Dynamics of microbiota and antimicrobial resistance in on-farm dairy processing plants using metagenomic and culture-dependent approaches.

On-farm dairy processing plants, which are situated close to farms and larger dairy processing facilities, face unique challenges in maintaining environmental hygiene. This can impact various stages of dairy processing. These plants operate on smaller scales and use Low-Temperature-Long-Time (LTLT) pasteurization, making them more susceptible to microbial contamination through direct and indirect contact. Antimicrobial-resistant bacteria found on dairy farms pose risks to human health by potentially transferring resistance via dairy products. Our study aimed to investigate microbial distribution and antimicrobial resistance at four key stages: the farm, pre-pasteurization, post-pasteurization, and processing environments. We assessed microbial distribution by quantifying indicator bacteria and conducting metagenomic analysis. Antimicrobial resistance was examined by identifying resistance phenotypes and detecting resistance genes in bacterial isolates and metagenomes. Our results showed that the indicator bacteria were detected at all stages of on-farm dairy processing. We observed a significant reduction in aerobic microbes and coliforms post-pasteurization. However, contamination of the final dairy products increased, suggesting potential cross-contamination during post-pasteurization. Metagenomic analysis revealed that Pseudomonas, a representative psychrotrophic bacterium, was predominant in both the farm (24.1 %) and pre-pasteurization (65.9 %) stages, indicating microbial transfer from the farms to the processing plants. Post-pasteurization, Pseudomonas and other psychrotrophs like Acinetobacter and Enterobacteriaceae remained dominant. Core microbiota analysis identified 74 genera in total, including 13 psychrotrophic bacteria, across all stages. Of the 59 strains isolated from these plants, 49 were psychrotrophic. Antimicrobial resistance analysis showed that 74.6 % (44/59) of isolates were resistant to at least one antibiotic, with cefoxitin-, ampicillin-, amoxicillin-, and ticarcillin-resistant bacteria present at all stages. Identical antimicrobial resistance patterns were observed in isolates from serial stages of the same farm and season, suggesting bacterial transmission across stages. Additionally, 27.1 % (16/59) of isolates carried plasmid-mediated resistance genes, which were also detected in the metagenomes of non-isolated samples, indicating potential antimicrobial resistance gene transmission and their presence in uncultured bacteria. These findings reveal the persistence of antimicrobial-resistant psychrotrophic bacteria in on-farm dairy processing plants, which pose potential health risks via dairy consumption. Our study underscores the importance of both culture-dependent and culture-independent methods to fully understand their distribution and impact.

Tracking the contamination sources of microbial population and characterizing Listeria monocytogenes in a chicken slaughterhouse by using culture-dependent and -independent methods.

Listeria monocytogenes is the etiologic agent of listeriosis, a foodborne disease that poses a threat to public health globally. Chicken meat exhibits heightened susceptibility to L. monocytogenes contamination during butchery. The persistence of this pathogen in the slaughterhouse environment enables recurring contamination of meat products. This study aimed at identifying the sources and transmission routes of L. monocytogenes contamination within an abattoir where it was consistently detected for three consecutive years (2019-2021). Furthermore, the environmental factors aiding contamination along chicken processing lines were determined by surveying the microbiome within the facility. Samples collected in 2019 to 2021 were subjected to culture-dependent analysis to assess the prevalence, serotypes, and multi-locus sequence typing (MLST) of L. monocytogenes. Additionally, the specimens collected in 2021 underwent culture-independent analysis via real-time quantitative polymerase chain reaction (qPCR) and 16S rRNA gene amplicon sequencing to identify the contamination sources and characterize the entire microbial community within the slaughterhouse. L. monocytogenes was isolated only from the clean zone, where the final slaughtering stage occurs. Most strains isolated from the final carcasses showed the same genetic cluster as the isolate in the chilling water and were assigned to MLST profile ST3. Culture-independent qPCR confirmed L. monocytogenes contamination in all samples, excluding post-scalding carcasses, prewashed post-evisceration carcasses, and the bleeding areas. Consequently, qPCR enabled more comprehensive identification of L. monocytogenes contamination points than culture-dependent approaches. Moreover, 16S rRNA gene amplicon sequencing demonstrated that psychro-tolerant and spoilage-related bacteria with L. monocytogenes-like attributes exhibited enhanced viability in the clean zone and immersion-chilling water. Metagenomics-based source tracking analysis further revealed that the shackles and chilling waters represent predominant sources of cross-contamination between different slaughterhouse zones, whereas the grading and packaging workstations and chilling water in the clean zone were deemed crucial sources affecting final carcass contamination. Collectively, these findings demonstrate through culture-dependent and -independent methods that L. monocytogenes spreads along the slaughter line, contaminating the slaughterhouse. Moreover, by investigating changes in microbial community and bacterial flow along the slaughter line within the facility, the sources influencing carcass contamination can be effectively traced.

