Whole-Genome Subtyping Reveals Population Structure and Host Adaptation of Salmonella Typhimurium from Wild Birds.

Within-host evolution of bacterial pathogens can lead to host-associated variants of the same species or serovar. Identification and characterization of closely related variants from diverse host species are crucial to public health and host-pathogen adaptation research. However, the work remained largely underexplored at a strain level until the advent of whole-genome sequencing (WGS). Here, we performed WGS-based subtyping and analyses of Salmonella enterica serovar Typhimurium (n = 787) from different wild birds across 18 countries over a 75-year period. We revealed seven avian host-associated S. Typhimurium variants/lineages. These lineages emerged globally over short timescales and presented genetic features distinct from S. Typhimurium lineages circulating among humans and domestic animals. We further showed that, in terms of virulence, host adaptation of these variants was driven by genome degradation. Our results provide a snapshot of the population structure and genetic diversity of S. Typhimurium within avian hosts. We also demonstrate the value of WGS-based subtyping and analyses in unravelling closely related variants at the strain level.

Low occurrence of multi-antimicrobial and heavy metal resistance in Salmonella enterica from wild birds in the United States.

Wild birds are common reservoirs of Salmonella enterica. Wild birds carrying resistant S. enterica may pose a risk to public health as they can spread the resistant bacteria across large spatial scales within a short time. Here, we whole-genome sequenced 375 S. enterica strains from wild birds collected in 41 U.S. states during 1978-2019 to examine bacterial resistance to antibiotics and heavy metals. We found that Typhimurium was the dominant S. enterica serovar, accounting for 68.3% (256/375) of the bird isolates. Furthermore, the proportions of the isolates identified as multi-antimicrobial resistant (multi-AMR: resistant to at least three antimicrobial classes) or multi-heavy metal resistant (multi-HMR: resistant to at least three heavy metals) were both 1.87% (7/375). Interestingly, all the multi-resistant S. enterica (n = 12) were isolated from water birds or raptors; none of them was isolated from songbirds. Plasmid profiling demonstrated that 75% (9/12) of the multi-resistant strains carried resistance plasmids. Our study indicates that wild birds do not serve as important reservoirs of multi-resistant S. enterica strains. Nonetheless, continuous surveillance for bacterial resistance in wild birds is necessary because the multi-resistant isolates identified in this study also showed close genetic relatedness with those from humans and domestic animals.

Comparative Genomic Analysis of Salmonella enterica Serovar Typhimurium Isolates from Passerines Reveals Two Lineages Circulating in Europe, New Zealand, and the United States.

Salmonella enterica serovar Typhimurium strains from passerines have caused wild bird deaths and human salmonellosis outbreaks in Europe, Oceania, and North America. Here, we performed comparative genomic analysis to explore the emergence, genetic relationship, and evolution of geographically dispersed passerine isolates. We found that passerine isolates from Europe and the United States clustered to form two lineages (EU and US passerine lineages), which were distinct from major S. Typhimurium lineages circulating in other diverse hosts (e.g., humans, cattle, pigs, chickens, and other avian hosts, such as pigeons and ducks). Further, passerine isolates from New Zealand clustered to form a sublineage (NZ passerine lineage) of the US passerine lineage. We inferred that the passerine isolates mutated at a rate of 3.2 × 10-7 substitutions/site/year, and the US, EU, and NZ passerine lineages emerged in approximately 1952, 1970, and 1996, respectively. Isolates from the three lineages presented genetic similarity, such as lack of antimicrobial resistance genes and accumulation of the same virulence pseudogenes. In addition, genetic diversity due to microevolution existed in the three passerine lineages. Specifically, pseudogenization in the type 1 fimbrial gene fimC (deletion of G at position 87) was detected only in the US and NZ passerine isolates, while single-base deletions in type 3 secretion system effector genes (i.e., gogB, sseJ, and sseK2) cooccurred solely in the EU passerine isolates. These findings provide insights into the evolution, host adaptation, and epidemiology of S. Typhimurium in passerines. IMPORTANCE Passerine-associated S. Typhimurium strains have been linked to human salmonellosis outbreaks in recent years. Here, we investigated the phylogenetic relationship of globally distributed passerine isolates and profiled their genomic similarity and diversity. Our study reveals two passerine-associated S. Typhimurium lineages circulating in Europe, Oceania, and North America. Isolates from the two lineages presented phylogenetic and genetic signatures that were distinct from those of isolates from other hosts. The findings shed light on the host adaptation of S. Typhimurium in passerines and are important for source attribution of S. Typhimurium strains to avian hosts. Further, we found that S. Typhimurium definitive phage type 160 (DT160) from passerines, which caused decades-long human salmonellosis outbreaks in New Zealand and Australia, formed a sublineage of the US passerine lineage, suggesting that DT160 might have originated from passerines outside Oceania. Our study demonstrates the importance of whole-genome sequencing and genomic analysis of historical microbial collections to modern epidemiologic surveillance.

