Rapid Detection of Hypervirulent Serovar 4h Listeria monocytogenes by Multiplex PCR

Listeria monocytogenes (L. monocytogenes) is a ubiquitous foodborne pathogen that comprises 14 serotypes, of which serovar 4h is a novel serotype recently reported. Serovar 4h L. monocytogenes belonging to hybrid sub-lineage II exhibit hypervirulent features. Conventional biochemical tests and widely used PCR-based serogrouping schemes could not distinguish serovar 4h strains. In this study, we developed a new multiplex PCR assay for rapid detection of serotype 4h L. monocytogenes. Three primer pairs based on the target genes, LMxysn_1095, lmo1083, and smcL, were designed. The multiplex PCR results showed that serovar 4h strains could be specifically identified from all tested strains, including various L. monocytogenes serovars, Listeria spp., and other species. The detection limits of the multiplex PCR were 291 fg/μL for genomic DNA and 5.5 × 106 CFU/mL for bacterial suspension. Furthermore, pork meat artificially contaminated with serovar 4h L. monocytogenes in a concentration of 1.8 × 103–1.8 × 100 CFU/10 g were successfully detected within 10–16 h. These results demonstrate that the multiplex PCR with high specificity and sensitivity is applicable for the rapid detection of L. monocytogenes serotype 4h strains.


INTRODUCTION
Listeria monocytogenes (L. monocytogenes) is a Gram-positive, facultative intracellular foodborne pathogen that is widely distributed in the environment (Vazquez-Boland et al., 2001;Liu et al., 2005). The bacterium is capable of causing spontaneous abortion, sepsis, and meningoencephalitis with a mortality rate of 20-30% in human (Farber and Peterkin, 1991;Barbuddhe and Chakraborty, 2009). Immunocompromised patients, pregnant women, newborns, and the elderly are at a greater risk of listeriosis (Lomonaco et al., 2009;Radoshevich and Cossart, 2017). Although the minimum infection dose of L. monocytogenes is at least 100 colony forming units (CFU) per gram of food (Yang et al., 2007), considering the possible serious consequences, many countries, including the United States, Australia, and China, have issued a zero-tolerance policy for L. monocytogenes in ready-to-eat (RTE) food (Churchill et al., 2006;Tao et al., 2016).
Serovar 4h was reported by Yin et al. (2019), that is, the 14th serotype of L. monocytogenes belonging to a hybrid sublineage of the major lineage II (HSL-II). L. monocytogenes serovar 4h strains are atypical because they fail to ferment rhamnose and cannot be recognized by the commonly accepted PCR serotyping method of Doumith et al. (2004). Strikingly, L. monocytogenes serotype 4h isolates harbor pan-species virulence genes acquiring from truncated pathogenicity island 2 (LIPI-2) of Listeria ivanovii (L. ivanovii) and gene cluster associated with galactosylation of wall teichoic acid (WTA) from Lineage I serovar 4b strains, which leads to hypervirulent properties of the bacteria (Yin et al., 2019). Therefore, it is urgent to implement robust monitoring and develop a rapid method of detecting L. monocytogenes serotype 4h strains.
In this study, we establish a multiplex PCR to distinguish L. monocytogenes serotype 4h from other serogroups and evaluate the specificity, sensitivity, and stability of this method. Besides, natural-occurring L. monocytogenes isolates and artificially contaminated meat samples were tested by the PCR assay. The multiplex PCR will be a useful tool for detection of serotype 4h L. monocytogenes from different sources of samples.

Bacterial Strains and Culture Conditions
The strains used in this study were commercially available or isolated by our laboratory, including 143 strains of Listeria spp. and 14 strains of other species. All tested strains were grown in brain heart infusion (BHI) (BD) or Luria-Bertani medium (LB) (OXOID), respectively, at 37 • C overnight under constant shaking.

Target Genes
To differentiate serotype 4h L. monocytogenes from other L. monocytogenes serogroups by multiplex PCR, we downloaded various L. monocytogenes serotypes and non-L. monocytogenes genome sequences from the GenBank database 1 . Three target genes, LMxysn_1095 (GenBank accession no. CP007583.1 segment 1113983-1115773), lmo1083 (Gene ID: 986257), and smcL (GenBank accession no. CP007583.1 segment 1245812-1246819), were selected by comparative genomics analysis. The corresponding primers were designed based on the conserved region of target genes by Primer Premier 5.0 software (PREMIER Biosoft International, Palo Alto, CA, United States) and determined by NCBI Primer-BLAST tool ( Table 1).

Genomic DNA Extraction
The extraction of Listeria genomic DNA was conducted by an optimized boiling lysis method. Bacteria culture (0.5-1 × 10 9 CFU) was rinsed with phosphate-buffered saline and then centrifuged at 8000 rpm for 2 min. The pellet was mixed thoroughly with 88 µL of sterile distilled water and 10 µL of lysozyme (20 mg/mL) and incubated at 37 • C for 10 min; after that, 1 µL of protease K (20 mg/mL) and 1 µL of RNase A (10 mg/mL) were added and incubated at 58 • C for 10 min. Finally, the suspension was heated at 95 • C for 5 min and centrifuged at 12,000 rpm for 2 min. The supernatant was used as the DNA template for multiplex PCR. The concentration of genomic DNA was measured by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, United States).

