ABSTRACT
A 5-year survey of public access surface waters in an agricultural region of the Central California Coast was done to assess the prevalence of the foodborne pathogen Listeria monocytogenes. In nature, L. monocytogenes lives as a saprophyte in soil and water, which are reservoirs for contamination of preharvest produce. Moore swabs were deployed biweekly in lakes, ponds, streams, and rivers during 2011 to 2016. L. monocytogenes was recovered in 1,224 of 2,922 samples, resulting in 41.9% prevalence. Multiple subtypes were isolated from 97 samples, resulting in 1,323 L. monocytogenes isolates. Prevalence was higher in winter and spring and after rain events in some waterways. Over 84% of the isolates were serotype 4b. Whole-genome sequencing was done on 1,248 isolates, and in silico multilocus sequence typing revealed 74 different sequence types (STs) and 39 clonal complexes (CCs). The clones most isolated, CC639, CC183, and CC1, made up 27%, 19%, and 13%, respectively, of the sequenced isolates. Other types were CC663, CC6, CC842, CC4, CC2, CC5, and CC217. All sequenced isolates contained intact copies of core L. monocytogenes virulence genes, and pathogenicity islands LIPI-3 and LIPI-4 were identified in 73% and 63%, respectively, of the sequenced isolates. The virulence factor internalin A was predicted to be intact in all but four isolates, while genes important for sanitizer and heavy metal resistance were found in <5% of the isolates. These waters are not used for crop irrigation directly, but they are available to wildlife and can flood fields during heavy rains.
IMPORTANCE Listeria monocytogenes serotype 4b and 1/2a strains are implicated in most listeriosis, and hypervirulent listeriosis stems from strains containing pathogenicity islands LIPI-3 and LIPI-4. The waters and sediments in the Central California Coast agricultural region contain widespread and diverse L. monocytogenes populations, and all the isolates contain intact virulence genes. Emerging clones CC183 and CC639 were the most abundant clones, and major clones CC1, CC4, and CC6 were well represented. CC183 was responsible for three produce-related outbreaks in the last 7 years. Most of the isolates in the survey differ from those of lesser virulence that are often isolated from foods and food processing plants because they contain genes encoding an intact virulence factor, internalin A, and most did not contain genes for sanitizer and heavy metal resistance. This isolate collection is important for understanding L. monocytogenes populations in agricultural and natural regions.
KEYWORDS: Listeria monocytogenes, food safety, microbial ecology, sediment, water quality
INTRODUCTION
Listeria monocytogenes causes foodborne illness on a spectrum from febrile gastroenteritis to systemic infection, yet the incidence of illness in the general population is low. Listeriosis occurs most often in the elderly and immunocompromised populations and causes miscarriage in pregnant women. The severity of the illness in susceptible populations is striking, and listeriosis is a major concern in the United States and Europe. Out of 2,953 foodborne outbreaks in the United States with known etiologic agents between 2009 and 2015, 35 were attributed to L. monocytogenes. The outbreak that resulted in the most deaths (33, or 22% of the cases in that outbreak) in that time period was a multistate cantaloupe-related outbreak, followed by another multistate outbreak of listeriosis traced to caramel apples (1). During 2009 to 2015, listeriosis resulted in 6% of all the hospitalizations and 52% of the deaths resulting from foodborne illness (1–6). Between 2010 and 2020, several high-profile outbreaks, hospitalizations, and deaths due to L. monocytogenes contamination were attributed to raw and processed produce (1, 7–12). The resulting surveillance has generated more awareness and costly produce recalls (2, 13, 14). Awareness incentivizes food manufacturers to invest in preventative controls that work, and those efforts may have decreased the number of illnesses attributed to L. monocytogenes in the United States (15).
The species L. monocytogenes is genetically heterogeneous and is comprised of 13 serotypes, four genetic lineages, and an increasing number of sequence types (STs) and clonal complexes (CCs) have been discovered as new genome information is revealed (16–19). Over 95% of the cases of human illness are caused by serotypes 1/2a (lineage II), 1/2b (lineage I), and 4b (mostly lineage I and some lineage III or IV) (20, 21). L. monocytogenes has a low rate of homologous recombination; thus, clones evolve slowly (22–24). As a result, some outbreak strains that belong to the same sequence types are isolated worldwide (19, 22, 25). A subtyping system defined by a seven-gene multilocus sequence typing (MLST) method differentiates groups into STs. CCs are comprised of STs that differ by no more than one allele from at least one other ST in the group (19, 26). Deeper similarity is determined through whole-genome sequencing (WGS) and core genome MLST (cgMLST) analysis (16). Analysis of virulence gene complements of outbreak strains revealed the hypervirulent clones CC1, CC2, CC4, and CC6 in Europe and the United States (27–29). All virulent L. monocytogenes strains carry the core virulence genes (prfA, plcA, hly, mpl, actA, and plcB) that make up Listeria pathogenicity island 1 (LIPI-1) (30). Hypervirulent strains may also carry the additional islands LIPI-3 and/or LIPI-4, which encode a bacteriocin (listeriolysin S) and a putative cellobiose family phosphotransferase system, respectively, and are more often associated with neurovirulence (27, 31, 32). Genome analysis of historically relevant strains indicates these hypervirulent groups are overrepresented among clinical isolates, as opposed to food isolates, and are more strongly associated with maternal-neonatal and central nervous system listeriosis in patients with and without immunosuppressive comorbidities (27).
Some clones, such as CC1, are distributed internationally, but there is also some geographical and niche bias among clones. The hypervirulent clone CC4 was recognized first in Europe (27). Maury et al. concluded that the abundant hypervirulent clone CC1 is more associated with dairy products, and CC9 and CC121 were more associated with meat products (33). Two emerging sequence types from North America, CC183 (encompassing ST382) and the singleton ST639, have a propensity for surface water (28). Interestingly ST382 (part of CC183) was involved in several produce-related outbreaks in the last few years (10, 34–37).
Because of its ability to grow at refrigeration temperatures, to endure acid and osmotic stress, and to join biofilms, L. monocytogenes is a particular problem as a postharvest contaminant in food processing facilities and packing houses (25, 38). A diverse array of physical and chemical tolerances have been used to characterize L. monocytogenes isolates, and some of these, such as cold tolerance, are encoded in the core genome (16). Other traits, such as resistance to various stresses, including arsenic, cadmium, antibiotics, and benzalkonium chloride (BC) sanitizers, are often encoded on plasmids, transposons, or genomic islands, such as stress survival islets 1 and 2 (SSI-1 and SSI-2) and Listeria genomic island-1 (LGI-2), which are subject to horizontal transfer (39, 40). A notable genetic difference between strains sourced from clinical illness and foods or food processing locations is the truncation of the gene encoding the virulence factor internalin A, encoded by inlA. An intact version of this gene is needed for full virulence. However, in surveys of isolates from foods and food processing facilities, about 40 to 50% of the strains carry premature stop codons (PMSCs) in inlA, resulting in truncated alleles that produce reduced virulence, in contrast to clinical strains that contain the intact gene (41–45). In the limited number of assessments of environmental L. monocytogenes strains, the inlA genes are mostly intact (46–49).
In the environment, L. monocytogenes is a saprophyte; however, most of the isolates that are studied are derived from clinical cases, outbreak investigations, or surveys of foods and food processing facilities. Nevertheless, environmental strains can be a source of produce contamination, which can occur preharvest with subsequent travel of strains into packing facilities and processing plants. Public waterways, such as rivers, lakes, and ponds, act as a central focus of pathogen contamination between agriculture and wildlife. Intrusion of wildlife into produce fields is a potential source of preharvest contamination, so growers in some regions are encouraged to use fencing or to clear riparian areas close to produce fields to discourage wildlife nesting (50, 51). In some areas with protected wetlands, such clearing is not allowed, so growers are encouraged not to plant in these areas. In the Central California Coast, surface waters are infrequently used for crop irrigation, especially for leafy greens; however, the waters are available to wildlife and can flood produce fields during rain events. Environmental surveys for L. monocytogenes conducted in New York in the United States and Nova Scotia and Ontario in Canada show prevalences of 10% to 30% in urban and agricultural water settings, with serotype 1/2a making up most of the isolates (52–55). Recently we concluded a 5-year survey of public access waterways along the Central California Coast to assess the prevalence of L. monocytogenes. This large agricultural region is known for leafy green vegetable production. Preliminary results of L. monocytogenes prevalence from the first 2 years of this study were published previously, along with prevalence data for Salmonella and Shiga toxin-producing Escherichia coli in the same region (56). Data from the first 2 years of the survey indicated that this California region had an L. monocytogenes prevalence of 43% (in 1,405 samples), with 85% of the isolates belonging to serotype 4b (56). A subset (n = 32) of the L. monocytogenes isolates from this study were analyzed to assess the source distribution of serotype 4b strains isolated in North America (28). In another study with 112 of these survey strains, 90% contained intact alleles of inlA (49). The current report contains the results for the full 5 years of sampling ponds, lakes, rivers, and streams on a biweekly basis and includes genomic subtyping data, virulence gene content, and content of stress resistance genomic regions. Through 5 years, over 2,900 samples were collected and enriched for L. monocytogenes to assess the prevalence, ecology, and genomic subtypes present in this region.
Our goals for this study were to (i) measure the prevalence of L. monocytogenes in this agricultural region by season and location, (ii) establish what serotypes and clonal subtypes of L. monocytogenes were present in the region, (iii) determine the complement of virulence genes and common stress resistance genes among these natural environmental isolates, and (iv) learn if isolates persist in the region over extended time periods. This survey was part of a larger project to collect prevalence data to develop a predictive geospatial risk assessment model (PGRAM) for improved spatial and temporal resolution of foodborne pathogens (56).
