Draft genome sequences of Listeria monocytogenes strains from human listeriosis in Sweden harboring premature stop codons in the virulence determinant inlA.

Premature stop codons in the internalin virulence determinant inlA are common in serotype 1/2a Listeria monocytogenes from food/food processing environments but rare among human clinical isolates. Here, we report the genome sequences of serotype 1/2a (STs 121 and 3258) human listeriosis isolates from Sweden harboring such mutations in inlA.

Draft genome sequences of two wide-host-range phages of Listeria monocytogenes from food processing environments in the United States.

Listeria monocytogenes is notorious for persistence in food facilities. Phages can significantly impact the ecology of Listeria, but there is a dearth of genome sequence data for Listeria phages from food processing ecosystems. We report the genome sequences of two Listeria phages from turkey processing facilities in the USA.

Genomic Analysis Points to Multiple Genetic Mechanisms for Non-Transformable Campylobacter jejuni ST-50.

Campylobacter jejuni and Campylobacter coli are well known for their natural competence, i.e., their capacity for the uptake of naked DNA with subsequent transformation. This study identifies non-transformable C. jejuni and C. coli strains from domestic animals and employs genomic analysis to investigate the strain genotypes and their associated genetic mechanisms. The results reveal genetic associations leading to a non-transformable state, including functional DNase genes from bacteriophages and mutations within the cts-encoded DNA-uptake system, which impact the initial steps of the DNA uptake during natural transformation. Interestingly, all 38 tested C. jejuni ST-50 strains from the United States exhibit a high prevalence of non-transformability, and the strains harbor a variety of these genetic markers. This research emphasizes the role of these genetic markers in hindering the transfer of antimicrobial resistance (AMR) determinants, providing valuable insights into the genetic diversity of Campylobacter. As ST-50 is a major clone of C. jejuni globally, we additionally determined the prevalence of the genetic markers for non-transformability among C. jejuni ST-50 from different regions of the world, revealing distinct patterns of evolution and a strong selective pressure on the loss of competence in ST-50 strains, particularly in the agricultural environment in the United States. Our findings contribute to a comprehensive understanding of genetic exchange mechanisms within Campylobacter strains, and their implications for antimicrobial resistance dissemination and evolutionary pathways within specific lineages.

Draft genome sequence of Exiguobacterium sp. from whole cantaloupe, with inhibition capacity against Listeria monocytogenes.

We report the draft genome sequence of a novel species, Exiguobacterium sp., isolated from a freshly harvested and untreated cantaloupe in North Carolina. The strain Exiguobacterium wild type exhibited inhibitory activity against the foodborne pathogen Listeria monocytogenes, including strains of diverse serotypes and genotypes, both on agar media and in biofilms.

Draft genome sequences of Listeria monocytogenes strains of sequence type 1733 which constituted a pseudo-outbreak due to contaminated blood agar media.

We report genome sequences of Listeria monocytogenes sequence type (ST) 1733 from a 2013 pseudo-outbreak, where L. monocytogenes isolation from non-sterile sites (urine, wound, or abscess) was an artifact from contaminated sheep blood in the isolation media. Two ST1733 strains from wound and urine in 2005 are also reported.

Tetracycline resistance in Listeria monocytogenes and L. innocua from wild black bears (Ursus americanus) in the United States is mediated by novel transposable elements.

IMPORTANCE: Listeria monocytogenes causes severe foodborne illness and is the only human pathogen in the genus Listeria. Previous surveys of AMR in Listeria focused on clinical sources and food or food processing environments, with AMR in strains from wildlife and other natural ecosystems remaining under-explored. We analyzed 185 sequenced strains from wild black bears (Ursus americanus) from the United States, including 158 and 27 L. monocytogenes and L. innocua, respectively. Tetracycline resistance was the most prevalent resistance trait. In L. monocytogenes, it was encountered exclusively in serotype 4b strains with the novel Tn916-like element Tn916.1039. In contrast, three distinct, novel tetracycline resistance elements (Tn5801.UAM, Tn5801.551, and Tn6000.205) were identified in L. innocua. Interestingly, Tn5801.551 was identical to elements in L. monocytogenes from a major foodborne outbreak in the United States in 2011. The findings suggest the importance of wildlife and non-pathogenic Listeria species as reservoir for resistance elements in Listeria.

