Some can cause especially severe illnesses when they infect people; while others cause milder illnesses. New technology is transforming how we detect and investigate outbreaks. Watch the video to learn more. Some groups of people , such as older adults, people with weakened immune systems, and children under five years old have a higher risk for Salmonella infection.
Infections in these groups can be more severe, resulting in long-term health consequences or death. More than 2, serotypes have been described for Salmonella ; but, because they are rare, scientists know very little about most of them. Less than serotypes account for most human infections. What we learn about the more common serotypes can help us better understand illness and the natural history of all the Salmonella strains.
Serotyping is a subtyping test based on differences in microbial e. Serology refers to the antibodies that form because of a viral or bacterial infection. Serotyping is sometimes referred to as serology, but this is technically inaccurate.
Since the s, public health scientists in the US have used serotyping to help find Salmonella outbreaks and track them to their sources. Prevalence of Salmonella serotypes in humans.
The distribution of Salmonella serotypes among 88 isolates from humans a , including 61 isolates from asymptomatic people b and 27 isolates from patients c. Besides, Antimicrobial resistance of human Salmonella isolates. The percentage of strains with resistance to each antimicrobial agent. These top three antimicrobials were also seen in the 61 isolates from asymptomatic people. However, ampicillin Resistance to all of the three antimicrobials was seen in Among the 88 isolates, the S.
Meleagridis F2—6 and S. Rissen F5—5 strain showed resistance to amikacin and imipenem, respectively Fig. Four isolates belonging to three serotypes S. Parytyphi A, S. Enteritidis, S. Typhimurium were resistant against polymyxin E, an antibiotic medication used as a last-resort treatment for MDR gram-negative infections, and all four strains were isolated from patients.
Enteritidis F6—1 strain displayed resistance to both polymyxin E and polymyxin B. The 88 strains were then subjected to the PFGE analysis with the restriction enzyme Xba I for inter-serotype differentiation to reveal their genetic relationship.
Rissen strains isolated from both patients and asymptomatic people Fig. Among the 23 strains belonging to E1 and E4 groups, 8 genetic profiles designated X01 to X08 were presented to show the genetic difference of 6 serotypes Fig. Meleagridis strains, respectively Fig. Except for one strain of S. PFGE analysis of Salmonella strains. Derby c , S. Typhimurium d , and S. Enteritidis e. The strains in red box were isolated from patients.
Typhimurium including S. Typhimurium monophasic variants divided by PFGE analysis. Among the 34 strains belonging to the B group, S. Derby took up Typhimurium strains Fig. Derby with smear patterns, the other 9 strains displayed 5 PFGE profiles X01 to X05 with X01 and X04 as predominant profiles, each of which are shared by 3 strains Fig. The only one strain F10—3 isolated from the patient belonged to the X04 genetic profile Fig. In our study, S. Enteritidis is the only detected serotype in the D group.
Enteritidis strains. Salmonella is one of the major pathogens causing human diarrhea, which is closely related to the consumption of bacterially contaminated foods. Typhimurium monophasic variants have been the top 2 serotypes causing human salmonellosis [ 6 , 7 ].
Our study showed that S. Typhimurium is the predominant serotype from diarrhea patients in Nantong city, followed by S. Enteritidis, correspondent to the previous reports in China [ 8 ].
However, in asymptomatic people, S. London became the predominant serotype as well as S. Typhimurium, followed by S. Derby and S. The difference in serotype distribution in patients and asymptomatic people reflected that many NTS serotypes could infect humans without any symptom, but these serotypes were underestimated in the existing surveillance system for mostly patients.
Additionally, these NTS serotypes have frequently been isolated from pig, chicken, and their associated meat products [ 14 , 21 ], implying the potential transmission of Salmonella from animal foods to humans. Except for S. Typhimurium causing disease or no symptoms, S. Rissen displayed a similar characteristic to S. Typhimurium in human infection Table 2.
In , S. Rissen were in the asymptomatic state as well as in our study Table 2. However, some serotypes have been mainly isolated from patients, such as the typhoidal S.