RESULTS
After 131 sampling trips and 2,922 Moore swab enrichments, 1,224 samples tested positive for L. monocytogenes, resulting in an overall prevalence of 41.9%. Here, prevalence relates to presence/absence of L. monocytogenes, as the levels were not quantified. One isolate was selected from each positive sample. However, if multiple serotypes were detected among the 3 to 5 isolates screened, as detailed in Materials and Methods, then one of each serotype was kept. Ninety-seven samples yielded more than one unique isolate, including two samples that had three unique isolates, resulting in a total of 1,323 isolates. Table 1 shows the breakdown of the isolates by serotype. The Moore swabs were enriched in a nonselective primary enrichment in buffered Listeria enrichment broth (BLEB), followed by two different secondary enrichments (one in Fraser broth [FB] and another with immunomagnetic beads [IMS]), as detailed in Materials and Methods. Before settling upon this regimen, 79 samples were enriched only in 1× BLEB, subjected to IMS, and underwent secondary enrichment in FB, while various plating media were assessed for efficiency (see Materials and Methods). FB enrichment data were lost from 25 samples from the 9 December 2012 sampling due to an incubator malfunction. Therefore, a total of 2,818 samples were enriched with the dual IMS-direct plating and FB secondary enrichment, and 1,193 of those were positive for L. monocytogenes. In direct comparison of the two enrichment methods, 140 samples were positive only with the FB secondary enrichment method, and 333 were positive only by the IMS-direct plating method, while 720 samples were positive by both methods. Most of the isolates (84%) were serotype 4b, followed by serotype 1/2a (7.0%) and serotype 1/2b (5.3%). Full data for each isolate are presented in Table S1 in the supplemental material.
TABLE 1.
Serotypes of the isolates, their isolation sources, and the years they were detected
| Serotype | No. of isolates (% of total) | Waterway(s)/location(s)a | Yr(s) |
|---|---|---|---|
| 1/2a | 92 (6.9) | A, C, G, US, LS, T, X1, X3 | 2012–2016 |
| 1/2b | 70 (5.3) | A, C, G, LS, US, T, X2 | 2012–2016 |
| 1/2c | 4 (0.3) | T | 2014, 2016 |
| 4?b | 4 (0.3) | G | 2012, 2016 |
| 4a/4c | 17 (1.3) | A, G, LS, T | 2011–2012, 2014–2016 |
| 4b | 1,119 (84.5) | A, C, G, US, LS, T, X2, X3 | 2011–2016 |
| 4e | 1 (0.07) | T | 2011 |
| Unknown/unclearb | 17 (1.3) | G, LS, T | 2012, 2013, 2016 |
Waterway abbreviations are defined in Fig. 1 and in Materials and Methods.
The full serotype could not be resolved by standard methods.
L. monocytogenes prevalence varied by season in most waterways and was affected by rainfall.
Table 2 shows the overall prevalence by year and season. Due to the differences in water types, a direct statistical analysis comparing these numbers is not advisable. However, the data show that all the sampling trips gave similar levels of overall percentages of positive samples when compared by year. More samples tested positive in the winter and spring than in the summer and fall. The data in Table 2 indicate that L. monocytogenes was isolated more often during the winter (December through February) and spring (March through May), with just over 50% of all the Moore swabs collected then testing positive. In the drier and warmer summer (June through August) and fall (September through November) seasons, prevalence was approximately 30%. To conduct statistical analyses for seasonal effects and to account for missing values due to lack of water at some locations during the survey, the 30 sampling sites were grouped into waterways, as indicated in Fig. 1. These waterways are Alisal Creek, Carr Lake, Gabilan Creek, the Tembladero Slough, and the Salinas River. The Salinas River waterway was divided into Upper and Lower regions, both because of its size and because there is often little flow between the Upper and Lower regions in the summer and fall months due to low water levels. Three sites were not part of these existing waterways and were labeled X1, X2, and X3. Site X1, a dry riverbed, was sampled only once after a big storm. That sample was positive and yielded two different strains. Table 3 reveals that overall prevalences of L. monocytogenes ranged from 25% to 30% for the locations in the Salinas River and from 38% in the Alisal waterway to 74% for the locations in the Gabilan. Because the tested waterways are different types (creeks, a lake, a river, and a stream), they are not directly comparable and cannot be compared statistically. However, comparisons within waterways by season can be done. All the waterways except for the Gabilan (P = 0.08) had statistical differences in prevalence between seasons. Carr Lake and the Tembladero Slough (each at P < 0.0001) showed higher prevalence in winter than in summer (each at P < 0.0001). The Alisal Creek sites had higher prevalence in winter than in fall (P = 0.01). The Upper Salinas locations had higher prevalence in the spring than in the fall (P = 0.03). The Lower Salinas had higher levels in winter than in fall (P = 0.01). Seasonal prevalence did not vary at locations X2 (P = 0.48) and X3 (P = 0.55). Among the individual locations, 12 tested positive over 50% of the time. Those locations included sites from the Gabilan, Tembladero, Carr Lake, Alisal, and Upper Salinas (see Table S2 in the supplemental material). Three locations (S1 and S2 in the Upper Salinas and S9 in the Lower Salinas) tested positive less than 10% of the time. Location X3 was sampled 120 times and tested positive 68% of the time, making X3 among the sites with the highest prevalence.
TABLE 2.
Prevalence of L. monocytogenes by seasona and year in Moore swab samples
| Yr | Prevalence, % (no. of sampling trips)a |
||||
|---|---|---|---|---|---|
| Fall | Winter | Spring | Summer | All | |
| 2011 | 16.7 (1) | 22.5 ± 11.7 (2) | ND | ND | 20.8 ± 7.7 (3) |
| 2012 | 26.4 ± 15.8 (6) | 39.0 ± 15.3 (6) | 60.8 ± 20.0 (8) | 36.5 ± 13.6 (7) | 45.2 ± 20.5 (27) |
| 2013 | 27.2 ± 18.0 (5) | 62.9 ± 10.2 (7) | 47.2 ± 10.8 (7) | 27.8 ± 9.7 (6) | 40.4 ± 17.5 (26) |
| 2014 | 26.8 ± 18.3 (7) | 36.3 ± 18.8 (7) | 42.3 ± 12.2 (7) | 22.1 ± 10.2 (6) | 37.7 ± 22.5 (28) |
| 2015 | 36.4 ± 18.7 (6) | 63.0 ± 20.9 (8) | 47.5 ± 17.9 (6) | 24.6 ± 13.8 (7) | 41.0 ± 20.2 (26) |
| 2016 | 17.5 ± 3.5 (2) | 64.5 ± 12.9 (8) | 49.3 ± 22.1 (6) | 31.3 ± 11.6 (8) | 43.7 ± 21.8 (21) |
| All | 27.5 ± 16.7 | 53.7 ± 20.8 | 51.2 ± 18.9 | 29.8 ± 13.8 | |
Fall, September through November; winter, December through February; spring, March through May; summer, June through August. Prevalence represents the percentage of total samples positive in the given time period. Values in parentheses are the number of sampling trips for that time period. ND, not done (no sampling occurred at these times).
FIG 1.
(A) Map of region indicating sampling sites along the Upper and Lower Salinas River. Sampling sites are labeled with a letter and number. The Upper Salinas area is indicated in the blue-shaded region. The Lower Salinas area is in the yellow-shaded region. Sites labeled X1 and X2 are not part of any of the designated waterways. (B) Map of the sampling region and the Alisal, Carr Lake, Gabilan, and Tembladero waterway areas. Waterways are indicated on the map by blue dotted lines. Sampling sites indicated with letter corresponding to the waterway to which they were assigned. A, Alisal (yellow-shaded region); C, Carr Lake (pink-shaded region); G, Gabilan (brown-shaded region); T, Tembladero (blue-shaded region); S, Salinas River. The map backgrounds are from The National Map courtesy of the U.S. Geological Survey and are in the public domain.
TABLE 3.
Prevalence by waterway, locations X2 and X3, and season for the entire survey
| Waterway/location (no. of sites)a | Prevalence, %b |
||||
|---|---|---|---|---|---|
| Overall | Fall | Winter | Spring | Summer | |
| Alisal (4) | 38.6 ± 8.9 | 16.5 ± 27.0 X | 50.8 ± 30.6 Y | 52.8 ± 25.8 XY | 28.9 ± 22.4 XY |
| Carr Lake (5) | 43.4 ± 7.5 | 32.3 ± 33.6 XZ | 59.2 ± 30.3 Y | 50.1 ± 19.6 XY | 17.1 ± 20.6 Z |
| Gabilan (4) | 74.2 ± 16.0 | 53.5 ± 43.8 | 75.0 ± 20.0 | 77.7 ± 27.4 | 79.6 ± 21.5 |
| Upper Salinas (5) | 30.0 ± 22.9 | 9.1 ± 16.6 | 29.7 ± 33.0 | 30.8 ± 33.3 | 19.0 ± 24.0 |
| Lower Salinas (4) | 26.8 ± 16.2 | 14.1 ± 17.9 | 31.4 ± 25.4 | 28.6 ± 26.3 | 16.0 ± 23.1 |
| Tembladero (5) | 47.0 ± 10.2 | 26.5 ± 25.3 X | 69.3 ± 25.1 Y | 57.8 ± 25.4 Y | 21.6 ± 19.5 X |
| Location X2 | 34.7 | 25.0 ± 30.7 | 41.3 ± 35.1 | 35.4 ± 26.7 | 21.8 ± 20.5 |
| Location X3 | 68.3 | 58.3 ± 37.6 | 75.4 ± 8.3 | 71.4 ± 20.3 | 61.2 ± 14.5 |
Information in parentheses indicates the number of sampling sites making up that waterway.
Prevalence represents the percentage of total samples positive in the given time period. The letters XY indicate statistical similarities among the values in the row for comparison of season. If the letters are not present, then all values are statistically similar.
The time frame for this survey included a period of severe drought in the region (57). During normal years, rain falls from approximately October through April, but in 2013 and 2014, minimal measurable precipitation occurred. Using the California Water Resources Control Board as a guide, a significant rain event was defined as an average precipitation accumulation of all the sampling sites of ≥0.5 in. up to 5 days before sampling (58). Out of 131 sampling trips, 14 sampling dates (four in 2012, none in 2013, five in 2014, two in 2015, and three in 2016) met those qualifications. The effect of those rain events on prevalence was waterway dependent and is shown in Table 4. Alisal Creek, Carr Lake, the Lower Salinas River, and the Tembladero Slough all had significantly higher prevalences in samplings taken after rain events (P < 0.03), with prevalences at least double those of samplings not affected by rain events. On the other hand, prevalences in the Gabilan and Upper Salinas River were not affected by rain (P > 0.1).
TABLE 4.
Prevalence of L. monocytogenes in waterways in samplings affected and not affected by rain events
| Waterway | Prevalence, %a |
P valueb | |
|---|---|---|---|
| Non-rain | Rain events | ||
| Alisal | 33.1 ± 9.7 | 71.0 ± 19.8 | 0.03 |
| Carr Lake | 37.8 ± 8.9 | 83.3 ± 11.3 | 0.004 |
| Gabilan | 72.2 ± 19.4 | 83.8 ± 11.8 | 0.41 |
| Upper Salinas | 27.8 ± 22.0 | 49.3 ± 39.8 | 0.12 |
| Lower Salinas | 22.2 ± 15.5 | 70.8 ± 29.0 | 0.006 |
| Tembladero | 42.4 ± 11.5 | 87.7 ± 6.9 | 0.003 |
Prevalence represents the percentage of total samples positive under the given circumstance.
