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ABSTRACT: Objectives: Klebsiella aerogenes is a largely understudied opportunistic pathogen that can cause sepsis and lead to high mortality rates. In this study, we reported the occurrence of carbapenem-resistant blaOXA-181-carrying Klebsiella aerogenes from swine in China and elucidate their genomic characteristics. Methods: A total of 126 samples, including 109 swine fecal swabs, 14 environmental samples, and three feed samples were collected from a pig farm in China. The samples were enriched with LB broth culture and then inoculated into MacConkey agar plates for bacterial isolation. After PCR detection of carbapenemases genes, the blaOXA-181-carrying isolates were subjected to antimicrobial susceptibility testing, and whole-genome sequence analysis. Results: Four Klebsiella aerogenes isolates carrying the blaOXA-181 gene were obtained from swine faecal samples. All the 4 strains were belonged to ST438. The blaOXA-181 genes were located in IncX3-ColKP3 hybrid plasmids with the core genetic structure of IS26-ΔIS3000-ΔISEcp1-blaOXA-181-ΔlysR-ΔereA-ΔrepA-ISKpn19-tinR-qnrS1-ΔIS2-IS26, which suggests the potential for horizontal transfer and further dissemination of this resistance gene among Enterobacteriaceae and other sources. Conclusions: This study represents the first instance of OXA-181-producing K. aerogenes being identified from swine faeces in China. It is crucial to maintain continuous monitoring and ongoing attention to the detection of K. aerogenes carrying blaOXA-181 and other resistance genes in pigs.

ABSTRACT: Salmonellosis in poultry is detrimental to the advancement of the breeding industry and poses hazards to human health. Approximately 2,600 Salmonella varieties exist, among which S. Enteritidis, S. Pullorum, S. Typhimurium, and S. Infantis are prevalent serotypes in the poultry industry in recent years. They can also infect humans by contaminating poultry eggs and meat. Therefore, identifying these serotypes is crucial for successful preventive and control interventions. The White–Kauffmann–Le Minor scheme is time-consuming and requires expensive reagents. Whole-genome sequencing (WGS) and other molecular biology techniques require skilled technical staff. In comparison, the polymerase chain reaction (PCR) is more accurate, rapid, and inexpensive, thus proving suitable for widespread application in the poultry industry. Here, we selected 4 specific primers: lygD, mdh, ipaJ, and SIN_02055, which correspond to detecting S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Infantis, respectively. They were integrated into a 1-step multiplex PCR method. We optimized the PCR method by utilizing specificity test results to determine the optimal annealing temperature (57°C). The PCR method exhibited excellent sensitivity for genomic DNA and bacterial cultures. We used the developed method to determine 157 clinical Salmonella isolates from various stages of the poultry production chain. The results aligned with serotype data generated via WGS analysis, demonstrating the method's excellent accuracy. In conclusion, this study developed a 1-step multiplex PCR method that simultaneously identifies S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Infantis, allowing routine mass detection in the grass-root poultry industry.

ABSTRACT: Objectives: The emergence of the florfenicol resistance gene fexA in Campylobacter poses a serious threat to public health, but the extent of the spread of fexA in Campylobacter from various hosts has not been well understood. This study aimed to investigate the fexA in Campylobacter isolates from different hosts. Methods: PCR was used to identify fexA-positive Campylobacter from different hosts during 2008–2019 in China, and the fexA-positive isolates were characterized by susceptibility tests, whole-genome sequencing, and natural transformation. Results: A total of 69 (2.54%, 69/2721) fexA-positive Campylobacter were identified, and the fexA-positive isolates increased remarkably (0.42%–16.90%) since it was first detected in 2010. By source, the 69 isolates were obtained from chickens (3.57%, 57/1595), geese (3.43%, 7/204), ducks (1.02%, 2/197), and environments (2.86%, 3/105); the fexA-positive isolates were not isolated in humans and pigs. In addition to fexA, these isolates also carried other antimicrobial resistance genes and exhibited multidrug resistance. Whole-genome sequencing analysis showed the fexA gene can disseminate clonally or horizontally via either multidrug resistance genomic islands or insertion sequences among the Campylobacter. The genetic structure IS1216-∆ISEfa11-hp-fexA-NAD(P)H-∆ISEfa11-IS1216 was conserved and widespread in the Campylobacter of various origins, and the IS1216 can form fexA-carrying circular intermediates, emphasizing that IS1216 plays an important role in the spread of fexA in Campylobacter. Conclusions: This study indicates the wide spread of fexA-positive Campylobacter in poultry and environments. Because multidrug resistance genomic islands and IS1216 can facilitate the transmission of fexA, systematic surveillance should be implemented to prevent the spread of fexA to humans.

ABSTRACTAlthough metals are essential for life, they are toxic to bacteria in excessive amounts. Therefore, the maintenance of metal homeostasis is critical for bacterial physiology and pathogenesis. Vibrio parahaemolyticus is a significant food-borne pathogen that mainly causes acute gastroenteritis in humans and acute hepatopancreatic necrosis disease in shrimp. Herein, we report that ZntA functions as a zinc (Zn) and cadmium (Cd) homeostasis mechanism and contributes to oxidative stress resistance and virulence in V. parahaemolyticus. zntA is remarkably induced by Zn, copper, cobalt, nickel (Ni), and Cd, while ZntA promotes V. parahaemolyticus growth under excess Zn/Ni and Cd conditions via maintaining Zn and Cd homeostasis, respectively. The growth of ΔzntA was inhibited under iron (Fe)-restricted conditions, and the inhibition was associated with Zn homeostasis disturbance. Ferrous iron supplementation improved the growth of ΔzntA under excess Zn, Ni or Cd conditions. The resistance of ΔzntA to H2O2-induced oxidative stress also decreased, and its virulence was attenuated in zebrafish models. Quantitative real-time PCR, mutagenesis, and β-galactosidase activity assays revealed that ZntR positively regulates zntA expression by binding to its promoter. Collectively, the ZntR-regulated ZntA is crucial for Zn and Cd homeostasis and contributes to oxidative stress resistance and virulence in V. parahaemolyticus.

