Relevance of genetic causes and environmental adaptation of Cronobacter spp. isolated from infant and follow-up formula production factories and retailed products in China: A 7-year period of continuous surveillance based on genome-wide analysis.

The possible contamination routes, environmental adaptation, and genetic basis of Cronobacter spp. in infant and follow-up formula production factories and retailed products in mainland China have been determined by laboratory studies and whole-genome comparative analysis in a 7-year nationwide continuous surveillance spanning from 2012 to 2018. The 2-year continuous multicenter surveillance of the production process (conducted in 2013 and 2014) revealed that the source of Cronobacter spp. in the dry-blending process was the raw dry ingredients and manufacturing environment (particularly in the vibro sieve and vacuum cleaner), while in the combined process, the main contamination source was identified as the packing room. It is important to note that, according to the contamination control knowledge obtained from the production process surveillance, the contamination rate of retail powdered infant formula (PIF) and follow-up formula (FUF) products in China decreased significantly from 2016 onward, after improving the hygiene management practices in factories. The prevalence of Cronobacter spp. in retailed PIF and FUF in China in 2018 was dramatically reduced from 1.55 % (61/3925, in 2012) to an average as low as 0.17 % (13/7655 in 2018). Phenotype determination and genomic analysis were performed on a total of 90 Cronobacter spp. isolates obtained from the surveillance. Of the 90 isolates, only two showed resistance to either cefazolin or cefoxitin. The multilocus sequence typing results revealed that C. sakazakii sequence type 1 (ST1), ST37, and C. malonaticus ST7 were the dominant sequence types (STs) collected from the production factories, while C. sakazakii ST1, ST4, ST64, and ST8 were the main STs detected in the retailed PIF and FUF nationwide. One C. sakazakii ST4 isolate (1.1 %, 1/90) had strong biofilm-forming ability and 13 isolates (14.4 %, 13/90) had weak biofilm-forming ability. Genomic analysis revealed that Cronobacter spp. have a relatively stable core-genome and an increasing pan-genome size. Plasmid IncFIB (pCTU3) was prevalent in this genus and some contained 14 antibacterial biocide- and metal-resistance genes (BMRGs) including copper, silver, and arsenic resistant genes. Plasmid IncN_1 was predicted to contain 6 ARGs. This is the first time that a multi-drug resistance IncN_1 type plasmid has been reported in Cronobacter spp. Genomic variations with respect to BMRGs, virulence genes, antimicrobial resistance genes (ARGs), and genes involved in biofilm formation were observed among strains of this genus. There were apparent differences in copies of bcsG and flgJ between the biofilm-forming group and non-biofilm-forming group, indicating that these two genes play key roles in biofilm formation. The findings of this study have improved our understanding of the contamination characteristics and genetic basis of Cronobacter spp. in PIF and FUF and their production environment in China and provide important guidance to reduce contamination with this pathogen during the production of PIF and FUF.

Prevalence and characterization of foodborne pathogens isolated from fresh-cut fruits and vegetables in Beijing, China.

Pre-cut fresh fruits and vegetables are highly appealing to consumers for their convenience, however, as they are highly susceptible to microbial contamination in processing, the potential risks of foodborne illnesses to public health are not negligible. This study aimed to assess the prevalence, antibiotic susceptibility and molecular characteristics of major foodborne pathogens (Listeria monocytogenes, Escherichia coli, Staphylococcus aureus and Salmonella) isolated from fresh-cut fruits and vegetables in Beijing, China. 86 stains were isolated from 326 samples, with S. aureus being the highest prevalence (15.38 %), followed by E. coli (9.23 %) and L. monocytogenes (1.85 %), while no Salmonella was detected. The prevalence by type of food indicated that fruit trays and mixed vegetables were more susceptible to contamination by pathogens. 98 % of S. aureus were resistant to at least of one antibiotic, and showed a high resistance rate to benzylpenicillin (90 %) and oxacillin (48 %). Among 25 E. coli isolates, 57.67 % of which exhibited multi-drug resistance, with common resist to trimethoprim/sulfamethoxazole (66.67 %) and ampicillin (63.33 %). A total of 9 sequence types (STs) and 8 spa types were identified in 35 S. aureus isolates, with ST398-t34 being the predominant type (42.86 %). Additionally, analysis of 25 E. coli isolates demonstrated significant heterogeneity, characterized by 22 serotypes and 18 STs. Genomic analysis revealed that 5 and 44 distinct antibiotic resistance genes (ARGs) in S. aureus and E. coli, respectively. Seven quinolone resistance-determining regions (QRDRs) mutations were identified in E. coli isolates, of which GyrA (S83L) was the most frequently detected. All the S. aureus and E. coli isolates harbored virulence genes. ARGs in S. aureus and E. coli showed a significant positive correlation with plasmids. Furthermore, one L. monocytogenes isolate, which was ST101 and serogroupIIc from watermelon sample, harbored virulence genes (inlA and inlB) and LIPI-1 pathogenic islands (prfA, plcA, hly and actA), which posed potential risks for consumer's health. This study focused on the potential microbial risk of fresh-cut fruits and vegetables associated with foodborne diseases, improving the scientific understanding towards risk assessment related to ready-to-eat foods.