Among the used 26 antimicrobial agents belonging to 8 different types, Among the 61 Salmonella isolates from asymptomatic people, This demonstrated that the NTS organisms from asymptomatic people showed strong resistance to antimicrobials as well as the human diarrheal or bloodborne isolates, which is similar to the report that However, the result was different from the recently reported fewer MDR NTS isolates in asymptomatic children than in symptomatic individuals in Vietnamese [ 26 ].
The emergence of ESBL-producing Salmonella may cause a substantial increase in treatment costs and prolonged treatment periodicity [ 27 ]. Although the whole-genome sequencing WGS -based typing methods have been considered highly discriminative epidemiological tools, PFGE has a relatively high concordance with epidemiological relatedness [ 28 ]. Newport, and S. Mabandaka, which is potentially caused by the integration of new genetic elements for adaption to adverse conditions [ 29 ].
Derby is another frequently reported serotype isolated from both human and animal or animal foods. Most isolates were obtained from asymptomatic people, revealing that it is not the predominant serotype causing severe infections in humans. Derby strains were divided into 5 profiles by PFGE analysis, which has been confirmed as a molecular subtyping method used to differentiate S.
Derby strains Fig. Derby strains of human origin with 9 antimicrobial resistance patterns [ 30 ]. With difference to S. Typhimurium is the predominant serotype causing either human diarrhea or asymptomatic infection [ 30 ]. PCR analysis according to Woods et al. Choleraesuis yielded the serotype Choleraesuis 3. In this case, SeqSero 1. SeqSero 2. We conclude that the rough phenotype of this particular isolate had a genetic basis.
We found five cases of prediction failure when using SeqSero 1. Choleraesuis and S. Except for isolate ERR, the remaining four cases had an intact wzy allele but only few reads mapped to the O-7 locus Figs. S4 and S5. When we performed the analysis with SeqSero 2. In summary, SeqSero 1. Currently an alpha test version of SeqSero 2. The reason for initial miscorrelation was mainly low data quality, which could be resolved by repeating the sequencing Fig. We conclude that with the exception of atypical monophasic variants of S.
Typhimurium and other serovars and genetically rough strains i. Notably, all six rough isolates were assigned to a sequence type ST and a corresponding serotype.
As expected, there is a clear correlation between serotype and one or more closely related STs for the majority of isolates Fig. The S. Colors are based on ST. ND means no official eBG number available from Enterobase. It is notable that for the majority of isolates in Enterobase the antigenic formula is not provided by the user. Nevertheless, the majority of Enterobase strains belonging to ST 34, which had an antigenic formula provided, represented monophasic S.
Typhimurium out of isolates as of May Therefore we assigned all ST 34 strains to monophasic Typhimurium. Enterobase strains belonging to ST 19 were a mix of monophasic and biphasic Typhimurium. We opted to classify all ST 19 isolates as biphasic Typhimurium although this would result in a high error rate.
We preferred this to no classification at all. We obtained correlating results between MLST and classical serotyping for 17 out of 19 Typhimurium strains. Only 32 out of 52 biphasic S. Typhimurium belonged to ST 19 We also checked whether the classification of monophasic and biphasic S. Typhimurium would be improved by clustering according to core genome MLST.
Figure 2 depicts a minimum spanning tree of only monophasic and biphasic S. The isolates cluster according to their ST rather than to their flagellin expression. Minimal Spanning tree of monophasic and biphasic S. The tree reveals that S. Typhimurium isolates cluster according to ST rather than expression of flagellin. Colors are based on phase and STs. Choleraesuis isolates of our collection, phenotypically lacking FliC were also not correctly classified by MLST typing. In Enterobase monophasic S.
Kunzendorf predominantly belonged to ST 66, whereas our isolates belonged to ST Paratyphi B var. Java as such, since ST 42 mostly consists of monophasic var. Java entries in Enterobase. Different but related STs can be merged to eBurst groups eBG by an algorithm, which identifies the relationship of isolates with highly similar genotypes The majority of our serotypes could each be assigned to a single eBG: e.
