Lineage II (Serovar 1/2a and 1/2c) Human Listeria monocytogenes Pulsed-Field Gel Electrophoresis Types Divided into PFGE Groups Using the Band Patterns Below 145.5 kb

L. monocytogenes isolates

Five hundred four isolates of L. monocytogenes lineage II from cases of human invasive listeriosis in Sweden during 1958 to 2010 were selected for the study. All isolates had been previously characterized by serotyping and PFGE with restriction enzyme AscI according to Graves and Swaminathan (2001) with some modifications (Lopez-Valladares et al., 2014). Among these 504 L. monocytogenes isolates, 483 serovar 1/2a isolates were identified and based on the number and distribution of all fragments in each DNA profile, partitioned into 114 PFGE types. A further 21 serovar 1/2c isolates yielded 9 PFGE types (Table 1). The isolates were kept at −70°C in brain–heart infusion broth (Merck, Darmstadt, Germany) with 20% glycerol.

Grouping of L. monocytogenes isolates

The DNA profiles were analyzed visually and the position of each restriction fragment of each PFGE type was sized with both a concatenated Lambda ladder and Salmonella Braenderup H9812 (digested with XbaI) as reference standards. The PFGE groups were established based on the number and distribution of small bands below 145.5 kb. PFGE profiles with identical banding patterns below 145.5 kb were considered the same PFGE group. For each PFGE group, a reference profile was selected for creating a reference collection with groups being denoted by capital letters. Each L. monocytogenes isolate was then assigned a numerical code. For example, the designation A:1/2a:4A denoted the isolate that belonged to PFGE group A. The second figure indicated that the isolate belonged to serovar 1/2a, the third figure represented the consecutive number of PFGE type for serogroup 1/2 (i.e., 4th AscI profile), and the fourth figure denoted the isolate belonged to variant A (i.e., closely related variants were termed A, B, C, etc.: Tenover et al., 1995). The AscI profiles of L. monocytogenes isolates representing the different PFGE groups are presented in Figure 1.

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FIG. 1.

AscI profiles of Listeria monocytogenes lineage II, human strains. Electrophoretic conditions: 21 h, pulse times 4–40 s, 120° angle, and 14°C. Lane 1 (Lambda ladder PFG Marker No. 340 S); lane 2 (Salmonella Braenderup H9812); lane 3 (group A); lane 4 (group B); lane 5 (group C); lane 6 (group E); lane 7 (group F); lane 8 (group H); lane 9 (group K); lane 10 (group L); lane 11 (group M); lane 12 (group N); lane 13 (group S); lane 14 (group V); lane 15 (group W); lane 16 (group Y); lane 17 (group Ö-10); lane 18 (group Ö-12); lane 19 (group Ö-11); lane 20 (group Ö-6); lane 21 (group Ö-8); lane 22 (group Ö-7); lane 23 (group Ö-9); lane 24 (Salmonella Braenderup H9812); and lane 25 (Lambda ladder PFG Marker No. 340 S).

In silico analyses

In silico means “research is conducted by computer simulations with models closely reflecting the real world” (Apache Software Foundation, 2016). Some sequenced L. monocytogenes strains belonging to lineage II were analyzed in silico with PFGE and restriction enzyme AscI (Bikandi et al., 2004). By selecting a specific strain of L. monocytogenes with known sequence (http://insilico.ehu.es), it is possible to make a theoretical cleavage (in silico) by selecting a desired restriction enzyme, for example, AscI, on the same site.