Antimicrobial susceptibility and genomic characterization of Vibrio parahaemolyticus isolated from aquatic foods in 15 provinces, China, 2020.

Prevalent in marine, estuarine and coastal environments, Vibrio parahaemolyticus is one of the major foodborne pathogens which can cause acute gastroenteritis through consumption of contaminated food. This study encompassed antimicrobial resistance, molecular characteristics and phylogenetic relationships of 163 V. parahaemolyticus isolated from aquatic foods across 15 provinces in China. The isolates showed high resistance rates against ampicillin (90.80 %, 148/163) and cefazolin (72.39 %, 118/163). Only 5 isolates demonstrated multi-drug resistance (MDR) phenotypes. A total of 37 different antibiotic resistance genes (ARGs) in correlation with seven antimicrobial categories were identified. tet(34) and tet(35) were present in all 163 isolates. Other most prevalent ARGs were those conferring resistance to ß-lactams, with prevalence rate around 18.40 % (30/163). The virulence genes tdh and trh were found in 17 (10.43 %) and 9 (5.52 %) isolates, respectively. Totally 121 sequence types (STs) were identified through whole genome analysis, among which 60 were novel. The most prevalent sequence type was ST3 (9.20 %, 15/163), which shared the same genotype profile of trh_, tdh+ and blaCARB-22+. Most of the tdh+V. parahaemolyticus isolates was clustered into a distinctive clade by the phylogenetic analysis. Our study showed that the antimicrobial resistance of V. parahaemolyticus in aquatic foods in China was moderate. However, the emerging of MDR isolates implicate strengthened monitoring is needed for the better treatment of human V. parahaemolyticus infections. High genetic diversity and virulence potential of the isolates analyzed in this study help better understanding and evaluating the risk of V. parahaemolyticus posed to public health.

Convergence of resistance and evolutionary responses in Escherichia coli and Salmonella enterica co-inhabiting chicken farms in China.

Sharing of genetic elements among different pathogens and commensals inhabiting same hosts and environments has significant implications for antimicrobial resistance (AMR), especially in settings with high antimicrobial exposure. We analysed 661 Escherichia coli and Salmonella enterica isolates collected within and across hosts and environments, in 10 Chinese chicken farms over 2.5 years using data-mining methods. Most isolates within same hosts possessed the same clinically relevant AMR-carrying mobile genetic elements (plasmids: 70.6%, transposons: 78%), which also showed recent common evolution. Supervised machine learning classifiers revealed known and novel AMR-associated mutations and genes underlying resistance to 28 antimicrobials, primarily associated with resistance in E. coli and susceptibility in S. enterica. Many were essential and affected same metabolic processes in both species, albeit with varying degrees of phylogenetic penetration. Multi-modal strategies are crucial to investigate the interplay of mobilome, resistance and metabolism in cohabiting bacteria, especially in ecological settings where community-driven resistance selection occurs.

Machine learning and metagenomics reveal shared antimicrobial resistance profiles across multiple chicken farms and abattoirs in China.