Red Ginseng Dietary Fiber Shows Prebiotic Potential by Modulating Gut Microbiota in Dogs.

Red ginseng, widely used in traditional medicine for various conditions, imparts health benefits mainly by modulating the gut microbiota in humans. Given the similarities in gut microbiota between humans and dogs, red ginseng-derived dietary fiber may have prebiotic potential in dogs; however, its effects on the gut microbiota in dogs remain elusive. This double-blinded, longitudinal study investigated the impact of red ginseng dietary fiber on the gut microbiota and host response in dogs. A total of 40 healthy household dogs were randomly assigned to low-dose (n = 12), high-dose (n = 16), or control (n = 12) groups and fed a normal diet supplemented with red ginseng dietary fiber (3 g/5 kg body weight per day, 8 g/5 kg per day, or no supplement, respectively) for 8 weeks. The gut microbiota of the dogs was analyzed at 4 weeks and 8 weeks using 16S rRNA gene sequencing of fecal samples. Alpha diversity was significantly increased at 8 and 4 weeks in the low-dose and high-dose groups, respectively. Moreover, biomarker analysis showed that short-chain fatty acid producers such as Sarcina and Proteiniclasticum were significantly enriched, while potential pathogens such as Helicobacter were significantly decreased, indicating the increased gut health and pathogen resistance by red ginseng dietary fiber. Microbial network analysis showed that the complexity of microbial interactions was increased by both doses, indicating the increased stability of the gut microbiota. These findings suggest that red ginseng-derived dietary fiber could be used as a prebiotic to modulate gut microbiota and improve gut health in dogs. IMPORTANCE The canine gut microbiota is an attractive model for translational studies, as it responds to dietary interventions similarly to those in humans. Investigating the gut microbiota of household dogs that share the environment with humans can produce highly generalizable and reproducible results owing to their representativeness of the general canine population. This double-blind and longitudinal study investigated the impact of dietary fiber derived from red ginseng on the gut microbiota of household dogs. Red ginseng dietary fiber altered the canine gut microbiota by increasing diversity, enriching short-chain fatty acid-producing microbes, decreasing potential pathogens, and increasing the complexity of microbial interactions. These findings indicate that red ginseng-derived dietary fiber may promote canine gut health by modulating gut microbiota, suggesting the possibility of its use as a potential prebiotic.

Effect of biofilm formation by antimicrobial-resistant gram-negative bacteria in cold storage on survival in dairy processing lines.

