Listeria monocytogenes ubiquitous in the environment and is a major foodborne pathogen affecting public health (de Noordhout et al., 2014). It causes listeriosis, a severe infection characterized by sepsis, meningitis, pregnancy loss, and can even be fatal to immuno-compromised or older patients. Listeriosis accounts for a disproportionate share of the foodborne disease burden, with high hospitalization and fatality rates (Scallan et al., 2011; de Noordhout et al., 2014), ranging from 15 to 30%, the highest among all foodborne infectious diseases (Barton Behravesh et al., 2011; Scallan et al., 2011; Hernandez-Milian and Payeras-Cifre, 2014). As a special pilot project of the National Foodborne Disease Surveillance Plan, human listeriosis surveillance has been implemented in 2013, and the fatality rate of listeriosis was 26.1% in China during 2013−2017 (Li et al., 2019).

L. monocytogenes causes sporadic cases or protracted outbreaks, and even multi-country outbreaks, and often the specific source may not be known (Halbedel et al., 2018). A subtyping method with high resolution, reproducibility, and exchangeability is required for international surveillance and investigation. Different methods are used to subtype L. monocytogenes. Pulsed-field gel electrophoresis (PFGE), the typing golden standard, has been used internationally, but lacks comparability between different networks and sufficient discriminatory power, and does not reflect the evolutionary relationships among strains (Gerner-Smidt et al., 2006). The other standardized genotyping method, multilocus sequence typing (MLST) based on seven genes, is an internationally comparable subtyping method, but lacks the discriminatory power required for epidemiological surveillance (Ragon et al., 2008; Chenal-Francisque et al., 2011; Haase et al., 2014). Whole-genome sequencing (WGS), a powerful epidemiological typing tool, can differentiate isolates that are indistinguishable by other typing methods (Harris et al., 2010; Mutreja et al., 2011; Maury et al., 2016; Moura et al., 2016). Thus, WGS has been widely applied for investigating outbreaks and contamination of food production plants (Schmid et al., 2014; Stasiewicz et al., 2015; Bergholz et al., 2016; Kwong et al., 2016). WGS can also help to predict pathogenic loci in virulent or hypervirulent strains. Furthermore, WGS can reveal population structure and infer evolutionary relationships among strains from a wide range of geographic, temporal and epidemiological origins (Moura et al., 2016).

L. monocytogenes encodes internalins and genomic islands (Listeria pathogenicity islands, LIPIs), that play important roles in pathogen virulence. Internalins help L. monocytogenes invade host cells (Gaillard et al., 1991). Some L. monocytogenes strains isolated from environment and food sources produce a truncated form of the InlA protein, and virulence is reduced (Jacquet et al., 2004; Nightingale et al., 2008). LIPI-1, the main pathogenicity island, is well conserved across strains independent of lineage (Gouin et al., 1994). LIPI-3, encoding a second haemolysin known as listeriolysin S (LLS), is strongly associated with lineage I strains (Cotter et al., 2008). LIPI-4, which includes a cellobiose family phosphotransferase system, is strongly associated with certain lineage I strains that are associated with invasion of the central nervous system (Maury et al., 2016).

In this study, we used WGS to assess the genomic diversity of L. monocytogenes in Beijing, China, to compare methods (PFGE, MLST, cgMLST, and wgSNP) for determining relatedness of the isolates. We also aim to characterize the distribution of virulence determinants of L. monocytogenes, to detect the absence/presence of antimicrobial resistance-encoding genes and their relationship with antimicrobial resistance profiles.