Tigecycline serves as a last-resort antimicrobial agent against severe infections caused by multidrug-resistant bacteria. Tet(X) and its numerous variants encoding flavin-dependent monooxygenase can confer resistance to tigecycline, with tet(X4) being the most prevalent variant. This study aims to investigate the prevalence and characterize tigecycline resistance gene tet(X) in E. coli isolates from various origins in Yangzhou, China, to provide insights into tet(X) dissemination in this region. In 2022, we tested the presence of tet(X) in 618 E. coli isolates collected from diverse sources, including patients, pig-related samples, chicken-related samples, and vegetables in Yangzhou, China. The antimicrobial susceptibility of tet(X)-positive E. coli isolates was conducted using the agar dilution method or the broth microdilution method. Whole genome sequencing was performed on tet(X)-positive strains using Illumina and Oxford Nanopore platforms. Four isolates from pig or pork samples carried tet(X4) and exhibited resistance to multiple antimicrobial agents, including tigecycline. They were classified as ST542, ST10, ST761, and ST48, respectively. The tet(X4) gene was located on IncFIA8-IncHI1/ST17 (n=2), IncFIA18-IncFIB(K)-IncX1 (n=1), and IncX1 (n=1) plasmids, respectively. These tet(X4)-carrying plasmids exhibited high similarity to other tet(X4)-bearing plasmids with the same incompatible types found in diverse sources in China. They shared related genetic environments of tet(X4) associated with ISCR2, as observed in the first identified tet(X4)-bearing plasmid p47EC. In conclusion, although a low prevalence (0.65%) of tet(X) in E. coli strains was observed in this study, the horizontal transfer of tet(X4) among E. coli isolates mediated by pandemic plasmids and the mobile element ISCR2 raises great concerns. Thus, heightened surveillance and immediate action are imperative to curb this clinically significant resistance gene and preserve the efficacy of tigecycline.

ABSTRACT: Salmonella, which is widely distributed in nature, is an important zoonotic pathogen affecting humans, livestock, and other animals. Salmonella infection not only hinders the development of livestock and poultry-related industries but also poses a great threat to human health. In this study, we collected 1,537 samples including weak chicks, dead embryos, fecal samples and environmental samples from 2020 to 2023 (for a period of 1 to 2 months per year) to keep a long-term monitor the prevalence of Salmonella in an intensive laying hen farm, 105 Salmonella strains were isolated with an isolation rate of 6.83% (105/1,537). It revealed a significant decrease in prevalence rates of Salmonella over time (P < 0.001). Before 2020, the predominant serotype was S. Enteritidis. S. Kentucky was first detected in November 2020 and its proportion was gradually found to exceed that of S. Enteritidis since then. S. Kentucky isolates were distributed in various links of the four regions in the poultry farm. A total of 55 S. Kentucky strains, were assigned to ST198 based on whole genome sequencing. Among them, 54 strains were resistant to 12 to 16 antibiotics, indicating that they were extensively drug-resistant (XDR). Seventeen antimicrobial resistance genes were detected in 55 S. Kentucky isolates. For most of these isolates, antibiotic resistance phenotypes were concordant with their genotypes. All S. Kentucky strains isolated from this farm in 2020 to 2023 showed a high similarity based on their core-genome SNP-based phylogeny. The traceability analysis revealed that S. Kentucky was introduced to the farm through newly purchased flocks. The long-term existence of XDR S. Kentucky ST198 poses a substantial risk because of the multiage management and circulation of workers in this poultry farm. Thus, this study is the first to report extensively drug-resistant S. Kentucky ST198 detected in this intensive poultry farm in China.

Abstract Background mcr-1-positive Escherichia coli has emerged as a significant threat to human health, veterinary health, and food safety in recent years. After the prohibition of colistin as a feed additive in animal husbandry in China, a noticeable reduction in both colistin resistance and the prevalence of mcr-1 was observed in E. coli from animals and humans. Objectives To assess the prevalence of the colistin resistance gene mcr-1 and characterize its genetic context in E. coli strains derived from fecal and meat samples from food-producing animals in China. Methods A total of 1,353 fecal samples and 836 food samples were collected between 2019 and 2020 in China. E. coli isolates were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and their susceptibility to colistin were determined using the broth microdilution method. The colistin-resistant E. coli isolates were screened for the presence of mcr by PCR analysis and sequencing. The minimal inhibitory concentrations (MICs) of 15 antimicrobial agents against the mcr-1-positive strains were further tested using the agar dilution method, conjugation assays were performed, and whole genome sequencing was performed using Illumina HiSeq. Results In total, 1,403 E. coli strains were isolated. Thirteen isolates from chicken meat (n = 7), chickens (n = 3), and pigs (n = 3) were resistant to colistin with MIC values of 4 to 16 mg/L, and carried mcr-1. All mcr-1-positive strains, except for isolate AH20PE105, contained multiple resistance genes and exhibited multidrug-resistant phenotypes. They belonged to 10 sequence types (STs), including a novel ST (ST14521). mcr-1 was located on IncI2 (n = 9), IncX4 (n = 2), and IncHI2 (n = 2) plasmids, which were highly similar to other mcr-1-carrying plasmids sharing the same incompatibility type. Seven mcr-1-carrying plasmids could be successfully conjugally transferred to E. coli C600. Conclusions While the low prevalence of mcr-1 (0.93%) identified in this study may not immediately seem alarming, the very emergence of this gene merits attention given its implications for colistin resistance and public health. Hence, ongoing surveillance of mcr-1 in E. coli remains crucial.

ABSTRACT: Biofilms formed by pathogenic or spoilage microorganisms have become serious issues in the dairy industry, as this mode of life renders such microorganisms highly resistant to cleaning-in-place (CIP) procedures, disinfectants, desiccation, and other control strategies. The advent of omics techniques, especially the integration of different omics tools, has greatly improved our understanding of the features of microbial biofilms, and provided in-depth knowledge on developing effective methods that are directly against deleterious biofilms. This review provides novel insights into the single use of each omics tool and the application of multiomics tools to unravel the mechanisms of biofilm formation, specific molecular phenotypes exhibited by biofilms, and biofilm control strategies. Challenges and future perspective on the integration of omics tools for biofilm studies are also addressed.

Abstract The haemagglutinin-neuraminidase (HN) protein plays a crucial role in the infectivity and virulence of Newcastle disease virus (NDV). In a previous study, the mutant HN protein was identified as a crucial virulence factor for the velogenic variant NDV strain JS/7/05/Ch, which evolved from the prototypic vaccine strain Mukteswar. Furthermore, macrophages are the main susceptible target cells of NDV. However, the possible involvement of cellular molecules in viral infectivity remains unclear. Herein, we elucidate the crucial role of vimentin, an intermediate filament protein, in regulating NDV infectivity through targeting of the HN protein. Using LC‒MS/MS mass spectrometry and coimmunoprecipitation assays, we identified vimentin as a host protein that differentially interacted with prototypic and mutant HN proteins. Further analysis revealed that the variant NDV strain induced more significant rearrangement of vimentin fibres compared to the prototypic NDV strain and showed an interdependence between vimentin rearrangement and virus replication. Notably, these mutual influences were pronounced in HD11 chicken macrophages. Moreover, vimentin was required for multiple infection processes of the variant NDV strain in HD11 cells, including viral internalization, fusion, and release, while it was not necessary for those of the prototypic NDV strain. Collectively, these findings underscore the pivotal role of vimentin in NDV infection through targeting of the HN protein, providing novel targets for antiviral treatment strategies for NDV.