China is the largest global consumer of antimicrobials and improving surveillance methods could help to reduce antimicrobial resistance (AMR) spread. Here we report the surveillance of ten large-scale chicken farms and four connected abattoirs in three Chinese provinces over 2.5 years. Using a data mining approach based on machine learning, we analysed 461 microbiomes from birds, carcasses and environments, identifying 145 potentially mobile antibiotic resistance genes (ARGs) shared between chickens and environments across all farms. A core set of 233 ARGs and 186 microbial species extracted from the chicken gut microbiome correlated with the AMR profiles of Escherichia coli colonizing the same gut, including Arcobacter, Acinetobacter and Sphingobacterium, clinically relevant for humans, and 38 clinically relevant ARGs. Temperature and humidity in the barns were also correlated with ARG presence. We reveal an intricate network of correlations between environments, microbial communities and AMR, suggesting multiple routes to improving AMR surveillance in livestock production.

Correlation Analysis of Microbial Contamination and Alkaline Phosphatase Activity in Raw Milk and Dairy Products.

Microbial contamination in raw milk and dairy products can detrimentally affect product quality and human health. In this study, the aerobic plate count, aerobic Bacillus abundance, thermophilic aerobic Bacillus abundance, and alkaline phosphatase activity were determined in 435 raw milk, 451 pasteurized milk, and 617 sterilized milk samples collected from 13 Chinese provinces (or municipalities). Approximately 9.89% and 2.22% of raw milk and pasteurized milk samples exceeded the threshold values for the aerobic plate count, respectively. The proportions of aerobic Bacillus in raw milk, pasteurized milk, and sterilized milk were 54.02%, 14.41%, and 1.30%, respectively. The proportions of thermophilic aerobic Bacillus species were 7.36% in raw milk and 4.88% in pasteurized milk samples, and no bacteria were counted in sterilized milk. Approximately 36.18% of raw milk samples contained >500,000 mU/L of alkaline phosphatase activity, while 9.71% of pasteurized milk samples contained >350 mU/L. For raw milk, there was a positive correlation between the aerobic plate count, the aerobic Bacillus abundance, and the alkaline phosphatase activity, and there was a positive correlation between the aerobic Bacillus abundance, the thermophilic aerobic Bacillus count, and the alkaline phosphatase activity. For pasteurized milk, there was a positive correlation between the aerobic plate count, the aerobic Bacillus abundance, and the thermophilic aerobic Bacillus count; however, the alkaline phosphatase activity had a negative correlation with the aerobic plate count, the aerobic Bacillus abundance, and the thermophilic aerobic Bacillus abundance. These results facilitate the awareness of public health safety issues and the involvement of dairy product regulatory agencies in China.

Multifunctional Electrospinning Polyhydroxyalkanoate Fibrous Scaffolds with Antibacterial and Angiogenesis Effects for Accelerating Wound Healing.

To treat large-scale wounds or chronic ulcers, it is highly desirable to develop multifunctional wound dressings that integrate antibacterial and angiogenic properties. While many biomaterials have been fabricated as wound dressings for skin regeneration, few reports have addressed the issue of complete skin regeneration due to the lack of vasculature and hair follicles. Herein, an instructive poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB) fibrous wound dressing that integrates an antibacterial ciprofloxacin (CIP) and pro-angiogenic dimethyloxalylglycine (DMOG) is successfully prepared via electrospinning. The resultant dressings exhibit suitable flexibility with tensile strength and elongation at break up to 4.08 ± 0.18 MPa and 354.8 ± 18.4%, respectively. The in vitro results revealed that the groups of P34HB/CIP/DMOG dressings presented excellent biocompatibility on cell proliferation and significantly promote the spread and migration of L929 cells in both transwell and scratch assays. Capillary-like tube formation is also significantly enhanced in the P34HB/CIP/DMOG group dressings. Additionally, dressings from the P34HB/CIP and P34HB/CIP/DMOG groups show a broad spectrum of antimicrobial action against Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. In vivo studies further demonstrated that the prepared dressings in the P34HB/CIP/DMOG group not only improved wound closure, increased re-epithelialization and collagen formation, as well as reduced inflammatory response but also increased angiogenesis and remodeling, resulting in complete skin regeneration and hair follicles. Collectively, this work provides a simple but efficient approach for the design of a versatile wound dressing with the potential to have a synergistic effect on the rapid stimulation of angiogenesis as well as antibacterial activity in full-thickness skin repair.

