Characterization of NMCR-3, NMCR-4 and NMCR-5, three novel non-mobile colistin resistance determinants: Implications for MCR-3, MCR-7, and MCR-5 progenitors, respectively.

In this study, the progenitors of MCR-3, MCR-7 and MCR-5, namely NMCR-3, NMCR-4 and NMCR-5, were firstly discovered and indicating Aeromonas was a natural reservoir for MCR-3 and MCR-7. Furthermore, different evolutionary models for MCR-3, MCR-7 and MCR-5 were proposed.

Surface Activation by Single Ru Atoms for Enhanced High-Temperature CO2 Electrolysis.

Cathodic CO2 adsorption and activation is essential for high-temperature CO2 electrolysis in solid oxide electrolysis cells (SOECs). However, the component of oxygen ionic conductor in the cathode displays limited electrocatalytic activity. Herein, stable single Ruthenium (Ru) atoms are anchored on the surface of oxygen ionic conductor (Ce0.8 Sm0.2 O2-δ , SDC) via the strong covalent metal-support interaction, which evidently modifies the electronic structure of SDC surface for favorable oxygen vacancy formation and enhanced CO2 adsorption and activation, finally evoking the electrocatalytic activity of SDC for high-temperature CO2 electrolysis. Experimentally, SOEC with the Ru1 /SDC-La0.6 Sr0.4 Co0.2 Fe0.8 O3-δ cathode exhibits a current density as high as 2.39 A cm-2 at 1.6 V and 800 °C. This work expands the application of single atom catalyst to the high-temperature electrocatalytic reaction in SOEC and provides an efficient strategy to tailor the electronic structure and electrocatalytic activity of SOEC cathode at the atomic scale.

PlyKp104, a Novel Phage Lysin for the Treatment of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Other Gram-Negative ESKAPE Pathogens.

Klebsiella pneumoniae and Pseudomonas aeruginosa are two leading causes of burn and wound infections, pneumonia, urinary tract infections, and more severe invasive diseases, which are often multidrug resistant (MDR) or extensively drug resistant. Due to this, it is critical to discover alternative antimicrobials, such as bacteriophage lysins, against these pathogens. Unfortunately, most lysins that target Gram-negative bacteria require additional modifications or outer membrane permeabilizing agents to be bactericidal. We identified four putative lysins through bioinformatic analysis of Pseudomonas and Klebsiella phage genomes in the NCBI database and then expressed and tested their intrinsic lytic activity in vitro. The most active lysin, PlyKp104, exhibited >5-log killing against K. pneumoniae, P. aeruginosa, and other Gram-negative representatives of the multidrug-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, K. pneumonia, Acinetobacter baumannii, P. aeruginosa, and Enterobacter species) without further modification. PlyKp104 displayed rapid killing and high activity over a wide pH range and in high concentrations of salt and urea. Additionally, pulmonary surfactants and low concentrations of human serum did not inhibit PlyKp104 activity in vitro. PlyKp104 also significantly reduced drug-resistant K. pneumoniae >2 logs in a murine skin infection model after one treatment of the wound, suggesting that this lysin could be used as a topical antimicrobial against K. pneumoniae and other MDR Gram-negative infections.

Genomic analyses of Staphylococcus aureus isolated from yaks in Ganzi Tibetan Autonomous Prefecture, China.

OBJECTIVES: To investigate the transmission and origination of MRSA in livestock with limited antimicrobial use. Yak (Bos grunniens) herds in Ganzi Tibetan Autonomous Prefecture, China were chosen for sampling. METHODS: The yaks from all 18 districts of Ganzi were sampled (anal swabs, n = 657; nasal swabs, n = 634). Based on the WGS data of 83 Staphylococcus aureus isolates, the novel structure of the yak S. aureus population was described. Phylogenetic analyses were utilized for determining the origin of the MRSA lineage in yaks. RESULTS: The yak S. aureus population consisted of 11 STs, 6 of which were previously undescribed, with ST6267 being the predominant novel ST. These isolates were generally susceptible to most of the tested antibiotics and lacked the associated antimicrobial resistance genes (ARGs) but showed high penicillin MIC values (MIC90 = 32 mg/L), which were consistent with the high positivity rate for blaZ (61/83). The MRSA identified in yaks were all ST59 and most likely of human origin. The yak ST59 MRSA each carried the human immune evasion cluster (IEC) while lacking the ARGs that are identified in the majority of reported Chinese human ST59 MRSA isolates [erm(B), ant6-Ia and aph(3″)-III]. CONCLUSIONS: The yak herds living on the Qinghai-Tibet Plateau are important livestock and follow the traditional free-grazing farming model. We surveyed the yak S. aureus population and found that all the yak MRSA isolates belonged to the lineage that might originate from the prevalent community-acquired MRSA ST59 in China. From a 'One Health' perspective, the transmission of human MRSA to farming animals with limited antimicrobial exposure highlights the multiple roles of animals in the expansion of MRSA.