Livestock-associated Staphylococcus aureus (LA-MRSA) has been of increasing concern due to its potential risk to humans. This study investigated the prevalence of MRSA in pig production in Eastern China and determined the genomic characteristics of pig-associated MRSA isolates by whole-genome sequencing (WGS). A total of 1,318 samples were collected from pig farms and pig slaughterhouses, and 150 S. aureus were identified, including 63 MRSA isolates and 87 MSSA isolates. MRSA was detected in all pig farms and pig slaughterhouses. The antimicrobial susceptibility test revealed that all MRSA isolates were multidrug-resistant. The WGS and MLST analysis demonstrated that 56 MRSA isolates belonged to clonal complex (CC) 398, and seven MRSA isolates belonged to CC9. All LA-MRSA isolates were absent of phiSa3 phage containing immune evasion cluster (IEC) and possessed an intact hlb gene. In addition, genes associated with Panton-Valentine leukocidin, typically indicative of human adaptation, were not detected. The analysis of antibiotic resistance genes (ARGs) demonstrated that all MRSA isolates contained multiple ARGs. All MRSA isolates had Plthe mecA gene and at least one tetracycline resistance gene. Both tetM and tetK were detected in all MRSA CC398 isolates, while tetL was detected in all MRSA CC9 isolates. The phenicol resistance gene fexA was detected in 51 MRSA isolates, while the linezolid resistance gene cfr was detected in 60 MRSA isolates. The emergence of LA-MRSA CC398 in four pig farms and one slaughterhouse in this study indicates the spread of this clonal complex in the pig production sector in Eastern China. Further investigations are required to understand the potential transmission routes of LA-MRSA CC398 within the pork production chain in China and to assess the potential risks to humans.

ABSTRACT Campylobacter is one of the leading causes of bacterial foodborne diseases globally. Poultry is considered a major reservoir for the transmission of Campylobacter to humans. The occurrence and epidemiology of Campylobacter in healthy poultry have been extensively studied, but the prevalence in diseased poultry has rarely been reported. In this study, a total of 1,563 intestinal samples, 547 gallbladder samples, and 3,729 parenchyma organ samples were collected from diseased chickens, ducks, and geese in Yangzhou Veterinary Hospital between December 2016 and September 2017. The bacterial isolates were analyzed for genotype, resistance, and virulence genes. The overall prevalence of Campylobacter was 17.9%, and a significantly higher prevalence was observed in the diseased chickens (21.8%) than in the diseased ducks (12.1%) and geese (11.9%). Campylobacter was significantly more prevalent in the intestinal tract (39.6%), followed by the gallbladder (11.7%) and parenchyma organ (9.7%). Among 84 whole-genome sequenced Campylobacter isolates, we identified 47 multi-locus sequence typing types, of which ST-51 and ST-825 were the most predominant sequence types (STs) for Campylobacter jejuni and Campylobacter coli, respectively. Phylogenetic analysis showed that Campylobacter isolates from different tissues from the same host tended to cluster together with the same STs, which indicated Campylobacter infection in different tissues in vivo. The antimicrobial resistance genes most frequently identified were gyrA (T86I) (97.6%) and tet (O) (65.5%). Our results revealed the high prevalence of Campylobacter in diseased poultry in China, which will help the farms take the necessary action to develop effective mitigation strategies for reducing Campylobacter infection in poultry. IMPORTANCE Campylobacter is a major cause of campylobacteriosis worldwide, and poultry is the main reservoir for its transmission. Campylobacter was generally considered to be a harmless commensal organism in poultry without pathogenic properties. However, it was proposed that a Campylobacter-like organism may be the cause of vibrionic hepatitis, which poses a significant public health risk. The occurrence and epidemiology of Campylobacter in healthy poultry have been studied systematically, but little is known about the epidemiology of Campylobacter isolates from diseased poultry in China. Therefore, this study determined the prevalence and molecular characterization of Campylobacter from diseased chickens, ducks, and geese in Yangzhou Veterinary Hospital between December 2016 and September 2017, which was critical for improving the diagnosis and prevention of Campylobacter infections.

ABSTRACT The development of safe and efficient vaccines against coronavirus disease 2019 (COVID-19) is necessary for global public health. The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major target for the COVID-19 vaccines. Among the available vaccine types, DNA vaccines are a promising alternative to conventional vaccines. Here, we developed a novel DNA vaccine containing the SARS-CoV-2 S1 subunit and an optimized flagellin adjuvant (FliCΔD2D3) from Salmonella typhimurium. The S1 gene and S1-fliCΔD2D3 fusion gene were codon optimized and then cloned into the antibiotic-resistance-gene-free vector asd-pVAX1, producing asd-pVAX1-S1 and asd-pVAX1-S1-fliCΔD2D3, respectively. The expression of S1 and S1-FliCΔD2D3 proteins was confirmed after transient expression in HEK293T cells. After in vitro characterization of the vaccine candidates, we evaluated the humoral and cellular immune responses induced in BALB/c mice. The DNA vaccine induced a long-lasting humoral response for approximately 4 months, and the elicited serum antibodies efficiently blocked the binding of the SARS-CoV-2 S1 receptor-binding domain to its entry receptor (angiotensin-converting enzyme 2). Moreover, the DNA vaccine induced strong Th1/Th2-mixed immune responses, as demonstrated by the similar levels of S1-specific immunoglobulin (Ig) G1 and IgG2a in the serum and the significantly elevated Th1-type (IFN-γ and TNF-α) and Th2-type (IL-4 and IL-6) cytokine expression from splenic lymphocytes. Importantly, asd-pVAX1-S1-fliCΔD2D3 immunization in mice could significantly enhance the levels of immune responses compared to asd-pVAX1-S1. This study provides crucial information regarding the selection of a safer DNA vector and a new adjuvant for the vaccine development of SARS-CoV-2 and other infectious diseases. IMPORTANCE The development of safe and effective vaccines is needed to control the transmission of coronavirus disease 2019 (COVID-19). Synthetic DNA vaccines represent a promising platform in response to such outbreaks. Here, DNA vaccine candidates were developed using an optimized antibiotic-resistance gene-free asd-pVAX1 vector. An optimized flagellin (FliC) adjuvant was designed by fusion expression to increase the immunogenicity of the S1 antigen. S1 and S1-FliCΔD2D3 proteins were strongly expressed in mammalian cells. The FliCΔD2D3-adjuvanted DNA vaccine induced Th1/Th2-mixed immune responses and high titers of neutralizing antibodies. This study provides crucial information regarding the selection of a safer DNA vector and adjuvant for vaccine development. Our FliCΔD2D3-adjuvanted S1 DNA vaccine is more potent at inducing both humoral and cellular immune responses than S1 alone. This finding provides a new idea for the development of novel DNA vaccines against COVID-19 and could be further applied for the development of other vaccines.