Dissecting microbial communities and resistomes for interconnected humans, soil, and livestock.

A debate is currently ongoing as to whether intensive livestock farms may constitute reservoirs of clinically relevant antimicrobial resistance (AMR), thus posing a threat to surrounding communities. Here, combining shotgun metagenome sequencing, machine learning (ML), and culture-based methods, we focused on a poultry farm and connected slaughterhouse in China, investigating the gut microbiome of livestock, workers and their households, and microbial communities in carcasses and soil. For both the microbiome and resistomes in this study, differences are observed across environments and hosts. However, at a finer scale, several similar clinically relevant antimicrobial resistance genes (ARGs) and similar associated mobile genetic elements were found in both human and broiler chicken samples. Next, we focused on Escherichia coli, an important indicator for the surveillance of AMR on the farm. Strains of E. coli were found intermixed between humans and chickens. We observed that several ARGs present in the chicken faecal resistome showed correlation to resistance/susceptibility profiles of E. coli isolates cultured from the same samples. Finally, by using environmental sensing these ARGs were found to be correlated to variations in environmental temperature and humidity. Our results show the importance of adopting a multi-domain and multi-scale approach when studying microbial communities and AMR in complex, interconnected environments.

Enhanced bone regeneration via PHA scaffolds coated with polydopamine-captured BMP2.

The hierarchical three-dimensional (3D)-printing scaffolds based on microbial polyester poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB) were designed and used for bone tissue engineering via surface functionalization on 3D-printed (P34HB) scaffolds using polydopamine (PDA)-mediated recombinant human bone morphogenetic protein-2 (BMP2), leading to enhanced bone formation in a rat model with a calvarial critical-size bone defect. Taking advantage of the adhesive property of PDA under alkaline and aerobic conditions, osteogenic BMP2 was captured on the surface of PHA scaffolds, resulting in their enhanced osteogenic bioactivity, better stem cell adhesion and proliferation, and sustainable release of a bioactive substance over a period of 30 days. These contributed to notable differences in alkaline phosphatase (ALP) activity, mineralization, expressions of osteogenesis-related genes, as well as finally enhanced in vivo bone formation in rats. The functionalized 3D-printed P34HB scaffolds via the PDA-mediated osteogenic activity were developed as a versatile platform for bone tissue regeneration.

Antibacterial Peptide NP-6 Affects Staphylococcus aureus by Multiple Modes of Action.

Our previous study extracted and identified an antibacterial peptide that was named NP-6. Herein, we investigated the physicochemical properties of NP-6, and elucidated the mechanisms underlying its antimicrobial activity against Staphylococcus aureus. The results showed that the hemolysis activity of NP-6 was 2.39 ± 0.13%, lower than Nisin A (3.91 ± 0.43%) at the same concentration (512 µg/mL). Negligible cytotoxicity towards RAW264.7 cells was found when the concentration of NP-6 was lower than 512 µg/mL. In addition, it could keep most of its activity in fetal bovine serum. Moreover, transmission electron microscopy, confocal laser scanning microscopy, and flow cytometry results showed that NP-6 can destroy the integrity of the bacterial cell membrane and increase the membrane permeability. Meanwhile, NP-6 had binding activity with bacterial DNA and RNA in vitro and strongly inhibited the intracellular ß-galactosidase activity of S. aureus. Our findings suggest that NP-6 could be a promising candidate against S. aureus.

Antimicrobial-Resistant Evolution and Global Spread of Enterococcus faecium Clonal Complex (CC) 17: Progressive Change from Gut Colonization to Hospital-Adapted Pathogen.

For a long time, Enterococcus faecium (E. faecium) was thought to be a commensal strain in human and animal digestive tracts. However, over the past three decades, some unique E. faecium clones rapidly acquired multiple antimicrobial resistance (AMR), which led these clones to survive hospital environments and become a hospital-adapted E. faecium clonal complex (CC) 17. Since the adaptation of these clones to changes in habitat, vancomycin-resistant E. faecium CC17 has emerged as the leading cause of hospital-acquired infections worldwide. This epidemic hospital-adapted lineage has diverged from other populations approximately 75 years ago. The CC17 lineage originated from animal strains, but not human commensal lines. We reviewed the evolutionary progress and the molecular mechanisms of E. faecium CC17 from a gut commensal to a multi-antimicrobial resistant nosocomial pathogen.