Molecular characterization of Glaesserella parasuis strains isolated from North America, Europe and Asia by serotyping PCR and LS-PCR.

Glaesserella parasuis strains were characterized by serotyping PCR, vtaA virulence marker Leader Sequence (LS)-PCR, clinical significance, and geographic region. Overall, the serovars 4, 5/12, 7, 1, and 13 were the most commonly detected. Serovars of greatest clinical relevance were systemic isolates that had a higher probability of being serovar 5/12, 13, or 7. In comparison, pulmonary isolates had a higher likelihood of being serovars 2, 4, 7, or 14. Serovars 5/12 and 13 have previously been considered disease-associated, but this study agrees with other recent studies showing that serovar 7 is indeed associated with systemic G. parasuis disease. Serovar 4 strains illustrated how isolates can have varying degrees of virulence and be obtained from pulmonary, systemic, or nasal sites. Serovars 8, 9, 15, and 10 were predominantly obtained from nasal samples, which indicates a limited clinical significance of these serovars. Additionally, most internal G. parasuis isolates were classified as virulent by LS-PCR and were disease-associated isolates, including serovars 1, 2, 4, 5/12, 7, 13, and 14. Isolates from the nasal cavity, including serovars 6, 9, 10, 11, and 15, were classified as non-virulent by LS-PCR. In conclusion, the distribution of G. parasuis serovars remains constant, with few serovars representing most of the strains isolated from affected pigs. Moreover, it was confirmed that the LS-PCR can be used for G. parasuis virulence prediction of field strains worldwide.

Bio-inspired and assembled fungal hyphae/carbon nanotubes aerogel for water-oil separation.

Carbon nanotube (CNT)-based materials have attracted tremendous interest for their high performance in oil separation. However, the preparation of CNT based materials always require harmful and expensive chemicals. Here, a biological assembly route was applied to assemble CNTs onto a fungal hyphae (FH) to produce FH/CNTs composites, followed by pyrolysis to obtain a hydrophobic CNT based aerogel for oil separation, which is a more environmentally friendly process. The as-prepared FH/CNTs-800 aerogel (pyrolyzed at 800 °C) showed hydrophobicity with a water contact angle of 143° and high specific surface area (1041.2 m2 g-1). The oil absorption results showed that the as-prepared FH/CNTs aerogels could absorb a wide range of oils with high absorption capacities ranging from 48 to 138 times their own weight. Furthermore, the oil-loaded aerogel was recycled through burning with little reduction in the oil absorption capacity. In addition, FH/CNTs-800 provided a high specific capacitance of 232 F g-1 at 1 A g-1 and maintained a capacity retention of 70.62% at 20 A g-1. Therefore, this study offers a simple, low-cost and environmentally friendly bioassembly route for large-scale assembly of CNTs into macroscopic 3D hydrophobic aerogels for highly efficient water-oil separation.

Effects of Environmental and Management-Associated Factors on Prevalence and Diversity of Streptococcus suis in Clinically Healthy Pig Herds in China and the United Kingdom.

