). This strategy maximized MFI signal while minimizing the need for different PCR primers to amplify both fliC and fljB alleles.
In the evaluation of the assay on 500 strains, all probes were specific for the antigen targets for which they were intended; no cross-reactions were observed (). About 10% of the isolates in the evaluation were not fully serotyped, because they possessed an H antigen not detected in the assay. This is higher than what might be expected during routine use because the National Salmonella
Reference Laboratory tends to receive atypical strains for serotype identification or confirmation. Only 381 (76%) of the 500 strains in the evaluation panel were a “top 100” serotype. Among the strains that did not react as expected in the assay (), the majority possessed the H:m,t, -g,s,t, or -1,5 flagellar antigen, each of which is known to exhibit allelic diversity (see Fig. S2 and S3 in the supplemental material). Several other strains that did not react as expected belonged to subspecies II or IIIb; flagellin alleles in non-subspecies I serotypes were shown to be genetically distinct for H:k and -i (26
). Allelic diversity may also be a factor for the flagellar antigen not detected here (H:z in subspecies IIIb and H:z35 and -1,5,7 in subspecies II). Two isolates, serotype I 9,12:l,v:−, did not react with the H:v probe. Sequencing of the fliC
genes from isolates that did not react as expected is needed in order to determine why these strains did not react with the probes as expected.
DNA sequencing of fliC and fljB revealed multiple alleles for some flagellar antigen types. In particular, genes encoding antigens H:a, H:1,5, and H:t consisted of multiple genetic lineages and posed challenges in probe design (see Fig. S1 to S3 in the supplemental material). When a single probe sequence that detected all alleles of a particular antigenic type could not be found, more than one probe, representing the different alleles, was used. All H:a and EN-complex alleles were detected using two probes each. The H:1,5 antigen is encoded by at least five different alleles (see Fig. S2 in the supplemental material). The H:5 probe in the assay detected the most-common H:1,5 lineages. In the evaluation of the assay, six H:1,5 isolates did not react with the 5-1 probe. These were all rare serotypes and may possess one of the H:1,5 alleles not yet detected in the assay.
The G-complex secondary antigens H:m and H:t also had more than one genetic lineage, based on DNA sequence alignment and phylogenetic analysis (J. McQuiston, unpublished observations). The antigenic diversity of H:m is well recognized, and commercially available antiserum is commonly a pool of two different antisera for detection of both H:m types. The different lineages of H:t have been less problematic for traditional serotyping, as they are typically detected with a single, polyclonal rabbit antiserum. Kauffmann described antigenic diversity for H:t that correlates with the allele distribution observed here (17
); however, the specifics of this observation have been lost in the literature over the years. In the evaluation, 19 isolates possessing an H:t allele reacted with the G-complex probe but did not react with the t-1 probe, including all Salmonella enterica
serotype Senftenberg isolates. This result was expected based on sequences of fliC
in these serotypes (see Fig. S3 in the supplemental material); additional probes detecting these alleles are currently being evaluated.
A less common antigen in the EN complex, H:e,n,x,z15, is genetically distinct from the more-common antigens H:e,n,x and H:e,n,z15; however, the secondary complex antigens H:x and H:z15 of H:e,n,x,z15 react with standard H:x and -z15 antisera. H:e,n,x,z15 alleles reacted with the EN-complex probe, but they did not react with the H:x and H:z15 probes; thus, full characterization of H:e,n,x,z15 strains will require additional probes. Similarly, the secondary antigens H:z13 and H:z28 of antigen H:l,z13,z28 are genetically distinct from both H:l,z13 and H:l,z28. H:l,z13,z28 strains reacted only with the L-complex probes and will require additional probes for full characterization as well.