Antimicrobial-resistant gram-negative bacteria in dairy products can transfer antimicrobial resistance to gut microbiota in humans and can adversely impact the product quality. In this study, we aimed to investigate their distribution in dairy processing lines and evaluate biofilm formation and heat tolerance under dairy processing line-like conditions. Additionally, we compared the relative expression of general and heat stress-related genes as well as spoilage-related gene between biofilm and planktonic cells under consecutive stresses, similar to those in dairy processing lines. Most species of gram-negative bacteria isolated from five different dairy processing plants were resistant to one or more antimicrobials. Biofilm formation by the bacteria at 5 °C increased with the increase in exposure time. Moreover, cells in biofilms remained viable under heat treatment, whereas all planktonic cells of the selected strains died. The expression of heat-shock-related genes significantly increased with heat treatment in the biofilms but mostly decreased in the planktonic cells. Thus, biofilm formation under raw milk storage conditions may improve the tolerance of antimicrobial-resistant gram-negative bacteria to pasteurization, thereby increasing their persistence in dairy processing lines and products. Furthermore, the difference in response to heat stress between biofilm and planktonic cells may be attributed to the differential expression of heat stress-related genes. Therefore, this study contributes to the understanding of how gram-negative bacteria persist under consecutive stresses in dairy processing procedures and the potential mechanism underlying heat tolerance in biofilms.

Circular intermediate-mediated horizontal transfer of the chromosome-encoded cfr(C) gene in multi-drug resistant Campylobacter coli from swine sources.

Campylobacter is a major zoonotic pathogen that causes gastrointestinal and, rarely, immune diseases in humans. The antimicrobial-resistance gene cfr(C) carried by Campylobacter and is a cfr-like gene that targets bacterial 23S rRNA through A2503 methylation. cfr(C) confers cross-resistance to five antimicrobial classes (PhLOPSA), including lincosamide, streptogramin A, and pleuromutilin, which are classified as critically important antimicrobials to human by the World Health Organization. To elucidate the genetic variation and horizontal transfer mechanism of cfr(C), we analyzed the genetic background and horizontal transfer unit of Campylobacter-derived cfr(C) through comparative genomic analysis. We identified nine cfr(C)-positive C. coli strains of 157 strains isolated from swine sources. Three novel cfr(C) gene single nucleotide polymorphism (SNP) sites (19delA, 674C > A, and 890 T > C) were identified from nine cfr(C)-positive strains. Among six identified cfr(C) SNP variant types (SNP-I to -VI), five types of randomly inserted cfr(C)-cassettes on chromosome and one type of plasmid-like element were identified, their gene cassette composition differing depending on the cfr(C) variants. Three of six cfr(C) cassette types contained aminoglycoside-streptothricin resistance cluster "aphA3-sat4-aadE." The cfr(C) gene cassette with pcp gene (GC-1, GC-4, and GC-5) formed a pcp-mediated circular intermediate "pcp-hp-cfr(C)-aphA3," which has not been previously reported. Other two cfr(C) cassette-types with ISChh1 formed circular intermediate "ISChh1-aphA3-cfr(C)-lnu (G)-pnp-ant1-hp-ATPase" and "ISChh1-aphA3-cfr(C)-hp." In conjugation assay, the pcp-mediated circular intermediate was naturally transferred to the plasmid of recipient C. coli wild-type strain from swine source, and comparative genomic analysis revealed that cfr(C) encoded in pcp-mediated circular intermediate was inserted into the plasmid of recipient by homologous recombination with pcp and aphA3. This study revealed that novel multidrug resistance gene cfr(C) carried by C. coli from swine sources can be highly genetically diverse and transferable. Moreover, we suggest that the transferability of chromosomal cfr(C) may contribute to the global spread of multidrug resistance against clinically important antimicrobials.

Environmental Perturbations during the Rehabilitation of Wild Migratory Birds Induce Gut Microbiome Alteration and Antibiotic Resistance Acquisition.