Multiplex PCR
The multiplex PCR mixture contained 12.5 µL of 2× Taq Master mix (Vazyme Biotech Co., Ltd., China), 0.4 µM of each primer, bacterial genomic DNA, and ultrapure water to fill the final volume to 25 µL. The PCR procedure consisted of an initial denaturation step at 95 • C for 5 min; 30 cycles of quantification at 95 • C for 0.5 min, 55 • C for 0.5 min, and 72 • C for 1 min, and a final cycle at 72 • C for 10 min. The amplicons were separated on 1% agarose gels (ethidium bromide dyed) in 1× TAE buffer and visualized using a Gel Doc XR system (Bio-Rad, Hercules, CA, United States).

Specificity Evaluation of Multiplex PCR
The specificity of multiplex PCR for detection of serovar 4h L. monocytogenes was evaluated. The genomic DNA was extracted from 14 strains of L. monocytogenes (including three serovar 4h strains) and 14 strains of other species (Table 2), and 1 µL of genomic DNA was used as the PCR template.

Detection Limits Evaluation of Multiplex PCR
For detection limit evaluation, genomic DNA of serovar 4h L. monocytogenes strain XYSN was extracted and serially diluted 10-fold from 291 ng/µL to 291 ag/µL. Meanwhile, bacteria suspension of the strain XYSN was serially diluted 10-fold from 5.5 × 10 9 to 5.5 × 10 0 CFU/mL, and 1 µL of genomic DNA and 5 µL of bacteria suspension as the PCR template were added to the PCR mixture, respectively. The detection limits of the multiplex PCR were judged by electrophoresis.

Listeria monocytogenes Isolates Testing
Naturally contaminated samples between September 2018 to September 2019 were collected from goat farms, slaughterhouses, and markets in Jiangsu, China, according to the testing methodology for Listeria species or L. monocytogenes in environmental samples (Anonymous, 2015). Briefly, all samples were incubated in UVM broth (BD) at 30 • C for 24 h. Then, the UVM enrichment (100 µL) was transferred to Fraser broth (BD) at 37 • C for 24 h. Afterward, the culture was streaked onto CHROMagar TM plates and incubated at 37 • C for 36 h. Colonies with a blue/green sheen and opaque halos were purified on BHI agar plates and confirmed by VITEK-2 system (bioMérieux, France). To evaluate the efficacy of the multiplex PCR for identifying serotype 4h strains, all L. monocytogenes isolates were further subjected to the developed multiplex PCR assay. Meanwhile, the serovar of isolates was determined by traditional slide agglutination test (Denka-Seiken Co., Ltd., Japan). The results obtained by multiplex PCR and the agglutination test were compared according to ISO 16140:2003(Anonymous, 2003.

Artificial Contamination Experiment
The artificial contamination experiment was performed based on the procedure by Sheng et al. (2018). Briefly, L. monocytogenes serotype 4h strain XYSN was cultured overnight at 37 • C and then prepared at different concentrations ranging from 10 1 to 10 5 CFU/mL. The raw pork was fully sterilized by UV for half an hour in advance. Each dilution of bacteria (100 µL) was sprayed separately on the upper surface of 10 g meat (around 10 cm 2 ). When the bacteria suspension on the surface was completely dry, the artificially contaminated pork was put into sampling bags containing 90 mL BHI medium and incubated at 37 • C for 4, 6, 8, 10, and 12 h. Genomic DNA were extracted from 1 mL of BHI medium and detected by multiplex PCR. The experiment was repeated three times.

Specificity of Multiplex PCR
Twenty-eight bacteria strains were used to detect the specificity of multiplex PCR. The results showed that strains of L. monocytogenes serotype 4h generated two amplicons of 602 and 889 bp for the LMxysn_1095 and smcL, respectively. Strains of L. monocytogenes serovars 4b and 4d amplified the 602 bp band for LMxysn_1095. L. ivanovii generated the 889 bp band for smcL. Strains of L. monocytogenes serovars 1/2a, 1/2b, 1/2c, 3a, 3c, and 7 and L. seeligery generated the 390 bp band for lmo1083. The remaining strains presented no amplicon ( Table 2). It was concluded that the multiplex PCR can specifically differentiate serovar 4h L. monocytogenes from other strains through the characteristic PCR band patterns.

Detection Limits of Multiplex PCR
To determine the detection limits of the multiplex PCR, serially diluted genomic DNA and bacteria suspension of serotype 4h L. monocytogenes were used as the PCR template. As shown in Figures 1A,B, the 602 and 889 bp bands could be observed when the genomic DNA concentration ranged from 291 ng/µL to 291 fg/µL and the bacteria suspension concentration decreased from 5.5 × 10 9 to 5.5 × 10 6 CFU/mL. The results indicate that at least 291 fg/µL of genomic DNA or 5.5 × 10 6 CFU/mL of bacteria suspension are needed for multiplex PCR detection of serovar 4h L. monocytogenes.