Streptococcus suis, a global zoonosis of pigs, shows regional differences in the prevalence of human-associated disease for Asian and non-Asian countries. The isolation rates and diversities of S. suis on tonsils of healthy slaughter pigs in China and the United Kingdom were studied for effects of geography, temperature, pig age, and farm type. Isolates underwent analysis of molecular serotype and multilocus sequence type and virulence-associated genotyping. Although we found no significant difference in positive isolation rates between Chinese and UK farms, the prevalences of serotypes previously associated with human disease were significantly greater in the Chinese collection (P = 0.003). A significant effect of temperature was found on the positive isolation rate of the Chinese samples and the prevalence of human disease-associated serotypes in the UK S. suis population (China, P = 0.004; United Kingdom, P = 0.024) and on the prevalence of isolates carrying key virulence genes in China (P = 0.044). Finally, we found marked diversity among S. suis isolates, with statistically significant temperature effects on detection of multiple strain types within individual pigs. This study highlighted the high carriage prevalence and diversity of S. suis among clinically healthy pig herds of China and the United Kingdom. The significant effect of temperature on prevalence of isolation, human disease-associated serotypes, and diversity carried by individual pigs may shed new light on geographic variations in human S. suis-associated disease.IMPORTANCEStreptococcus suis is a global zoonotic pathogen and also a normal colonizer mainly carried on the tonsil of pigs. Thus, it is important to study the effect of environmental and management-associated factors on the S. suis populations in clinically healthy pigs. In this research, we investigated the similarities and differences between the S. suis populations obtained from different pig ages, seasons, and farm management systems and discovered the relationship between high climatic temperature and the prevalence of S. suis.

Prevalence, quantification and isolation of pathogenic shiga toxin Escherichia coli O157:H7 along the production and supply chain of pork around Hubei Province of China.

Shiga toxin Escherichia coli (STEC) O157:H7 is an important zoonotic food borne pathogen causing gastroenteritis that may lead to life threatening hemorragic colitis (HC) and hemorrhagic uremic syndrome (HUS). 325 meat and tissue samples were tested for enumeration of O157:H7 strains using most probable number (MPN)-PCR targeting their specific genes flicH7 and rfbO157 followed by isolation, sereotyping and pathogenicity testing. The overall prevalence of O157:H7 was 41.3% (134/325) along the production and supply chain of pork (PSCP), being higher in supply chain (59%, 118/200) as compared to pig farms (12.8%, 16/125). Along the PSCP, the highest prevalence was found in slaughter houses (86.25%, 69/80) followed by wet- (53.3%, 32/60) and super-markets (28.3%, 17/60). The MPN values ranged from 3 to 1100 MPN/g in overall positive samples, being higher in slaughter houses followed by wet and super markets. Except from intestine and meat samples of slaughter house, the MPN was found higher in summer as compared to winter samples. Eight STEC O157:H7 isolated from meat and liver samples were tested in Balb/C mice for pathogenicity. After development of clinical signs and symptoms, 50-83.3% mortality was produced in the infected mice. Histopathological investigations revealed visible necrosis of intestinal epithelial cells, shedding of cellular debris in the intestine, while in the kidney, necrosis of renal cortical portion of tubular epithelial cells was observed. STEC O157:H7 is prevalent along PSCP around Hubei of China in different proportions being alarmingly higher in supply chain and markets which is a matter of concern for public health.

Overexpression of Porcine Beta-Defensin 2 Enhances Resistance to Actinobacillus pleuropneumoniae Infection in Pigs.

To reduce the need for antibiotics in animal production, alternative approaches are needed to control infection. We hypothesized that overexpression of native defensin genes will provide food animals with enhanced resistance to bacterial infections. In this study, recombinant porcine beta-defensin 2 (PBD-2) was overexpressed in stably transfected PK-15 porcine kidney cells. PBD-2 antibacterial activities against Actinobacillus pleuropneumoniae, an important respiratory pathogen causing porcine contagious pleuropneumonia, were evaluated on agar plates. Transgenic pigs constitutively overexpressing PBD-2 were produced by a somatic cell cloning method, and their resistance to bacterial infection was evaluated by direct or cohabitation infection with A. pleuropneumoniae. Recombinant PBD-2 peptide that was overexpressed in the PK-15 cells showed antibacterial activity against A. pleuropneumoniae. PBD-2 was overexpressed in the heart, liver, spleen, lungs, kidneys, and jejunum of the transgenic pigs, which showed significantly lower bacterial loads in the lungs and reduced lung lesions after direct or cohabitation infection with A. pleuropneumoniae. The results demonstrate that transgenic overexpression of PBD-2 in pigs confers enhanced resistance against A. pleuropneumoniae infection.

Discovery of Antimicrobial Lysins from the "Dark Matter" of Uncharacterized Phages Using Artificial Intelligence.