Deletion of a 261-base segment of fliC
has been shown to convert flagellar antigen H:d to H:j in Salmonella enterica
serotype Typhi (7
). The deleted region is flanked by direct repeat sequences, suggesting that the deletion occurs via recombination. Two probes were designed to differentiate H:d and H:j alleles. The H:d probe corresponded to a sequence within the 261-base deletion and detected most H:d alleles. The H:j probe corresponds to a region of fliC
that appears to be unique to the H:d allele found in all Salmonella
serotype Typhi isolates characterized to date. Thus, reactivity with only the H:d probe would be indicative of H:d in Salmonella
serotypes other than Typhi, reactivity with only the H:j probe would be indicative of an H:j variant allele likely in Salmonella
serotype Typhi, and reactivity with both would be indicative of H:d alleles likely in Salmonella
serotype Typhi ().
has been shown to be unique among the Enterobacteriaceae
in that it possesses two different flagellar antigens that are coordinately expressed via a phase variation mechanism (30
). However, many Salmonella
serotypes possess only one flagellar antigen (e.g., Salmonella enterica
serotypes Enteritidis and Typhi); these are commonly referred to as monophasic serotypes. Monophasic variants of typically diphasic serotypes have also been described, where expression of one flagellar antigen in a typically diphasic serotype has been lost (e.g., Salmonella enterica
serotype I 4,5,12:i:−) (8
). Monophasic variants result from loss or inactivation of the flagellin gene itself, from loss of expression of a flagellar antigen gene, or from a mutation in the phase inversion mechanism which prevents switching between the two flagellar types. Loss or inactivation of a flagellar gene can occur through deletion of all or part of the gene and has been described only for fljB
). Nonmotile variants arise through a variety of different mutations that result in the inability to produce functional flagella; the flagellin genes are almost always still intact in these isolates.
In the evaluation, nine variants identified as monophasic by traditional methods were identified as diphasic by molecular methods. Depending on the genetic basis for a monophasic serotype or serotype variant, the isolate may or may not type the same by traditional and molecular methods. When fljB
is absent, as with most commonly recognized monophasic serotypes and many monophasic variants, the isolate will type the same by both traditional and molecular methods. Isolates that still possess a particular flagellar antigen gene but do not express the antigen will be identified by molecular methods, provided that the antigen is represented in the assay. For example, Salmonella
serotype Paratyphi A (antigenic formula, I 2,12:a:−) contains a fljB
H:1,5 allele but commonly does not express it (23
). This allele is detected in the molecular assay. Isolates that become monophasic through the deletion of part of fljB
react differently in the molecular method, depending on the nature of the fljB
deletion. If the portion of fljB
that is homologous to a particular probe is still present enough so that the primers will amplify the target, this portion will react with that probe even though the antigen is not expressed and cannot be detected by traditional methods. Since nonmotile isolates typically still have at least one intact flagellin gene, they can usually be typed as the serotype or partial serotype from which they were derived in the molecular assay. In the evaluation, four isolates that were nonmotile by traditional methods were typed as common serotypes in the molecular method ().
Additional DNA targets are being planned for future versions of the assay in order to improve the accuracy and completeness of results. A PCR-probe combination that detects the conserved regions of fljB
will indicate that fljB
is present when the specific flagellar antigen type encoded by fljB
is not detected in the assay or indicate that fljB
is absent in monophasic strains that have lost the entire gene. A PCR-probe combination targeting sdf
), shown to be specific for Salmonella
serotype Enteritidis, will differentiate Salmonella
serotype Enteritidis from Salmonella enterica
serotypes Gallinarum and Pullorum. A PCR-probe combination for detection of viaB
, encoding the Vi antigen, will help confirm the identification of Salmonella
serotype Typhi, and a three-gene assay will differentiate the Salmonella enterica
biovars Paratyphi B and Paratyphi B var. tartrate+ (formerly Salmonella enterica
Use of the liquid microsphere array format allows for flexibility in assay development. Additional probes for antigens not yet covered by the assay can be added one by one as they become available without disruption or redesign of the assay. At this time, this H-antigen assay cannot completely replace traditional methods, since it does not detect all flagellar antigen types. However, it will greatly reduce the time needed to subtype common serotypes, therefore enhancing surveillance activities for Salmonella
, and dramatically increase the speed for complete serotyping, as phase reversal would no longer be required. The greatest impact of this assay will result from its use in combination with the O-group assay of Fitzgerald et al. (6
). We have found it relatively easy to perform 100 or more O- and H-antigen assays in a single working day. During outbreaks, this assay has been extremely helpful in ruling out nonoutbreak isolates within hours after they are obtained, greatly reducing the workload for traditional methods. The combined O- and H-antigen assay should greatly enhance the serotyping ability of public health laboratories throughout the world.