Salmonella enterica Serovar Typhimurium Isolates from Wild Birds in the United States Represent Distinct Lineages Defined by Bird Type.

Salmonella enterica serovar Typhimurium is typically considered a host generalist; however, certain isolates are associated with specific hosts and show genetic features of host adaptation. Here, we sequenced 131 S. Typhimurium isolates from wild birds collected in 30 U.S. states during 1978-2019. We found that isolates from broad taxonomic host groups including passerine birds, water birds (Aequornithes), and larids (gulls and terns) represented three distinct lineages and certain S. Typhimurium CRISPR types presented in individual lineages. We also showed that lineages formed by wild bird isolates differed from most isolates originating from domestic animal sources, and that genomes from these lineages substantially improved source attribution of Typhimurium genomes to wild birds by a machine learning classifier. Furthermore, virulence gene signatures that differentiated S. Typhimurium from passerines, water birds, and larids were detected. Passerine isolates tended to lack S. Typhimurium-specific virulence plasmids. Isolates from the passerine, water bird, and larid lineages had close genetic relatedness with human clinical isolates, including those from a 2021 U.S. outbreak linked to passerine birds. These observations indicate that S. Typhimurium from wild birds in the United States are likely host-adapted, and the representative genomic data set examined in this study can improve source prediction and facilitate outbreak investigation. IMPORTANCE Within-host evolution of S. Typhimurium may lead to pathovars adapted to specific hosts. Here, we report the emergence of disparate avian S. Typhimurium lineages with distinct virulence gene signatures. The findings highlight the importance of wild birds as a reservoir for S. Typhimurium and contribute to our understanding of the genetic diversity of S. Typhimurium from wild birds. Our study indicates that S. Typhimurium may have undergone adaptive evolution within wild birds in the United States. The representative S. Typhimurium genomes from wild birds, together with the virulence gene signatures identified in these bird isolates, are valuable for S. Typhimurium source attribution and epidemiological surveillance.

Alginate-based antimicrobial coating reduces pathogens on alfalfa seeds and sprouts.

Foodborne illness associated with the consumption of contaminated sprouts has been a significant food safety risk. While seed disinfection with chemical sanitizers has been used as an intervention approach, its efficacy to reduce bacterial load has not been always satisfactory. In this study, a newly developed alginate-based, antimicrobial seed coating treatment was evaluated for its efficacy to reduce foodborne pathogens from alfalfa seeds and sprouts. Alfalfa seeds were inoculated with Listeria monocytogenes or Salmonella enterica serovar Typhimurium, air-dried, and thereafter subjected to different treatments. The treated seeds were analyzed for bacterial cell populations over 28 days of storage. Meanwhile, treated seeds after one day of storage were sprouted for three days and the populations of pathogens on sprouts were determined. The results showed that the alginate coating in the presence of lactic acid (alginate coating/LA) reduced both pathogens to an undetectable level one day after the treatment. With sprouts, alginate coating/LA resulted in significantly lower (P < 0.05) populations of Listeria and Salmonella than chlorine or lactic acid treatment. While the germination rate of seeds was reduced due to the use of lactic acid, the impact of alginate coating on germination was not significant. In general, this study indicated the effect of alginate coating on reducing the bacterial load from alfalfa seeds and sprouts, and further study is needed to select antimicrobial compounds and coating materials to reduce the adverse impact on germination rate.