Whole-genome sequencing and gene sharing network analysis powered by machine learning identifies antibiotic resistance sharing between animals, humans and environment in livestock farming.

Anthropogenic environments such as those created by intensive farming of livestock, have been proposed to provide ideal selection pressure for the emergence of antimicrobial-resistant Escherichia coli bacteria and antimicrobial resistance genes (ARGs) and spread to humans. Here, we performed a longitudinal study in a large-scale commercial poultry farm in China, collecting E. coli isolates from both farm and slaughterhouse; targeting animals, carcasses, workers and their households and environment. By using whole-genome phylogenetic analysis and network analysis based on single nucleotide polymorphisms (SNPs), we found highly interrelated non-pathogenic and pathogenic E. coli strains with phylogenetic intermixing, and a high prevalence of shared multidrug resistance profiles amongst livestock, human and environment. Through an original data processing pipeline which combines omics, machine learning, gene sharing network and mobile genetic elements analysis, we investigated the resistance to 26 different antimicrobials and identified 361 genes associated to antimicrobial resistance (AMR) phenotypes; 58 of these were known AMR-associated genes and 35 were associated to multidrug resistance. We uncovered an extensive network of genes, correlated to AMR phenotypes, shared among livestock, humans, farm and slaughterhouse environments. We also found several human, livestock and environmental isolates sharing closely related mobile genetic elements carrying ARGs across host species and environments. In a scenario where no consensus exists on how antibiotic use in the livestock may affect antibiotic resistance in the human population, our findings provide novel insights into the broader epidemiology of antimicrobial resistance in livestock farming. Moreover, our original data analysis method has the potential to uncover AMR transmission pathways when applied to the study of other pathogens active in other anthropogenic environments characterised by complex interconnections between host species.

[Microbial contamination in dried fruit products in China in 2019].

OBJECTIVE: To investigate the microbial contamination in dried fruit products in China. METHODS: In 2019, 2917 samples of dried fruit products on the market were collected, and examined for aerobic bacterial count, coliforms, molds, yeasts, Salmonella and Listeria monocytogenes according to the method specified in GB 4789. RESULTS: A total of 34.42%(1004/2917)of the samples had molds above 50 CFU/g and 9.46%(276/2917)of the samples had yeast above 50 CFU/g. The occurrence of aerobic plate count above 10~4 CFU/g and coliforms above 10~2 CFU/g was 5.01%(146/2917)and 2.98%(87/2917), respectively. The detection rate of Salmonella and Listeria monocytogenes were 0.14%(4/2917) and 0.03%(1/2917), respectively. Microbial contamination in different kinds of dried fruit products varied widely, with dried wolfberries and dried durian having the worst overall hygiene. There were differences in microbial contamination of dried fruit products in different regions. In general, samples collected in South China, Southwest China and Central China had more serious microbial contamination. There was no significant difference in microbial contamination between dried fruit products with different packaging and sampling places. CONCLUSION: The hygienic condition of dried fruit products is generally poor in 2019.

Genome-Scale Metabolic Models and Machine Learning Reveal Genetic Determinants of Antibiotic Resistance in Escherichia coli and Unravel the Underlying Metabolic Adaptation Mechanisms.