Typhimurium to eBG 1, S. Enteritidis to eBG 4, S. Typhi to eBG 13 and S. Choleraesuis to eBG 6 Table 2. This is also reflected in the phylogenetic tree, where the different STs, which comprise the same serovar and belong to the same eBG are located in the same branch Fig. This indicates that German strains belonging to these serovars originate from a common ancestor 4 , Different serovars with the same antigenic formula split into distinct eBurst groups e.
Choleraesuis eBG 6 and S. Paratyphi C eBG MLST additionally provided important phylogenetic information, e. Derby strains in our collection were of a polyphyletic nature as they split into three different eBG Table 2 and Fig. In conclusion, MLST-based serotype prediction also proved to be very successful with the draw-back of not being able to distinguish between monophasic and biphasic S. Typhimurium as well as between S.
Choleraesuis and monophasic S. After performing both analyses independently, we combined SeqSero 1. In general, there was good agreement between the two methods. In case of disagreement, we evaluated the sequences individually. Since our findings indicated that MLST was not suited for identification of phase variation and SeqSero generally performed better in this regard, we rated the SeqSero result as more adequate.
Typhimurium isolates, which were also not correctly classified by MLST. In summary the combination of both independent methods enabled the identification of potential misclassifications where a closer analysis was necessary and thus reduced the rate of error.
In this study we evaluated two genome-based in silico approaches and their combination for predicting Salmonella serotypes and their suitability for replacing classical serotyping. Table 3 summarizes the advantages and drawbacks of the three typing methods.
We found that both tested prediction methods, the in silico serotyping approach by SeqSero 1. Since our collection lacked a representative selection of strains of rare serotypes or higher subspecies we cannot rate the performance in this regard. Nonetheless it was representative of the most common clinical strains in Germany.
Our collection also included a novel serovar, derived from an outbreak related to sesame seeds Interestingly, the antigenic formula of this novel serovar was correctly identified by SeqSero demonstrating its effectiveness for classifying novel serovars.
However, the correlation rate found by Zhang and colleagues dropped to Recently, the developers of SeqSero presented a new version of the program named SeqSero 2. We did not test the assembly mode since it required the additional program SalmID, which we did not include in our assessment.
We tested SeqSero 2. We found that with the default settings, SeqSero 2. Food-borne outbreaks are epidemiologically investigated and reported Hugas and Beloeil, Food safety management systems including good manufacturing practices are put in place to ensure food safety. Salmonella detection is tested by a reference method ISO , or validated alternative methods. Once a presumptive Salmonella is detected, the isolate must be confirmed and often the serotype is identified.
Serotyping remains the first step to characterize Salmonella isolates although it does not provide sufficient discriminatory subtyping for outbreaks investigation. The traditional method to determine a Salmonella serotype is a phenotypic method, based on the WKL scheme Grimont and Weill, The serotype is determined by agglutination of the bacteria with specific antisera to identify variants of somatic O and flagella H antigens.
This provides the antigenic formula of the strain associated to the name and subspecies of the serotype. To date, 46 O antigens and H antigens are identified that, in various combinations, characterize more than reported serotypes Issenhuth-Jeanjean et al. The drawback of the traditional phenotypic method is that it requires the availability of more than specific antisera and well-trained personnel to correctly interpret the results Wattiau et al.
Consequently, it is not possible for all laboratories to carry out this method in-house, and often, laboratories have to send the isolates to a national reference laboratory, an expert laboratory or a commercial laboratory.
This process can significantly delay the time to result. In terms of performance, the method may give false positive reactions due to weak or non-specific agglutination. Autoagglutination or loss of antigen expression, as observed for rough and non-motile isolates, results in unidentified serotypes Wattiau et al. Numerous alternative methods have been developed Wattiau et al. With the advent of NGS and the decrease in sequencing cost, WGS is becoming increasingly more affordable and represents a powerful tool for pathogen subtyping, source tracking and characterization such as virulence and antimicrobial resistance gene profiling Deng et al.