Genomic analysis of Salmonella Typhimurium from humans and food sources accurately predicts phenotypic multi-drug resistance.

INTRODUCTION: Salmonella Typhimurium is the leading cause of foodborne illnesses in the U.S., causing over a million cases each year. In recent years, whole-genome sequencing (WGS) has become a standard tool for routine epidemiological subtyping. OBJECTIVES: The objectives of this study are 1) to compare the phenotypic and genotypic antimicrobial resistance (AMR) profiles of multidrug resistant (MDR) S. Typhimurium isolates, 2) to examine the genetic relatedness of a historic collection of MDR and pan-susceptible isolates from retail chickens. METHODS: We used data on Salmonella Typhimurium isolates in the publicly available NARMS national clinical and retail meat datasets from 2016 to 2018. Staramr (0.5.1) was used to identify AMR determinants and predictive resistance from genomes submitted to NCBI. Sensitivity and specificity of the WGS method were calculated with phenotypic resistance results as the reference. SNP-based cluster analysis was used to examine the genetic relatedness of MDR resistant and pan-susceptible isolates from retail chickens. RESULTS: The overall sensitivity of WGS as a predictor of clinical resistance was 96.47% and the overall specificity was 100.00%. The disagreement between phenotypic and genotypic results were mostly related to streptomycin. The MDR isolates differed by an average of 73.1 SNPs, while the pan-susceptible isolates differed by an average of 473.1 SNPs (p < 0.0001). The nearest distance between a pan-susceptible and an MDR isolate was 547 SNPs. CONCLUSION: WGS can reliably predict AMR in S. Typhimurium isolates and it can reveal genetic determinants to elucidate the evolution of antimicrobial resistance.

Antimicrobial-coated films as food packaging: A review.

Antimicrobial food packaging involves packaging the foods with antimicrobials to protect them from harmful microorganisms. In general, antimicrobials can be integrated with packaging materials via direct incorporation of antimicrobial agents into polymers or application of antimicrobial coating onto polymer surfaces. The former option is generally achieved through thermal film-making technology such as compression molding or film extrusion, which is primarily suitable for heat-stable antimicrobials. As a nonthermal technology, surface coating is more promising compared to molding or extrusion for manufacturing food packaging containing heat-sensitive antimicrobials. In addition, it also has advantages over direct incorporation to preserve the packaging materials' bulk properties (e.g., mechanical and physical properties) and minimize the amount of antimicrobials to reach sufficient efficacy. Herein, antimicrobial food packaging films achieved through surface coating is explored and discussed. The two components (i.e., film substrate and antimicrobials) consisting of the antimicrobial-coated films are reviewed as plastic/biopolymer films; and synthetic/naturally occurring antimicrobials. Furthermore, special emphasis is given to different coating technologies to deposit antimicrobials onto film substrate. Laboratory coating techniques (e.g., knife coating, bar coating, and spray coating) commonly applied in academic research are introduced briefly, and scalable coating methods (i.e., electrospinning/spraying, gravure roll coating, flexography coating) that have the potential to bring laboratory-developed antimicrobial-coated films to an industrial level are explained in detail. The migration profile, advantages/drawbacks of antimicrobial-coated films for food applications, and quantitative analyses of the reviewed antimicrobial-coated films from different aspects are also covered in this review. A conclusion is made with a discussion of the challenges that remain in bringing the production of antimicrobial-coated films to an industrial level.

Pathogen biofilm formation on cantaloupe surface and its impact on the antibacterial effect of lauroyl arginate ethyl.