Draft genome sequences of a historical collection of Listeria monocytogenes from humans and other sources, 1926-1964.

Listeria monocytogenes can persistently contaminate food processing environments and tolerate sanitizers. Most sequenced strains are from clinical and environmental sources in the contemporary era, with relatively few prior to extensive food processing and sanitizer use. We report the genome sequences of a diverse panel of 83 strains from 1926 to 1964.

Draft Genome Sequences of Heavy Metal-Resistant Listeria monocytogenes Strains of Sequence Type 14 (Clonal Complex 14) from Human Listeriosis Cases in Sweden.

Listeria monocytogenes of clonal complex 14 (CC14) is a potentially hypervirulent clone of serotype 1/2a but remains poorly characterized. We report the genome sequences of five sequence type 14 (ST14) (CC14) strains from human listeriosis cases in Sweden, which harbor a chromosomal heavy metal resistance island that is generally uncommon in serotype 1/2a.

Draft Genome Sequences of 158 Listeria monocytogenes Strains Isolated from Black Bears (Ursus americanus) in the United States.

Listeria monocytogenes is responsible for severe foodborne disease and major economic losses, but its potential reservoirs in natural ecosystems remain poorly understood. Here, we report the draft genome sequences of 158 L. monocytogenes strains isolated from black bears (Ursus americanus) in the southeastern United States between 2014 and 2017.

Draft Genome Sequences of Closely Related Listeria monocytogenes Lineage III Strains Isolated from a Food Processing Environment and a Case of Human Listeriosis.

Listeria monocytogenes lineage III is genetically highly diverse, and closely related lineage III strains from food facilities and human listeriosis have not been reported. Here, we report the genome sequences of three closely related lineage III strains from Hawaii, namely, one isolated from a human case and two isolated from a produce storage facility.

Investigation of a Listeria monocytogenes Chromosomal Immigration Control Region Reveals Diverse Restriction Modification Systems with Complete Sequence Type Conservation.

Listeria monocytogenes is a Gram-positive pathogen responsible for the severe foodborne disease listeriosis. A chromosomal hotspot between lmo0301 and lmo0305 has been noted to harbor diverse restriction modification (RM) systems. Here, we analyzed 872 L. monocytogenes genomes to better understand the prevalence and types of RM systems in this region, designated the immigration control region (ICR). Type I, II, III and IV RM systems were found in 86.1% of strains inside the ICR and in 22.5% of strains flanking the ICR. ICR content was completely conserved within the same multilocus sequence typing-based sequence type (ST), but the same RM system could be identified in diverse STs. The intra-ST conservation of ICR content suggests that this region may drive the emergence of new STs and promote clone stability. Sau3AI-like, LmoJ2 and LmoJ3 type II RM systems as well as type I EcoKI-like, and type IV AspBHI-like and mcrB-like systems accounted for all RM systems in the ICR. A Sau3AI-like type II RM system with specificity for GATC was harbored in the ICR of many STs, including all strains of the ancient, ubiquitous ST1. The extreme paucity of GATC recognition sites in lytic phages may reflect ancient adaptation of these phages to preempt resistance associated with the widely distributed Sau3AI-like systems. These findings indicate that the ICR has a high propensity for RM systems which are intraclonaly conserved and may impact bacteriophage susceptibility as well as ST emergence and stability.

Optimization of Plasma-activated Water and Validation of a Potential Surrogate for Salmonella for Future Egg Washing Processes.