Wild migratory birds are essential for sustaining healthy ecosystems, but the effects of a rehabilitation period on their gut microbiomes are still unclear. Here, we performed longitudinal sampling, 16S rRNA sequencing, and antibiotic resistance monitoring of the gut microbiome of six species of wild migratory birds protected as natural monuments in South Korea that are subject to short- or long-term rehabilitation periods. Overall, gut microbiome diversity was significantly decreased in the early stages of rehabilitation, and it did not recover to a level comparable to that of wild birds. Moreover, while the abundance of short-chain fatty acid-producing bacteria decreased, that of zoonotic pathogens increased, indicating rehabilitation-induced dysbiosis. The metabolic pathways involved in the degradation of aromatic pollutants were significantly downregulated, suggesting the depletion of pollutant-degrading microorganisms. Antibiotic resistance of Escherichia coli significantly increased during rehabilitation, particularly ciprofloxacin and tetracycline resistance, and seven of the rehabilitated wild birds acquired multidrug resistance. The diet and habitat changes experienced by wild migratory birds during rehabilitation may have induced the observed gut microbiome dysbiosis and acquisition of antibiotic resistance. These rehabilitation-induced alterations might affect the adaptability of wild birds to their natural environments and contribute to the spread of antibiotic resistance after their release. IMPORTANCE Wild migratory birds are key for ecosystem health but highly sensitive to anthropogenic activities. Therefore, wild migratory birds often undergo rehabilitation to prevent species extinction or biodiversity monitoring. However, the impact of rehabilitation on the gut microbiome of wild migratory birds, which is closely associated with host fitness, remains unclear. For the migratory bird species considered natural monuments in South Korea evaluated here, such impacts could include rehabilitation-induced gut microbiome dysbiosis and acquisition of antibiotic resistance, with possible repercussions on the adaptability of wild birds and spread of antibiotic resistance in the environment after their release. Therefore, the dynamics of the gut microbiome and antibiotic resistance should be considered for implementing sustainable rehabilitation strategies.

Prevalence, Characteristics, and Clonal Distribution of Escherichia coli Carrying Mobilized Colistin Resistance Gene mcr-1.1 in Swine Farms and Their Differences According to Swine Production Stages.

Global spread of Escherichia coli strains carrying the mobilized colistin resistance gene mcr-1.1 (MCR1-EC) poses serious threats to public health. Colistin has been generally prescribed for swine colibacillosis, having made swine farms as major reservoirs of MCR1-EC. The present study aimed to understand characteristic differences of MCR1-EC, including prevalence, antimicrobial resistance, and virulence, according to swine production stages. In addition, genetic relatedness was evaluated between MCR1-EC isolated from this study as well as pig-, human-, and chicken-derived strains published in the National Center for Biotechnology Information (NCBI), based on the multi-locus sequence types (MLSTs) and whole-genome sequences (WGS). Individual fecal samples (n = 331) were collected from asymptomatic weaning-piglets, growers, finishers, and sows from 10 farrow-to-finishing farms in South Korea between 2017 and 2019. The weighted prevalence of MCR1-EC was 11.6% (95% CI: 8.9%-15.0%, 55/331), with the highest prevalence at weaning stage. The 96.2% of MCR1-EC showed multi-drug resistance. Notably, weaning stage-derived MCR1-EC showed higher resistance rates (e.g., against extended-spectrum ß-lactams or quinolones) than those from other stages. MCR1-EC with virulence advantages (e.g., intestinal/extraintestinal pathogenic E. coli or robust biofilm formation) were identified from all pig stages, accounting for nearly half of the total strains. WGS-based in-depth characterization showed that intestinal pathogenic MCR1-EC harbored multi-drug resistance and multiple virulence factors, which were highly shared between strains isolated from pigs of different stages. The clonal distribution of MCR1-EC was shared within swine farms but rarely across farms. The major clonal type of MCR1-EC from swine farms and NCBI database was ST10-A. Core genomes of MCR1-EC isolated from individuals within closed environments (same farms or human hospitals) were highly shared (genetic distance < 0.01), suggesting a high probability of clonal expansion of MCR1-EC within closed environments such as livestock husbandry. To the best of our knowledge, this is the first study to analyze the differences in the characteristics and clonal distribution of MCR1-EC according to production stages in swine farms, an important reservoir of MCR1-EC. Our results highlight the need to establish MCR1-EC control plans in swine farms based on an in-depth understanding of MCR1-EC characteristics according to swine production stages, focusing especially on the weaning stages.

Hyper-aerotolerant Campylobacter coli, an emerging foodborne pathogen, shows differential expressions of oxidative stress-related genes.