L. monocytogenes PFGE groups

In a previous study (Lopez-Valladares et al., 2014), 483 human L. monocytogenes serovar 1/2a isolates were distributed into 114 PFGE types and the 21 serovar 1/2c isolates were distributed into 9 PFGE types. In this study, all the PFGE types were further divided into 21 PFGE groups based on the number and distribution of small bands below 145.5 kb. The 114 PFGE types of L. monocytogenes serovar 1/2a could be divided into 20 PFGE groups and the 9 PFGE types of serovar 1/2c into two PFGE groups (Table 1). PFGE group E harbored both 1/2a and 1/2c isolates (Table 2). Small restriction fragments with sizes <33.3 kb were visible in all L. monocytogenes isolates (Fig. 1). Seven PFGE types represented by only one isolate were identified from among the 114 different PFGE types belonging to serovar 1/2a (Table 2). These seven PFGE types all had different configuration, even of small bands below 145.5 kb; thus, forming seven PFGE groups (Ö-6 to Ö-12, Table 2). Although L. monocytogenes sharing PFGE group Ö-8 was isolated in 1987, this group had not been subsequently identified: the other Ö groups have been isolated in recent years (Table 3).

L. monocytogenes isolates representing PFGE groups A, C, and E have been common among human cases over six decades in Sweden, that is, the groups remain. PFGE groups A and E are still the dominant PFGE groups within lineage II and isolates from these groups have been responsible for 294 cases (out of 932) of invasive listeriosis in Sweden during 1958–2010 (Table 3). In addition, two of the most common PFGE types (1/2a:4A and 1/2a:4B) belonging to PFGE group A have been identified in various countries within Europe (unpublished data). PFGE groups A, C, and E have persisted over a few decades and may signify that there is a common marker among the strains within each of the PFGE groups in the small fragments and the strains have a common ancestor. Isolates representing six different PFGE groups (M, N, S, V, W, and Y) were observed sporadically throughout the period studied. Isolates representing PFGE group H, first identified in 1988, have appeared with an increasing trend (Table 3) and since 2000, only three new PFGE groups (M, V, and Y) have been identified, each represented by two isolates per group. Therefore, it is reasonable to assume that the most important PFGE groups of L. monocytogenes in food-borne listeriosis have been identified in Sweden as new PFGE groups rarely appear (Table 3). Most clonal complexes (CC) of L. monocytogenes have persisted over decades and only a small number of new CC have been identified within lineages I and II (Haase et al., 2014). A 1/2a/CC8 strain was causing listeriosis throughout Canada for at least two decades (Knabel et al., 2012).

Association between PFGE groups and serovars

There was an association between PFGE groups and serovars. L. monocytogenes isolates belonging to PFGE groups A, B, C, E, F, H, K, L, M, S, V, W, Y, Ö-6, Ö-7, Ö-8, Ö-9, Ö-10, Ö-11, and Ö-12 shared serovar 1/2a, with one exception. PFGE group E also included two PFGE types sharing serovar 1/2c (1/2c:36 and 1/2c:95) and four PFGE types belonging to either serovar 1/2a or 1/2c (1/2:12A, 1/2:12B, 1/2:93, and 1/2:105, Table 2). All isolates from PFGE group N shared serovar 1/2c (Table 2). However, a few L. monocytogenes isolates with indistinguishable PFGE profiles displaying different serovars are reported (Nadon et al., 2001; Lukinmaa et al., 2003; Revazishvili et al., 2004; Gianfranceschi et al., 2009; Lopez-Valladares et al., 2015).

L. monocytogenes isolates belonging to lineages I and II versus PFGE groups

PFGE band patterns (AscI) <145.5 kb have been used to identify groups within L. monocytogenes lineage I (serovars 1/2b and 4b), with 334 serovar 4b isolates clustered into 30 PFGE types and 8 PFGE groups (Lopez-Valladares et al., 2015). In this study, 483 serovar 1/2a isolates clustered into 114 PFGE types and 20 PFGE groups. Division of serovar 4b (lineage I) isolates in a smaller number of clusters, unlike serovar 1/2a (lineage II) isolates, has also been observed (Bibb et al., 1989; Aarts et al., 1999). In a comparison of the grouping of lineage I isolates and the grouping of lineage II isolates, there was a difference between the two lineages based on the small bands below 145.5 kb. In contrast to the PFGE banding pattern for lineage I isolates, small fragments <33.3 kb were visible in all L. monocytogenes isolates belonging to lineage II. Thus, lineage I isolates and lineage II isolates did not share any PFGE group. Hence, the small bands were able to confirm the relatedness of strains and differentiate lineages I and II. Some isolates from the L. monocytogenes collection were analyzed by multiple-locus variable-number tandem-repeat analysis (MLVA) and the clusters were generally in good agreement with data generated by both PFGE groups and serotypes (Lindstedt et al., 2008).