Plasma-activated water (PAW) is considered a novel sanitizer for the food industry due to the antimicrobial mechanisms exhibited by reactive oxygen and nitrogen species. The plasma operation parameters can affect the chemistry of PAW and can therefore influence its microbial inactivation efficacy. This study statistically optimized the operating conditions of PAW (activation time, distance from nozzle, and volume of water) using response surface methodology. Two optimized conditions of PAW were identified for the inactivation of planktonic cells of the avirulent strain of Salmonella Typhimurium MHM112 providing a minimum reduction of 6.3 log. All three operating parameters significantly affected the physicochemical characteristics (pH, ORP, EC, nitrite, and nitrate) and microbial inactivation efficacy of PAW. Mixing of small batches using the two optimized conditions to obtain larger volumes did not significantly change the microbial inactivation. However, there were significant reductions in nitrite and nitrate concentrations in PAW due to the mixing of batches while the pH and ORP values remained unaffected. The storage of large volumes of PAW for 25 min at 40-46°C, which is the commercial egg washing temperature in the United States, did not significantly impact S. Typhimurium MHM112 inactivation or the physicochemical characteristics of PAW. A validation study using a cocktail of six pathogenic strains of Salmonella revealed no significant differences in inactivation between the avirulent S. Typhimurium MHM112 and the pathogenic strains, suggesting that the avirulent S. Typhimurium MHM112 may serve as a surrogate for sanitation of S. enterica at the optimized conditions of PAW. The results obtained from this study are useful for our long-term goal of evaluating PAW efficacy in surface egg washing to inactivate Salmonella.

Contrasting Genetic Diversity of Listeria Pathogenicity Islands 3 and 4 Harbored by Nonpathogenic Listeria spp.

Listeria monocytogenes causes the severe foodborne disease listeriosis. Several clonal groups of L. monocytogenes possess the pathogenicity islands Listeria pathogenicity island 3 (LIPI-3) and LIPI-4. Here, we investigated the prevalence and genetic diversity of LIPI-3 and LIPI-4 among 63 strains of seven nonpathogenic Listeria spp. from the natural environment, i.e., wildlife (black bears [Ursus americanus]) and surface water. Analysis of the whole-genome sequence data suggested that both islands were horizontally acquired but differed considerably in their incidence and genetic diversity. LIPI-3 was identified among half of the L. innocua strains in the same genomic location as in L. monocytogenes (guaA hot spot) in a truncated form, with only three strains harboring full-length LIPI-3, and a highly divergent partial LIPI-3 was observed in three Listeria seeligeri strains, outside the guaA hot spot. Premature stop codons (PMSCs) and frameshifts were frequently noted in the LIPI-3 gene encoding listeriolysin S. On the other hand, full-length LIPI-4 without any PMSCs was found in all Listeria innocua strains, in the same genomic location as L. monocytogenes and with ~85% similarity to the L. monocytogenes counterpart. Our study provides intriguing examples of genetic changes that pathogenicity islands may undergo in nonpathogenic bacterial species, potentially in response to environmental pressures that promote either maintenance or degeneration of the islands. Investigations of the roles that LIPI-3 and LIPI-4 play in nonpathogenic Listeria spp. are warranted to further understand the differential evolution of genetic elements in pathogenic versus nonpathogenic hosts of the same genus. IMPORTANCE Listeria monocytogenes is a serious foodborne pathogen that can harbor the pathogenicity islands Listeria pathogenicity island 3 (LIPI-3) and LIPI-4. Intriguingly, these have also been reported in nonpathogenic L. innocua from food and farm environments, though limited information is available for strains from the natural environment. Here, we analyzed whole-genome sequence data of nonpathogenic Listeria spp. from wildlife and surface water to further elucidate the genetic diversity and evolution of LIPI-3 and LIPI-4 in Listeria. While the full-length islands were found only in L. innocua, LIPI-3 was uncommon and exhibited frequent truncation and genetic diversification, while LIPI-4 was remarkable in being ubiquitous, albeit diversified from L. monocytogenes. These contrasting features demonstrate that pathogenicity islands in nonpathogenic hosts can evolve along different trajectories, leading to either degeneration or maintenance, and highlight the need to examine their physiological roles in nonpathogenic hosts.

Campylobacter Colonization and Diversity in Young Turkeys in the Context of Gastrointestinal Distress and Antimicrobial Treatment.