P < 0.05 denotes a significant difference determined by paired t tests.
Many of the most common serotypes and clones were isolated throughout the survey from all waterways.
Table 1 indicates the distribution of the serotypes in the region. The most common serotypes (4b, 1/2a, and 1/2b) were isolated from all the waterways throughout the course of the survey. A total of 1,248 isolates were subjected to in silico MLST. Based on data from other subtyping projects (49, 59), the remaining 75 isolates were presumed to be duplicates of other isolates in the collection, and therefore, their genomes were not sequenced. MLST analysis revealed the collection was comprised of 74 sequence types (STs) that belong to 39 clonal complexes (CCs) and 6 singletons. Figure 2 contains a dendrogram of all the STs. The number and distribution of the sequence types, which were determined only for the 1,248 sequenced isolates, in the region are shown in Table 5. The five most common clonal complexes recovered were CC639 (332 isolates [26.6%]), followed by CC183 (233 isolates [18.7%]), CC1 (165 isolates [13.5%]), CC663 (81 isolates [6.5%]), and CC6 (63 isolates [5.0%]). These five CCs all belonged to serotype 4b of lineage I (Fig. 2). Serogroup assessment based on WGS indicated that all the CC183 isolates were a variant of serotype 4b termed 4b-v1, which is common for that genomic subtype. Serotype 4b-v1 strains contain a lineage II-specific gene cassette that includes lmo0737, which is detectable by PCR or WGS (60). The 4b-v1 serotype was also detected in isolates of ST784/CC5 and ST554/CC554. The five most abundant CCs were isolated from each waterway, although the distributions were not equivalent (Table 6). The Gabilan and Tembladero each were the source of 30% of CC639 isolates. Carr Lake was the source of 33.5% of the CC183 isolates, while the Tembladero Slough was the source of 22.7% of CC183 isolates. The Gabilan was the waterway where the highest numbers of CC1 and CC663 representatives were found (31.5% of all CC1 isolates and 38.6% of all CC663 isolates). CC6 isolates were isolated at similar rates from the Tembladero Slough, Carr Lake, Alisal, and the Gabilan, accounting for 23.8%, 20.6%, 19.0%, and 15.9%, respectively.
FIG 2.
Phylogenetic trees of sequenced isolates. L. monocytogenes isolates were grouped into their clonal complex or sequence type. The tree was constructed by the neighbor-joining algorithm using pairwise allelic differences generated by cgMLST and rooted at midpoint. The scale bar at the bottom represents the percentage of dissimilarity. Shown is the relatedness of CCs and/or STs, the presence or absence of genes associated with virulence (including LIPI-1, -2, -3, and -4), stress tolerance, biofilm formation, detergent and heavy metal resistance, and motility. Dark blue boxes indicate all isolates in that branch carrying intact genes. Light blue boxes indicate there is variability among the isolates in the branch on specific gene content. Orange boxes indicate that isolates in the branch are missing the labeled gene. LIPI-1 contains genes necessary for L. monocytogenes virulence (prfA, plcA, hly, mpl, actA, and plcB). LIPI-3 (which contains llsABDGHPXY, with llsX encoding lysteriolysin S) is an additional pathogenicity island present in some strains. LIPI-4, which contains genes encoding a cellobiose phosphor-transferase system (LM9005581_70009, -70010, -70012, -70013, and -70014), is an additional pathogenicity island present in some strains). Internalin genes inlEFGHKL are involved in virulence with inlL playing a role in initial adhesion for biofilm development. The brcABC genes encode resistance determinants for benzalkonium chloride. ladR and mdlL are involved in a multidrug efflux pump. qacA/C, ermE, ermC, and Tn1688 are involved in resistance to quaternary ammonium compounds. bapL is a gene coding for a biofilm-associated protein. lmo0673 represents lmo0673, lmo2504, luxS, and recO, which are involved in biofilm development. LGI-2 (Listeria genomic island 2) encodes stress determinants for cadmium and arsenic resistance on a genomic island. cadA1A2, cadA2C2, and cadA3C3 encode extrachromosomal cadmium resistance determinants. SSI-1 (stress survival islet 1) contains lmo0444, lmo0445, pva, gadD1, and gadT1 and is involved in response to low pH and high salt, and SSI-2 (stress survival islet 2) contains lin0464, lin0465, LM6179_0748, and yoaZ and is involved in alkaline and oxidative stress responses. The remaining columns are labeled as to what processes they have been shown to influence.
TABLE 5.
Genomic subtypes of sequenced 1,248 L. monocytogenes isolates with locations and years detected
| CC or singleton, group total, and diversity index | ST | Serotype (serogroup)a | Lineage | No. of isolates | Yr(s) | No. of isolates in waterway/location |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| A | C | G | US | LS | T | X1 | X2 | X3 | ||||||
| CC | ||||||||||||||
| CC1 | ST1 | 4b (IVb) | I | 164 | 2011–2016 | 14 | 28 | 53 | 13 | 14 | 29 | 0 | 5 | 8 |
| ST308 | 4b (IVb) | I | 3 | 2012–2014 | 0 | 2 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | |
| ST2132 | 4b (IVb) | I | 1 | 2015 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | |
| CC2 | ST2 | 4b (IVb) | I | 18 | 2012–2016 | 2 | 5 | 0 | 0 | 2 | 9 | 0 | 0 | 0 |
| ST782 | 4b (IIb) | I | 7 | 2012, 2014–2016 | 0 | 2 | 3 | 1 | 0 | 1 | 0 | 0 | 0 | |
| ST1039 | 4b (IVb) | I | 1 | 2012 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| ST2125 | 4b (IVb) | I | 1 | 2014 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | |
| CC3 | ST3 | 1/2b (IIb) | I | 1 | 2014 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 |
| ST1530 | 4b (IIb) | I | 1 | 2013 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | |
| CC4 | ST4 | 4b (IVb) | I | 10 | 2012–2016 | 0 | 4 | 0 | 2 | 2 | 2 | 0 | 0 | 0 |
| ST219 | 4b (IVb) | I | 3 | 2013 | 0 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| ST397 | 4b (IVb) | I | 3 | 2014–2015 | 0 | 1 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | |
| ST531 | 4b (IVb) | I | 4 | 2012, 2016 | 0 | 0 | 2 | 0 | 1 | 0 | 0 | 1 | 0 | |
| ST631 | 4b (IVb) | I | 15 | 2012–2013, 2015 | 0 | 9 | 0 | 1 | 1 | 4 | 0 | 0 | 0 | |
| ST1773 | 4b (IVb) | I | 3 | 2013–2014 | 0 | 0 | 0 | 0 | 0 | 3 | 0 | 0 | 0 | |
| ST2128 | 4b (IVb) | I | 1 | 2016 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| CC5 | ST5 | 1/2b (IIb) | I | 16 | 2012–2016 | 0 | 4 | 0 | 0 | 3 | 9 | 0 | 0 | 0 |
| ST363 | 1/2b (IIb) | I | 10 | 2012–2016 | 0 | 3 | 0 | 2 | 0 | 0 | 0 | 5 | 0 | |
| ST784 | 4b (IVb-v1)b | I | 10 | 2012, 2014–2016 | 0 | 2 | 0 | 0 | 2 | 5 | 0 | 1 | 0 | |
| ST1526 | 1/2b (IIb) | I | 1 | 2014 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| CC6 | ST6 | 4b (IVb) | I | 63 | 2011–2016 | 12 | 13 | 10 | 1 | 3 | 15 | 0 | 0 | 9 |
| CC7 | ST7 | 1/2a (IIa) | II | 14 | 2013–2015 | 9 | 1 | 0 | 0 | 1 | 2 | 0 | 0 | 1 |
| CC8 | ST8 | 1/2a (IIa) | II | 1 | 2014 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 |
| CC9 | ST9 | 1/2a [1], 1/2c [2] (IIc) | II | 3 | 2015–2016 | 0 | 0 | 0 | 0 | 0 | 3 | 0 | 0 | 0 |
| ST2134 | 1/2c (IIc) | II | 1 | 2014 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | |
| CC11 | ST11 | 1/2a (IIa) | II | 4 | 2014 | 0 | 1 | 0 | 0 | 0 | 3 | 0 | 0 | 0 |
| ST451 | 1/2a (IIa) | II | 2 | 2012, 2016 | 0 | 0 | 0 | 1 | 0 | 1 | 0 | 0 | 0 | |
| CC26 | ST26 | 1/2a (IIa) | II | 1 | 2015 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| CC29 | ST29 | 1/2a (IIa) | II | 1 | 2014 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 |
| CC37 | ST37 | 1/2a (IIa) | II | 2 | 2012 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| CC87 | ST87 | 1/2b (IIb) | I | 8 | 2013–2016 | 0 | 3 | 0 | 0 | 0 | 5 | 0 | 0 | 0 |
| CC88 | ST88 | 1/2b (IIb) | I | 2 | 2012, 2016 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 | 0 |
| ST296 | 1/2b (IIb) | I | 1 | 2015 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | |
| CC124 | ST124 | 1/2a (IIa) | II | 1 | 2012 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 |
| CC155 | ST155 | 1/2a (IIa) | II | 2 | 2012, 2015 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| CC183 | ST382 | 4b (IVb-v1) | I | 225 | 2012–2016 | 16 | 70 | 12 | 29 | 21 | 53 | 0 | 12 | 12 |
| ST2126 | 4b (IVb-v1) | I | 8 | 2012, 2014–2016 | 0 | 8 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| CC199 | ST199 | 1/2a (IIa) | II | 1 | 2014 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| CC217 | ST217 | 4b (IVb) | I | 28 | 2012–2016 | 3 | 3 | 9 | 4 | 6 | 3 | 0 | 0 | 0 |