Although Campylobacter, an obligate microaerophilic foodborne pathogen, is susceptible to oxygen, aerotolerant/hyper-aerotolerant (HAT) Campylobacter can survive under aerobic conditions. Here, we aimed to reveal what affects the enhanced aerotolerance in HAT Campylobacter coli at genome and gene expression levels. We compared the whole genomes between HAT and oxygen-sensitive (OS) C. coli isolates from swine and analyzed the relative expressions of oxidative stress-related (sodB, ahpC, katA, and trxB) and iron transport/uptake-related (cfbpA, ceuE, feuB, and feoB) genes. The comparative genomics showed no relation between the clustering of the strains and aerotolerance levels. The reactive oxygen species-related factors involved in respiration, stress response, and iron acquisition/uptake were similar among the strains, regardless of their aerotolerance levels. However, the expressions of the oxidative stress-related genes under aerobic conditions compared to that of microaerobic conditions increased in the HAT strains, while decreased in the OS strains. Our findings suggest that what influences differences in aerotolerance between HAT and OS C. coli may be due to the differential expressions of oxidative stress-related genes despite the similarities in genomic structure. This study provides insights into the genetic basis of aerotolerance in C. coli. Therefore, it could assist in managing HAT C. coli that has the potential to be easily transmitted to humans through the food chain.

Hyper-Aerotolerant Campylobacter coli From Swine May Pose a Potential Threat to Public Health Based on Its Quinolone Resistance, Virulence Potential, and Genetic Relatedness.

Campylobacter, a major foodborne pathogen, is susceptible to oxygen. Recently, aerotolerant Campylobacter with enhanced tolerance to aerobic stress has become a major concern in food safety. However, the aerotolerance of Campylobacter coli from pigs has not been studied extensively. Here, we sought to investigate the prevalence of C. coli across multiple swine groups in farms, including weaning, growing, and fattening pigs in production stages and pregnant sows. Additionally, we analyzed C. coli aerotolerance, quinolone resistance, virulence potential, and multilocus sequence typing (MLST) genotypes. Finally, we compared the characteristics of C. coli according to the aerotolerance levels. In total, we obtained 124 (66.3%) C. coli isolates from 187 swine fecal samples across six swine farms. The pathogen was prevalent in weaning (45.5%), growing (68.3%), and fattening (75.4%) pigs, and pregnant sows (66.7%). Hyper-aerotolerant HAT C. coli (13.7% of 124 isolates) was present in all swine groups, with the highest proportion in the pregnant sows (27.3%). All HAT isolates possessed diverse virulence-related genes such as flaA, cadF, pldA, ceuE, and cdtA. All C. coli isolates were resistant to quinolones, and 12 (10%) presented high-level ciprofloxacin resistance (MIC ≥ 32 µg/mL). The proportion of C. coli isolates with a high-level ciprofloxacin resistance was the highest in HAT C. coli (18.8%). Furthermore, six MLST sequence types (STs) (ST827, ST830, ST854, ST1016, ST1068, and ST1096) of swine-derived C. coli were in common with human-derived C. coli (PubMLST). The proportion of C. coli belonging to such shared STs at each aerotolerance level was the highest in HAT C. coli (HAT vs. oxygen-sensitive; OR = 3.13). In conclusion, quinolone resistance of C. coli may be distributed throughout in all swine groups in farms. HAT C. coli is likely to remain in pig farms and re-infect other pigs in the farms. Furthermore, swine-derived HAT C. coli could be transmitted to humans easily through the food chain owing to its aerotolerance, and it could pose a threat to public health owing to its high-level ciprofloxacin resistance and virulence. This study highlights the need to develop management practices that prevent the transmission of swine-derived HAT C. coli to humans.

Prevalence, Characteristics and Clonal Distribution of Extended-Spectrum ß-Lactamase- and AmpC ß-Lactamase-Producing Escherichia coli Following the Swine Production Stages, and Potential Risks to Humans.