Antimicrobial resistance (AMR) is becoming one of the largest threats to public health worldwide, with the opportunistic pathogen Escherichia coli playing a major role in the AMR global health crisis. Unravelling the complex interplay between drug resistance and metabolic rewiring is key to understand the ability of bacteria to adapt to new treatments and to the development of new effective solutions to combat resistant infections. We developed a computational pipeline that combines machine learning with genome-scale metabolic models (GSMs) to elucidate the systemic relationships between genetic determinants of resistance and metabolism beyond annotated drug resistance genes. Our approach was used to identify genetic determinants of 12 AMR profiles for the opportunistic pathogenic bacterium E. coli. Then, to interpret the large number of identified genetic determinants, we applied a constraint-based approach using the GSM to predict the effects of genetic changes on growth, metabolite yields, and reaction fluxes. Our computational platform leads to multiple results. First, our approach corroborates 225 known AMR-conferring genes, 35 of which are known for the specific antibiotic. Second, integration with the GSM predicted 20 top-ranked genetic determinants (including accA, metK, fabD, fabG, murG, lptG, mraY, folP, and glmM) essential for growth, while a further 17 top-ranked genetic determinants linked AMR to auxotrophic behavior. Third, clusters of AMR-conferring genes affecting similar metabolic processes are revealed, which strongly suggested that metabolic adaptations in cell wall, energy, iron and nucleotide metabolism are associated with AMR. The computational solution can be used to study other human and animal pathogens. IMPORTANCE Escherichia coli is a major public health concern given its increasing level of antibiotic resistance worldwide and extraordinary capacity to acquire and spread resistance via horizontal gene transfer with surrounding species and via mutations in its existing genome. E. coli also exhibits a large amount of metabolic pathway redundancy, which promotes resistance via metabolic adaptability. In this study, we developed a computational approach that integrates machine learning with metabolic modeling to understand the correlation between AMR and metabolic adaptation mechanisms in this model bacterium. Using our approach, we identified AMR genetic determinants associated with cell wall modifications for increased permeability, virulence factor manipulation of host immunity, reduction of oxidative stress toxicity, and changes to energy metabolism. Unravelling the complex interplay between antibiotic resistance and metabolic rewiring may open new opportunities to understand the ability of E. coli, and potentially of other human and animal pathogens, to adapt to new treatments.

Whole-Genome Sequencing and Machine Learning Analysis of Staphylococcus aureus from Multiple Heterogeneous Sources in China Reveals Common Genetic Traits of Antimicrobial Resistance.

Staphylococcus aureus is a worldwide leading cause of numerous diseases ranging from food-poisoning to lethal infections. Methicillin-resistant S. aureus (MRSA) has been found capable of acquiring resistance to most antimicrobials. MRSA is ubiquitous and diverse even in terms of antimicrobial resistance (AMR) profiles, posing a challenge for treatment. Here, we present a comprehensive study of S. aureus in China, addressing epidemiology, phylogenetic reconstruction, genomic characterization, and identification of AMR profiles. The study analyzes 673 S. aureus isolates from food as well as from hospitalized and healthy individuals. The isolates have been collected over a 9-year period, between 2010 and 2018, from 27 provinces across China. By whole-genome sequencing, Bayesian divergence analysis, and supervised machine learning, we reconstructed the phylogeny of the isolates and compared them to references from other countries. We identified 72 sequence types (STs), of which, 29 were novel. We found 81 MRSA lineages by multilocus sequence type (MLST), spa, staphylococcal cassette chromosome mec element (SCCmec), and Panton-Valentine leukocidin (PVL) typing. In addition, novel variants of SCCmec type IV hosting extra metal and antimicrobial resistance genes, as well as a new SCCmec type, were found. New Bayesian dating of the split times of major clades showed that ST9, ST59, and ST239 in China and European countries fell in different branches, whereas this pattern was not observed for the ST398 clone. On the contrary, the clonal transmission of ST398 was more intermixed in regard to geographic origin. Finally, we identified genetic determinants of resistance to 10 antimicrobials, discriminating drug-resistant bacteria from susceptible strains in the cohort. Our results reveal the emergence of Chinese MRSA lineages enriched of AMR determinants that share similar genetic traits of antimicrobial resistance across human and food, hinting at a complex scenario of evolving transmission routes. IMPORTANCE Little information is available on the epidemiology and characterization of Staphylococcus aureus in China. The role of food is a cause of major concern: staphylococcal foodborne diseases affect thousands every year, and the presence of resistant Staphylococcus strains on raw retail meat products is well documented. We studied a large heterogeneous data set of S. aureus isolates from many provinces of China, isolated from food as well as from individuals. Our large whole-genome collection represents a unique catalogue that can be easily meta-analyzed and integrated with further studies and adds to the library of S. aureus sequences in the public domain in a currently underrepresented geographical region. The new Bayesian dating of the split times of major drug-resistant enriched clones is relevant in showing that Chinese and European methicillin-resistant S. aureus (MRSA) have evolved differently. Our machine learning approach, across a large number of antibiotics, shows novel determinants underlying resistance and reveals frequent resistant traits in specific clonal complexes, highlighting the importance of particular clonal complexes in China. Our findings substantially expand what is known of the evolution and genetic determinants of resistance in food-associated S. aureus in China and add crucial information for whole-genome sequencing (WGS)-based surveillance of S. aureus.