Young turkeys are vulnerable to undifferentiated gastrointestinal distress, including "irritable and crabby syndrome" (ICS), which compromises flock performance and is typically treated with a combination of penicillin and gentamicin (P/G). However, the effects of ICS and P/G treatment on Campylobacter remain poorly understood. We investigated the impact of ICS and P/G treatment on Campylobacter levels and diversity in four flocks from three turkey farms. Cecum and jejunum samples were analyzed weekly from day of hatch to week 4-5. All four flocks became colonized with multidrug resistant (MDR) Campylobacter jejuni and C. coli by week 2-3, and two developed ICS. ICS and P/G treatment did not significantly impact total Campylobacter levels or strain genotypes but impacted species and antimicrobial resistance (AMR) profiles. One flock was raised under antibiotic-free (ABF) conditions while another flock at the same farm was raised conventionally. The ABF flock did not develop ICS while its counterpart did. However, Campylobacter strains, AMR profiles and sequence types were generally shared between these two flocks. Our findings suggest that ICS and P/G treatment impacted Campylobacter population dynamics in commercial young turkey flocks, and that ABF flocks may become readily colonized by MDR strains from non-ABF flocks at the same farm.

Campylobacter jejuni and Campylobacter coli from Houseflies in Commercial Turkey Farms Are Frequently Resistant to Multiple Antimicrobials and Exhibit Pronounced Genotypic Diversity.

Campylobacter is a leading foodborne pathogen, and poultry are a major vehicle for infection. Houseflies play important roles in colonization of broiler flocks with Campylobacter but comparable information for turkey farms is limited. Here, we investigated houseflies as potential vectors for Campylobacter in 28 commercial turkey flocks. We characterized species, genotypes, and the antimicrobial resistance (AMR) profiles of Campylobacter from turkey feces and houseflies in the same turkey house. Of the 28 flocks, 25 yielded Campylobacter from turkey droppings and houseflies, with an average of 6.25 and 3.11 Campylobacter log CFU/g feces and log CFU/fly, respectively. Three flocks were negative for Campylobacter both in turkey feces and in houseflies. Both C. coli and C. jejuni were detected in turkey feces and houseflies, with C. coli more likely to be recovered from houseflies than feces. Determination of Campylobacter species, genotypes, and AMR profiles revealed up to six different strains in houseflies from a single house, including multidrug-resistant strains. For the predominant strain types, presence in houseflies was predictive of presence in feces, and vice versa. These findings suggest that houseflies may serve as vehicles for dissemination of Campylobacter, including multidrug-resistant strains, within a turkey house, and potentially between different turkey houses and farms in the same region.

Horizontal Gene Transfer and Loss of Serotype-Specific Genes in Listeria monocytogenes Can Lead to Incorrect Serotype Designations with a Commonly-Employed Molecular Serotyping Scheme.

Listeria monocytogenes is a Gram-positive, facultative intracellular foodborne pathogen capable of causing severe, invasive illness (listeriosis). Three serotypes, 1/2a, 1/2b, and 4b, are leading contributors to human listeriosis, with 4b including the major hypervirulent clones. The multiplex PCR scheme developed by Doumith and collaborators employs primers targeting specific lineages (e.g., lineage II-specific lmo0737, lineage I-specific LMOf2365_2059) or serotypes (e.g., serotype 4b-specific LMOf2365_1900). The Doumith scheme (DS) is extensively employed for molecular serotyping of L. monocytogenes due to its high accuracy, relative ease, and affordability. However, for certain strains, the DS serotype designations are in conflict with those relying on antibody-based schemes or whole-genome sequence (WGS) analysis. In the current study, all 27 tested serotype 4b strains with sequence type 782 (ST782) within the hypervirulent clonal complex 2 (CC2) were designated 1/2b/3b using the DS. These strains lacked the serotype 4b-specific gene LMOf2365_1900, while retaining LMOf2365_2059, which, together with prs, yields the DS 1/2b/3b profile. Furthermore, 15 serotype 1/2a strains of four STs, mostly from water, were designated 1/2b/3b using the DS. These strains lacked the lmo0737 cassette but harbored genomic islands with LMOf2365_2059, thus yielding the DS 1/2b/3b profile. Lastly, we investigated a novel, dual 1/2a-1/2b profile obtained using the DS with 21 serotype 1/2a strains of four STs harboring both the lmo0737 cassette and genomic islands with LMOf2365_2059. The findings suggest that for certain strains and clones of L. monocytogenes the DS designations should be viewed with caution and complemented with alternative tools, e.g., traditional serotyping or WGS analysis. IMPORTANCE Listeria monocytogenes is a foodborne pathogen responsible for severe illness (listeriosis), especially in pregnant women and their fetuses, immunocompromised individuals, and the elderly. Three serotypes, 1/2a, 1/2b, and 4b, account for most human listeriosis, with certain serotype 4b clonal complexes (CCs) overrepresented in human disease. Serotyping remains extensively employed in Listeria epidemiologic investigations, and a multiplex PCR-based serotyping scheme is widely used. However, the PCR gene targets can be lost or gained via horizontal gene transfer, leading to novel PCR profiles without known serotype designations or to incorrect serotype assignments. Thus, an entire serotype 4b clone of the hypervirulent CC2 would be misidentified as serotype 1/2b, and several strains of serotype 1/2a would be identified as serotype 1/2b. Such challenges are especially common in novel clones from underexplored habitats, e.g., wildlife and surface water. The findings suggest caution in application of molecular serotyping, while highlighting Listeria's diversity and potential for horizontal gene transfer.