The worldwide spread of extended spectrum ß-lactamase (ESBL)- and AmpC ß-lactamase (AmpC)-producing Escherichia coli poses serious threats to public health. Swine farms have been regarded as important reservoirs of ESBL/AmpC-EC. This study aimed to determine the prevalence, ESBL/AmpC types, and clonal distribution of ESBL/AmpC-EC from swine farms and analyze the difference according to the swine production stages. In addition, we evaluated the potential risks of swine ESBL/AmpC-EC clones to humans. Individual fecal samples (n = 292) were collected from weaning, growing, finishing, and pregnant pigs in nine swine farms of South Korea between July 2017 and March 2020. In total, 161 ESBL/AmpC-EC isolates were identified (55.1%), with the highest prevalence detected in the weaning stage (86.3%). The dominant ESBL and AmpC types were CTX-M-55 (69.6%) and CMY-2 (4.3%), respectively. CTX-M found in all production stages, while CMY was only found in growing and finishing stages. In the conjugation assay, the high transferability of CTX-M gene (55.8%) was identified, while the transfer of CMY gene was not identified. The major clonal complexes (CCs) were CC101-B1 (26.8%), CC10-A (8.7%), and CC648-F (2.9%). There was similarity in clonal distribution between different swine production stages within swine farms, estimated using the k-means analysis, which suggested a clonal transmission between the different swine stages. Among swine ESBL/AmpC-EC sequence types (STs), seven STs (ST101, ST10, ST648, ST457, ST410, ST617, and ST744) were common with the human ESBL/AmpC-EC, which registered in National Center for Biotechnology Information database. The clonal population structure analysis based on the virulence factor (VF) presented that swine ESBL/AmpC-EC clones, especially ST101-B1, harbored a highly virulent profile. In conclusion, ESBL/AmpC-EC was distributed throughout the swine production stages, with the highest prevalence in the weaning stage. The CTX-M was present in all stages, while CMY was mostly found in growing-finishing stages. The swine ESBL/AmpC-EC was identified to harbor shared clone types with human ESBL/AmpC-EC and a virulent profile posing potential risk to humans. Considering the possibility of genetic and clonal distribution of ESBL/AmpC-EC among swine production stages, this study suggests the need for strategies considering the production system to control the prevalence of ESBL/AmpC-EC in swine farms.

Complete genome sequence and comparative genomic analysis of hyper-aerotolerant Campylobacter lari strain SCHS02 isolated from duck for its potential pathogenicity.

Campylobacter lari strain SCHS02, a novel hyper-aerotolerant strain that survives under aerobic conditions, was isolated from retail duck meat. The genome is a single chromosome of 1,520,838 base pairs, with a mean GC content of 29.7%. It harbors 1546 protein-coding sequences and 45 tRNA and 9 rRNA genes. Genes associated with the oxidative stress response, including perR, bcp, ahpC, and sodB, were identified in the genome. Furthermore, 68 virulence-related genes were identified and sorted into 9 classes and 14 subclasses. The virulence gene profile of SCHS02 was similar to those of two human clinical C. lari isolates. Comparative genomic analysis of strain SCHS02 and 18 C. lari strains retrieved from a public database revealed the core and accessory gene profiles of C. lari strains, as well as putative core gene involved in halotolerance. Phylogenetic analysis revealed that strain SCHS02 is genetically related to isolates from bird samples and human clinical isolates, rather than to isolates from other environmental sources. These findings reveal essential genomic information about the newly identified hyper-aerotolerant C. lari strain isolated from a duck source, providing a basis for future studies of the strain considering its potential threat to public health and further research of the pathogenicity of C. lari.

Metagenomic Analysis of the Gut Microbiota of Wild Mice, a Newly Identified Reservoir of Campylobacter.

