Newly Designed Primers for the Sequencing of the inlA Gene of Lineage I and II Listeria monocytogenes Isolates

Listeria monocytogenes is a major human foodborne pathogen responsible for listeriosis. The virulence factor Internalin A (inlA) has a key role in the invasion of L. monocytogenes into the human intestinal epithelium, and the presence of premature stop-codons (PMSC) mutations in the inlA gene sequence is correlated with attenuated virulence. The inlA sequencing process is carried out by dividing the gene into three sections which are then reassembled to obtain the full gene. The primers available however were only able to entirely amplify the lineage II isolates. In this study, we present a set of new primers which allow inlA sequencing of isolates belonging to both lineages, since lineage I isolates are the ones most frequently associated to clinical cases. Using newly designed primers, we assessed the presence of inlA PMSCs in food, food processing environments and clinical isolates.


Introduction
Listeria monocytogenes is a ubiquitous bacterium which colonizes several ecological niches, such as soil, water, food, and food processing plants [1,2]. In humans, it is responsible for inducing listeriosis, which can induce various clinical syndromes such as gastroenteritis in healthy individuals, or sepsis, meningitis, encephalitis in immunocompromised patients, the elderly, and neonates, and miscarriage or stillbirth in pregnant women [3,4].
L. monocytogenes exhibits different virulence factors, including the surface protein internalin A (inlA), which mediates the internalization of the bacterium in human intestinal cells through interaction with the E-cadherin receptor [7]. Several studies have reported that the inlA gene can carry premature stop codon (PMSC) mutations leading to a truncated form of the protein, which is secreted rather than being anchored to the bacterial cell wall, which is associated with its attenuated invasive capacity and to the hypovirulent phenotype of the bacterium [8][9][10][11]. Indeed, PMSCs in inlA occur in 35-45% of food and food-related isolates, but they are rarely observed in listeriosis clinical cases [11][12][13][14]. In addition, the truncated protein may influence stress tolerance, such as cold and desiccation sensibility, adhesion capability, and biofilm formation, giving a possible explanation for the persistence of certain L. monocytogenes strains in food processing environments [15,16]. Nowadays, a total of 29 PMSC mutation types have been found (https://bigsdb.pasteur.fr/listeria/; accessed on 7 June 2022).
In the literature three different approaches are reported to evaluate the presence of PMSCs in the inlA gene. The first consists of four pairs of primers which cover the whole 2 of 8 gene of isolates belonging to both lineages; the second one is based on a pair of primers for a real-time PCR assay which detects three specific PMSCs; the last approach uses three pairs of primers that have been tested on lineage II isolates only [17][18][19]. The latter has several advantages, with the major one being represented by having to sequence fewer fragments, which consequently decreases the cost for reagents, the time for the assembly and analysis of the inlA sequence, and the possibility of identifying all 29 PMSC mutations. The major drawback of this protocol is that sequencing of the whole gene is possible only for lineage II isolates.
Therefore, to overcome the issues linked to inlA amplification of lineage I isolates, the aim of the present report is to provide an updated primer set for the sequencing of the inlA gene of L. monocytogenes isolates belonging to both lineage I and lineage II to evaluate the presence of PMSCs.

Results
Primers found in the literature previously described by Gelbíčová et al. [19] allowed inlA sequencing of isolates belonging to lineage II ( Figure 1a) and the sequencing of the first two parts of the gene of isolates belonging to lineage I (Figures 1b and S1a).
Indeed, the sequencing of the whole gene of lineage I isolates (serotype 4b and serotype 1/2b) is not possible due to the presence of mismatches in the annealing region of the third pair of primers (Figures 2 and S2).
gene of isolates belonging to both lineages; the second one is based on a pair for a real-time PCR assay which detects three specific PMSCs; the last approac pairs of primers that have been tested on lineage II isolates only [17][18][19]. Th several advantages, with the major one being represented by having to sequ fragments, which consequently decreases the cost for reagents, the time for th and analysis of the inlA sequence, and the possibility of identifying all 29 P tions. The major drawback of this protocol is that sequencing of the whole gen only for lineage II isolates.
Therefore, to overcome the issues linked to inlA amplification of lineag the aim of the present report is to provide an updated primer set for the seque inlA gene of L. monocytogenes isolates belonging to both lineage I and lineage II the presence of PMSCs.