| ST2127 | 4b (IVb) | I | 1 | 2014 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| CC224 | ST224 | 1/2b (IIb) | I | 6 | 2013–2016 | 0 | 4 | 1 | 1 | 0 | 0 | 0 | 0 | 0 |
| ST845 | 1/2b (IIb) | I | 1 | 2015 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| CC288 | ST288 | 1/2b (IIb) | I | 8 | 2012–2013, 2015–2016 | 0 | 1 | 3 | 0 | 0 | 3 | 0 | 1 | 0 |
| CC321 | ST321 | 1/2a (IIa) | II | 6 | 2012–2014 | 0 | 0 | 1 | 0 | 0 | 5 | 0 | 0 | 0 |
| CC388 | ST388 | 4b (IVb) | I | 29 | 2012–2016 | 6 | 7 | 1 | 7 | 0 | 3 | 0 | 4 | 1 |
| ST824 | 4b (IVb) | I | 2 | 2015 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | |
| CC389 | ST389 | 4b (IVb) | I | 4 | 2012, 2014–2015 | 1 | 1 | 0 | 0 | 0 | 2 | 0 | 0 | 0 |
| CC392 | ST392 | 1/2b (IIb) | I | 4 | 2012, 2013, 2014, 2016 | 1 | 0 | 0 | 1 | 0 | 2 | 0 | 0 | 0 |
| CC412 | ST412 | 1/2a (IIa) | II | 12 | 2012–2015 | 1 | 1 | 0 | 5 | 3 | 2 | 0 | 0 | 0 |
| CC426 | ST426 | 1/2b (IIb) | I | 1 | 2012 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| CC429 | ST429 | 1/2b (IIb) | I | 2 | 2014, 2016 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 0 |
| ST2141 | 1/2b (IIb) | I | 1 | 2013 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | |
| CC506 | ST506 | 1/2b (IIb) | I | 2 | 2012, 2013 | 0 | 1 | 0 | 0 | 0 | 1 | 0 | 0 | 0 |
| CC554 | ST554 | 4b (IVb-v1) | I | 11 | 2012–2016 | 1 | 2 | 0 | 1 | 1 | 5 | 0 | 1 | 0 |
| CC639 | ST639 | 4b (IVb) | I | 317 | 2011–2016 | 31 | 22 | 94 | 7 | 33 | 97 | 0 | 3 | 30 |
| ST2129 | 4b (IVb) | I | 4 | 2012, 2014, 2015, 2016 | 0 | 1 | 0 | 0 | 0 | 3 | 0 | 0 | 0 | |
| ST2130 | 4b (IVb) | I | 10 | 2012–2013 | 0 | 0 | 3 | 0 | 0 | 0 | 0 | 0 | 7 | |
| ST2133 | 4b (IVb) | I | 1 | 2016 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | |
| CC663 | ST663 | 4b (IVb) | I | 83 | 2012–2016 | 10 | 4 | 32 | 3 | 4 | 12 | 0 | 6 | 12 |
| CC666 | ST666 | 4b (IVb) | I | 1 | 2012 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| CC688 | ST688 | 4b (IVb) | I | 8 | 2012–2015 | 0 | 6 | 0 | 0 | 0 | 1 | 0 | 0 | 1 |
| CC736 | ST736 | 1/2b (IIb) | I | 4 | 2015 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 |
| CC842 | ST1004 | 1/2a (?)c | II | 6 | 2013–2016 | 1 | 0 | 5 | 0 | 0 | 0 | 0 | 0 | 0 |
| ST2138 | 4b (?) | II | 1 | 2012 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | |
| ST2139 | 1/2a [29], 4b [6] (?) | II | 35 | 2011–2016 | 0 | 2 | 15 | 7 | 5 | 5 | 1 | 0 | 0 | |
| ST2140 | 1/2a (?) | II | 2 | 2013, 2016 | 0 | 0 | 1 | 1 | 0 | 0 | 0 | 0 | 0 | |
| CC1000 | ST1000 | 1/2b (IIb) | I | 1 | 2015 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| CC1800 | ST2136 | 1/2a (IIa) | II | 1 | 2012 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 |
| Singletons | ||||||||||||||
| ST2131 | ST2131 | 4b (IVb) | I | 1 | 2013 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 |
| ST2135 | ST2135 | 4b (?) | II | 1 | 2012 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 |
| ST2137 | ST2137 | 1/2a (?) | II | 4 | 2012–2013 | 0 | 0 | 0 | 2 | 1 | 0 | 1 | 0 | 0 |
| ST2142 | ST2142 | 4a/4c [17], 4 [4], ? [13], 1/2b [1] (?) | II | 35 | 2011–2016 | 1 | 0 | 32 | 0 | 1 | 1 | 0 | 0 | 0 |
| ST2143 | ST2143 | 1/2a (IIa) | II | 1 | 2016 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| ST2144 | ST2144 | 1/2a (IIa) | II | 1 | 2016 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Total no. of: | ||||||||||||||
| Sequenced isolates | 116 | 223 | 281 | 93 | 106 | 304 | 2 | 41 | 82 | |||||
| STs | 20 | 38 | 21 | 23 | 20 | 42 | 2 | 12 | 10 | |||||
| Clones | 19 | 26 | 16 | 20 | 16 | 29 | 2 | 11 | 9 | |||||
| Diversity index | ||||||||||||||
| Shannon’s | 2.35 | 2.70 | 2.11 | 2.46 | 2.26 | 2.62 | 0.18 | 2.12 | 1.82 | |||||
| Simpson’s | 0.87 | 0.87 | 0.82 | 0.86 | 0.84 | 0.85 | 1.00 | 0.85 | 0.80 | |||||
The serotype was determined by ELISA to assign the O antigen and serotype 4 strains. PCR was used to assign the H antigen for serotype 1/2 and 3 strains. Values in parentheses are the serogroup determined through cgMLST. Values in brackets are the number of isolates of that serotype. If there are no values in brackets, then all the isolates of the subtype displayed the shown serotype.
The 4b-v1 serotype cannot be determined by the ELISA serotyping method, so this grouping was assigned through the serogroup.
The serogroup and/or lineage was not clearly discernible.
TABLE 6.
Distribution of the 10 most common clonal complexes and sequence types among the waterway areas and locations
| CC/ST | No. of isolates | % of subtype in waterway/locationa |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| A | C | G | US | LS | T | X1 | X2 | X3 | ||
| CC639 | 332 | 9.3 | 6.9 | 29.2 | 2.1 | 9.9 | 30.4 | 0 | 0.9 | 11.1 |
| CC183 | 233 | 6.9 | 33.5 | 5.2 | 12.4 | 9.0 | 22.7 | 0 | 5.2 | 5.2 |
| CC1 | 168 | 8.3 | 17.9 | 31.5 | 8.3 | 8.3 | 17.9 | 0 | 3.0 | 4.8 |
| CC663 | 83 | 12.5 | 4.8 | 38.6 | 3.6 | 4.8 | 14.5 | 0 | 7.2 | 14.5 |
| CC6 | 63 | 19.0 | 20.6 | 15.9 | 1.6 | 4.8 | 23.8 | 0 | 0 | 14.3 |
| CC842 | 44 | 2.3 | 4.5 | 47.7 | 18.2 | 13.6 | 11.4 | 2.3 | 0 | 0 |
| CC4 | 39 | 0 | 46.2 | 5.1 | 7.7 | 10.3 | 28.2 | 0 | 2.6 | 0 |
| ST2142 | 35 | 2.9 | 0 | 91.4 | 0 | 2.9 | 2.9 | 0 | 0.0 | 0 |
| CC388 | 31 | 19.4 | 22.6 | 3.2 | 22.6 | 0 | 16.1 | 0 | 12.9 | 3.2 |
| CC217 | 29 | 13.8 | 10.3 | 31.0 | 13.8 | 20.7 | 10.3 | 0 | 0 | 0 |
The values shown are the percentage of the total number of each subtype.
The sixth most common subtype was CC842 (44 isolates [3.5% of the total]). This group contained several isolates that were atypical, because traditional serotyping methods revealed them to be serotypes 1/2a and 4b, and in silico serogroup assignment through WGS was inconclusive for 15 isolates. Isolates in the CC842 group contained ORF2110, which is specific to serotypes 4b, 4d, and 4e, but they did not contain ORF2819, which should be present in serotypes 1/2b, 3b, 4b, 4d, and 4e (18). Thus, the isolates in CC842 could not be assigned to a molecular serogroup. However, they were determined to be lineage II based on WGS phylogeny (Fig. 2). CC842 and two singletons, ST2135 and ST2137, shared the same molecular serogroup signature (containing open reading frame 2110 [ORF2110] but not ORF2819), and these groups formed a large monophyletic clade within lineage II (Fig. 2; Table S1). These three clones differed from each other by 539 to 877 alleles, while other clones in lineage II differed by 1,116 to 1,567 alleles. Thus, the three clones with this atypical serogroup signature were relatively close phylogenetically, compared to other lineage II clones. These isolates also were distant from lineage III isolates of L. monocytogenes (unpublished data). CC842 was found throughout the survey and in all the waterways, but 47.7% of the isolates were detected in the Gabilan (Table 6).
Following CC842, the next most isolated clone was CC4 (39 isolates [3.1% of total] of serotype 4b), which was detected in each of the waterways. CC4 isolates were isolated most often from Carr Lake (46.2% of CC4 isolates), followed by the Tembladero Slough (28.2% of CC4 isolates). The eighth most common MLST type was the singleton ST2142 (35 isolates [2.8% of the total]). The ST2142 isolates were atypical, and the isolates in this group had serotypes, based on enzyme-linked immunosorbent assay (ELISA) and PCR results, of 1/2b, 4a, or 4c, and serotype 4 with an unclear H antigen. The serogroup of these isolates also could not be assigned by in silico analysis because none of the serogroup gene targets (18) were found in these isolates (Table 5; Table S1). Such a molecular signature, which may also be found among lineage III isolates, was assigned serogroup L (60, 61). Analysis by cgMLST showed that the ST2142 isolates belonged to lineage II (Fig. 2). These isolates were distant from lineage III isolates of L. monocytogenes (unpublished data). ST2142 isolates were detected throughout the survey and, in all but one case, were isolated from samples in the Gabilan. The ninth and tenth most common clones were CC388 (30 isolates [2.4%]) and CC217 (29 isolates [2.3%]), both serotype 4b and isolated throughout the survey. The CC388 isolates were found in most of the waterways, but most often in Carr Lake, the Upper Salinas River, and Alisal Creek (23%, 23%, and 20%, respectively, of all the CC388 isolates). The CC217 isolates were more evenly dispersed among the waterways; however, they were recovered most often from the Gabilan (31% of CC217 isolates).