ABSTRACTClade 2.3.4.4 H5N6 avian influenza virus (AIV) has been predominant in poultry in China, and the circulating haemagglutinin (HA) gene has changed from clade 2.3.4.4h to clade 2.3.4.4b in recent years. In 2021, we isolated four H5N6 viruses from ducks during the routine surveillance of AIV in China. The whole-genome sequencing results demonstrated that the four isolates all belonged to the currently prevalent clade 2.3.4.4b but had different internal gene constellations, which could be divided into G1 and G2 genotypes. Specifically, G1 possessed H9-like PB2 and PB1 genes on the H5-like genetic backbone while G2 owned an H3-like PB1 gene and the H5-like remaining internal genes. By determining the characteristics of H5N6 viruses, including growth performance on different cells, plaque-formation ability, virus attachment ability, and pathogenicity and transmission in different animal models, we found that G1 strains were more conducive to replication in mammalian cells (MDCK and A549) and BALB/c mice than G2 strains. However, G2 strains were more advantageously replicated in avian cells (CEF and DF-1) and slightly more transmissible in waterfowls (mallards) than G1 strains. This study enriched the epidemiological data of H5 subtype AIV to further understand its dynamic evolution, and laid the foundation for further research on the mechanism of low pathogenic AIV internal genes in generating novel H5 subtype reassortants.

ABSTRACT Metals are nutrients essential for almost all lifeforms. Bacteria have evolved several mechanisms to overcome the metal restrictions imposed by the host. Vibrio parahaemolyticus causes severe threats to public health and significant economic losses in shrimp aquaculture. Herein, we report that ZrgA contributes to zinc acquisition in this pathogen. The operon VP_RS01455 to VP_RS01475 of V. parahaemolyticus encodes the putative Zn transporter ZrgABCDE, whose homologs are widely distributed in Vibrionaceae. RNA sequencing analysis revealed that V. parahaemolyticus modulates the transcriptome in response to Zn limitation. Genes in the Zinc uptake regulator (Zur) regulon are upregulated during Zn limitation, including three genes annotated to encode Zn-binding proteins. Significant upregulation of these three genes during Zn limitation was also confirmed by quantitative real-time PCR (qRT-PCR) analysis. However, only the mutants containing a VP_RS01470 (zrgA) deletion exhibited impaired growth under Zn-deficient conditions, indicating that VP_RS01470 plays the predominant role in V. parahaemolyticus Zn acquisition. The VP_RS01470 deletion mutant displayed a false appearance of decreased swimming motility under Zn-deficient conditions, as revealed by the fact that the polar flagellar-related genes were not downregulated in the mutant. Moreover, VP_RS01470 deletion produced no noticeable impact on the swarming motility and virulence in mice. qRT-PCR analysis and β-galactosidase activity assays indicated that Zur negatively regulates VP_RS01470 expression in V. parahaemolyticus. Collectively, our findings suggest that ZrgA is required for Zn acquisition in V. parahaemolyticus and highlight the importance of detecting the expression of flagellar genes during analysis of motility of a mutant deficient in growth.

Background: Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an important pathogen causing healthcare-associated infections. In recent years, an increasing number of CA-MRSA clones have emerged and rapidly spread in the community and hospital settings in China. Objectives: To investigate the molecular epidemiology and resistance of CA-MRSA in the respiratory tracts of Chinese adults with community-acquired pneumonia (CAP). Methods: A total of 243 sputum samples were collected from adult patients with CAP at the Nantong Hospital in China between 2018 and 2021. S. aureus was identified using PCR, and its susceptibility to 14 antimicrobials was tested using the broth dilution method. Genomic characterization of respiratory CA-MRSA and our previously collected intestinal CA-MRSA isolates was performed using whole-genome sequencing, and the evolutionary relationships of these isolates were assessed using phylogenetic analysis. Results: The CA-MRSA colonization rate among adults with CAP in China was 7.8 % (19/243). Antimicrobial resistance analysis revealed that the proportion of multidrug-resistant respiratory CA-MRSA isolates (100 %) was higher than that of intestinal CA-MRSA isolates (6.3 %). Among the 35 CA-MRSA isolates, 10 MLST types were identified and clustered into five clone complexes (CCs). CC5 (48.6 %) and CC88 (20 %) were predominant CA-MRSA clones. Notably, the CC5 clone ST764/ST6292-MRSA-II-t002 was identified as the major lineage causing respiratory tract infections in Chinese adults with CAP. Conclusions: The prevalence of CA-MRSA among Chinese adults with CAP is high and often involves ST764/ST6292-MRSA-II-t002 as the causal pathogen.

The flagellin (FliC) of Salmonella typhimurium is a potential vaccine adjuvant as it can activate innate immunity and promote acquired immune responses. Macrophages are an important component of the innate immune system. The mechanism of flagellin’s adjuvant activity has been shown to be related to its ability to activate macrophages. However, few studies have comprehensively investigated the effects of Salmonella flagellin in macrophages using transcriptome sequencing. In this study, RNA-Seq was used to analyze the expression patterns of RAW264.7 macrophages induced by FliC to identify novel transcriptomic signatures in macrophages. A total of 2204 differentially expressed genes were found in the FliC-treated group compared with the control. Gene ontology and KEGG pathway analyses identified the top significantly regulated functional classification and canonical pathways, which were mainly related to immune responses and regulation. Inflammatory cytokines (IL-6, IL-1β, TNF-α, etc.) and chemokines (CXCL2, CXCL10, CCL2, etc.) were highly expressed in RAW264.7 cells following stimulation. Notably, flagellin significantly increased the expression of interferon (IFN)-β. In addition, previously unidentified IFN regulatory factors (IRFs) and IFN-stimulated genes (ISGs) were also significantly upregulated. The results of RNA-Seq were verified, and furthermore, we demonstrated that flagellin increased the expression of IFN-β and IFN-related genes (IRFs and ISGs) in bone marrow-derived dendritic cells and macrophages. These results suggested that Salmonella flagellin can activate IFN-β-related immune responses in macrophages, which provides new insight into the immune mechanisms of flagellin adjuvant.

ABSTRACT Salmonella are intracellular bacterial pathogens for which, as with many of the other Enterobacteriaceae, antibiotic resistance is becoming an increasing problem. New antibiotics are being sought as recommended by the World Health Organization and other international institutions. These must be able to penetrate macrophages, and infect the major host cells and the Salmonella-containing vacuole. This study reports screening a small library of Food and Drug Administration (FDA)-approved drugs for their antibacterial effect in macrophages infected with a rapid-multiplying mutant of Salmonella Enteritidis. The most effective drug that was least toxic for macrophages was Nifuratel, a nitrofuran antibiotic already in use for parasitic infections. In mice, it provided 60% protection after oral infection with a lethal S. Enteritidis dose with reduced bacterial numbers in the tissues. It was effective against different serovars, including multidrug-resistant strains of Salmonella Typhimurium, and in macrophages from different host species and against Listeria monocytogenes and Shigella flexneri. It reduced IL-10 and STAT3 production in infected macrophages which should increase the inflammatory response against Salmonella. IMPORTANCE Salmonella can keep long-term persistence in host's macrophages to evade cellular immune defense and antibiotic attack and exit in some condition and reinfect to cause salmonellosis again. In addition to multidrug resistance, this infection circle causes Salmonella clearance difficult in the host, and so there is a great need for new antibacterial agents that reduce intramacrophage Salmonella survival to block endogenous Salmonella reinfection.

