Performance of MALDI-TOF Mass Spectrometry (VITEK MS) in the Identification of Salmonella Species

Salmonella is a major pathogen causing foodborne infections in humans. Salmonella isolates are identified using biochemical and serological tests, including automated systems such as the VITEK2 system. However, there are few reports on Salmonella identification using VITEK MS. Therefore, we aimed to evaluate the usefulness of MALDI-TOF VITEK MS for Salmonella identification. A total of 1389 Salmonella isolates were identified using VITEK MS ver3.0 or ver3.2. All Salmonella isolates were confirmed by serotyping using the Kauffmann-White scheme, and the results were compared with the VITEK MS results. A total of 1389 Salmonella isolates, including 66 serotypes, were correctly identified at the genus level by VITEK MS. However, these systems failed to correctly identify typhoidal Salmonella. Among the five Salmonella enterica ssp. diarizonae isolates, only one was correctly identified, whereas one and three isolates were partially identified and misidentified, respectively. On the other hand, the VITEK2 system successfully identified all typhoidal Salmonella (Typhi and Paratyphi A) and Salmonella enterica ssp. diarizonae isolates. VITEK MS was useful for identifying Salmonella species isolated from clinical specimens; however, additional biochemical tests, such as the VITEK2 System, should be considered to accurately identify Salmonella ser. Typhi, and Salmonella ser. Paratyphi A.


Introduction
Salmonella is a major pathogen causing foodborne infections, including gastroenteritis and enteric fever, in humans [1]. The genus Salmonella includes two species, S. enterica and S. bongori, which have similar phenotypes and genotypes [2]. Salmonella enterica is the most frequently isolated species of Salmonella and is closely associated with human infections [3].
Salmonella identification is routinely performed using biochemical and serological tests. Biochemical identification using automated systems, such as the VITEK2 system (bioMérieux, Lyon, France), is commonly performed in clinical microbiology laboratories [4]. Owing to advantages of speed and accuracy, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently become a routine identification system [5]. Two common MALDI-TOF MS systems, MALDI Biotyper ® (Bruker Daltonik GmbH, Bremen, Germany) and VITEK MS system (bioMérieux, Lyon, France), are used in clinical laboratories.
The performance of VITEK MS in identifying bacteria, fungi, and mycobacteria has been steadily evaluated [6][7][8][9][10]. However, only a few studies have evaluated the performance of VITEK MS in identifying Salmonella strains and serotypes [11][12][13]. This study aimed to evaluate the usefulness of MALDI-TOF VITEK MS for Salmonella identification using more than 1000 Salmonella strains, including 60 serotypes, isolated from human specimens.

Clinical Isolates
In total, 1389 Salmonella strains isolated from various human specimens such as stool, blood, urine, body fluids, and tissues were included in this study.

Final Identification by Serotyping
Salmonella strains were identified by serotyping using the White-Kauffmann-Scheme with the slide agglutination test for somatic antigens and the tube agglutination test for flagella antigens, as in our previous report [14,15].

Identification by VITEK MS and VITEK2 Systems
All Salmonella strains were identified using the VITEK MS system (bioMérieux, Lyon, France), according to the manufacturer's instructions. The results were interpreted using VITEK MS v3.0, (n = 1167) until October 2019, and VITEK MS v3.2 (n = 222) was used thereafter. A fresh colony was smeared onto a 48-wall target plate and covered with 1 µL of α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution. After drying, the target plate was loaded into the MALDI-TOF VITEK MS system [4]. The developed MS fingerprint was automatically compared to the VITEK MS database v3.0 and v3.2. Escherichia coli ATCC 8739 was used as the quality control strain. Additionally, we tested the VITEK2 system for 93 typhoidal Salmonella, including 20 Salmonella ser. Paratyphi A, 9 Salmonella ser. Paratyphi B, 64 Salmonella ser. Typhi, and 5 Salmonella enterica subsp. diarizonae. The results of the VITEK MS were compared with those of the final identification by serotyping. The difference in the results between the VITEK2 system and VITEK MS for 93 typhoidal Salmonella and 5 Salmonella enterica ssp. diarizonae were analyzed.
We compared the results of VITEK MS v3.0 and 3.2 with those of serotyping and the VITEK2 system for 98 Salmonella isolates (Table 4). VITEK MS v3.0. did not correctly identify typhoidal Salmonella and Salmonella enterica ssp. diarizonae, although these were included in the ver3.0. Only 3 of the 14 Salmonella ser. Paratyphi A isolates were correctly identified, and the other three isolates were partially identified as Salmonella ser. Paratyphi A/Salmonella group. The remaining eight isolates were assigned to the Salmonella group. Most of the Salmonella ser. Typhi was reported to belong to the Salmonella group, although three isolates were partially identified as Salmonella ser. Typhi/Salmonella group. For two Salmonella enterica ssp. diarizonae isolates, Salmonella ser. IIIb 47:r:z was reported as Salmonella group and Salmonella ser. IIIb 48:k:z was reported as Salmonella enterica subsp. arizonae/Salmonella enterica subsp. diarizonae. However, all Salmonella ser. Paratyphi A, Salmonella ser. Typhi, and Salmonella enterica ssp. diarizonae were correctly identified using the VITEK2 system.  There were a few changes in the VITEK MS v3.2 as described in the Methods. VITEK MS v3.2 system identified all six Salmonella ser. Paratyphi A and 13 Salmonella ser. Typhi as Salmonella enterica subsp. enterica. Two Salmonella enterica ssp. diarizonae, including one Salmonella ser. IIIb 47:r:z and one Salmonella ser. IIIb 48:k:z were misidentified as Salmonella enterica subsp. enterica. The remaining one Salmonella enterica ssp. diarizonae was partially identified as Salmonella enterica ssp. enterica/Salmonella enterica ssp. arizonae/Salmonella enterica ssp. diarizonae. All Salmonella isolates were correctly identified using the VITEK2 system.