Results
Primers found in the literature previously described by Gelbíčová [19] in the four major serotypes of lineage I and II.
Considering this, new primers were designed using degenerate bases (Y, V, R, S) to allow for the sequencing of the third inlA fragment in isolates belonging to both lineages (Figures 1c,d and S1b).
The new primers from all 40 isolates, of which 17 isolates (42%) were collected from Considering this, new primers were designed using degenerate bases (Y, V, R, S) to allow for the sequencing of the third inlA fragment in isolates belonging to both lineages (Figures 1c,d and S1b).
The new primers from all 40 isolates, of which 17 isolates (42%) were collected from food, 10 isolates (25%) were collected from food-related environments, and 13 isolates (33%) were collected from clinical cases, were successfully sequenced. Among the sequenced isolates, 75% (n = 30) presented a full-length inlA, while 25% (n = 10) exhibited PMSCs. PMSCs were found only in food and food processing plants isolates belonging to lineage II, in particular in 41% (n = 7) of food isolates and in 30% (n = 3) of environmental isolates. All clinical isolates (n = 13) presented a full-length inlA.

Discussion
In this study we designed a new set of primers able to sequence isolates belonging to both lineage I and II in only three fragments. The analysis of lineage I isolates is crucial because they are mainly related to clinical cases and are found less often in food and environmental isolates. In fact, previous studies have demonstrated that strains carrying inlA PMSCs are rarely found in lineage I, and they are usually collected from food and environmental sources rather than clinical cases [20,21]. In light of this, inlA sequencing of isolates belonging to lineage I can be important in order to increase knowledge on the effect of PMSCs on attenuated virulence.
Unlike the primers previously designed, this set of primers enables full inlA amplification of isolates belonging to both lineages and has the advantage of reducing costs and analysis time as three fragments are generated instead of four. Moreover, while whole genome sequencing (WGS) is becoming a common typing approach, a Sanger sequencing protocol for inlA sequencing can still be convenient and straightforward for those settings in which WGS is not yet routinely applied.
It is well-known that isolates from lineage I are generally more virulent than isolates belonging to lineage II. Indeed, our results are in line with the literature as most clinical isolates linked to invasive disease belonged to lineage I [22]. However, as previously reported, 56% (n = 15) of food-origin isolates and environmental isolates were from lineage II underling that this lineage is more related to possible food contaminations than to clinical cases [23]. In the set of isolates selected for this study, inlA PMSCs were found only in L. monocytogenes lineage II isolates, confirming a possible reason for the high prevalence of this lineage in food-correlated products and for the low frequency among clinical cases [11]. These findings are consistent with previous studies carried out in France, the United States, and Italy, in which a significant proportion (30-40%) of L. monocytogenes isolates from food and from food processing plants presented a truncated inlA, while all isolates from human listeriosis cases had a full-length protein [11,24,25].

Isolates Collection
Among L. monocytogenes isolates previously typed with MLST and collected from food, food processing environments, and clinical cases during routine surveillance between 2013-2022 in Lombardy, 40 were selected for sequencing to obtain a set of isolates representative of the different lineages (20 isolates belonging to lineage I and 20 isolates of lineage II), sequence types (STs), and origin (food, environment, and clinical) ( Table 1).

PCR Assay
PCR reactions contained: HotStarTaq Master Mix Kit (1X) (Qiagen, Hilden, Germany), DNase-RNase-free water, and forward and reverse primers (0.6 µM). The cycling conditions were as follows: a denaturation step at 96 • C for 15 min and 35 cycles of 30 s at 94 • C, 90 s at 55 • C, and 90 s at 72 • C and a final extension step at 72 • C for 10 min. The successful amplification of PCR products was visualized with capillary electrophoresis on a QIAxcel Advanced System (Qiagen, Hilden, Germany). The PCR products were purified with ExoSAP-IT™ Express PCR Product Cleanup Reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the instructions. Cycle sequencing, for forward and reverse sequences, was performed using BigDye™ Terminator v1.1 Cycle Sequencing (Thermo Fisher Scientific, Waltham, MA, USA) in a GeneAmp ® PCR System 9700 (Thermo Fisher Scientific, Waltham, MA, USA). The products were then purified using the BigDye Xterminator™ Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the instructions. Then, the samples were run on an Applied Biosystems Seqstudio Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) with the long_BDX run module. After sequencing, the three consensus sequences for each sample were aligned and assembled in a single sequence of 2400 bp with software MEGA version 6 (Molecular Evolutionary Genetics Analysis Version 6.0) [26] using the inlA sequence as a template with accession number MG922918.1.

Conclusions
The assessment of L. monocytogenes pathogenicity is a discussed concern and inlA analysis has a key role to play in the virulence evaluation. Given the high prevalence of inlA mutations in food and environmental isolates rather than in clinical isolates, the detection of PMSCs may provide information to support studies that assess virulence variability among isolates.
In light of this, the set of primers presented in this study allows for the efficient amplification and, consequently, the sequencing of the entire inlA gene of L. monocytogenes isolates belonging to both lineage I, and lineage II and may prove to be useful for assessing the pathogenic potential of L. monocytogenes isolates from different origins.