Campylobacter, the most common etiologic agent of zoonotic gastroenteritis in humans, is present in many reservoirs including livestock animals, wildlife, soil, and water. Previously, we reported a novel Campylobacter jejuni strain SCJK02 (MLST ST-8388) from the gut of wild mice (Micromys minutus) using culture-dependent methods. However, due to fastidious growth conditions and the presence of viable but non-culturable Campylobacter spp., it is unclear whether M. minutus is a Campylobacter reservoir. This study aimed to: 1) determine the distribution and proportion of Campylobacter spp. in the gut microbiota of wild mice using culture-independent methods and 2) investigate the gut microbiota of wild mice and the relationship of Campylobacter spp. with other gut microbes. The gut microbiota of 38 wild mice captured from perilla fields in Korea and without any clinical symptoms (18 M. minutus and 20 Mus musculus) were analyzed. Metagenomic analysis showed that 77.8% (14 of 18) of the captured M. minutus harbored Campylobacter spp. (0.24-32.92%) in the gut metagenome, whereas none of the captured M. musculus carried Campylobacter spp. in their guts. Notably, 75% (6 of 8) of M. minutus determined to be Campylobacter-negative using culture-dependent methods showed a high proportion of Campylobacter through metagenome analysis. The results of metagenome analysis and the absence of clinical symptoms suggest that Campylobacter may be a component of the normal gut flora of wild M. minutus. Furthermore, linear discriminant analysis (LDA) showed that Campylobacter was the most enriched genus in the gut microbiota of M. minutus (LDA score, 5.37), whereas Lactobacillus was the most enriched genus in M. musculus (LDA score, -5.96). The differences in the presence of Campylobacter between the two species of wild mice may be attributed to the differential abundance of Campylobacter and Lactobacillus in their respective gut microbiota. In conclusion, the results indicate that wild M. minutus may serve as a potential Campylobacter reservoir. This study presents the first metagenomics analysis of the M. minutus gut microbiota to explore its possible role as an environmental Campylobacter reservoir and provides a basis for future studies using culture-independent methods to determine the role of environmental reservoirs in Campylobacter transmission.

Hyper-Aerotolerant Campylobacter coli from Duck Sources and Its Potential Threat to Public Health: Virulence, Antimicrobial Resistance, and Genetic Relatedness.

Campylobacter, a common foodborne human pathogen, is considered sensitive to oxygen. Recently, aerotolerant (AT) Campylobacter jejuni with the ability to survive under aerobic stress has been reported. Here, we investigated the prevalence of hyper-aerotolerant (HAT) Campylobacter coli from duck sources (118 carcasses and meat) and its characteristics to assess potential impacts on public health. Half of 56 C. coli isolates were HAT and most harbored various virulence genes including flaA, cadF, cdtA, ceuB, and wlaN. Moreover, 98.2% of C. coli isolates showed resistance to quinolones, including ciprofloxacin (CIP), and nine (16.1%) showed high-level resistance to ciprofloxacin (Minimum Inhibitory Concentration, MIC ≥ 32 µg/mL) and most of these were HAT. Based on genetic relatedness between C. coli from duck sources and those from human sources (PubMLST and NCBI), HAT isolates sharing the same MLST sequence types were significantly more prevalent than those not sharing the same sequence types as those from human sources. Therefore, HAT C. coli is prevalent in duck sources, and is most likely transmitted to humans through the food chain given its aerotolerance. This being so, it might pose a threat to public health given its virulence and antimicrobial resistance (AMR). This study will assist in improving control strategies to reduce farm-to-table HAT C. coli transmission to humans.

Correction to: Metagenomic analysis of isolation methods of a targeted microbe, Campylobacter jejuni, from chicken feces with high microbial contamination.

Following publication of the original article [1], the authors reported an error in Fig. 2. The correct figure is shown below.

Metagenomic analysis of isolation methods of a targeted microbe, Campylobacter jejuni, from chicken feces with high microbial contamination.

