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.

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 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.

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.

Search for Campylobacter spp. Reveals High Prevalence and Pronounced Genetic Diversity of Arcobacter butzleri in Floodwater Samples Associated with Hurricane Florence in North Carolina, USA.

In September 2018, Hurricane Florence caused extreme flooding in eastern North Carolina, USA, a region highly dense in concentrated animal production, especially swine and poultry. In this study, floodwater samples (n = 96) were collected as promptly post-hurricane as possible and for up to approximately 30 days and selectively enriched for Campylobacter using Bolton broth enrichment and isolation on modified charcoal cefoperazone deoxycholate agar (mCCDA) microaerobically at 42°C. Only one sample yielded Campylobacter, which was found to be Campylobacter jejuni with the novel sequence type 2866 (ST-2866). However, the methods employed to isolate Campylobacter readily yielded Arcobacter from 73.5% of the floodwater samples. The Arcobacter isolates failed to grow on Mueller-Hinton agar at 25, 30, 37, or 42°C microaerobically or aerobically but could be readily subcultured on mCCDA at 42°C microaerobically. Multilocus sequence typing of 112 isolates indicated that all were Arcobacter butzleri The majority (85.7%) of the isolates exhibited novel sequence types (STs), with 66 novel STs identified. Several STs, including certain novel ones, were detected in diverse waterbody types (channel, isolated ephemeral pools, floodplain) and from multiple watersheds, suggesting the potential for regionally dominant strains. The genotypes were clearly partitioned into two major clades, one with high representation of human and ruminant isolates and another with an abundance of swine and poultry isolates. Surveillance of environmental waters and food animal production systems in this animal agriculture-dense region is needed to assess potential regional prevalence and temporal stability of the observed A. butzleri strains as well as their potential association with specific types of food animal production.IMPORTANCE Climate change and associated extreme weather events can have massive impacts on the prevalence of microbial pathogens in floodwaters. However, limited data are available on foodborne zoonotic pathogens such as Campylobacter or Arcobacter in hurricane-associated floodwaters in rural regions with intensive animal production. With a high density of intensive animal production as well as pronounced vulnerability to hurricanes, eastern North Carolina presents unique opportunities in this regard. Our findings revealed widespread incidence of the emerging zoonotic pathogen Arcobacter butzleri in floodwaters from Hurricane Florence. We encountered high and largely unexplored diversity while also noting the potential for regionally abundant and persistent clones. We noted pronounced partitioning of the floodwater genotypes into two source-associated clades. The data will contribute to elucidating the poorly understood ecology of this emerging pathogen and highlight the importance of surveillance of floodwaters associated with hurricanes and other extreme weather events for Arcobacter and other zoonotic pathogens.

Draft Genome Sequence of Multidrug-Resistant Listeria innocua Strain UAM003-1A, Isolated from a Wild Black Bear (Ursus americanus).

There is currently limited knowledge of the genome sequences of nonpathogenic Listeria species, especially strains from wildlife. Here, we report the draft genome sequence and associated genome information of an antibiotic-resistant Listeria innocua strain, UAM003-1A, isolated from the feces of a black bear in California, USA.

Strain-Specific Differences in Survival of Campylobacter spp. in Naturally Contaminated Turkey Feces and Water.

Campylobacter jejuni and Campylobacter coli are leading causes of human foodborne illness, with poultry as a major vehicle. Turkeys are frequently colonized with Campylobacter, but little is known about Campylobacter survival in turkey feces, even though fecal droppings are major vehicles for Campylobacter within-flock transmission as well as for environmental dissemination. Our objective was to examine survival of Campylobacter, including different strains, in freshly excreted feces from naturally colonized commercial turkey flocks and in suspensions of turkey feces in water from the turkey house. Fecal and water suspensions were stored at 4°C, and Campylobacter populations were enumerated on selective media at 48-h intervals. C. jejuni and C. coli isolates were characterized for resistance to a panel of antibiotics, and a subset was subtyped using multilocus sequence typing. Campylobacter was recovered from feces and water for up to 16 days. Analysis of 548 isolates (218 C. jejuni and 330 C. coli) revealed that C. jejuni survived longer than C. coli in feces (P = 0.0005), while the reverse was observed in water (P < 0.0001). Strain-specific differences in survival were noted. Multidrug-resistant C. jejuni isolates of sequence type 1839 (ST-1839) and the related ST-2935 were among the longest-surviving isolates in feces, being recovered for up to 10 to 16 days, while multidrug-resistant C. coli isolates of ST-1101 were recovered from feces for only up to 4 days. Data on Campylobacter survival upon excretion from the birds can contribute to further understanding of the transmission dynamics of this pathogen in the poultry production ecosystem.IMPORTANCECampylobacter jejuni and Campylobacter coli are leading foodborne pathogens, with poultry as a major reservoir. Due to their growth requirements, these Campylobacter spp. may be unable to replicate once excreted by their avian hosts, but their survival in feces and the environment is critical for transmission in the farm ecosystem. Reducing the prevalence of Campylobacter-positive flocks can have major impacts in controlling both contamination of poultry products and environmental dissemination of the pathogens. However, understanding the capacity of these pathogens to survive in transmission-relevant vehicles such as feces and farmhouse water remains poorly understood, and little information is available on species- and strain-associated differences in survival. Here, we employed model conditions to investigate the survival of C. jejuni and C. coli from naturally colonized turkey flocks, and with diverse genotypes and antimicrobial resistance profiles, in turkey feces and in farmhouse water.