Among the isolates subjected to in silico MLST, the Tembladero Slough had 303 isolates and 42 different sequence types (STs), which was the largest number among the waterways (Table 5). This was followed by Carr Lake (38 STs), the Upper Salinas (23 STs), the Gabilan (21 STs), and the Lower Salinas and Alisal waterways (each with 20 STs). ST639 or ST382 was the dominant subtype in each waterway or location. Calculations of diversity within the waterways were similar. Shannon’s index of diversity, which gives an indication of the diversity of subtypes in the different areas, ranged from 2.11 to 2.70 (Table 5). Simpson’s index of diversity, which ranges between 0 and 1 and considers the numbers of different subtypes and the abundance of those subtypes, for all the waterways ranged from 0.82 to 0.88 (Table 5). Shannon’s index emphasizes rare subtypes, and Simpson’s index emphasizes dominant subtypes.
CC designations made by 7-gene in silico MLST had some inconsistency with WGS phylogeny and serogrouping. For example, CC5 contained ST5 (n = 16), ST363 (n = 10), ST1526 (n = 1), and ST784 (n = 10). ST363 and ST1526 belonged to a WGS clade, but they differed by 831 to 839 alleles, much higher than the typical diversity of a CC (i.e., ≤167 alleles) previously observed when using the same cgMLST scheme (15). ST5 and ST784 belonged to different clades (Fig. 2). Thus, ST363, ST1526, and ST784 would have been classified into different clones from ST5 by WGS diversity. ST5, which caused multiple recent listeriosis outbreaks, and ST363, and ST1526 belong to serogroup IIb; however, ST784 belongs to serogroup 4b-v1 and formed a clade with CC554, another 4b-v1 CC. Furthermore, ST5 and ST784 differed by 1,265 to 1,298 alleles, while ST784 and CC554 differed by 499 to 520 alleles. Another example of inconsistency between WGS, serogroup, and in silico MLST is CC2, which contained ST2, ST2125, ST782, and ST1039. ST2 and ST2125 formed a clade and differed by 66 to 96 alleles. However, while ST1039 (n = 1), ST2, and ST2125 formed a clade, ST1039 differs from ST2 by 1,156 alleles, indicating that ST1039 should belong to a different clone from ST2. ST782 (n = 7) falls into a different clade from other CC2 STs. In addition, based on allele differences, ST308/CC1 (n = 3), ST824/CC388 (n = 2), ST397/CC4 (n = 3), ST631/CC4 (n = 15), ST2128/CC4 (n = 1), ST219/CC4 (n = 3), ST824/CC388 (n = 2), ST296/CC88 (n = 1), one isolate of ST1004/CC842, 10 isolates of ST2139/CC842, and two isolates of ST2140/CC842 would have been assigned by WGS to a clone different from that assigned by 7-gene MLST, making the total number of such isolates to be 72. These isolates differed from other isolates in the same CC by ≥283 alleles. Among them, 21 of the CC5 isolates and seven CC2 isolates fall into clades different from most of the isolates in the same CC (Fig. 2).
Among the sequenced isolates, a total of 1,007 were determined to belong to clusters with high relatedness to each other, indicated they were closely related strains (see Table S3 in the supplemental material). Whole-genome sequencing was used to determine relatedness of isolates from different collection sites and times. It has been shown that isolates associated with an outbreak or belonging to a cluster could have variations in genetic diversity (16) (https://pubmed.ncbi.nlm.nih.gov/30042741/), so we combined cgMLST neighbor-joining phylogeny and seven allele differences as a threshold to identify isolates that were closely related. Previous studies showed that in a minimum-spanning tree, isolates that differ from neighboring isolates in the tree by ≤7 alleles often form a cluster in the whole-genome phylogeny, so we first identified a group of isolates that met this criterion and then defined the smallest clade containing these isolates as a cluster, even when some isolates in this clade could differ by >7 alleles. Ninety-five clusters were identified (Table S3). Among the 332 isolates of CC639, 321 (including those of ST639, ST2129, ST2130, and ST2133), belonged to one cluster (cluster 64), which was detected during all six calendar years and four seasons, and in all five waterways and locations X2 and X3 (Fig. 1). Among the 233 isolates of CC183, 215 isolates of ST382 belonged to one cluster (cluster 21), which was isolated in five calendar years (2012 to 2016), all four seasons, and all five waterways, as well as locations X2 and X3. Cluster 6 contained 61 ST1/CC1 isolates that were collected in all six calendar years and all four seasons, as well as in all five waterways and location X3. Cluster 28 contained 23 ST217/CC217 isolates that were collected in all six calendar years and all four seasons, as well as in all five waterways. Cluster 70 contained 34 ST663/CC663 isolates that were collected in five calendar years (2012 to 2016) and all four seasons, as well as in four waterways. Recurrence of isolates from the same cluster over time is an indication of persistence or continued reintroduction of these strains in the region. Among the 353 isolates in other clusters, 10 clusters (containing 106 isolates total) were collected during all four seasons, and another 11 clusters (64 isolates total) were collected in three of the four seasons. Five clusters (59 isolates total) were collected in five out of the 6 years. One cluster (cluster 38, 13 isolates) was detected in all five waterways, and one cluster (cluster 7, 8 isolates) was collected in four waterways. The appearance of these isolates in different areas is an indication of movement of isolates in the region. The Alisal, Carr Lake, Gabilan, Tembladero, Lower Salinas, and Upper Salinas waterways had 23, 35, 32, 47, 18, and 17 clusters of L. monocytogenes, respectively. The Tembladero Slough had the largest number of clusters, possibly due to the Alisal, Carr Lake and Gabilan waterways eventually migrating to the Tembladero Slough.
There were seasonal variations among L. monocytogenes genotypes from these waterways and locations. Among 94 clusters, 79 clusters from 24 clones were isolated in spring or winter; and 54 clusters from 17 clones were isolated in summer or fall. Forty clusters that contained 115 isolates from 22 CCs were only isolated in winter or spring, while 10 clusters that contained 15 isolates from 8 CCs were only isolated in summer or fall. Thus, L. monocytogenes isolates were more prevalent, and the population was more diverse in winter and spring than in summer and fall.
All the L. monocytogenes isolates contain core virulence genes, and a majority contain additional pathogenicity islands.
As shown in Fig. 2, all 1,248 sequenced isolates contained intact copies of the core L. monocytogenes virulence genes (actA, hlyA, mpl, plcA, plcB, and prfA) that make up Listeria pathogenicity island 1 (LIPI-1). Pathogenicity islands LIPI-3 and LIPI-4, which are found in strains that demonstrate more invasive listeriosis illness (33) were well represented among the strains. Intact LIPI-3 was detected in 913 (73.2%) of the sequenced isolates, and only in lineage I isolates (Fig. 2). Among the most common detected clonal complexes, LIPI-3 was identified in all isolates of CC639, CC183, CC1, and CC6; however, none of the CC663 isolates contained LIPI-3. LIPI-4 was detected in 785 (62.9%) of the sequenced isolates. The isolates containing LIPI-4 were all in lineage I, and the majority were clones of CC639, CC183, CC663, and CC4 (Fig. 2). The commonly found clones CC1 and CC6 did not contain LIPI-4. Among sequenced isolates, 639 isolates (51.2%) contained all three pathogenicity islands LIPI-1, LIPI-3, and LIPI-4, and most of them belonged to CC639, CC183, CC4, and CC217 (Table 5).
In addition to the pathogenicity islands, 61 different inlA alleles were identified among the sequenced isolates, and nearly all of them were predicted to encode intact, 800-amino-acid InlA proteins. Among CC6 isolates, 61 of 63 contained an inlA allele with a 9-nucleotide deletion, which would result in a 797-amino-acid InlA, which has been reported previously for other CC6 strains (28, 62). None of the other CCs contained this inlA allele. RM19528, a serotype 4b, CC183 strain isolated in 2012 from location T1 had a deletion of nucleotide G in the 1316th position of the inlA gene, which would result in a protein of 440 amino acids. This allele was not detected in any other of the isolates in the survey. Of the 232 remaining CC183 strains, 228 contained the same intact inlA allele. Three of the four serotype 1/2c CC9 isolates (RM20062, RM20406, and RM20422) and the single serotype 1/2a CC199 isolate (RM19882) had a deletion of nucleotide A in the 12th position of inlA; this would result in a protein of 8 amino acids.
All the sequenced isolates contain genes for biofilm formation and cold stress, but other stress resistance genes were less well represented.
All the sequenced isolates contained the genes cspBD, lisK, lmo0886, lmo1722, and yycG, which play roles in surviving cold stress (Fig. 2). Some genes important for biofilm formation, such as lmo0673, lmo2504, luxS, and recO, were detected in all the sequenced isolates. However, the presence of other genes implicated in biofilm formation varied. The bapL gene was present in 53 lineage II isolates, and the inlL gene was present in 42 lineage II isolates. Of note were the isolates associated with the singleton clone ST2142, which included isolates difficult to serotype and characterize for lineage. Most of the ST2142 isolates (33 of 35 [94%]) contained bapL, but none contained inlL.
Several gene clusters known to play roles in stress resistance were assessed (Fig. 2). Stress survival islet 1 (SSI-1), which aids growth under suboptimal pH and osmotic conditions, was present in 151 (12%) of the sequenced isolates and was present in both lineage I and II isolates, and those isolates were predominantly in Carr Lake and the Tembladero Slough. The clones containing the highest number of isolates with SSI-1 were CC5 and CC7. No isolates contained SSI-2, which is responsible for resistance to alkaline and oxidative stresses (63). Previous studies showed that SSI-2 is present predominantly in CC121 strains, and CC121 was not identified in this survey.
L. monocytogenes isolates from processing facilities often show resistance to commonly used sanitizers such as benzalkonium chloride (BC) (64–67). Resistance to the heavy metals arsenic and cadmium has been used as a criterion to subtype strains (68–70). Consequently, the strain collection was screened for the presence of brcABC and qacACH, as well as emrC and emrE. Resistance to cadmium and arsenic, in addition to benzalkonium chloride sanitizers, was poorly represented among the isolates. Cadmium resistance and arsenic resistance are encoded on Listeria genomic island 2 (LGI-2), and this region was detected in only 50 (4.0%) of the sequenced isolates. Additional genes involved in cadmium resistance, including cadA1C1, cadA2C2, and cadA3C3, were detected in 27 (2.2%), 17 (1.4%), and 10 (0.8%), respectively, of the sequenced isolates. The gene cluster brcABC, which is involved in resistance to benzalkonium chloride sanitizers, was detected in only 18 (1.4%) isolates (Fig. 2), and additional genes involved in benzalkonium chloride resistance, including qacACH, emrC, and emrE, were not detected in any of the isolates. The 18 isolates included 13 lineage II isolates of CC321 (n = 6), CC155 (n = 2), CC9 (n = 4), and CC199 (n = 1) and five lineage I isolates of CC2 (n = 1), CC5 (n = 3), and CC6 (n = 1). Similarly, genes involved in cadmium and arsenic resistance were present in only 77 isolates of both lineages I and II. The isolates carrying these resistance determinants were isolated in 2012 to 2016 and were represented in most of the waterways; however, the Tembladero Slough and Carr Lake were the sources of most of these isolates (P = 0.0057) (Table 7).