Bacteriophages Isolated From Turkeys Infecting Diverse Salmonella Serovars.

Salmonella is one of the leading causes of foodborne illnesses worldwide. The rapid emergence of multidrug-resistant Salmonella strains has increased global concern for salmonellosis. Recent studies have shown that bacteriophages (phages) are novel and the most promising antibacterial agents for biocontrol in foods because phages specifically kill target bacteria without affecting other bacteria, do not alter organoleptic properties or nutritional quality of foods, and are safe and environmentally friendly. Due to the vast variation in Salmonella serotypes, large numbers of different and highly virulent Salmonella phages with broad host ranges are needed. This study isolated 14 Salmonella phages from turkey fecal and cecal samples. Six phages (Φ205, Φ206, Φ207, ΦEnt, ΦMont, and Φ13314) were selected for characterization. These phages were from all three families in the Caudovirales order. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that each phage had a unique structural protein profile. Each phage had a distinct host range. Φ207 and ΦEnt are both siphophages. They shared eight hosts, including seven different Salmonella serovars and one Shigella sonnei strain. These two phages showed different restriction banding patterns generated through EcoRI or HindIII digestion, but shared three bands from EcoRI digestion. ΦEnt displayed the broadest and very unusual host range infecting 11 Salmonella strains from nine serovars and three Shigella strains from two species, and thus was further characterized. The one-step growth curve revealed that ΦEnt had a short latent period (10 min) and relatively large burst size (100 PFU/infected cell). ΦEnt and its host showed better thermal stabilities in tryptic soy broth than in saline at 63 or 72°C. In the model food system (cucumber juice or beef broth), ΦEnt infection [regardless of the multiplicity of infections (MOIs) of 1, 10, and 100] resulted in more than 5-log10 reduction in Salmonella concentration within 4 or 5 h. Such high lytic activity combined with its remarkably broad and unusual host range and good thermal stability suggested that ΦEnt is a novel Salmonella phage with great potential to be used as an effective biocontrol agent against diverse Salmonella serovars in foods.

Differences in the Propensity of Different Antimicrobial Resistance Determinants to Be Disseminated via Transformation in Campylobacter jejuni and Campylobacter coli.