Summary: Salmonella enterica subsp. enterica serovar 4,[5],12:i:- (Salmonella 4,[5],12:i:-), derived from S. Typhimurium, has become the dominant serotype causing human salmonellosis. In this study, we define the genetic mechanism of the generation of Salmonella 4,[5],12:i:- from S. Typhimurium through complicated transpositions and demonstrate that Salmonella 4,[5],12:i:- displays more efficient colonization and survival abilities in mice than its parent S. Typhimurium strain. We identified intermediate strains carrying both resistance regions (RRs) and the fljAB operon for the generation of Salmonella 4,[5],12:i:-. The insertion of RR3 into the chromosomal hin-iroB site of S. Typhimurium produced RR3-S. Typhimurium as a primary intermediate. Salmonella 4,[5],12:i:- was then produced by replacing the fljAB operon and/or its flanking sequences through intramolecular transpositions mediated by IS26 and/or IS1R elements in RR3-S. Typhimurium, which was further confirmed both in vitro and in vivo. Overall, we demonstrate the molecular mechanism underlying the origin, generation, and advantage of RRs-Salmonella 4,[5],12:i:- from S. Typhimurium.

ABSTRACT Polyphenols are abundant in nature, and their anaerobic biodegradation by gut and soil bacteria is a topic of great interest. The O2 requirement of phenol oxidases is thought to explain the microbial inertness of phenolic compounds in anoxic environments, such as peatlands, termed the enzyme latch hypothesis. A caveat of this model is that certain phenols are known to be degraded by strict anaerobic bacteria, although the biochemical basis for this process is incompletely understood. Here, we report the discovery and characterization of a gene cluster in the environmental bacterium Clostridium scatologenes for the degradation phloroglucinol (1,3,5-trihydroxybenzene), a key intermediate in the anaerobic degradation of flavonoids and tannins, which constitute the most abundant polyphenols in nature. The gene cluster encodes the key C-C cleavage enzyme dihydrophloroglucinol cyclohydrolase, as well as (S)-3-hydroxy-5-oxo-hexanoate dehydrogenase and triacetate acetoacetate-lyase, which enable phloroglucinol to be utilized as a carbon and energy source. Bioinformatics studies revealed the presence of this gene cluster in phylogenetically and metabolically diverse gut and environmental bacteria, with potential impacts on human health and carbon preservation in peat soils and other anaerobic environmental niches. IMPORTANCE This study provides novel insights into the microbiota’s anaerobic metabolism of phloroglucinol, a critical intermediate in the degradation of polyphenols in plants. Elucidation of this anaerobic pathway reveals enzymatic mechanisms for the degradation of phloroglucinol into short-chain fatty acids and acetyl-CoA, which are used as a carbon and energy source for bacterium growth. Bioinformatics studies suggested the prevalence of this pathway in phylogenetically and metabolically diverse gut and environmental bacteria, with potential impacts on carbon preservation in peat soils and human gut health.

ABSTRACT In this study, we screened the tmexCD-toprJ gene cluster among 1,541 samples obtained from patients, healthy individuals, companion animals, pigs, chicken, and pork and chicken meat in Yangzhou, China. As a result, nine strains from humans, animals, and foods were positive for tmexCD1-toprJ1, which was located on plasmids or the chromosome. Seven different sequence types (STs) were identified, including ST15 (n = 2), ST580, ST1944, ST2294, ST5982, ST6262 (n = 2), and ST6265. All the positive strains were clustered into two distinct clades, and they shared a 24,087-bp core structure of tmexCD1-toprJ1, bounded by IS26 in the same orientation. IS26 could facilitate rapid and wide dissemination of tmexCD1-toprJ1 in Enterobacteriaceae from various sources. IMPORTANCE Tigecycline has been regarded as one of the last-resort antibiotics available for the treatment of infections caused by carbapenem-resistant Enterobacterales. The plasmid-mediated resistance-nodulation-division–type efflux pump gene cluster tmexCD-toprJ is a newly identified tigecycline resistance determinant. In this study, we revealed that tmexCD-toprJ has disseminated among Klebsiella pneumoniae strains from poultry, food markets, and patients. It is critical to strengthen continuous monitoring, and control measures should be implemented to prevent the further dissemination of tmexCD-toprJ.

Most cases of chicken salmonellosis are caused by Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum, which lead to a significant morbidity and fatality rate. Although the conventional Kaufmann-White scheme is the reliable method for the serotyping of Salmonella, it does not distinguish between closely related biotypes like S. Pullorum and S. Gallinarum. Herein, we conducted a single one-step multiplex PCR assay that can identify and distinguish between S. Pullorum and S. Gallinarum in an accurate manner. This PCR method was based on three genes, including torT for S. Pullorum identification, I137_14430 for S. Gallinarum identification, and stn as the genus-level reference gene for Salmonella. By comparing S. Pullorum to S. Gallinarum and other serovars of Salmonella, in silico study revealed that only the former has a deletion of 126 bp-region in the carboxyl terminus of torT. The I137_14430 gene does not exist in S. Gallinarum. However, it is present in all other Salmonella serotypes. The multiplex PCR approach utilizes unique sets of primers that are intended to specifically target these three different genes. The established PCR method was capable of distinguishing between the biovars Pullorum and Gallinarum from the 29 distinct Salmonella serotypes as well as the 50 distinct pathogens that are not Salmonella, showing excellent specificity and exclusivity. The minimal amount of bacterial cells required for PCR detection was 100 CFU, while the lowest level of genomic DNA required was 27.5 pg/μL for both S. Pullorum and S. Gallinarum. After being implemented on the clinical Salmonella isolates collected from a poultry farm, the PCR test was capable of distinguishing the two biovars Pullorum and Gallinarum from the other Salmonella strains. The findings of the PCR assay were in line with those of the traditional serotyping and biochemical identification methods. This new multiplex PCR could be used as a novel tool to reinforce the clinical diagnosis and differentiation of S. Pullorum and S. Gallinarum, particularly in high-throughput screening situations, providing the opportunity for early screening of infections and, as a result, more effective management of the illness among flocks.