In study on Staphylococcus aureus (Blanc et al., 2002), the configuration of small PFGE bands (10–85 kb) correlated better with the phylogenetic typing method RAPD (multiprimer random amplified polymorphic DNA) than with large PFGE bands (>85–700 kb). The authors (Blanc et al., 2002) conclude that larger bands, because of their size, should have a greater propensity to change at a faster rate than smaller bands, and proposed this is due to a greater propensity for size instability in larger fragments, as the frequency of random genetic events increases with the size of the DNA fragment. In another staphylococci study (Hallin et al., 2007), PFGE had good concordance with multilocus sequence typing (MLST), suggesting that PFGE is a reliable method for long-term, nationwide epidemiological surveillance studies.

Some sequenced L. monocytogenes strains belonging to lineage II analyzed in silico with PFGE and restriction enzyme AscI (Bikandi et al., 2004) are presented in Table 4. The in silico analyses indicated that the AscI restriction fragments below the size of 145.5 kb were more preserved than the larger fragments. This observation supported the approach of using small fragments for grouping PFGE types into PFGE groups based on the number and distribution of small bands below 145.5 kb.

Several studies covering cases and outbreaks have observed closely related PFGE types (with AscI) that are identical in the region below 145.5 kb. Tham et al. (2007) report two L. monocytogenes strains isolated from a blood sample of one human case of invasive listeriosis; the strains belonged to different PFGE types, but shared the same PFGE group E. In a second case of listeriosis, another two strains were isolated from the bloods simple; similarly, the two strains belonged to different PFGE type, but shared the same PFGE group F. In the United States, two outbreaks of listeriosis are associated with ready-to-eat meat products contaminated with L. monocytogenes serovar 4b occurring in 1998–99 (hot dogs) and 2002 (turkey deli meats: Kathariou et al., 2006). The PFGE profiles (AscI) of the isolates from the 1998–1999 and 2002 outbreaks were closely related (Kathariou et al., 2006). The closely related isolates appeared identical in the AscI-restricted fragments <145.5 kb, that is, the isolates belonged to the same PFGE group. In Denmark, between 2002 and 2012, two closely related PFGE types among 122 L. monocytogenes clinical isolates cases represent a possible outbreak (Kvistholm Jensen et al., 2016). The two PFGE types both belong to clonal complex 8 and sequence type 8 and these closely related isolates are identical in the AscI-restricted fragments <145.5 kb, that is, the isolates belong to the same PFGE group. Although these data suggest a common ancestor among strains sharing a PFGE group, ultimately this must be confirmed through methods such as whole genome sequencing (WGS).

Several typing methods have been compared with PFGE including, MLVA, MLST, and multivirulence-locus sequence typing (MvLST). PFGE has the advantage that it can be performed in the absence of detailed information about the genome (Cooper and Feil, 2004). Although, PFGE typing is labor intensive, it probes the entire genome, whereas, for example, MLST only analyzes nucleotides within specific genes (Revazishvilli et al., 2004). In a comparison of results from PFGE, MLST, and MvLST among L. monocytogenes clonal groups, Cantinelli et al. (2013), observed that MvLST did not provide more discrimination than MLST, and PFGE has a higher discriminatory power than MLST. Besides WGS, all available molecular approaches to genomic comparison represent incomplete information (Goering, 2010). Three different PFGE protocols have been compared by Michelon et al. (2015) with all PFGE profiles being similar in quality and indistinguishable except one profile: the PFGE profiles “are both similar and comparable” (Michelon et al., 2015). Even though WGS is an appropriate subtyping tool for replacing all other subtyping methods, it is not accessible in all reference laboratories. Therefore, PFGE is still the gold standard method.