Comparative Genomic Analysis of the Foodborne Pathogen Burkholderia gladioli pv. cocovenenans Harboring a Bongkrekic Acid Biosynthesis Gene Cluster.

The environmental bacterium Burkholderia gladioli pv. cocovenenans (B. cocovenenans) has been linked to fatal food poisoning cases in Asia and Africa. Bongkrekic acid (BA), a mitochondrial toxin produced by B. cocovenenans, is thought to be responsible for these outbreaks. While there are over 80 species in the Burkholderia genus, B. cocovenenans is the only pathovar capable of producing BA and causing human death. However, the genomic features of B. gladioli and the evolution of the BA biosynthesis gene cluster, bon, in B. cocovenenans remain elusive. In this study, 239 whole genome sequences (WGSs) of B. gladioli, isolated from 12 countries collected over 100 years, were used to analyze the intra-species genomic diversity and phylogenetic relationships of B. gladioli and to explore the origin and evolution of the bon gene cluster. Our results showed that the genome-wide average nucleotide identity (ANI) values were above 97.29% for pairs of B. gladioli genomes. Thirty-six of the 239 (15.06%) B. gladioli genomes, isolated from corn, rice, fruits, soil, and patients from Asia, Europe, North America, and South America, contained the bon gene cluster and formed three clades within the phylogenetic tree. Pan- and core-genome analysis suggested that the BA biosynthesis genes were recently acquired. Comparative genome analysis of the bon gene cluster showed that complex recombination events contributed to this toxin biosynthesis gene cluster's evolution and formation. This study suggests that a better understanding of the genomic diversity and evolution of this lethal foodborne pathovar will potentially contribute to B. cocovenenans food poisoning outbreak prevention.

Complex Internal Microstructure of Feather Follicles on Chicken Skin Promotes the Bacterial Cross-Contamination of Carcasses During the Slaughtering Process.

Chicken skin is considered the most susceptible to bacterial contamination during slaughter. It is rich in bushy feather follicles with complex internal structures that can absorb bacteria via cross-contamination during slaughter. Until now, the microstructural changes and local bacterial composition of feather follicles during slaughter have not been thoroughly investigated. This study used hematoxylin-eosin (HE) staining of the tissue paraffin section to investigate the structure of the feather follicles on chicken skin. In addition, the biopsy sampling method was employed for the high-throughput sequencing of 16S RNA genes to study the composition and source of bacterial contamination during slaughter. The results show that the feather follicles on chicken skin form a closed cavity structure during the slaughtering process. The volume of the irregular follicle cavity was about Ø: 200 µm × D: 1040 µm, which provides a place for the bacteria to absorb and resist the cleaning and disinfection during the slaughtering process. The composition of bacteria in the feather follicle was mainly Acinetobacter (37%), Psychrobacter (8%), Macrococcus (5%), and Comamonas (2%). The heat map obtained via the species abundance analysis of the feather follicle samples as well as the slaughter environment samples suggests that the gastrointestinal feces contaminated the feather follicles on the chicken skin mainly during the evisceration, defeathering, and chilling processes, and the last-stage chilling water also caused severe cross-contamination to the feather follicles during the chilling process.

[Establishment of a method with real-time reverse transcription-polymerase chain reaction for hepatitis E virus in swine feces and its molecular epidemiology].