TABLE 7.
Percentage of isolates carrying resistance determinants
| Resistance determinanta | % of isolates in waterway or location |
|||||||
|---|---|---|---|---|---|---|---|---|
| A | C | G | US | LS | T | X2 | X3 | |
| LGI-2 (50) | 10.0 | 18.0 | 24.0 | 18.0 | 6.0 | 20.0 | 4.0 | 0 |
| cadA1C1 (27) | 0 | 11.1 | 7.4 | 0 | 11.1 | 66.7 | 3.7 | 0 |
| cadA2C2 (17) | 0 | 41.2 | 0 | 0 | 11.8 | 47.1 | 0 | 0 |
| cadA3C3 (10) | 0 | 60.0 | 0 | 10.0 | 0 | 30.0 | 0 | 0 |
| brcABC (18) | 0 | 27.8 | 5.6 | 0 | 16.7 | 50.0 | 0 | 0 |
| SSI-1 (156) | 12.2 | 14.7 | 23.1 | 5.1 | 5.8 | 23.1 | 7.1 | 8.3 |
LGI-2 (Listeria genomic island 2) encodes stress determinants for cadmium and arsenic resistance on a genomic island. cadA1A2, cadA2C2, and cadA3C3 encode extrachromosomal cadmium resistance determinants. The brcABC genes encode resistance determinants for benzalkonium chloride. SSI-1 (stress survival islet 1) is involved in growth under stressful conditions of low pH and high salt. The number in parentheses following the genetic identification is the total number of isolates carrying intact copies of the genes.
DISCUSSION
Based on a relatively high prevalence and frequent isolation of highly related isolates from six waterways over multiple seasons, the results of this survey indicate that L. monocytogenes is persistent in the surface waters of this Central California region. The preponderance of isolates and clones were of lineage I. This finding differs from environmental surveys of other geographical regions. Haase et al. reported that 369 environmental isolates of L. monocytogenes from Finland, the United States, Austria, and Ireland were equally distributed among lineages I and II (26). Other recent surveys and retrospective analyses of old culture collections of environmental isolates report a preponderance of isolates from lineage II (52, 54, 71, 72). However, a recent survey of ruminants revealed that 69% of the L. monocytogenes isolates were of lineage I (73). The enrichment and selection protocols used can influence the types of isolates that result (74, 75). At the start of this survey, there was literature regarding selective enrichment of L. monocytogenes from foods, but not much comparison of methods for isolation from environmental sources. We chose to use a nonselective primary enrichment in BLEB (without added antibiotics and acriflavine), a buffered medium that contains sodium pyruvate to aid in the recovery of stressed cells and to potentially minimize the competition between L. monocytogenes and other Listeria spp. There are several reports in the literature regarding the ability of Listeria innocua to outgrow L. monocytogenes in selective enrichment culture (76–79). However, the literature regarding direct comparisons of nonselective versus selective media in the enrichment of L. monocytogenes is mixed. Duffy et al. reported no enhancement of L. monocytogenes detection when comparing selective and nonselective enrichment media with meat that also contained L. innocua (80). However, studies with injured cells reported decreased recovery when plated onto selective agar compared with the nonselective Trypticase soy agar (TSA) (81, 82). Sheridan et al. reported improved recovery of L. monocytogenes from meat when using a nonselective enrichment medium (83). Previously published work on 62 samples from this survey that tested positive by both the FB and IMS methods indicated that the FB method was more likely to result in isolation of serotype 1/2a strains (59). The two enrichment methods used in this survey resulted in the isolation of more L. monocytogenes strains than either method used alone. However, the selection of enrichment method will always bias the types of strains recovered. Those recovered in this survey were strains able to survive a nonselective enrichment and compete with other microbiota present in the samples. Similarly, the use of Moore swabs as a sampling technique can bias the recovery. Moore swabs were selected as the means of sample collection because their ease of use was conducive with the number and frequency of sampling events planned over a 5-year survey. A recent study indicates that Moore swabs yield L. monocytogenes less often than 10-L grab samples (84).
The region surveyed in the current study is a large, mostly agricultural region, with definite contributions from urban runoff. This mixture of rural, urban, agricultural, and riparian may contribute to the high levels of lineage I isolates and clones of L. monocytogenes in this region. In environments impacted by urban runoff, L. monocytogenes strains of lineage I are more prevalent (72). For example, the sampling locations in Carr Lake are inside the city of Salinas, and the Tembladero Slough lies north of the city of Salinas, in the direction of stream runoff (Fig. 1). In addition to urban runoff and seepage from septic systems, these waterways regularly receive water from the Gabilan and Alisal Creeks, which are more heavily agricultural. Along with agriculture and wildlife in riparian regions, the region also has some impact from cow/calf cattle operations (mostly in the Gabilan area), some of which occur in elevated areas. The headwaters of the Salinas River lie in Paso Robles (roughly 40 miles south of the map in Fig. 1A), and the river flows north and ends at the Monterey Bay. At the time of this survey, along this path were hundreds of farms and ranches, eight small municipalities, large riparian regions on both sides, and sporadic spots of cattle grazing. The region is a focus of a large amount of crop production. Typically, crops are grown throughout the year. Large amounts of leafy greens are sown starting in late December and through September and then harvested from March until mid-November. Broccoli is harvested in February through late December. The timing of other crops varies through the year, but at least 34 different types of produce are grown in the region (85). The moderate climate allows year-round production. The first frost in the region varies, but is typically in late October, and the last frost is generally in May or April. The dominant crops include strawberries, lettuce, spinach, and broccoli (86).
Several investigators have hypothesized that lineage I and II isolates evolved separately with recombination and positive selection driving the evolution, and there is evidence that lineage II isolates have undergone more horizontal gene transfer (87, 88). This increased genome plasticity among lineage II isolates may play a role in it being predominant in surveys of food processing plants and in other environmental surveys. It would be interesting to compare L. monocytogenes subtype distributions between the Central California Coastal area and other heavy agriculture and urban/agriculture areas to determine if the lineage I predominance in this survey is unique to the location or can be found in other areas that mix agriculture with small urban areas. It is intriguing that no lineage III isolates were found, which was unexpected since lineage III isolates are primarily obtained from animals raised for production (89, 90). The reason for this may be the reduced presence of lineage III in the region or lack of fitness of lineage III isolates in the enrichment scheme used, such that they were missed in the protocol.
While overall prevalence was 42%, this number may be an underestimation. Weller and colleagues reported that recovery of L. monocytogenes was predicted to be lower from Moore swabs rather than 10-L grab samples (84). Prevalence in the current survey was higher in winter and spring, which are the cooler and wetter seasons. In four of the six waterways, prevalence was higher after significant rain events. Chapin et al. reported prevalences of 33 and 34% in natural and produce production environments of New York State (91) that were positively affected by soil moisture and proximity to water. Mohammed et al. reported that incidence in winter and summer was higher than in fall on dairy farms in New York State (92). Weller and colleagues reported on relationships between L. monocytogenes detection, rain events, and stream flow rates in New York State and showed higher prevalence after rain events and during higher stream flow (84, 93). In our study, the winter/spring levels remained higher even during years of drought, indicating that factors other than rainfall contribute to incidence and persistence in the California region. Notably, water is released periodically from the San Antonio and Nacimiento Reservoirs south of San Ardo (i.e., south of location S1 in Fig. 1A) into the Salinas River, which runs south to north, during the non-rainy season to provide water for recharging aquifers and for waste processing downstream. Temperature may play a role in L. monocytogenes persistence in the region. Studies done in New York State indicated that L. monocytogenes was likely to be isolated from Moore swabs deployed in streams where the air temperature (10 to 15°C) was consistent with that in the Salinas region during winter and spring (84). Sharma et al. reported that a nontidal river in the mid-Atlantic region with lower water temperature than those of other sampled waters supported higher levels of L. monocytogenes (94). However, a recent study by Gu et al. indicated no relation to abiotic factors, including temperature, on the incidence of L. monocytogenes in irrigation ponds sampled monthly in Virginia over the course of a single year (95).
It is possible that had more colonies been selected from Brilliance plates and/or if different enrichment methods were used, the genomic subtypes isolated would differ. However, based on the methods used in this survey, the resulting array of genomic subtypes isolated indicates that there are a variety of sources for overall pathogen persistence in the region. Also, genome sequence comparisons indicated recurring isolates or very similar isolates in the region and over the survey time. These data suggest that the L. monocytogenes population in the region is diverse, and there are isolates that were able to persist. While the Tembladero Slough had the largest number of different clones and WGS clusters, Simpson’s diversity index indicated that each waterway had a high level of clonal diversity. The clones and sequence types isolated most often were scattered throughout the region. Isolates of CC1, CC6, CC183, CC217, CC388, CC639, and CC842 were found throughout the course of the survey from nearly every waterway, indicating widespread prevalence of these groups in the area. Fifteen clones (CC639, CC183, CC1, CC663, CC6, CC842, CC4, ST2142, CC388, CC217, CC2, CC5, CC639, CC412, and CC7) were represented more than 1% of the time among the 1,248 sequenced isolates and were isolated throughout the 5 years. Taken together, these data, combined with the distribution and timing of isolations of the various genomic clusters described above, indicate that many different L. monocytogenes strains were distributed widely in the sampling locations. These strains either persisted or were reintroduced to sites throughout the survey. Several sequence types and clones showed notable location bias. Approximately 75% of CC4 isolates were isolated from Carr Lake and the Tembladero Slough. Similarly, 78% of ST2/CC2 isolates and 81% of ST5/CC5 isolates were collected from Carr Lake and the Tembladero Slough. CC4 and ST2/CC2 were hypervirulent clones that have caused multiple listeriosis outbreaks (16, 17), and ST5/CC5 has also caused multiple listeriosis outbreaks (96). These clones might be expected to reside in areas most impacted by urban runoff. In contrast, 94% of the ST2142 isolates were enriched from Gabilan Creek, which is the site of cow/calf grass grazing. The ST2142 group contained atypical isolates difficult to serotype, but many appeared by ELISA serotyping to be 4a/4c, which are more commonly associated with agricultural animals and lineage III (97). However, WGS analysis indicated they were serogroup L and clustered with lineage II isolates. These atypical isolates require further study to fully characterize. While ST2142 was isolated repeatedly during the survey, its confinement to the Gabilan area indicates that it might be less fit outside that immediate area. Water from Gabilan Creek migrates eventually to other sampled sites (such as Carr Lake and the Tembladero Slough) where ST2142 was rarely detected. Results of other studies related to those presented here include a recent report of small produce farm environments, with Belias et al. describing eight different sigB allelic types and 22 pulsed-field gel electrophoresis (PFGE) pulsotypes among 86 L. monocytogenes isolates (98). Another report of a survey of surface waters in Switzerland revealed 25 L. monocytogenes isolates that belonged predominantly to CC1, CC4, and CC6 (62).