In this study, analysis of 5 years of data (2014−2018) on clinical L. monocytogenes isolates in Beijing, China, showed that pregnancy-associated cases and non-pregnancy-associated cases accounted for half each. Listeriosis is an important disease that needs attention and continuous surveillance. Some listeriosis and deaths might go undiagnosed and unreported (Anand et al., 2016; Kylat et al., 2016; Fan et al., 2019). Serogroups 1/2a,3a and 1/2b,3b,7 were the dominant serogroups in our study, consistent with food source strains in China, but different from clinical source strains from other countries (Mammina et al., 2009; Centers for Disease Control and Prevention, 2014; Chen et al., 2015; Maury et al., 2016; Wu et al., 2016; Jennison et al., 2017). In many countries such as the United States, Australia, France and Italy, serotype 4b is the most commonly identified serotype of clinical L. monocytogenes (Mammina et al., 2009; Centers for Disease Control and Prevention, 2014; Jennison et al., 2017). The distribution of STs in clinical L. monocytogenes strains in Beijing, China differs from those in other countries (Maury et al., 2016; Bergholz et al., 2018; Halbedel et al., 2018). The three most common STs were ST8, ST87 and ST5. While ST8 is distributed globally and ST5 caused several outbreaks in the United States in recent years (Lomonaco et al., 2013; Centers for Disease Control and Prevention, 2015; Jackson et al., 2015), ST87 is seldom linked to human infection in other countries. Approximately 7.5% of STs were ST619 (9 isolates), which has been seldom reported in other countries. The possible reasons for these differences in serogroups and STs are that some clonal groups may not be disseminated widely because of their own characteristics, also there are different types of circulating food, production links, processing techniques and eating habits among regions, resulting in relatively unique clonal groups in different regions. However, some STs prevalent worldwide such as ST1, ST2, ST9, ST121 and ST155 were also identified in this study.

We present a collection of genome-sequenced L. monocytogenes isolates from listeriosis patients in Beijing, China, over 5 years. Typing resolution increased from serogroups (3) to MLST (25 STs) to PFGE (59 PTs) to cgMLST (116 CTs), which is consistent with other studies (Kwong et al., 2016; Moura et al., 2016; Halbedel et al., 2018). In our study, most PTs with ≥ 2 isolates could be further differentiated by cgMLST. Only two isolates with the same CTs were further discriminated by PFGE, with one pattern difference. Genomic variation outside the core genome or variations in intergenic region may account for this variability in isolates with identical CTs. In our study, a maximum of one cgMLST locus and two wgSNP loci were found in isolates from the same case isolated in different sample sources or at different sample times that were expected to be highly related. Each of seven CTs included two or three cases, indicating that isolates in the same CTs are likely to be phylogenetically linked. However, following further retrospective investigation, no epidemiological links between the cases in the same CTs could be determined. Previous research (Moura et al., 2016) showed that most isolates sampled during investigations of single outbreaks had seven or fewer allelic mismatches, and isolates with no documented epidemiological links typically differed by more than 10 mismatches. In our study, we used cgMLST ≤ 7 as a cutoff for the classification of closely linked isolates, and 16 complexes were identified. When wgSNP ≤ 12 was used as a cutoff, eight complexes were found. These results imply that strains in the same complex should arise the potential for common source outbreaks. CgMLST is supposed to become a universal tool for cluster detection and international communication during regional or global listeriosis outbreaks because it improves L. monocytogenes typing and reduces unnecessary epidemiological investigations (Moura et al., 2016). CgMLST typing results do not require a multiple sequence alignment step, and they are easier to be interpreted by microbiologists, epidemiologists and public health professionals (Moura et al., 2016). Simplified cgMLST data can be readily exchanged between global laboratories (Halbedel et al., 2018). However, linking L. monocytogenes isolates to listeriosis outbreaks without epidemiological data is not feasible (Hilliard et al., 2018).

The use of cgMLST analysis helps to determine the population structure, and indicates cross-country and intercontinental transmission of L. monocytogenes (Moura et al., 2016). To date, only a few cross-country outbreaks have been recognized (Schmid et al., 2014; Wang et al., 2018), and most listeriosis outbreaks have gone unreported (Leclercq et al., 2014). In our study, we compared isolates with those from the United States. No closely linked isolates were found between the United States and the isolates in our study, suggesting lack of the transmission behavior of L. monocytogenes. The results revealed some isolates located in the same cluster with those from the United States, suggesting long-standing widespread dispersion. The results also demonstrated that ST87 and ST169 were seldom linked to human listeriosis in the United States. Consistent with our results, a study comparing ST87 isolates from various regions of the world showed that the core gene sequence-based phylogeny grouped the majority of clinical and food isolates from China into a unified clade (Yin et al., 2020).