The rapid rise of antibiotic resistance and slow discovery of new antibiotics have threatened global health. While novel phage lysins have emerged as potential antibacterial agents, experimental screening methods for novel lysins pose significant challenges due to the enormous workload. Here, the first unified software package, namely DeepLysin, is developed to employ artificial intelligence for mining the vast genome reservoirs ("dark matter") for novel antibacterial phage lysins. Putative lysins are computationally screened from uncharacterized Staphylococcus aureus phages and 17 novel lysins are randomly selected for experimental validation. Seven candidates exhibit excellent in vitro antibacterial activity, with LLysSA9 exceeding that of the best-in-class alternative. The efficacy of LLysSA9 is further demonstrated in mouse bloodstream and wound infection models. Therefore, this study demonstrates the potential of integrating computational and experimental approaches to expedite the discovery of new antibacterial proteins for combating increasing antimicrobial resistance.

ESKtides: a comprehensive database and mining method for ESKAPE phage-derived antimicrobial peptides.

'Superbugs' have received increasing attention from researchers, such as ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.), which directly led to about 1 270 000 death cases in 2019. Recently, phage peptidoglycan hydrolases (PGHs)-derived antimicrobial peptides were proposed as new antibacterial agents against multidrug-resistant bacteria. However, there is still a lack of methods for mining antimicrobial peptides based on phages or phage PGHs. Here, by using a collection of 6809 genomes of ESKAPE isolates and corresponding phages in public databases, based on a unified annotation process of all the genomes, PGHs were systematically identified, from which peptides were mined. As a result, a total of 12 067 248 peptides with high antibacterial activities were respectively determined. A user-friendly tool was developed to predict the phage PGHs-derived antimicrobial peptides from customized genomes, which also allows the calculation of peptide phylogeny, physicochemical properties, and secondary structure. Finally, a user-friendly and intuitive database, ESKtides (http://www.phageonehealth.cn:9000/ESKtides), was designed for data browsing, searching and downloading, which provides a rich peptide library based on ESKAPE prophages and phages. Database URL:  10.1093/database/baae022.

Improving the safety and efficacy of phage therapy from the perspective of phage-mammal interactions.

Phage therapy has re-emerged as a promising solution for combating antimicrobial-resistant bacterial infections. Increasingly, studies have revealed that phages possess therapeutic potential beyond their antimicrobial properties, including regulating the gut microbiome and maintain intestinal homeostasis, as a novel nanocarrier for targeted drug delivery. However, the complexity and unpredictability of phage behavior during treatment pose a significant challenge in clinical practice. The intricate interactions established between phages, humans, and bacteria throughout their long coexistence in the natural ecosystem contribute to the complexity of phage behavior in therapy, raising concerns about their efficacy and safety as therapeutic agents. Revealing the mechanisms by which phages interact with the human body will provide a theoretical basis for increased application of promising phage therapy. In this review, we provide a comprehensive summary of phage-mammal interactions, including signaling pathways, adaptive immunity responses, and phage-mediated anti-inflammatory responses. Then, from the perspective of phage-mammalian immune system interactions, we present the first systematic overview of the factors affecting phage therapy, such as the mode of administration, the physiological status of the patient, and the biological properties of the phage, to offer new insights into phage therapy for various human diseases.

Encapsulation and delivery of phage as a novel method for gut flora manipulation in situ: A review.

Intestinal flora regulation is an effective method to intervene and treat diseases associated with microbiome imbalance. In addition to conventional probiotic supplement, phage delivery has recently exhibited great prospect in modifying gut flora composition and regulating certain gene expression of gut bacteria. However, the protein structure of phage is vulnerable to external factors during storage and delivery, which leads to the loss of infection ability and flora regulation function. Encapsulation strategy provides an effective solution for improving phage stability and precisely controlling delivery dosage. Different functional materials including enzyme-responsive and pH-responsive polymers have been used to construct encapsulation carriers to protect phages from harsh conditions and release them in the colon. Meanwhile, diverse carriers showed different characteristics in structure and function, which influenced their protective effect and delivery efficiency. This review systematically summarizes recent research progress on the phage encapsulation and delivery, with an emphasis on function properties of carrier systems in the protection effect and colon-targeted delivery. The present review may provide a theoretical reference for the encapsulation and delivery of phage as microbiota modulator, so as to expedite the development of functional material and delivery carrier, as well as the advances in practical application of intestinal flora regulation.

