Rapid screening of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg and Typhimurium using a serologically-correlative allelotyping PCR targeting the O and H antigen alleles

Background Classical Salmonella serotyping is an expensive and time consuming process that requires implementing a battery of O and H antisera to detect 2,541 different Salmonella enterica serovars. For these reasons, we developed a rapid multiplex polymerase chain reaction (PCR)-based typing scheme to screen for the prevalent S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Results By analyzing the nucleotide sequences of the genes for O-antigen biosynthesis including wba operon and the central variable regions of the H1 and H2 flagellin genes in Salmonella, designated PCR primers for four multiplex PCR reactions were used to detect and differentiate Salmonella serogroups A/D1, B, C1, C2, or E1; H1 antigen types i, g, m, r or z10; and H2 antigen complexes, I: 1,2; 1,5; 1,6; 1,7 or II: e,n,x; e,n,z15. Through the detection of these antigen gene allele combinations, we were able to distinguish among S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. The assays were useful in identifying Salmonella with O and H antigen gene alleles representing 43 distinct serovars. While the H2 multiplex could discriminate between unrelated H2 antigens, the PCR could not discern differences within the antigen complexes, 1,2; 1,5; 1,6; 1,7 or e,n,x; e,n,z15, requiring a final confirmatory PCR test in the final serovar reporting of S. enterica. Conclusion Multiplex PCR assays for detecting specific O and H antigen gene alleles can be a rapid and cost-effective alternative approach to classical serotyping for presumptive identification of S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.


Background
There are approximately 15 cases of salmonellosis per 100,000 persons annually in the United States, more than double the 2010 Healthy People goal of 6.8 cases/ 100,000 individuals per year [1]. In order to reduce human illnesses, epidemiological measures have been implemented to reduce the source(s) of infection. Because food animals and poultry are recognized as important reservoirs of Salmonella [2,3], the United States Department of Agriculture (USDA) Food Safety Inspection Service (FSIS) implemented an "in plant" Hazard Analysis and Critical Control Point (HACCP) program to reduce the prevalence of foodborne pathogen contamination in meats, eggs, and milk. Although in-plant HACCP programs have been successful, further reductions in Salmonella contamination may require application of a risk reduction strategy to the farm environment. On-farm control programs have been effective in the past when they have been directed against vertically-transmitted S. enterica serovars (such as S. enterica serovar Enteritidis and S. enterica serovar Gallinarum) [4], but it is unclear whether this approach could be effective against all serovars. A more achievable goal may be to mitigate those S. enterica serovars that are most frequently associated with severe human illness. To further reduce Salmonella contamination in or on the final food product, producers may need to reduce its prevalence in animals brought into the meat processing plant. Producers may also need to accurately identify the source of Salmonella within a specific setting, in order to identify the points where an intervention [5] may be effective. Such an approach would require knowing whether these serovars are present on the farm. Also, determining the appropriate S. enterica serovar is a necessary first step in any epidemiological investigation of foodborne outbreaks; followed then by strain typing, using molecular based methods including pulsed-field gel electrophoresis (PFGE) [6] or amplified fragment length polymorphism that is needed to match patient strain to source [7]. Serotyping can be a formidable task because of the numerous antisera required and the expertise necessary for interpreting the agglutination reactions, thereby limiting its efficacy as a large scale screening tool.
Salmonella serotyping is based on the identification of the variable O and H antigens. Because the antigenic composition of the O, H1 and H2 antigens are a reflection of their unique DNA sequence alleles [11,12], PCR and similar nucleotide-based methods have made it possible to accelerate the identification of serotypes based upon the identification of unique genes or gene arrangements [13][14][15][16][17][18] and use as a diagnostic tool [19]. We report here on the development and validation of a serologically-correlative PCRbased assay that could solve a number of the logistical challenges faced by diagnostic and food microbiology labs.