Of note was the wide distribution of virulence genes and pathogenicity islands among the isolates. Every sequenced isolate contained intact LIPI-1. Furthermore, 73.2% of the sequenced isolates contained LIPI-3, and 62.9% contained LIPI-4, which are attributed to more severe listeriosis. The most prevalent clones, CC639 and CC183, contained both LIPI-3 and LIPI-4, and the third most prevalent clone, CC1, contained LIPI-3, and this explains the high percentage of isolates containing both pathogenicity islands in the present study. Information on the incidence of these pathogenicity islands in environmental isolates is just beginning to be assessed. In a report on 25 environmental isolates from surface waters in Switzerland, Raschle et al. (62) described 12 lineage I isolates that contained LIPI-3 and four CC4 isolates that contained LIPI-4. Intact LIPI-3 and LIPI-4 were identified in 51% and 31%, respectively, of L. monocytogenes isolates from a survey of ready-to-eat retail foods in the United States (61). In a collaborative study, Lee et al. (28) reported on the incidence of LIPI-3 and LIPI-4 among environmental strains; however, 32 of the 36 environmental isolates in that study came from the survey collection described here. Studies characterizing isolates from foods and food processing facility surveillance generally report low incidence of LIPI-3 and LIPI-4 (99–101). Among wildlife, the 19 L. monocytogenes strains (15 of CC388, 1 of CC1, and 3 of CC155) isolated from wild deer and boar over the course of 15 months in a Mediterranean forest in Spain all contained LIPI-1, while the CC1 strain also contained LIPI-3, and several of the CC388 strains carried LIPI-4 (102). Another recent survey described 130 isolates of L. monocytogenes from ruminants in a dairy environment, and 50% and 32.3% carried LIPI-3 and LIPI-4, respectively (73).
There are many properties that microbes need to live, survive, and persist in nature, and two that would be important for L. monocytogenes in this environment are survival of cold temperature stress and growth on surfaces. All isolates contained some genes shown to affect biofilm formation, including the predicted exoprotein gene lmo2504 and the luxS gene encoding autoinducer 2 (103, 104). All sequenced isolates contained genes shown to be important in cold stress. Biofilm and cold tolerance genes were also present among isolates from ready-to-eat retail foods, indicating potential uses for these systems in both environments (61).
The transition from saprophytic to virulent lifestyles is complex for L. monocytogenes and is affected by temperature and carbon source through the global transcriptional regulator PrfA (encoded by prfA as part of LIPI-1), the alternative sigma factor SigB, as well as antisense RNAs and riboswitches (105, 106). Maintenance in the natural environment may include cycling between agricultural animals and wildlife. Important in this regard is the finding that nearly all the sequenced isolates were predicted to encode intact internalin A, which would allow for attachment to host cells. Intact inlA genes and other virulence factors also were described for isolates from wild deer and boars, indicating persistence of virulence among this population over a 15-month survey (102). In surveys of isolates from foods and food processing facilities, about 40 to 50% of the isolates carry premature stop codons (PMSCs) in the gene encoding internalin A, resulting in truncated inlA alleles that cause reduced virulence (41–45). In surveys of strains isolated from ruminants and farm animals and their environments, inlA alleles were predominantly intact, similar to the strains in this study (46–48, 73). Analysis of the full isolate collection reinforces the preliminary reporting of 112 strains from this study indicating inlA alleles were mostly intact in L. monocytogenes isolates from this region (49).
Strains of CC1 have been implicated in outbreaks around the world since the 1980s, and it was suggested that continued involvement of CC1 in different outbreaks implies it has reservoirs in which to reside between outbreaks (25). While isolates of CC1 are more commonly found from clinical samples, strains of CC1 have also been found in agricultural animals (25, 27, 33, 73). CC1 was the third most common clone isolated in this survey from all the waterways, indicating that the water/sediment environment, tracked through the Moore swabs, may be another reservoir. Another clone of interest is CC183, which contains ST382, which has been implicated in three produce-related outbreaks since 2013. This clone has recently emerged in the United States and is estimated to have diverged from its ancestor just over 30 years ago (34). As the second most common clone isolated, CC183 appears widespread in the region surveyed here. CC183 was identified as a recently emerged clone of L. monocytogenes with limited genetic diversity (34). The CC183 isolates collected in these waters were indeed closely related. Considering that nearly all the CC183 isolates contained all three LIPI pathogenicity islands, this finding is of concern. A recent survey of L. monocytogenes isolates from wild bears in North Carolina revealed that over 50% of the serotype 4b isolates contained the 4b-v1 profile found in CC183 and CC554 isolates (107). The relationship between the 4b-v1 serogroup, environmental fitness, emerging clones, and the impact on food safety will be topics of future study.
CC639 (which contains ST639), the most frequent clone in the region, is another emerging clone. In a collaborative study, ST639 was concluded to have a greater association with water/sediment than with food or clinical sources (28). However, 11 of the 15 strains of ST639 in that study were from this isolate collection. While not implicated in any widespread outbreaks, strains of ST639 have been the cause of at least three invasive, sporadic cases of listeriosis in the United States (28, 108). All the CC639 isolates in this survey contain all three LIPI pathogenicity islands.
While virulence genes and pathogenicity islands were common among the isolates, resistance genes for heavy metals and benzalkonium were infrequently seen. It should be noted that the genes for resistance to heavy metals and BC often are carried on plasmids and other mobile elements (40, 109, 110). The poor representation of genes involved in responses to these stresses may indicate no fitness advantage in this water/sediment environment. Gelbicova et al. (110) reported that genes for cadmium, arsenic, and benzalkonium chloride resistance in L. monocytogenes were less common in environmental strains than in isolates from humans and foods. It is expected that fitness determinants would differ between food processing facilities and natural water/sediment environments.
The 7-gene MLST-defined CCs have been established and proven to be a valuable tool to study the clonal diversity and population structure of L. monocytogenes. A 1,748-gene MLST scheme found that isolates in most CCs identified prior to 2016 differed by ≤150 alleles. A new nomenclature, sublineage, was used to define L. monocytogenes clones identified using this cutoff by the 1,748-gene MLST, and the profiles are curated in a database (17). The analysis of a moderate number of CCs identified prior to 2016 using the 1,827-gene cgMLST scheme employed in the present study showed that isolates in most CCs differed by ≤167 alleles. In the present study, a total of 72 isolates (5.8% of all isolates) differed from other isolates in the same CC by at least 283 cgMLST alleles, and thus, these isolates would have been classified as a clone different from the MLST-defined CC. In the future, isolates in the present study could be classified as WGS-defined clones. Nonetheless, only 5.8% of all the sequenced isolates in this study would be affected. Therefore, the big picture of isolates analyzed in this study would remain unchanged.
In conclusion, this Central California Coast agricultural region is rich with L. monocytogenes diversity. The 5-year survey revealed that several genomic clones were commonly found in the region during 2012 to 2016. The isolates recovered in this survey will serve as a baseline for L. monocytogenes presence and diversity in the region and serve as benchmarks for future traceback investigations. Repeated isolation of clinically important subtypes and isolates indicates these strains were either persistent or regularly reintroduced to the region. The locations of the strains in relation to crop production will be important information for growers, processors, and regulators. The high levels of virulence genes among the isolates suggest that these genomic determinants may provide useful fitness advantages to the pathogen in this environment. The lack of stress resistance determinants, such as resistance to sanitizers used in food processing plants, implies that such genes may not be needed for fitness in a natural environment. Of note is the finding that 97 of the 1,224 positive Moore swab samples yielded more than one type of L. monocytogenes, as determined solely by serotype. Deeper initial subtyping by MLST or cgMLST of enrichments would likely have revealed even more isolates. The mixtures of subtypes in the region and the high levels of the emerging clones CC183 and CC639, as well as significant levels of other emerging groups, such as CC217 and CC4, and high levels of the established groups CC1 and CC6, indicate that further study of this region can enhance our understanding of L. monocytogenes population structure and environmental fitness as it relates to food safety.
MATERIALS AND METHODS
Sampling and enrichment of L. monocytogenes.
All sampling sites were in the Salinas Valley in California and are indicated in Fig. 1. Sites were selected based on previous knowledge of the region from previous surveys searching for Shiga toxin-producing Escherichia coli and Salmonella (111–113). The current survey was part of a larger project to collect pathogen prevalence data by collaborators at the Food and Drug Administration and the National Aeronautics and Space Administration to develop a predictive geospatial risk assessment model (PGRAM) (56, 114). Due to lack of water at locations during some sampling dates, not all locations were sampled on each trip. To perform statistical analysis, sample sites were grouped by geographical characteristics into their respective waterways. Locations along the same creek, slough, river, or lake were named for that regional waterway: Alisal, Carr Lake, Gabilan, Upper and Lower Salinas River, and Tembladero Slough. Depending on the season, the waterways are sometimes hydrologically interconnected. For example, Carr Lake is upstream of the Tembladero Slough. Carr Lake is a seasonal lake contained within the city of Salinas, CA. The Alisal and Gabilan waterways are in more agricultural areas of the region, whereas the other waters are more affected by urban and human influences. The locations in the Alisal waterway are either along Alisal Creek or in tributaries that drain into Alisal Creek. Gabilan Creek is in the Gabilan mountains. Because of its length, the Salinas River was broken up into two areas: the Upper and Lower. The Lower Salinas River area has more human influence than the Upper Salinas River, which is affected by a small cattle feedlot approximately 4 miles from location S1 (Fig. 1A) and has agricultural operations along both sides of the river. Three locations (X1 along the Arroyo Seco River, X2 in Chualar Creek, and X3 along Santa Rita Creek) were standalone sites not affected by any of the waterways indicated above and are discussed as discrete locations. The waterways are distinct from each other such that previous surveys indicate differences in incidences and prevalences of Shiga toxin-producing E. coli (STEC) and Salmonella between them (56, 111–113).