Heterogeneity and transmission of food safety-related enterotoxigenic Staphylococcus aureus in pig abattoirs in Hubei, China.

The dissemination of Staphylococcus aureus in the pork production chain is a major food safety concern. Abattoirs can serve both as disruptor and transmitter for S. aureus. In this study, we conducted a systematic genomic epidemiology research on the prevalence, heterogeneity, and transmission of S. aureus in 3,638 samples collected from four pig abattoirs in Hubei province, China. Our findings revealed substantial heterogeneity between S. aureus recovered from samples collected at upstream (from stunning step to head-removal step) and downstream (from splitting step to chilling step) locations within the slaughter process. Overall, 966 (26.6%) samples were positive for S. aureus, with significantly higher overall prevalence for upstream samples (29.0%, 488/1,681) compared to downstream samples (24.4%, 478/1,957). Antimicrobial susceptibility testing demonstrated that the isolates from the upstream exhibited significantly higher resistance proportions to different antimicrobials than those from the downstream. Whole-genome sequencing of 126 isolates revealed that ST398 (32.9%, 23/70) and ST9 (22.9%, 16/70) were more common among upstream isolates, while ST7 (35.7%, 20/56) and ST97 (28.6%, 16/56) were most frequently observed among downstream isolates. Additionally, molecular characterization analysis demonstrated that upstream isolates possessed significantly higher enterotoxigenic potential, more antimicrobial resistance genes, and S. aureus pathogenicity islands than downstream isolates. Notably, we discovered that enterotoxigenic S. aureus could be transmitted across different slaughter stages, with knives, water, and air serving as vectors. Although slaughtering processes had a substantial effect on reducing the food safety risk posed by enterotoxigenic S. aureus, the possibility of its widespread transmission should not be disregarded. IMPORTANCE Staphylococcus aureus (S. aureus) is one of the most important foodborne pathogens, and can cause foodborne poisoning by producing enterotoxins. Pork is a preferable reservoir and its contamination often occurs during the slaughter process. Our findings revealed significant differences in the prevalence, antimicrobial resistance, and enterotoxigenic potential between the upstream and downstream isolates within the slaughter process. Also, it is imperative not to overlook enterotoxigenic S. aureus transmitted across all stages of the slaughter process, with notable vectors being knives, water, and air. These findings hold significant implications for policy-makers to reassess their surveillance projects, and underscore the importance of implementing effective control measures to minimize the risk of S. aureus contamination in pork production. Moreover, we provide a more compelling method of characterizing pathogen transmission based on core-SNPs of bacterial genomes.

Combine thermal processing with polyvalent phage LPEK22 to prevent the Escherichia coli and Salmonella enterica contamination in food.

Thermal processing is the most frequently used method to destruct bacteria in food processing. However, insufficient thermal processing may lead to the outbreak of foodborne illness. This study combined thermal processing with thermostable phage to prevent food contamination. The thermostable phages were screened which can retain activity at 70 °C for 1 h. Among them, the polyvalent phage LPEK22 was obtained to lyse Escherichia coli and Salmonella enterica, especially several multi-drug resistant bacteria. In milk (liquid food matrix), LPEK22 significantly reduced the E. coli by 5.00 ± 0.18 log10 CFU/mL and S. enterica by 4.20 ± 0.23 log10 CFU/mL after thermal processing at 63 °C for 30 min. For beef sausage (solid food matrix), LPEK22 significantly reduced the E. coli by 2.34 ± 0.17 log10 CFU/cm2 and S. enterica by 1.54 ± 0.13 log10 CFU/cm2 after thermal processing at 66 °C for 90 s. Genome analysis revealed that LPEK22 was a novel phage with a unique tail spike protein belonging to the family of Ackermannviridae. LPEK22 did not contain lysogenic, drug-resistant, and virulent genes that may compromise the safety of food application. These results determined that LPEK22, a novel polyvalent Ackermannviridae phage, could combine with thermal processing to prevent drug-resistant E. coli and S. enterica both in vitro and in foods.

Application of a novel phage LPCS28 for biological control of Cronobacter sakazakii in milk and reconstituted powdered infant formula.