BACKGROUND: Originating from poultry, particularly chickens, Campylobacter jejuni is the leading foodborne pathogen worldwide and a major cause of campylobacteriosis. Isolating C. jejuni is difficult due to its specific growth requirements, the presence of viable but non-culturable bacteria, and because it is often masked by competing flora. Currently, there is no optimized method for isolating C. jejuni from chicken feces. Here, we evaluated the method for isolating C. jejuni from chicken feces using culture-independent sequence-based metagenomics and culture-dependent tools. Further, we assessed changes in microbial communities during microbe isolation to determine how the process can be improved. RESULTS: Fourteen different variations of C. jejuni isolation procedures were applied to all 35 chicken fecal samples. These variations included using different enrichment broths (without enrichment or enrichment in Bolton or Preston broth), different ratios of sample-to-enrichment broth (1:101, 1:102, and 1:103), and different selective agars (modified charcoal-cefoperazone-deoxycholate agar (mCCDA) or Preston agar). Enrichment during isolation of C. jejuni was evaluated on the basis of microbial diversity and taxonomic composition using metagenomics tools. The effect of selective media was evaluated using a combination of metagenomics and culture-dependent tools. Microbial diversity significantly decreased during the enrichment process, regardless of the type of enrichment broth, with the most significant decrease observed at a feces-to-broth ratio of 1:103. Particularly, in 103-Preston broth, the relative abundance of Campylobacter increased, while extended-spectrum beta-lactamase-producing Escherichia coli, which interfere with Campylobacter isolation, decreased. Metagenomics results were validated by quantitative PCR and culture-dependent analysis. Additionally, selective media affected the isolation results, although microbes with high relative abundance during enrichment were also frequently isolated using culture-dependent methods. Significantly more C. jejuni was isolated from mCCDA than from Preston agar enriched in 103 Preston broth. CONCLUSIONS: Enrichment in Preston broth at a ratio of 1:103 followed by spreading onto mCCDA was the most effective method for isolating C. jejuni. This is the first study to apply metagenomics to evaluate a method for isolating a targeted microbe, C. jejuni, from chicken feces, a source with high microbial contamination. Thus, metagenomics can be applied to improve methods for isolating bacteria that are difficult to separate.

The Wild Mouse (Micromys minutus): Reservoir of a Novel Campylobacter jejuni Strain.

Campylobacter jejuni is one of the most common zoonotic pathogens worldwide. Although the main sources of human C. jejuni infection are livestock, wildlife can also affect C. jejuni transmission in humans. However, it remains unclear whether wild mice harbor C. jejuni and are involved in the "environment-wildlife-livestock-human" transmission cycle of C. jejuni in humans. Here, we characterized C. jejuni from wild mice and identified genetic traces of wild mouse-derived C. jejuni in other hosts using a traditional approach, along with comparative genomics. We captured 115 wild mice (49 Mus musculus and 66 Micromys minutus) without any clinical symptoms from 22 sesame fields in Korea over 2 years. Among them, M. minutus were typically caught in remote areas of human houses and C. jejuni was solely isolated from M. minutus (42/66, 63.6%). We identified a single clone (MLST ST-8388) in all 42 C. jejuni isolates, which had not been previously reported, and all isolates had the same virulence/survival-factor profile, except for the plasmid-mediated virB11 gene. No isolates exhibited antibiotic resistance, either in phenotypic and genetic terms. Comparative-genomic analysis and MST revealed that C. jejuni derived from M. minutus (strain SCJK2) was not genetically related to those derived from other sources (registered in the NCBI genome database and PubMLST database). Therefore, we hypothesize that C. jejuni from M. minutus is a normal component of the gut flora following adaptation to the gastro-intestinal tract. Furthermore, M. minutus-derived C. jejuni had different ancestral lineages from those derived from other sources, and there was a low chance of C. jejuni transmission from M. minutus to humans/livestock because of their habitat. In conclusion, M. minutus may be a potential reservoir for a novel C. jejuni, which is genetically different from those of other sources, but may not be involved in the transmission of C. jejuni to other hosts, including humans and livestock. This study could form the basis for further studies focused on understanding the transmission cycle of C. jejuni, as well as other zoonotic pathogens originating from wild mice.