Pathogen biofilm at fruit surface may pose a particular risk to food safety. In this study, the biofilms of Listeria monocytogenes V7 and Salmonella enterica serovar Typhimurium ATCC 13311 on cantaloupe fruit surface were visualized, and the resistance of biofilms against lauroyl arginate ethyl (LAE, an antibacterial compound) was evaluated. Each bacterium was inoculated on isolated cantaloupe rind surfaces at 105-106 CFU/cm2 and after incubation for 2, 12, 24, and 48 h, the surfaces were imaged using cryo-scanning electron microscopy (Cryo-SEM). The images showed that both pathogens formed biofilms on rind surfaces, with S. Typhimurium forming biofilm in 12 h and L. monocytogenes cells starting to aggregate in 2 h. For the inoculated rind surfaces treated with LAE, the cell counts were affected by both the incubation time and LAE concentration. For rind surface with 2 h incubation of S. Typhimurium, 400 and 800 µg/mL LAE was able to achieve >2.00 log reduction; however, 12 h incubation required 1600 and 2000 µg/mL LAE for >2.00 log reduction. In contrast, even the highest LAE concentration (2000 µg/mL) was unable to cause 1.00 log reduction for L. monocytogenes regardless the incubation time applied. The results showed that the biofilms of both bacteria substantially reduced LAE efficacy, and that the biofilm of L. monocytogenes was more resistant than that of S. Typhimurium.

A Comparative Study on Antimicrobial Resistance in Escherichia coli Isolated from Channel Catfish and Related Freshwater Fish Species.

Antimicrobial resistance (AMR) trends in 114 generic Escherichia coli isolated from channel catfish and related fish species were investigated in this study. Of these, 45 isolates were from commercial-sized channel catfish harvested from fishponds in Alabama, while 69 isolates were from Siluriformes products, accessed from the U.S. Department of Agriculture Food Safety and Inspection Service' (FSIS) National Antimicrobial Resistance Monitoring System (NARMS) program. Antibiotic susceptibility testing and whole genome sequencing were performed using the GenomeTrakr protocol. Upon analysis, the fishpond isolates showed resistance to ampicillin (44%), meropenem (7%) and azithromycin (4%). The FSIS NARMS isolates showed resistance to tetracycline (31.9%), chloramphenicol (20.3%), sulfisoxazole (17.4%), ampicillin (5.8%) and trimethoprim-sulfamethoxazole, nalidixic acid, amoxicillin-clavulanic acid, azithromycin and cefoxitin below 5% each. There was no correlation between genotypic and phenotypic resistance in the fishpond isolates, however, there was in NARMS isolates for folate pathway antagonists: Sulfisoxazole vs. sul1 and sul2 (p = 0.0042 and p < 0.0001, respectively) and trimethoprim-sulfamethoxazole vs. dfrA16 and sul1 (p = 0.0290 and p = 0.013, respectively). Furthermore, correlations were found for tetracyclines: Tetracycline vs. tet(A) and tet(B) (p < 0.0001 each), macrolides: Azithromycin vs. mph(E) and msr(E) (p = 0.0145 each), phenicols: Chloramphenicol vs. mdtM (p < 0.0001), quinolones: Nalidixic acid vs. gyrA_S83L=POINT (p = 0.0004), and ß-lactams: Ampicillin vs. blaTEM-1 (p < 0.0001). Overall, we recorded differences in antimicrobial susceptibility testing profiles, phenotypic-genotypic concordance, and resistance to critically important antimicrobials, which may be a public health concern.

New Techniques in Structural Tailoring of Starch Functionality.

Inherent characteristics of native starches such as water insolubility, retrogradation and syneresis, and instability in harsh processing conditions (e.g., high temperature and shearing, low pH) limit their industrial applications. As starch properties mainly depend on starch composition and structure, structural tailoring of starch has been important for overcoming functional limitations and expanding starch applications in different fields. In this review, we first introduce the basics of starch structure, properties, and functionalities and then describe the interactions of starch with lipids, polysaccharides, and phenolics. After reviewing genetic, chemical, and enzymatic modifications of starch, we describe current progress in the areas of porous starch and starch-based nanoparticles. New techniques, such as using the CRISPR-Cas9 technique to tailor starch structures and using an emulsion-assisted approach in forming functional starch nanoparticles, are only feasible when they are established based on fundamental knowledge of starch.

Evidence for common ancestry and microevolution of passerine-adapted Salmonella enterica serovar Typhimurium in the UK and USA.