ABSTRACT The host-specific Salmonella serovar S. Pullorum (SP) modulates the chicken immune response to a Th2-biased response associated with persistent infection. This is different from the Th1-biased immune response induced by the genetically close serovar, S. Enteritidis (SE). Based on core genome differences between SP and SE, we used three complementary bioinformatics approaches to identify SP genes, which may be important for stimulation of the immune response. Defined mutants were constructed in selected genes, and the infection potential and ability of mutants to stimulate cytokine production in avian derived HD11 macrophages were determined. Deletion of large genomic regions unique to SP did not change infection potential nor immune stimulation significantly. Mutants in genes with conserved single nucleotide polymorphisms (SNPs) between the two serovars in the region 100 bp upstream of the start codon (conserved upstream SNPs [CuSNPs]) such as sseE, osmB, tolQ, a putative immune antigen, and a putative persistent infection factor, exhibited differences in induction of inflammatory cytokines compared to wild-type SP, suggesting a possible role of these CuSNPs in immune regulation. Single nucleotide SP mutants correcting for the CuSNP difference were constructed in the upstream region of sifA and pipA. The SNP corrected pipA mutant expressed pipA at a higher level than the wild-type SP strain, and the mutant differentially caused upregulation of proinflammatory cytokines. It suggests that this CuSNP is important for the suppression of proinflammatory responses. In conclusion, this study has identified putative immune stimulating factors of relevance to the difference in infection dynamics between SP and SE in avian macrophages. IMPORTANCE Salmonella Pullorum is host specific to avian species, where it causes life-threatening infection in young birds. It is unknown why it is host restricted and causes systemic disease, rather than gastroenteritis normally seen with Salmonella. In the present study, we identified genes and single nucleotide polymorphisms (SNPs; relative to the broad-host-range type Salmonella Enteritidis), which affected survival and immune induction in macrophages from hens suggesting a role in development of the host specific infection. Further studies of such genes may enable understanding of which genetic factors determine the development of host specific infection by S. Pullorum. In this study, we developed an in silico approach to predict candidate genes and SNPs for development of the host-specific infection and the specific induction of immunity associated with this infection. This study flow can be used in similar studies in other clades of bacteria.

We investigated genetic and biologic characteristics of 2 Eurasian avian-like H1N1 swine influenza viruses from pigs in China that belong to the predominant G4 genotype. One swine isolate exhibited strikingly great homology to contemporaneous human Eurasian avian-like H1N1 isolates, preferential binding to the human-type receptor, and vigorous replication in mice without adaptation.

ABSTRACT: Bovine tuberculosis (bTB) caused by Mycobacterium bovis is an important zoonotic disease. This infection is difficult to control because of the limited ability of the tuberculin skin test (TST) and ancillary IFN-γ release assay to detect all infected animals. In this study, we aimed to develop an efficient assay based on the enzyme-linked immunospot (ELISpot) technique for the diagnosis of bTB, with IFN-γ monoclonal antibodies 3E9 and Bio-labeled 6F8 used as capture and detection antibodies, respectively. As expected, there were significantly more M. bovis-specific spot-forming units (SFU) in bTB-infected cattle than in healthy cattle when an M. bovis-specific antigen, CFP-10-ESAT-6 fusion protein (CE protein), was used. The M. bovis IFN-γ ELISpot assay demonstrated a high level of agreement (90.83%) with the BOVIGAM ELISA test (Thermo Fisher Scientific) for detecting bTB. Furthermore, 3 of 109 cattle tested negative by both the TST and the BOVIGAM ELISA tests, but positive by the ELISpot assay (TST− ELISA− ELISpot+). During subsequent long-term monitoring, these 3 cattle became TST+ ELISA+ ELISpot+. These results suggest that the M. bovis IFN-γ ELISpot assay we established could detect infected cattle earlier than the BOVIGAM ELISA test.

Salmonella Enteritidis is an important intracellular pathogen, which can cause gastroenteritis in humans and animals and threaten life and health. S. Enteritidis proliferates in host macrophages to establish systemic infection. In this study, we evaluated the effects of Salmonella pathogenicity island-1 (SPI-1) and SPI-2 to S. Enteritidis virulence in vitro and in vivo, as well as the host inflammatory pathways affected by SPI-1 and SPI-2. Our results show that S. Enteritidis SPI-1 and SPI-2 contributed to bacterial invasion and proliferation in RAW264.7 macrophages, and induced cytotoxicity and cellular apoptosis of these cells. S. Enteritidis infection induced multiple inflammatory responses, including mitogen-activated protein kinase (ERK-mediated) and Janus kinase-signal transducer and activator of transcript (STAT) (STAT2-mediated) pathways. Both SPI-1 and SPI-2 were necessary to induce robust inflammatory responses and ERK/STAT2 phosphorylation in macrophages. In a mouse infection model, both SPIs, especially SPI-2, resulted in significant production of inflammatory cytokines and various interferon-stimulated genes in the liver and spleen. Activation of the ERK- and STAT2-mediated cytokine storm was largely affected by SPI-2. S. Enteritidis ΔSPI-1-infected mice displayed moderate histopathological damage and drastically reduced bacterial loads in tissues, whereas only slight damage and no bacteria were observed in ΔSPI-2- and ΔSPI-1/SPI-2-infected mice. A survival assay showed that ΔSPI-1 mutant mice maintained a medium level of virulence, while SPI-2 plays a decisive role in bacterial virulence. Collectively, our findings indicate that both SPIs, especially SPI-2, profoundly contributed to S. Enteritidis intracellular localization and virulence by activating multiple inflammatory pathways.

Inflammasome activation is an essential innate immune defense mechanism against Salmonella infections. Salmonella has developed multiple strategies to avoid or delay inflammasome activation, which may be required for long-term bacterial persistence. However, the mechanisms by which Salmonella evades host immune defenses are still not well understood. In this study, Salmonella Enteritidis (SE) random insertion transposon library was screened to identify the key factors that affect the inflammasome activation. The type I secretion system (T1SS) protein SiiD was demonstrated to repress the NLRP3 inflammasome activation during SE infection and was the first to reveal the antagonistic role of T1SS in the inflammasome pathway. SiiD was translocated into host cells and localized in the membrane fraction in a T1SS-dependent and partially T3SS-1-dependent way during SE infection. Subsequently, SiiD was demonstrated to significantly suppress the generation of mitochondrial reactive oxygen species (mtROS), thus repressing ASC oligomerization to form pyroptosomes, and impairing the NLRP3 dependent Caspase-1 activation and IL-1β secretion. Importantly, SiiD-deficient SE induced stronger gut inflammation in mice and displayed NLRP3-dependent attenuation of the virulence. SiiD-mediated inhibition of NLRP3 inflammasome activation significantly contributed to SE colonization in the infected mice. This study links bacterial T1SS regulation of mtROS-ASC signaling to NLRP3 inflammasome activation and reveals the essential role of T1SS in evading host immune responses.

The emergence and spread of carbapenemase genes, colistin resistance genes mcr-1, and tigecycline resistance gene tet(X) represent a significant threat to clinical therapy and public health. In this study, we investigated the presence of carbapenemase genes, mcr-1, and tet(X) in 298 Escherichia coli strains obtained from a teaching hospital in China. In total, eight (2.68%), six (2.01%), and one (0.34%) E. coli isolates carried blaNDM, mcr-1, and tet(X4), respectively. The blaNDM gene was located on IncX3 (n = 4), F2:A-:B- (n = 3), and F2:A1:B1 (n = 1) plasmids, with high similarity to multiple plasmids belonging to the same incompatibility type from Enterobacteriaceae. Six MCR-producing strains contained mcr-1-carrying IncI2 plasmids, organized similarly to other mcr-1-bearing IncI2 plasmids from animals in China. The blaCTX−M−55/64/132/199 gene located within a typical transposition unit (ISEcp1-blaCTX−M-orf477Δ) was inserted near dnaJ to generate 5-bp direct repeats in four mcr-1-positive plasmids. The tet(X) and another four resistance genes [aadA2, tet(A), floR, and Δlnu(F)] were co-located on an IncX1 plasmid, highly similar to other tet(X4)-carrying IncX1 plasmids from Escherichia and Klebsiella of animal or food origin, except that the conjugative transfer region of IncX1 plasmids was absent in our plasmid. Although a low prevalence of blaNDM, mcr-1, and tet(X) was observed in E. coli from patients in this study, their dissemination associated with some successful pandemic plasmids is of great concern. The continued surveillance of these crucial resistance genes in patients should be strengthened.