Contamination of infant formula with Cronobacter sakazakii (C. sakazakii) can cause fatal infections in neonates. Phages have emerged as promising antibacterial agents for food safety, but their effectiveness may be limited by thermal processing. In this study, we isolated 27 C. sakazakii phages from environmental water samples and selected LPCS28 due to its broad lysis spectrum. The titer of LPCS28 will not be significantly affected by heating at a temperature of 60 °C for one hour. In both reconstituted powdered infant formula (RPIF) and liquid milk, the pre-added LPCS28, after the thermal processing at 63 °C for 30 min, significantly inhibited the post-contaminated C. sakazakii (103 CFU/mL) and eventually reduced the number of C. sakazakii to below the limit of detection (<10 CFU/mL) within 9 h at 37 °C and significantly delayed the increase of bacterial concentration in the samples at 23 °C. The phylogenetic analysis revealed that LPCS28 belonged to a new genus, we proposed as Nanhuvirus, under the family Straboviridae. These findings suggest that phage LPCS28 is a promising biological control agent for pathogenic C. sakazakii in the dairy industry.

High-Throughput Mutagenesis Reveals a Role for Antimicrobial Resistance- and Virulence-Associated Mobile Genetic Elements in Staphylococcus aureus Host Adaptation.

Methicillin-resistant Staphylococcus aureus (MRSA) clonal-complex 398 (CC398) is the dominant livestock-associated (LA) MRSA lineage in European livestock and an increasing cause of difficult-to-treat human disease. LA-CC398 MRSA evolved from a diverse human-associated methicillin-sensitive population, and this transition from humans to livestock was associated with three mobile genetic elements (MGEs). In this study, we apply transposon-directed insertion site sequencing (TraDIS), a high-throughput transposon mutagenesis approach, to investigate genetic signatures that contribute to LA-CC398 causing disease in humans. We identified 26 genes associated with LA-CC398 survival in human blood and 47 genes in porcine blood. We carried out phylogenetic reconstruction on 1,180 CC398 isolates to investigate the genetic context of all identified genes. We found that all genes associated with survival in human blood were part of the CC398 core genome, while 2/47 genes essential for survival in porcine blood were located on MGEs. Gene SAPIG0966 was located on the previously identified Tn916 transposon carrying a tetracycline resistance gene, which has been shown to be stably inherited within LA-CC398. Gene SAPIG1525 was carried on a phage element, which in part, matched phiSa2wa_st1, a previously identified bacteriophage carrying the Panton-Valentine leucocidin (PVL) virulence factor. Gene deletion mutants constructed in two LA-CC398 strains confirmed that the SAPIG0966 carrying Tn916 and SAPIG1525 were important for CC398 survival in porcine blood. Our study shows that MGEs that carry antimicrobial resistance and virulence genes could have a secondary function in bacterial survival in blood and may be important for host adaptation. IMPORTANCE CC398 is the dominant type of methicillin-resistant Staphylococcus aureus (MRSA) in European livestock and a growing cause of human infections. Previous studies have suggested MRSA CC398 evolved from human-associated methicillin-sensitive Staphylococcus aureus and is capable of rapidly readapting to human hosts while maintaining antibiotic resistance. Using high-throughput transposon mutagenesis, our study identified 26 and 47 genes important for MRSA CC398 survival in human and porcine blood, respectively. Two of the genes important for MRSA CC398 survival in porcine blood were located on mobile genetic elements (MGEs) carrying resistance or virulence genes. Our study shows that these MGEs carrying antimicrobial resistance and virulence genes could have a secondary function in bacterial survival in blood and may be important for blood infection and host adaptation.

Screening for Virulence-Related Genes via a Transposon Mutant Library of Streptococcus suis Serotype 2 Using a Galleria mellonella Larvae Infection Model.

Streptococcus suis (S. suis) is a zoonotic bacterial pathogen causing lethal infections in pigs and humans. Identification of virulence-related genes (VRGs) is of great importance in understanding the pathobiology of a bacterial pathogen. To identify novel VRGs, a transposon (Tn) mutant library of S. suis strain SC19 was constructed in this study. The insertion sites of approximately 1700 mutants were identified by Tn-seq, which involved 417 different genes. A total of 32 attenuated strains were identified from the library by using a Galleria mellonella larvae infection model, and 30 novel VRGs were discovered, including transcription regulators, transporters, hypothetical proteins, etc. An isogenic deletion mutant of hxtR gene (ΔhxtR) and its complementary strain (CΔhxtR) were constructed, and their virulence was compared with the wild-type strain in G. mellonella larvae and mice, which showed that disruption of hxtR significantly attenuated the virulence. Moreover, the ΔhxtR strain displayed a reduced survival ability in whole blood, increased sensitivity to phagocytosis, increased chain length, and growth defect. Taken together, this study performed a high throughput screening for VRGs of S. suis using a G. mellonella larvae model and further characterized a novel critical virulence factor.