The evolution of Salmonella enterica serovar Typhimurium (S. Typhimurium) within passerines has resulted in pathoadaptation of this serovar to the avian host in Europe. Recently, we identified an S. Typhimurium lineage from passerines in North America. The emergence of passerine-adapted S. Typhimurium in Europe and North America raises questions regarding its evolutionary origin. Here, we demonstrated that the UK and US passerine-adapted S. Typhimurium shared a common ancestor from ca. 1838, and larids played a key role in the clonal expansion by disseminating the common ancestor between North America and Europe. Further, we identified virulence gene signatures common in the passerine- and larid-adapted S. Typhimurium, including conserved pseudogenes in fimbrial gene lpfD and Type 3 Secretion System (T3SS) effector gene steC. However, the UK and US passerine-adapted S. Typhimurium also possessed unique virulence gene signatures (i.e. pseudogenes in fimbrial gene fimC and T3SS effector genes sspH2, gogB, sseJ and sseK2), and the majority of them (38/47) lost a virulence plasmid pSLT that was present in the larid-adapted S. Typhimurium. These results provide evidence that passerine-adapted S. Typhimurium share a common ancestor with those from larids, and the divergence of passerine- and larid-adapted S. Typhimurium might be due to pseudogenization or loss of specific virulence genes.

Abrasive brushing reduces pathogen biofilms at cantaloupe rind surface.

Foodborne pathogens constitute a major food safety risk for cantaloupe, and pathogen biofilms formed are particularly difficult to remove. The goal of this study was to evaluate abrasive brushing in removing biofilms from cantaloupe surface using Listeria monocytogenes V7 and Salmonella enterica serovar Typhimurium ATCC 13311 as models. Cantaloupe rind pieces were inoculated and then subjected to 30 min or 24 h incubations. The incubated rind pieces were subject to different washing and/or brushing procedures and then the bacterial cells were enumerated. Cryo-SEM showed planktonic cells after 30 min incubation, whereas biofilms formed at rind surfaces after 24 h incubation. For L. monocytogenes 30 min incubation group, the log reductions were 1.4 for brushing, 2.1 for brushing with diatomaceous earth (DE), 2.8 for peroxyacetic acid (PAA) washing, 4.2 for brushing with PAA, and 4.0 for brushing with DE and PAA. In contrast, for the 24 h incubation group, the log reductions were 0.4 for brushing, 1.5 for brushing with DE, 1.1 for PAA washing, 1.6 for brushing with PAA, and 3.0 for brushing with DE and PAA. Similar results were observed for the S. Typhimurium group. These outcomes showed the pathogen-removal efficacy of brushing with DE (abrasive brushing) in the presence of biofilms, suggesting the potential application of abrasive brushing in cleaning cantaloupe for improved food safety.

Biofilm of Escherichia coli O157:H7 on cantaloupe surface is resistant to lauroyl arginate ethyl and sodium hypochlorite.

Biofilms formed by Escherichia coli O157:H7 on cantaloupe rind were characterized in this study. Cantaloupe rind pieces inoculated with E. coli O157:H7 B6-914 was sampled after 2, 12, and 24h incubation for imaging with cryo-scanning electron microscopy (Cryo-SEM) or treating with lauroyl arginate ethyl (LAE) or sodium hypochlorite (SHC). Cryo-SEM images showed that E. coli O157:H7 formed a biofilm within 12h on the rind surface. For rind samples treated with LAE or SHC, the residual cell counts were significantly different (p<0.05) between 2 and 12h incubation, and between 2 and 24h of incubation. For the 2h incubation samples, E. coli O157:H7 was undetectable (>5-log reduction) after treatment with 2000µg/mL of LAE or SHC. In contrast, for 12h incubation samples, 2000µg/mL of LAE or SHC could only achieve 1.74 or 1.86-log reduction, respectively. The study showed the low efficacy of LAE and SHC on cantaloupe rind surface to reduce the E. coli biofilm, suggesting the needs for cantaloupe cleaning methods beyond washing with conventional antimicrobial agents.