Multiplex PCR differentiation of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium
We developed multiplex PCRs targeted to the O, H1, and H2 alleles associated with four S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific PCR primers to identify specific Salmonella serogroups, H1 and H2 alleles were designed based on the divergence of the glycosyl synthase genes, the unique linkage between two genes for a specific O-antigen of Salmonella, or allele-specific sequences within the hypervariable region of H1 and H2 antigen genes. In the primer design, a unique amplicon size was selected in order to facilitate development of a multiplex PCR (Table 1, Fig. 1 &2). The ability of the multiplex PCR to correctly identify serogroups ( Fig. 1) was evaluated for 239 Salmonella isolates representing fortythree different serotypes which belonged to one of the six major serogroups, A, B, C1, C2, D1 and E1. With the exception of serogroups A and D1, which produce the same size amplicons (Kappa = 0.98), the multiplex PCR accurately distinguished salmonellae belonging to serogroups B, C1, C2, and E1 (Kappa = 1.00) ( Table 2). The inability to distinguish serogroups A and D1 is due to the high degree of nucleotide sequence homology between the prt (paratose synthase) genes [20]. The fliC multiplex PCRs successfully detected the H1, i, r, or z 10 , alleles ( Fig.  2A) and no amplicons were produced for serovars with other H1, flagellins (Kappa = 1.00) ( Table 2). However, the fliC g,m primer set produced the same size amplicon only for salmonellae that possessed both the g and m, or g alone, or either epitope, g or m, in combination with other serotype-specific epitopes, or non-motile salmonellae that possess the fliC g,m allele [21] and therefore it did not have the specificity of the other H1 primer sets (Kappa = 0.58 vs. 1.00) ( Table 2). To complement our PCR-based H allelotyping, a fljB multiplex PCR was designed to detect the H2 antigen alleles by targeting conserved regions within fljB alleles encoding the antigen complexes I: 1,2; 1,5; 1,6; 1,7 or II: e,n,x; e,n,z 15 and producing unique size amplicons ( Table 1, Fig. 2B). The expected size amplicons were produced for only those S. enterica serovars belonging to H2 antigen complexes I: 1,2; 1,5; 1,6: 1,7 and. II: e,n,x; e,n,z 15 (Fig. 2B). The H2 multiplex PCR however could not distinguish H2 1,2 allele (Kappa = 0.75) or e,n,x (Kappa = 0.54) among the different H2 alleles within each antigen complex; for example indistinguishable amplicons were produced for Salmonella isolates bearing 1,2 vs 1,5; 1,6; or 1,7 ( Table 2).

Comparison of multiplex PCR allelotyping of O, H1, and H2 genes with conventional serotyping in differentiating S. enterica serovars Enteritidis, Hadar, Heidelberg and Typhimurium
Validation of the allelotyping method is important for its integration with conventional Salmonella culture and typing methods used in diagnostic and food microbiology [22][23][24][25]. We therefore assessed the allelotyping multiplex PCR against the standard conventional Salmonella serotyping method in identifying Salmonella O, H1 and H2 antigens for 43 different serovars of salmonellae isolated mainly from chicken carcasses and poultry environments (Tables 2 and 3).
The allelotyping PCR scheme for identifying S. enterica serovars Enteritidis, Hadar, Heidelberg and Typhimurium is envisioned to work as follows. An initial multiplex PCR is performed to determine which O antigen allele that an isolate possesses and a serogroup designation is given or unknown, based on PCR results. If the isolate possesses O alleles for serogroups B, C2, or A/D1, then a 2 nd allelotyping PCR is done to determine the presence of H2 alleles: i; g,m; r; or z 10 . Based on the results of this 2 nd allelotyping PCR, an H1 allele type can be given an isolate as either being i; g,m; r; z 10 or unknown, if no amplicons with the expected size for the H1 allelotyping PCR are produced. If both O and H1 allelotyping PCR detects O and H1 alleles associated with S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium, then a 3 rd final H2 allelotyping PCR is performed to further differentiate the isolate to serovar level. Therefore, identifying one allele for each O, H1, and H2 allelotyping PCR, as listed in Table 3; it is pos-   sible to discern the serovar for isolates typed using this PCR-based scheme. For example, identification of serogroup B, H1 i, and H2 I antigen complex by multiplex PCR presumptively identifies the isolate as S. enterica serovar Typhimurium (Sensitivity = 1.00; Specificity = 1.00) (  [8,9], it is still a possibility, and where a reporting laboratory may require confirmatory testing there are additional PCR based tests that can discern these allelic differences to make a final, definitive serovar designation possible [15,16]. Alternatively, the H2 amplicons can be sequenced to definitively identify the H2 allele.
Although several multiplex PCRs have been developed to assist laboratories in identification of S. enterica serovars [15][16][17]22], our results are the first to focus on, validate and describe a PCR-based scheme for assisting diagnostic labs in differentiating S. enterica serovars: Enteritidis, Hadar, Heidelberg, and Typhimurium.