A Combinatorial Vaccine Containing Inactivated Bacterin and Subunits Provides Protection Against Actinobacillus pleuropneumoniae Infection in Mice and Pigs.

Actinobacillus pleuropneumoniae (APP) is the etiological agent of porcine contagious pleuropneumonia (PCP) that causes great economic losses in the swine industry. Currently, vaccination is still a commonly used strategy for the prevention of the disease. Commercially available vaccines of this disease, including inactivated bacterins and subunit vaccines, have clinical limitations such as side effects and low cross-protection. In this study, a combinatorial vaccine (Bac-sub) was developed, which contained inactivated bacterial cells of a serovar 1 strain and three recombinant protoxins (rApxIA, rApxIIA, and rApxIIIA). Its side effects, immune protection, and cross-protection were evaluated and compared with a commercial subunit vaccine and a commercial trivalent bacterin in a mouse infection model. The results revealed that the Bac-sub vaccine showed no obvious side effects, and induced higher levels of Apx toxin-specific IgG, IgG1, and IgG2a than the commercial vaccines after booster. After a challenge with virulent strains of serovars 1, 5, and 7, the Bac-sub vaccine provided greater protection (91.76%, 100%, and 100%, respectively) than commercial vaccines. Much lower lung bacterial loads (LBLs) and milder lung lesions were observed in the Bac-sub-vaccinated mice than in those vaccinated with the other two vaccines. The protective efficacy of the Bac-sub vaccine was further evaluated in pigs, which showed that vaccinated pigs displayed significantly milder clinical symptoms and lung lesions than the unvaccinated pigs after the challenge. Taken together, Bac-sub is a safe and effective vaccine that could provide high protection against A. pleuropneumoniae infection in both mice and pigs.

Phage controlling method against novel freshwater-derived Vibrio parahaemolyticus in ready-to-eat crayfish (Procambarus clarkii).

Vibrio parahaemolyticus is a halophilic foodborne pathogen majorly isolated from seafood, threatening public health worldwide. However, our recent study reported the presence of this bacterium in freshwater crayfish, which were rarely identified and investigated. Its pathogenicity and genomic features remain unclear, and the controlling method to inhibit this bacterium in ready-to-eat (RTE) crayfish is not developed. Compared with a clinical strain (ATCC17802), the representative strain (LVP1) from freshwater crayfish showed higher pathogenicity in a zebrafish model, indicating its hypervirulence and foodborne infection risk. Unlike most clinical V. parahaemolyticus isolates that carried tdh and (or) trh, two classic virulence factor genes associated with clinical infections, the hypervirulent LVP1 lacked these two genes, indicating it is a novel strain and other unknown virulence factors play key roles in its pathogenicity. Genomic and phylogenetic analyses demonstrated this strain is V. parahaemolyticus, while it is phylogenetically distant from other global isolates. Therefore, LVP1 is considered a novel V. parahaemolyticus strain from freshwater crayfish, being hypervirulent, and lacking virulence marker genes. The antimicrobial resistant genes drfA6 and qnrV1 were unique in LVP1 and absent in other reference V. parahaemolyticus strains. Antimicrobial susceptibility testing confirms it as multidrug resistance, with resistance to ampicillin, amoxicillin/clavulanate, trimethoprim, and colistin. To control this novel and multidrug-resistant V. parahaemolyticus strain in food production chains, we developed a phage cocktail and applied it to the surface of RTE crayfish meat, resulting in a significant decrease in the bacterial load by 2.36 log10 CFU/mL. These data highlight freshwater food products pose threats to public health through 'farm-to-fork' transmission of hypervirulent V. parahaemolyticus and reveal the phage cocktail as a promising method to attenuate this bacterium in RTE food, emphasizing the necessity to prevent food contamination caused by this bacterium from freshwater products.