As discussed above, WGS can be used to identify the presence of genes or pathogenicity islands associated with hypervirulence or particular modes of pathogenesis (Maury et al., 2016). In our study, LIPI-1 was present in almost all isolates, consistent with other studies (Hilliard et al., 2018). LIPI-3 plays a role in gastrointestinal colonization (Quereda et al., 2016), and is strongly associated with lineage I strains (Hilliard et al., 2018). In our study, LIPI-3 was detected in 26 lineage I strains, including CC224, CC3 and ST619 of serotype 1/2b,3b,7, and CC1 of serotype 4b,4d,4e. As shown in other studies (Hilliard et al., 2018), there was no LIPI-3 in CC2 of 4b,4d,4e strains. LIPI-4 was recently described as a gene cluster involved in neural and placental infection (Maury et al., 2016), and it appears to be strongly associated with CC4 isolates (Maury et al., 2016; Hilliard et al., 2018). In our study, there were no CC4 isolates, and this island appears to be associated with CC87 and ST619 isolates, and neural and placental infection. Among 19 pregnancy-associated patients with isolates possessing LIPI-4, 6 (31.6%) patients had abortion or fetal death, 11 (57.9%) patients were cured or infants survived, and the outcome of two cases was unknown. Among 44 pregnancy-associated patients with isolates without LIPI-4, 22 (50.0%) patients had abortion or fetal death, 21 (47.7.0%) patients were cured or infants survived, and the outcome of two cases was unknown. The outcomes of patients with isolates possessing LIPI-4 were different, suggesting that other factors contributing to virulence remain to be characterized. Clinical ST619 isolates were especially specific to China (Wang et al., 2018; Zhang et al., 2019), and harbored the most virulence genes. ST619 isolates carried many virulence genes, including llsX and ptsA that were also found in various food products in China (Wang et al., 2018, 2021; Chen et al., 2019a,b, 2020). However, to date, little information is available on the pathogenicity of ST619 strains, which should be focused on in future studies.

In this study, 13 (8.6%) of the 151 L. monocytogenes isolates were resistant to at least one of the tested antibiotics that are commonly used to treat listeriosis in animals and humans in China. Using WGS, antibiotic resistance phenotypes were established. Antibiotic resistance of L. monocytogenes is not as serious as that in Salmonella, E. coli, Campylobacter and some other organisms. Previous study showed that the frequency of acquired resistance in clinical isolates is low, such as in France since 1926 (1.27%), in Poland between 1997 and 2013 (0.29%), resistance is more commonly observed in animal and food isolates (Morvan et al., 2010; Wieczorek and Osek, 2017; Kuch et al., 2018). In a study on 2,862 L. monocytogenes isolates from food surveillance in China, the resistance rate for tetracycline (8.7%) was the highest, followed by erythromycin (2.2%), trimethoprim/sulfamethoxazole (0.98%) and chloramphenico (0.8%), similar to our current study (Yan et al., 2019). In this previous study, 11 MDR isolates were identified belonging to ST9, and 13 of the other 17 isolates resistant to trimethoprim/sulfamethoxazole belonged to ST155. In our study, six of the 11 antibiotic resistance isolates belonged to ST155. Two isolates were resistant to MRP, but no genes known to encode resistance to MRP were found. More research is therefore needed.

WGS of L. monocytogenes isolates from cases of human listeriosis in Beijing, China, between 2014 and 2018 has provided an overview of locally circulating clinical strains of the pathogen. This work identified particular STs responsible for disease in Beijing, China. CgMLST analysis revealed that isolates from the same patient were highly related, and indicated possible outbreaks, although retrospective follow-up failed to prove any clear epidemiological links. WGS also confirmed the presence of pathogenicity genes or islands and resistance genes.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI BioProject, PRJNA759341.

XZ, QC, and XM conceived and designed the research study. YN and XM performed the sample collection. XZ and YL performed the experiments. XZ, PZ, and YN analyzed the data. XZ and XM wrote the manuscript. All authors read and approved the final version of the article.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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