Discussion
More than 2600 serotypes of Salmonella, including 46 type O serogroups and 114 type H serogroups, have been reported [2]. The two major antigens that determine the serotype are bacterial somatic antigens corresponding to O-polysaccharide and flagella antigens corresponding to flagellin proteins. Accurate serotype identification is crucial because the virulence of Salmonella, especially typhoidal Salmonella, varies depending on the serotype [15].
Salmonella identification has been performed using biochemical and serological tests. Automated identification systems such as the VITEK2 system provide rapid, reliable, and highly reproducible results [16]. Recently, MALDI-TOF MS has been commonly used as a routine identification method [5,6]. However, there are a few previous reports on the performance of VITEK MS in identifying Salmonella [11][12][13]. Guo et al. [17] using 1025 bacteria isolated from clinical specimens, reported that VITEK MS exhibited good performance; however, only two strains of Salmonella were included in the report. Richter et al. [6] reported that VITEK MS exhibited good performance in identifying Enterobacteriaceae at the genus and species levels. They reported that 35 isolates of Salmonella enterica ssp. enterica were included, and 33 and 2 strains were correctly identified at the species and genus levels, respectively, using VITEK MS v2.0. Therefore, they concluded that VITEK MS would be appropriate for the identification of Salmonella at the genus and species levels with high accuracy. Wattal et al. [18] evaluated VITEK MS using 12,003 microbial isolates, including Enterobacterales, other gram-negative bacteria, gram-positive bacteria, yeast, fungi, and mycobacteria. They reported that VITEK MS correctly identified 95.8% of the isolates at the species level. Among the 145 Salmonella isolates, 138 isolates were identified as Salmonella; however, seven Salmonella ser. Typhi isolates showed no identification results. Interestingly, they reported that all 18 Salmonella spp. Paratyphi A and 44/51 Salmonella ser. Typhi were correctly identified at the serotype level. In our study, we evaluated the performance of VITEK MS with numerous Salmonella isolates consisting of 66 confirmed serotypes, and all 1389 Salmonella isolates were correctly identified as Salmonella, consistent with the above studies. Collectively, these findings demonstrate that VITEK MS is suitable for the identification of Salmonella at the genus level.
In our study, only three Salmonella ser. Paratyphi A isolates were correctly identified using the VITEK MS v3.0 among 14 Salmonella ser. Paratyphi A and 51 Salmonella ser. Typhi isolates, which is in contrast with the above report by Wattal et al. [18]. Moreover, three Salmonella ser. Paratyphi A and three Salmonella ser. Typhi isolates were partially identified, whereas the others were identified as Salmonella group using VITEK MS v3.0. In the updated VITEK MS v3.2, the serotypes of Salmonella ser. Paratyphi A, Salmonella ser. Typhi, and Salmonella ser. Gallinarum were removed from the database. Another MALDI-TOF MS device, MALDI Biotyper ® (Bruker Daltonik GmbH, Germany) is also widely used in clinical laboratories. Bastin et al. [19] reported that the MALDI Biotyper could identify 100% of Salmonella isolates at the genus level; however, it failed to correctly identify the serotype for typhoidal Salmonella.
Using the VITEK2 system, Salmonella ser. Typhi isolates were correctly identified at the serotype level. Therefore, additional biochemical tests, such as the VITEK2 system should be performed for the accurate identification of Salmonella ser. Paratyphi A and Salmonella ser. Typhi. Nevertheless, Salmonella ser. Paratyphi B cannot be correctly identified at the serotype level by either the VITEK2 system or VITEK MS, and additional tests are needed.
There are few reports on the identification of Salmonella enterica ssp. arizonae/Salmonella enterica ssp. diarizonae using VITEK MS. In this study, we found that the results for Salmonella enterica ssp. arizonae/S. enterica ssp. diarizonae using VITEK MS v3.0 and v3.2 were not suitable for the final identification of the pathogens (Table 4). Only one Salmonella enterica ssp. diarizonae isolate was correctly identified by VITEK MS. In contrast, one and three Salmonella enterica ssp. diarizonae isolates were partially identified and misidentified, respectively. Nevertheless, these five isolates were correctly identified as Salmonella enterica ssp. diarizonae using the VITEK2 system. Therefore, we believe that biochemical identification systems, such as the VITEK2 system, are useful for the accurate identification of Salmonella enterica subsp. diarizonae.

Conclusions
In this study, we demonstrated that VITEK MS can identify most of the common serotypes of Salmonella in the Salmonella group or Salmonella enterica subsp. enterica with 100% sensitivity. However, additional tests, such as the VITEK2 system, are required to confirm the presence of typhoidal Salmonella spp. (Salmonella ser. Typhi, and Salmonella ser. Paratyphi A).

Institutional Review Board Statement:
This study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board of Inje University Busan Paik Hospital (2022-08-054) with consent exemption.

Informed Consent Statement: Not applicable.
Data Availability Statement: Patient consent was waived because personal information was not used.

Conflicts of Interest:
The authors declare no conflict of interest.