Macrophages can participate in immune responses by altering their metabolism, and play important roles in controlling bacterial infections. However, Salmonella Enteritidis can survive and proliferate in macrophages. After the deletion of DNA adenine methylase (Dam), the proliferation of Salmonella Enteritidis in macrophages decreased, the molecular mechanism is still unclear. After infecting macrophages with Salmonella Enteritidis wild type and dam gene deletion strains, intracellular metabolites were extracted and detected by non-targeted metabolomics and fatty acid targeted metabolomics. We found Dam had significant effects on arachidonic acid and related metabolic pathways in macrophages. The dam gene can promote the proliferation of Salmonella Enteritidis in macrophages by inhibiting the metabolic pathway of cytosolic phospholipase A2-mediated arachidonic acid production and conversion to prostaglandin E2 in macrophages, reducing the secretion of the pro-inflammatory factors IL-1β and IL-6. In addition, inhibition of arachidonic acid-related pathways in macrophages by Arachidonyl trifluoromethyl ketone could restore the proliferation of dam gene deletion strains in macrophages. This study explored the role of Dam in the process of Salmonella Enteritidis invading host cells from the perspective of host cell metabolism, and provides new insights into the immune escape mechanism of Salmonella Enteritidis.

Abstract Background Salmonella Derby is one of the most predominant Salmonella serotypes that seriously threatens food safety. This bacterium can be further differentiated to sub-populations with different population sizes; however, whether and how the S. Derby–gut microbiota interactions affect epidemic patterns of S. Derby sub-populations remain largely unknown. Results We selected two representative strains, 14T and 14C, which represent rarely distributed and prevalent sub-populations of the S. Derby ST40 group, respectively, to address this question using a mouse model. Effects of oral administration of both strains was monitored for 14 days. Alpha diversity of gut microbiota at early stages of infection (4 h post infection) was higher in 14C-treated mice and lower in 14T-treated mice compared with controls. Strain 14T triggered stronger inflammation responses but with lower pathogen titer in spleen compared with strain 14C at 14 days post infection. Certain known probiotic bacteria that can hinder colonization of Salmonella, such as Bifidobacteriaceae and Akkermansiaceae, exhibited increased relative abundance in 14T-treated mice compared with 14C-treated mice. Our results also demonstrated that Ligilactobacillus strains isolated from gut microbiota showed stronger antagonistic activity against strain 14T compared with strain 14C. Conclusions We identified how S. Derby infection affected gut microbiota composition, and found that the 14T strain, which represented a rarely distributed S. Derby sub-population, triggered stronger host inflammation responses and gut microbiota disturbance compared with the 14C strain, which represented a prevalent S. Derby sub-population. This study provides novel insights on the impacts of gut microbiota on the epidemic patterns of Salmonella populations.

A growing body of evidence suggests the pivotal role of long non-coding RNA (lncRNA) in influenza virus infection. Based on next-generation sequencing, we previously demonstrated that Lnc45 was distinctively stimulated by H5N1 influenza virus in mice. In this study, we systematically investigated the specific role of Lnc45 during influenza A virus (IAV) infection. Through qRT-PCR, we first demonstrated that Lnc45 is highly up-regulated by different subtypes of IAV strains, including H5N1, H7N9, and H1N1 viruses. Using RNA-FISH and qRT-PCR, we then found that Lnc45 can translocate from nuclear to cytoplasm during H5N1 virus infection. In addition, forced Lnc45 expression dramatically impeded viral replication of H1N1, H5N1, and H7N9 virus, while abolish of Lnc45 expression by RNA interference favored replication of these viruses, highlighting the potential broad antiviral activity of Lnc45 to IAV. Correspondingly, overexpression of Lnc45 inhibited viral polymerase activity and suppressed IAV-induced cell apoptosis. Moreover, Lnc45 significantly restrained nuclear aggregation of viral NP and PA proteins during H5N1 virus infection. Further functional study revealed that the stem ring arms of Lnc45 mainly mediated the antiviral effect. Therefore, we here demonstrated that Lnc45 functions as a broad-spectrum antiviral factor to inhibit influenza virus replication probably through inhibiting polymerase activity and NP and PA nuclear accumulation via its stem ring arms. Our study not only advances our understanding of the complexity of the IAV pathogenesis but also lays the foundation for developing novel anti-IAV therapeutics targeting the host lncRNA.

Vibrio parahaemolyticus is a foodborne pathogen that can colonize the small intestine of the host and cause diarrhea. The alternative sigma factor RpoN plays a vital role in regulating motility, carbon utilization and affects host colonization in V. parahaemolyticus RIMD2210633. In this study, transcriptome and phenotypic analysis further expanded our understanding of the RpoN regulon in V. parahaemolyticus. A deletion mutant of rpoN (ΔrpoN) was subjected to RNA-seq for systemic identification of the RpoN-controlled genes. Compared with the wild-type (WT), 399 genes were differentially expressed in the ΔrpoN strain. Moreover, 264 genes were down-regulated in the ΔrpoN strain, including those associated with nitrogen utilization (VP0118), glutamine synthetase (VP0121), formate dehydrogenase (VP1511 and VP1513-VP1515), quorum sensing (opaR and luxZ), polar flagellar systems, and type VI secretion system 2 (T6SS2). Quantitative real-time reverse transcription PCR (qRT-PCR) and electrophoretic mobility shift assay (EMSA) further confirmed that RpoN could directly bind to the promoters of these genes associated with polar flagellar systems (flgB and fliE), lateral flagellar systems (flgB2 and lafA), T6SS2 (hcp2 and VPA1044) and glutamine synthetase (VP0121), and then positively regulate the expression of these systems. A RpoN-binding motif was identified in V. parahaemolyticus using the MEME suite and verified by the EMSA. Besides, the deletion of rpoN caused a significant decrease in hemolytic activity, adhesion, and cytotoxicity. Our results provide new cues to better understand the regulatory networks of RpoN protein to motility, T6SS2, and metabolism in V. parahaemolyticus.