Proximity to Other Commercial Turkey Farms Affects Colonization Onset, Genotypes, and Antimicrobial Resistance Profiles of Campylobacter spp. in Turkeys: Suggestive Evidence from a Paired-Farm Model.

Campylobacter is a leading foodborne pathogen, and poultry products are major vehicles for human disease. However, determinants impacting Campylobacter colonization in poultry remain poorly understood, especially with turkeys. Here, we used a paired-farm design to concurrently investigate Campylobacter colonization and strain types in two turkey breeds (Hybrid and Nicholas) at two farms in eastern North Carolina. One farm (the Teaching Animal Unit [TAU]) was a university teaching unit at least 40 km from commercial turkey farms, while the other (SIB) was a commercial farm in an area with a high density of turkey farms. Day-old birds were obtained from the same breeder flock and hatchery and placed at TAU and SIB on the same day. Birds were marked to identify turkey breed and then commingled on each farm. TAU birds became colonized 1 week later than SIB and had lower initial Campylobacter levels in the cecum. Interestingly, Campylobacter genotypes and antimicrobial resistance profiles differed markedly between the farms. Most TAU isolates were resistant only to tetracycline, whereas multidrug-resistant isolates predominated at SIB. Multilocus sequence typing revealed that no Campylobacter genotypes were shared between TAU and SIB. A bovine-associated genotype (sequence type 1068 [ST1068]) predominated in Campylobacter coli from TAU, while SIB isolates had genotypes commonly encountered in commercial turkey production in the region. One multidrug-resistant Campylobacter jejuni strain (ST1839) showed significant association with one of the two turkey breeds. The findings highlight the need to further characterize the impact of farm-specific factors and host genetics on antimicrobial resistance and genotypes of C. jejuni and C. coli that colonize turkeys.IMPORTANCE Colonization of poultry with Campylobacter at the farm level is complex, poorly understood, and critically linked to contamination of poultry products, which is known to constitute a leading risk factor for human campylobacteriosis. Here, we investigated the use of a paired-farm design under standard production conditions and in the absence of experimental inoculations to assess potential impacts of farm and host genetics on prevalence, antimicrobial resistance and genotypes of Campylobacter in commercial turkeys of two different breeds. Data suggest impacts of farm proximity to other commercial turkey farms on the onset of colonization, genotypes, and antimicrobial resistance profiles of Campylobacter colonizing the birds. Furthermore, the significant association of a specific multidrug-resistant Campylobacter jejuni strain with turkeys of one breed suggests colonization partnerships at the Campylobacter strain-turkey breed level. The study design avoids potential pitfalls associated with experimental inoculations, providing novel insights into the dynamics of turkey colonization with Campylobacter in actual farm ecosystems.

The Listeria monocytogenes Key Virulence Determinants hly and prfA are involved in Biofilm Formation and Aggregation but not Colonization of Fresh Produce.

Listeria monocytogenes has been extensively studied as a model facultative intracellular pathogen. While the roles of major virulence factors in host-pathogen interactions have been extensively characterized, recent work suggests that some of these factors can also contribute to environmental proliferation of this pathogen. In this study, we characterized two non-hemolytic transposon mutants of strain 2011L-2858 (serotype 1/2b), implicated in the 2011 listeriosis outbreak via whole cantaloupe, for their capacity to form biofilms on polystyrene, aggregate, and colonize cantaloupe rind. One mutant harbored a single mariner-based transposon insertion in hly, encoding the hemolysin Listeriolysin O, while the other harbored a single insertion in prfA, encoding PrfA, a master regulator for hly and numerous other virulence genes. Biofilm formation was significantly reduced in the prfA mutant, and to a lesser extent, in the hly mutant. Inactivation of either hly or prfA significantly reduced L. monocytogenes aggregation. However, both mutants adhered similarly to the wildtype parental strain on cantaloupe rind at either 25 or 37°C. Furthermore, growth and competitive fitness of the mutants on cantaloupe rind was not significantly impacted at either temperature. The findings suggest that, in spite of their involvement in biofilm formation and aggregation, these key virulence determinants may not be required for the ability of L. monocytogenes to colonize fresh produce.

Complete Genome Sequences of Multidrug-Resistant Campylobacter jejuni Strain 14980A (Turkey Feces) and Campylobacter coli Strain 14983A (Housefly from a Turkey Farm), Harboring a Novel Gentamicin Resistance Mobile Element.

Multidrug resistance (MDR) in foodborne pathogens is a major food safety and public health issue. Here we describe whole-genome sequences of two MDR strains of Campylobacter jejuni and Campylobacter coli from turkey feces and a housefly from a turkey farm. Both strains harbor a novel chromosomal gentamicin resistance mobile element.