Conclusion
The conventional Salmonella serological serotyping scheme is a time-consuming, labor-intensive and expensive procedure. With this PCR based allelotyping scheme, specific S. enterica serovars can be differentiated rapidly. The method is cost-effective and needs little technical training. This multiplex PCR allows large service laboratories to rapidly identify S. enterica serovars of public health importance including Enteritidis, Hadar, Heidelberg, and Typhimurium and focus conventional efforts towards identification of unusual serovars.

Bacterial strains
The S. enterica isolates used in this study were from multiple animal species, including human, poultry, livestock and wildlife [27][28][29][30], and serotyped by the National Veterinary Service Laboratory (NVSL; Ames, IA) using classical methods ( Table 2). The isolates were used to test the specificity of PCRs specific for O, H1 and H2 alleles described in Table 1. Additional Salmonella isolates of unknown serovars were obtained from two poultry farms in northeast Georgia [25,31]

Isolation and serotyping of Salmonella
We sampled the commercial chicken broiler house environment and chicken carcasses for Salmonella as previously described [31]. The processing, enrichment, isolation and final diagnostic confirmation of Salmonella from samples is described in detail elsewhere [31]. Sero-typing was done using standard serological typing procedures for Salmonella O, H1 and H2 antigens [32].

Multiplex allelotyping PCR for Salmonella O, H1, and H2 antigen genes and differentiating S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium
The O-antigen multiplex PCR was designed to detect serogroup A/D1, B, C1, C2, or E1 specific genes or alleles (  hringer Mannheim), and 1 μl of whole cell template. The PCR was performed with pre-amplification heating as described by D'Aquilla et al. [40]. The program parameters for PCR include an initial five minutes incubation at 85°C, to mix the two PCR reaction mixes, followed by 30 cycles of denaturation (94°C for 1 min), annealing (55°C for 1 min), and extension (72°C for 1 min). Amplicons were separated on 1.5% agarose gel with Tris-acetate-EDTA buffer [41] and ethidium bromide (0.2 μg/ml) at 100 V. The 100-bp ladder (GIBCO/BRL, Gaithersburg, MD) was used as a molecular weight (MW) standard for determining the MW of the PCR products. Various S. enterica serovars belonging to serogroups A/D1, B, C1, C2, E1 were used in the PCR to test the specificity of the primer sets.
The H1-1 multiplex PCR was used to identify isolates with antigens i or g, m; while the H1-2 multiplex PCR was designed to detect isolates with antigens r or z 10 . Finally, the H2 multiplex PCR was created to differentiate isolates with either H2 antigen complexes 1,2; 1,5; 1,6; 1,7; or e,n,x; e,n,z 15 . In order to identify the H1 and H2 alleles, capillary PCR reaction was performed to amplify the alleles of fliC and fljB by three multiplex PCRs with the Rapi-dycler™ hot-air thermocycler (Idaho Technologies; Idaho Falls, ID) [42] in 10-μl capacity capillary tubes. We sought to reduce the expense of reagents and reaction time by utilizing a capillary thermocycler that accommodates very low reaction volumes. The program parameters for the hot-air thermocycler were an initial heating step of 94°C for 1 min; 94°C for 1 sec, 55°C for 1 sec, and 72°C for 20 sec with a slope of 2.0 for 40 cycles; and a final extension at 72°C for 4 min. Amplicons were detected as described above. The specificity of the PCR detection was tested against various Salmonella serovars possessing the relevant fliC and fljB alleles ( Table 2). Escherichia coli LE392 served as a negative control. Whole cell template for all multiplex PCRs was prepared according to the procedures of Hilton et al. [43].

Statistics
Kappa statistics were calculated to evaluate the agreement between the classical serotyping systems and multiplex PCR for each of the antigen groups examined. Sensitivity and specificity of the allelotyping PCR scheme relative to conventional serotyping was calculated for S. enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.