Since serotyping remains the first step in Salmonella characterization, several in silico platforms utilizing WGS data to predict the serotypes have been developed Zhang et al. While WGS is increasingly used in routine by public health and regulatory agencies and authorities e. The use of WGS and its application including Salmonella serotyping in routine laboratories are becoming a viable option. In our study, we compared the traditional phenotypic method to the CTS assay, a proprietary method commercialized by Check-Points Netherlands and in silico platform SeqSero.
It provides a fast and easy-to-use platform for Salmonella enterica subsp. The latest version of the method also includes O and H gene markers and the current database contains patterns for over serotypes personal communication. SeqSero mainly targets genetic determinants of O and H antigens, including the fliC and fljB genes and the wzx or wzy genes in the rfb region Zhang et al.
This is the first study that compared the traditional serotyping using antisera to a rapid proprietary molecular method CTS and a WGS-based serotyping by serotype determinants SeqSero with the same set of Salmonella strains covering 45 serotypes. One hundred Salmonella enterica subsp. Only Salmonella enterica subsp. Typhimurium monophasic variants and its close antigenic formula related serotypes S. Lagos, S. Agama were also included to challenge the methods.
Where possible, each serotype was tested with three different strains Table 1. All strains previously have been part of the EURL- Salmonella Proficiency Testing schemes for serotyping, and originated from human, food or environmental sources 3. Strains were originally received on agar transport tubes and purified on blood agar before actual serotyping. Identification of a monophasic variant of S. Typhimurium was confirmed by PCR Tennant et al. After amplification, the PCR products are hybridized to a specific location on the microarray due to the presence of an unique DNA sequence, or zip, incorporated in the PCR primer that is complementary to a sequence on the microarray.
The proprietary genomic markers that were amplified then become visible on unique locations, creating a microarray hybridization profile that can identify and discriminate between different S.
The proprietary software of Check-Points B. Subsequently, the software compares the genovar code to a database and provides the end-user with a S. Salmonella strains were processed as described in version 9. The incubation of the exonuclease was performed for 30 min. These modifications are the updated protocol, designed to reduce overall time-to-result without eliminating steps or altering the chemical reactions.
Computer analysis was performed as described in the user manual version 9. Check-Points B. After incubation, 1 ml of BHI broth was transferred into an Eppendorf tube and centrifuged at g for 5 min. Supernatant was then discarded and the pellet was collected for DNA extraction. DNA was normalized at 0. A final AMPure beads purification at ratio 0. The equimolar pool was assembled using a Hamilton robotic platform. The pool of samples including positive and negative controls, was sequenced on a HiSeq platform Illumina using Rapid v2 chemistry in PE The FastQC software v0.
SeqSero analysis was performed using two versions of the software: published SeqSero 1 5 and the alpha test version SeqSero 2 6 from September for which new algorithms were implemented. SeqSero 1 and SeqSero 2 allow serotyping prediction from raw reads and genome assemblies. Both types of data for all the strains used in this study were tested with SeqSero 1 and SeqSero 2. In addition, SeqSero 2 provides a k-mer based algorithm which allows rapid serotype prediction from raw reads seconds per genome and improves serotype prediction from draft genome assemblies.
The allele microassembly workflow performs targeted assemblies of serotype determinant genes, instead of assembling the entire genomes for serotype prediction. Trimmomatic v. It was used with the options PE -phred33 leading: 20 trailing sliding window minlen SKESA v.
A total of Salmonella enterica subsp. Typhimurium and its monophasic variant and different O groups were included in this study. Overall results are presented in Table 2. Multiple prediction indicates that the alternative method proposed several serotype predictions, which included the serotype determined by the traditional phenotypic method. In the case of CTS assay, a genovar code is assigned.
Disagreement indicates that the result obtained with the alternative method is different from the traditional phenotypic method. All CTS results were obtained with a novel protocol, designed to reduce overall time-to-result without eliminating steps or altering the chemical reactions. Ninety three out of strains tested with the CTS assay were assigned with a unique serotype, were concordant with the traditional phenotypic method results.
Two disagreement PIR S. Brandenburg instead of S. Poona, PIR S. Typhimurium instead of 1,4,[5],i:- were observed.
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