OBJECTIVE: Real-time reverse transcription-polymerase chain reaction(real-time RT-PCR) assay based on Taqman and phylogenetic tree were developed for detecting hepatitis E virus in swine feces of pig farms from several provinces and city. METHODS: Designing prime and probe refering to HEV genotype sequences of Genbank, we developed a Taqman-based real-time RT-PCR assay and nested RT-PCR according to HEV conserved domain after optimizing reaction system, then detected the prevalence of HEV infection of pig farms. RESULTS: The sensitivity of the real-time RT-PCR assay established in this experiment was 19. 9 copies/µL, the amplification efficiency was 92. 9%-109. 1%, there was no cross reaction with Sapovirus, Norovirus and Hepatitis A. A total of 342 samples of swine feces were detected. There were two hundred and ten positive samples, and positive rate was 61. 4%. The positive rate of before-fattening was 56. 6%, and after-fattening was 66. 9%. The positive rate of before and after fattening samples had statistical difference(χ~2=24. 8, P<0. 05). The genotype identification system determined that the positive strains isolated in this study were HEV-4 type, and three subtypes of 4 b, 4 d and 4 h were detected. CONCLUSION: The pig farms of several provinces and city are contaminated by HEV extensively. The genotypes of the isolated strains are all HEV-4 type. The infection rate and infection subtype of pigs in different provinces and cities are different.

Susceptibility (re)-testing of a large collection of Listeria monocytogenes from foods in China from 2012 to 2015 and WGS characterization of resistant isolates.

OBJECTIVES: Our aim was to determine the antimicrobial susceptibilities of 2862 Listeria monocytogenes cultured from various foods in China and to use WGS to characterize the antimicrobial resistance and virulence genotypes of those expressing a resistance phenotype. METHODS: The susceptibilities of 2862 L. monocytogenes were determined by broth microdilution. Twenty-eight L. monocytogenes were found to be resistant to one to four antibiotics. All 28 resistant isolates were subsequently sequenced using short-read high accuracy protocols. The corresponding genomes were assembled and further analysis was carried out using appropriate bioinformatics pipelines. RESULTS: All 28 resistant L. monocytogenes were classified into five STs (ST3, ST8, ST9, ST155 and ST515). Both ST9 and ST155 were dominant and their genotypes correlated with their resistance phenotypes. All ST9 isolates were MDR and could be phylogenetically classified into two clusters. One was relatively close to clinical origins and one to food. Downstream analysis of the genetic contexts in which these resistance genotypes were found suggested that these may have been acquired from other bacteria by horizontal transfer or insertion into the chromosome. All isolates harboured Listeria pathogenicity island (LIPI)-1 and LIPI-2, and only two harboured LIPI-3. CONCLUSIONS: This study reported on the antimicrobial susceptibilities of 2862 foodborne L. monocytogenes along with the genomic characterization of 28 resistant isolates, 11 of which expressed an MDR phenotype. These data showed that this bacterium can acquire resistance by horizontal gene transfer in and between species. This study may necessitate a re-evaluation of risk to public health, associated with this bacterial species.

Effects of metal and metalloid pollutants on the microbiota composition of feces obtained from twelve commercial pig farms across China.

Understanding the metal and metalloid contamination and microbiota composition of pig feces is an important step required to support the design and implementation of effective pollution control and prevention strategies. A survey was implemented in 12 locations across China to investigate the content of metals and metalloids, and the main composition of the microbial communities of commercially reared pigs during two growth periods, defined as the early (Q group) and the later fattening growth phases (H group). These data showed widespread Al, Mn, Cu, Zn, and Fe pollution in pig feces. The concentration of Zn in the Q group feces was nearly two times higher than the levels measured in the H group. The microbial composition of the Q group exhibited greater richness of operational taxonomic units (OTUs) and fewer bacteria associated with zoonotic diseases compared with the microbial composition of the H group. Spearman rank correlation analysis showed that Cu and northern latitudes had a significant positive effect on the richness of bacterial communities in pig feces. Zn and Cd exhibited the biggest impact on microbial community composition based on canonical correspondence analysis. Functional metagenomic prediction indicated that about 0.8% genes present in the pig feces bacteria community are related to human diseases, and significantly more predicted pathogenic genes were detected in the H group than in the Q group. These results support the need to monitor heavy metal contamination and to control for zoonotic pathogens disseminated from pig feces in Chinese pig farms.