Identification of Listeria monocytogenes Isolates
All 129 L. monocytogenes isolates from food and environmental samples were identified by multiplex PCR and serotyped by slide agglutination in parallel (Table 3 and Supplementary Table S1).
These results were consistent with that of the slide agglutination test. All serovar 4h isolates were identified using the multiplex PCR method, and no amplification was found for non-serovar 4h strains, indicating that the relative sensitivity and specificity of the multiplex PCR were both 100%.
Considering the time required for other operation procedures (genomic DNA extraction, PCR, and electrophoresis), it can be speculated that meat samples with the contamination dose less than 1.8 × 10 3 CFU/10 g can be detected within 10-16 h.

DISCUSSION
Listeria monocytogenes is the main etiology of the fatal disease listeriosis, which poses a serious threat to human health. The vast majority of listeriosis clinical cases were caused by L. monocytogenes serotypes 1/2a, 1/2b, and 4b (Vines and Swaminathan, 1998). Hypervirulent serotype 4h L. monocytogenes were discovered and reported from goat listeriosis outbreaks and environments. Newly named serotype 4h can be identified by slide agglutination that reacts positively to O-antigens V/VI and VI and to H-antigens A and B (Yin et al., 2019), but this method is limited by the high cost of antisera and only if it is the pure colony. Thus, establishment of a rapid detection method for serotype 4h L. monocytogenes is of great significance for effective prevention and control of the pathogen. In this study, a multiplex PCR method comprising three primer pairs, LMxysn_1095, lmo1083, and smcL, was developed for detection of L. monocytogenes serotype 4h. The sensitivity of the multiplex PCR was evaluated using both serovar 4h L. monocytogenes pure culture and their genomic DNA. It was found that the detection limit of multiplex PCR using genomic DNA as the template is close to previously reported PCR methods for L. monocytogenes subtyping (Tao et al., 2016;Razei et al., 2017;Sheng et al., 2018). However, the detection limit from bacteria culture was lower than genomic DNA because of the impact of the cell wall, nuclease, and metabolites in bacterial suspension (Sheng et al., 2018). Low contamination levels in samples make it difficult to detect target pathogens; hence, enrichment steps were applied in the process of L. monocytogenes detection (Garrido et al., 2013;Ding et al., 2017;Parichehr et al., 2019). Target bacteria and background flora, such as E. coli and Salmonella, coexist in enrichment medium, which may interfere with the stability of detection methods. Nevertheless, the multiplex PCR was not affected by existence of other strains even though the genomic DNA concentration of background flora was significantly higher than the target bacteria. Contamination of meat products with L. monocytogenes is a public health concern because many human listeriosis outbreaks have been linked to contaminated meat products (Jacquet et al., 1995;Loncarevic et al., 1997;Jensen et al., 2016;Anna et al., 2018). Thus, rapid detection of L. monocytogenes from contaminated meat, etc., provides an effective guarantee for the food safety system. Conventional detection methods for L. monocytogenes are laborious and time-consuming, including procedures of primary isolation with enrichment and selective medium, biochemical analysis and serological tests, which take up to 5-10 days (Grady et al., 2008;Vanegas et al., 2009;Frece et al., 2010). In the artificial contamination experiment of this study, the multiplex PCR could detect serotype 4h L. monocytogenes in meat with a contaminated concentration of less than 1.8 × 10 3 CFU/10 g within 10-16 h, which dramatically shortened the detection time (10-16 h vs. 5-10 days). It is noteworthy that the optimized boiling lysis method was applied to extract genomic DNA directly from enrichment medium as the PCR template. The full process could be accomplished in 25-30 min, which is more proper for rapid extraction of genomic DNA from large-scale samples than the conventional extraction method by kit (25-30 min vs. 3 h).
In general, the multiplex PCR could detect serovar 4h L. monocytogenes in artificially contaminated meat rapidly and accurately, which is expected to be adopted for laboratory or clinical tests of naturally contaminated samples. Up to now, the collection of L. monocytogenes serovar 4h isolates was limited; five 4h strains have been isolated from goat listeriosis and environment. More L. monocytogenes serovar 4h strains are possible to be detected with the multiplex PCR assay established in this study.

CONCLUSION
In conclusion, a multiplex PCR method comprising three primer pairs, LMxysn_1095, lmo1083, and smcL, was proposed in this study. PCR results demonstrate that the multiplex PCR with high specificity, sensitivity, and stability is applicable to detect L. monocytogenes serotype 4h rapidly and effectively. Our research affords a useful detection and identification technique to enhance surveillance for the emergence of hypervirulent serovar 4h L. monocytogenes.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.

AUTHOR CONTRIBUTIONS
YY and XJ designed the experiments. YF, HY, SC, and XS collected all strains, performed the PCR assays, and analyzed the results. YY, XJ, and YF wrote the manuscript. All authors read and approved the final manuscript.