Moore swabs (cut cotton gauze tied and secured with fishing line) were deployed for 20 to 24 h in lakes, rivers, and streams at sites twice monthly from October 2011 to September 2016. This comprised 2,922 samples with a range of 1 to 130 samples per site and an average of 96 samples per site (Table S2). All 30 sampling sites were on public lands, with no permits required, and the study did not involve endangered or protected species. Not every location was sampled every time due to lack of water at the location or to loss of the swab. Cumulative precipitation amounts for 5 days up to sampling dates were acquired from the California Irrigation Management Information System (CIMIS) and are shown in Table S4 in the supplemental material. Precipitation affecting a particular sampling date was calculated as the average 5-day cumulative precipitation from CIMIS weather sites closest to the sample location. Moore swabs were collected by 1:00 p.m. on the day after deployment, placed into 2-gallon Whirl-Pak bags (Whirl-Pak, Madison, WI), and immediately placed on ice for transport back to the laboratory. Later in the afternoon, the swabs were washed in their collection bag by vigorous agitation for approximately 20 s with 0.5 L of sterile H2O, and 100 mL of rinsate was measured into a 500-mL Whirl-Pak bag. Sterile buffered Listeria enrichment broth base (BLEB) (Difco, Becton, Dickinson-BBL, Franklin Lakes, NJ) with no additives was prepared in a 5× concentration, and 25 mL of 5× BLEB was added to the aliquoted 100-mL rinsate, resulting in cultures in 1× BLEB. The remaining rinsate from the swab was used for other projects in the lab. The BLEB cultures were incubated at 30°C on a rotating shaker set at 150 rpm for 18 h, followed by a 4°C static hold until further processing. After incubation, 1 mL of culture was mixed with 20 μL of anti-L. monocytogenes magnetic beads (Dynal, Thermo Fisher Scientific, Waltham, MA, USA), the mixture processed in a Dynal bead retriever according to manufacturer’s instructions, and the beads recovered in 130 μL of phosphate-buffered saline with Tween (PBST) (10 mM sodium phosphate buffer [pH 7.2], 150 mM NaCl, 0.05% Tween 20). The suspension of recovered beads was divided and subcultured in two separate ways. In one method, 30 μL of enriched beads was streaked for isolation directly onto Brilliance Listeria agar (Oxoid, Remel, Lenexa, KS) and incubated at 37°C for 2 days. The remaining 100 μL of enriched beads was inoculated into 5 mL of Fraser broth (FB) (Difco) supplemented with ferric ammonium citrate at 0.5 g/L and incubated at 37°C. After 2 days of incubation 30 μL of any FB cultures that turned black was streaked for isolation onto Brilliance Listeria plates and incubated as described above. If present, three to five distinctive colonies (blue with a surrounding zone of clearing) were selected from Brilliance agar, spotted onto Trypticase soy agar (TSA) (Difco), and incubated overnight at 37°C. If fewer than three distinctive colonies were present, then we picked all that were visible. At the beginning of the survey project, 76 samples were enriched as described in 1× BLEB and subjected to IMS, and all the beads were subcultured into FB before plating onto Brilliance agar, Oxford agar (OX) (Difco), modified Oxford agar (MOX) (Difco) agar, and PALCAM agar (Difco) to assess different culture media for the most efficient L. monocytogenes detection. All the black colonies selected from these OX, MOX, and PALCAM plates were Listeria spp., but the majority were not L. monocytogenes. Therefore, only Brilliance agar was used moving forward, and direct plating of the beads without a secondary FB enrichment was added to the protocol.
L. monocytogenes confirmation and serotyping.
Whole-colony PCR (56) was done on all of the colonies selected from Brilliance plates to screen for the hlyA gene by using previously described primers and protocol (115). Those isolates that had a PCR product of 858 bp on a 1% agarose gel were restreaked onto MOX plates and incubated at 37°C to ensure pure cultures. Isolates were serotyped using a combination of the ELISA and multiplex PCR serotyping methods targeting the lmo0737, lmo1118, and prs genes, as well as the genes at ORF2819 and ORF2110 (18, 116), and then confirmed during WGS. The ELISA serotyping method was used to determine the O-antigen designation for all positive isolates, and for serotype 4 strains, this was the only method used, as it is sufficient for assigning any of the serotype 4 identities. The H antigens for strains that carried O antigen 1/2 or 3 were determined with the multiplex PCR serotyping method. This hierarchical method of serotyping was faster than using ELISA for O- and H-antigen detection for all the isolates since H-antigen detection by antisera requires several days of priming motile strains on soft agar. Similarly, the multiplex PCR serotyping method was used only for strains for which it was essential for assigning the H-antigen content. In our hands, screening isolates with ELISA was faster than using PCR. Any isolates that would not sort into the O-antigen groupings of 1/2, 3, or 4 were presumed not to be L. monocytogenes and were discarded. Such isolates were encountered <1% of the time. Chromosomal DNA was purified using the Wizard Genomic purification kit (Promega, Madison, WI). Cultures in Trypticase soy broth (TSB) (Difco) were grown overnight at 37°C, 1 mL was centrifuged for 5 min at 13,000 × g, and the pellet was resuspended in Tris-EDTA (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) plus 10 mg/mL lysozyme. These suspensions were incubated at 37°C for 45 min, centrifuged (13,000 × g) for 5 min, the supernatant discarded, and the pellet used for DNA extraction following manufacturer’s instructions for DNA purification.
Whole-genome sequencing, genomic analysis, in silico MLST, and serogrouping.
All isolates were sequenced either on an Illumina MiSeq platform (250-bp paired-end reads; Illumina, Inc., San Diego, CA) or on an Illumina NextSeq platform (150-bp paired-end reads; Illumina) using the Nextera XT library preparation kit per the manufacturer’s instructions. The genomic sequence contigs for each isolate were de novo assembled using Qiagen CLC Genomics Workbench 11.1 (Aarhus, Denmark). Genomes were analyzed by a previously developed cgMLST typing scheme utilizing the cgMLST tool built in Ridom SeqSphere+ (Ridom GmbH, Münster, Germany) targeting 1,827 core genes of L. monocytogenes (16). A neighbor-joining tree was constructed using pairwise allelic differences. The combination of clustering in the neighbor-joining tree and 7-allele cutoff was used to define a cluster. This cutoff was used in a cgMLST scheme targeting a similar number of genes (17). Isolates in the same cluster likely are the same strain.
In silico MLST implemented in the SeqSphere+ software was used to determine the sequence type of the isolates. Clonal complexes and singletons were then assigned based on the definition by Ragon et al. (22) and profiles curated in the Institut Pasteur MLST Listeria database (https://bigsdb.pasteur.fr/listeria/). In silico PCR serogroup identification was performed by determining the presence of targets used to define the four major PCR serogroups: (i) IIa, which was shown to correspond to serotypes 1/2a and 3a by antiserum based-serotyping; (ii) IIc, corresponding to serotypes 1/2c and 3c; (iii) IVb, corresponding to serotypes 4b, 4d, and 4e; and (iv) IIb, corresponding to serotypes 1/2b and 3b (18). Each isolate was determined to be lineage I or lineage II by using both serogroup information and cgMLST phylogeny. The inlA sequences were extracted from the whole-genome sequences using CLC Genomics Workbench and aligned using MEGA 7.0 (117). PMSCs in inlA were determined manually.
Nucleotide BLAST was performed to determine the presence of previously surveyed genes associated with virulence and stress response among isolates genetically confirmed as L. monocytogenes by WGS analyses (17, 33). A threshold of ≥70% query coverage with ≥80% sequence identity of BLAST alignment indicated the presence of a gene or genomic island (118).
Statistics.
To assess the effect of season on prevalence in the various waterways, the percentages of positive samples for each season and each year for each location were combined for their respective waterways. Averages and standard deviations were calculated, and a mixed model compound symmetry covariance matrix was fitted using restricted maximum likelihood (REML) with a Geisser-Greenhouse correction to determine seasonal effects. Nominal effects were waterway and season, and the measurement variable was the percentage of positive samples. Tukey’s multiple-comparison test was used to compare seasonal prevalences within each waterway. The REML was used for this assessment because it allows for missing values (due to lack of sampling for some locations due to lack of water) and gives the same P values and multiple-comparison tests as a repeated-measures one-way analysis of variance (ANOVA). To compare the effect of rain events on prevalence, average prevalence by location was calculated for sampling dates both affected and not affected by rain events (>0.5 in. of accumulation up to 5 days presampling). These data were grouped by their respective waterways, and paired t tests were performed comparing prevalences by using rain events as a measured variable. Simpson’s index of diversity was calculated as described previously (119). Shannon’s index of diversity and evenness were calculated as described previously (120). Prism v.9.3.1 (Graph Pad, San Diego, CA) was used for statistical calculations.
Data availability.
Genome sequences were deposited into NCBI with the sample IDs listed in Table S1.
ACKNOWLEDGMENTS
We are grateful for the technical assistance of Anita Liang, Jessica Govoni, and Kelly Romanolo.
This work was supported by funds from the U.S. Department of Agriculture, Agricultural Research Service CRIS project 2030-42000-052-00D, and grant 224-11-2044 from the Food and Drug Administration.
Footnotes
Supplemental material is available online only.
Contributor Information
Lisa Gorski, Email: lisa.gorski@usda.gov.
Edward G. Dudley, The Pennsylvania State University
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Table S1. Download aem.00357-22-s0001.xlsx, XLSX file, 0.5 MB (565.9KB, xlsx)
Tables S2 and S4. Download aem.00357-22-s0002.pdf, PDF file, 0.3 MB (281.8KB, pdf)
Table S3. Download aem.00357-22-s0003.xlsx, XLSX file, 0.1 MB (69.4KB, xlsx)
Data Availability Statement
Genome sequences were deposited into NCBI with the sample IDs listed in Table S1.