Campylobacter jejuni and Campylobacter coli are leading zoonotic foodborne pathogens, and the drugs of choice for human campylobacteriosis are macrolides (e.g., erythromycin) and fluoroquinolones. C. jejuni and C. coli are naturally competent for transformation via naked DNA uptake, but potential differences in transformation frequency (TF) for different antimicrobial resistance (AMR) markers remain poorly understood. We determined TFs for resistance to different antibiotics using as recipient a derivative of C. jejuni NCTC 11168 (strain SN:CM) with donor DNA from multidrug-resistant C. jejuni or C. coli. TF for nalidixic acid resistance ranked significantly highest (~1.4 × 10-3), followed by resistance to streptomycin and gentamicin. Tetracycline resistance via chromosomal tet(O) was less commonly transferred (~7.6 × 10-7), while transformation to erythromycin resistance was rare (≤4.7 × 10-8). We also determined TFs with the contemporary poultry-derived strains C. jejuni FSIS 11810577 and C. coli FSIS 1710488 as recipients. TFs to nalidixic acid and streptomycin resistance remained the highest (~7 × 10-4). However, TF for gentamicin resistance was remarkably low in certain recipient-donor combinations, while average TF for erythromycin resistance was noticeably higher (~3 × 10-6) than with SN:CM. Findings from this experimental model provide insights into factors that may impact transformation-mediated transfer of AMR leading to AMR dissemination in the agricultural ecosystem.

Genomic Analysis Reveals That Isolation Temperature on Selective Media Introduces Genetic Variation in Campylobacter jejuni from Bovine Feces.

Campylobacter jejuni is commonly isolated on selective media following incubation at 37 °C or 42 °C, but the impact of these temperatures on genome variation remains unclear. Previously, Campylobacter selective enrichments from the feces of steers before and after ceftiofur treatment were plated on selective agar media and incubated at either 37 °C or 42 °C. Here, we analyzed the whole genome sequence of C. jejuni strains of the same multilocus sequence typing (MLST)-based sequence type (ST) and isolated from the same sample upon incubation at both temperatures. Four such strain pairs (one ST8221 and three ST8567) were analyzed using core genome and whole genome MLST (cgMLST, wgMLST). Among the 1970 wgMLST loci, 7-25 varied within each pair. In all but one of the pairs more (1.7-8.5 fold) new alleles were found at 42 °C. Most frameshift, nonsense, or start-loss mutations were also found at 42 °C. Variable loci CAMP0575, CAMP0912, and CAMP0913 in both STs may regularly respond to different temperatures. Furthermore, frameshifts in four variable loci in ST8567 occurred at multiple time points, suggesting a persistent impact of temperature. These findings suggest that the temperature of isolation may impact the sequence of several loci in C. jejuni from cattle.

Whole-Genome Sequencing Reveals Multiple Subpopulations of Dominant and Persistent Lineage I Isolates of Listeria monocytogenes in Two Meat Processing Facilities during 2011-2015.

Listeria monocytogenes is a foodborne pathogen with a highly clonal population structure comprising multiple phylogenetic sub-groups that can persist within food processing environments and contaminate food. The epidemiology of L. monocytogenes is well-described in some developed countries; however, little is known about the prevalence and population structure of this pathogen in food and food processing environments located in less developed regions. The aim of this study was to determine the genetic characteristics and clonal relatedness of L. monocytogenes that were isolated from two Jamaican meat processing facilities. Of the 37 isolates collected between 2011 and 2015, only a single lineage II isolate was recovered (serotype 1/2c), and the remaining were lineage I isolates representing serotypes 4b, 1/2b, 3b, and two untypeable isolates. Pulsed-field gel electrophoresis (PFGE) delineated isolates into seven pulsotypes, and whole-genome sequencing (WGS) categorized most isolates within one of three clonal complexes (CC): CC2 (N = 12), CC5 (N = 11), and CC288 (N = 11). Isolates representing CC1 (N = 2) and CC9 (N = 1) were also recovered. Virulence-associated genes such as inlA and the LIPI-3 cluster were detected in multiple isolates, along with the stress survival islet cluster-1 (SSI-1), and benzalkonium (bcrABC) and cadmium (cad1, cad2, cad4) resistance cassettes. Multiple isolates that belong to the same CC and matching PFGE patterns were isolated from food and the environment from both facilities across multiple years, suggesting the presence of persistent strains of L. monocytogenes, and/or constant re-entry of the pathogens into the facilities from common sources. These findings highlight the ability of lineage I isolates of L. monocytogenes to colonize, persist, and predominate within two meat-producing environments, and underscores the need for robust surveillance strategies to monitor and mitigate against these important foodborne pathogens.