Invasion plasmid antigen J (IpaJ) is a protein with cysteine protease activity that is present in Salmonella and Shigella species. Salmonella enterica serovar Pullorum uses IpaJ to inhibit the NF-κB pathway and the subsequent inflammatory response, resulting in bacterial survival in host macrophages. In the present study, we performed a DNA pull-down assay and EMSA and identified ItrA, a new DeoR family transcriptional regulator that could control the expression of IpaJ by directly binding to the promoter of ipaJ. The deletion of itrA inhibited the transcription of ipaJ in Salmonella. Tn-Seq revealed that two regulators of Salmonella pathogenicity island 1 (SPI-1), namely HilA and HilD, regulated the secretion of IpaJ. The deletion of hilA, hilD or SPI-1 inhibited the secretion of IpaJ in both cultured medium and Salmonella-infected cells. In contrast, the strain with the deletion of ssrB (an SPI-2 regulator-encoding gene) displayed normal IpaJ secretion, indicating that IpaJ is an effector of the SPI-1-encoded type III secretion system (T3SS1). To further demonstrate the role of IpaJ in host cells, we performed quantitative phosphoproteomics and compared the fold changes in signaling molecules in HeLa cells infected with wild-type S. Pullorum C79-13 with those in HeLa cells infected with the ipaJ-deleted strain C79-13ΔpSPI12. Both phosphoproteomics and Western blot analyses revealed that p-MEK and p-ERK molecules were increased in C79-13ΔpSPI12- and C79-13ΔpSPI12-pipaJ(C45A)-infected cells; and Co-IP assays demonstrated that IpaJ interacts with Ras to reduce its ubiquitination, indicating that IpaJ can inhibit the activation of the MAPK signaling pathway.

ABSTRACT This study aimed to investigate the prevalence and genomic characteristics of the colistin resistance gene mcr in Salmonella enterica in China. In total, 445 S. enterica isolates from animals and food products were screened through PCR and sequencing for the presence of mcr. The mcr genes were detected in nine Salmonella strains (2.02%), with complete mcr-1 in S. enterica serovar Indiana (n = 1) and an S. Typhimurium monophasic variant (S. 4,[5],12:i:-; n = 1), mcr-4.3 in S. enterica serovar London (n = 1), and an incomplete mcr-1 in S. Indiana (n = 6). They exhibited MIC values of 0.25 to 8 mg/L to colistin and showed resistance to multiple antimicrobial agents. Whole-genome sequencing was performed on mcr-positive Salmonella strains using Illumina HiSeq or PacBio single-molecule real-time sequencing. The complete mcr-1 gene was located on conjugative IncN1-IncHI2 plasmid and IncX4 plasmid, respectively, with high similarity to other mcr-1-bearing plasmids belonging to the same incompatibility type. Together with an additional 13 antimicrobial resistance genes, the incomplete mcr-1 was embedded in an 81,442-bp multiresistance region on the chromosome in S. Indiana YZ20MCS6. The Δmcr-1-pap2 segment and a set of tellurite resistance determinants (terYXWZABCDEF) in six S. Indiana strains were similar to other IncHI2 plasmid backbones. The mcr-4.3 gene was located on an untyped plasmid pYULZMPS10. Although low prevalence of mcr was observed in Salmonella, continuous surveillance of this gene in Salmonella is required. Plasmids play an important role in mcr transmission, and mcr-1, although incomplete, can be captured by chromosomes with the help of mobile elements. IMPORTANCE Colistin is a last-resort antibiotic for severe infections caused by multidrug-resistant (MDR) Gram-negative pathogens. Colistin resistance genes mcr, particularly mcr-1, have been found in Enterobacteriaceae around the world, mainly in Escherichia coli and Salmonella. Salmonella enterica is a major foodborne pathogen, with MDR Salmonella being considered a “Serious Threat Level pathogen” by the Centers for Disease Control and Prevention. Therefore, the prevalence of mcr in Salmonella strains must be monitored. In this study, a low mcr prevalence (2.02%) was observed in Salmonella strains from animals and food products, with plasmid-borne mcr-1 in S. enterica serovar Indiana and an S. Typhimurium monophasic variant (S. 4,[5],12:i:-) and chromosomally located mcr-1 in S. Indiana. The mcr-4.3 gene was first identified in S. enterica serovar London associated with an untyped plasmid. Although this study reports a low mcr prevalence in Salmonella, the transmission ability of mcr-positive Salmonella strains to humans via the food chain is a public health concern.

Salmonella enterica serovar Enteritidis is the most prevalent serotype that causes human infections worldwide. Consumption of S. Enteritidis-contaminated animal foods is a major source of human infections; however, eradicating bacteria from animals remains difficult. Therefore, it is necessary to develop new measures to prevent and control salmonellosis. Here, we used the outer-membrane vesicles (OMVs) of S. Enteritidis and assessed their protective efficacy and immune response in mice. Deletion of tolR in S. Enteritidis increased the production and size of OMVs compared to those in the wild type (WT) and ΔrfaQ strains. Intramuscular immunization with OMVs conferred greater protection than intraperitoneal and intranasal immunization. Moreover, OMVs extracted from both WT and ΔtolR strains provided an 83.3% protective rate in mice challenged with S. Enteritidis, which was higher than that provided by OMVs extracted from the ΔrfaQ strain. However, compared with OMVs from the ΔtolR strain, OMVs from WT and ΔrfaQ strains rapidly eradicated S. Enteritidis colonizing the liver, spleen, ileum, and cecum of BALB/c mice after immunization. Immunization with OMVs from each of the three strains induced humoral immune responses and showed no side effects on the growth of mice. Our study revealed that OMVs from various S. Enteritidis strains could be developed for use as subunit vaccine candidates against nontyphoidal Salmonella infections in mammals.

Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum cause severe chicken salmonellosis, a disease associated with high mortality and morbidity among chickens worldwide. The conventional serotyping and biochemical reactions have been used to identify Salmonella serovars. However, the conventional methods are complicated, time-consuming, laborious, and expensive. Furthermore, it is challenging to distinguish S. Gallinarum and S. Pullorum via biochemical assays and serotyping because of their antigenic similarity. Although various PCR methods were established, a PCR protocol to detect and discriminate S. Gallinarum and S. Pullorum simultaneously is lacking. Herein, a one-step multiplex PCR method was established for the accurate identification and discrimination of S. Pullorum and S. Gallinarum. Three specific genes were used for the multiplex PCR method, with the I137_14445 and ybgL genes being the key targets to identify and differentiate S. Gallinarum and S. Pullorum, and stn being included as a reference gene for the Salmonella genus. In silico analysis showed that the I137_14445 gene is present in all Salmonella serovars, except for S. Gallinarum, and could therefore be used for the identification of S. Gallinarum. A 68-bp sequence deficiency in ybgL was found only in S. Pullorum compared to other Salmonella serovars, and this could therefore be used for the specific identification of S. Pullorum. The developed PCR assay was able to distinguish S. Gallinarum and S. Pullorum among 75 various Salmonella strains and 43 various non-Salmonella pathogens with excellent specificity. The detection limit for the genomic DNA of S. Gallinarum and S. Pullorum was 21.4 pg./μL, and the detectable limit for bacterial cells was 100 CFU. The developed PCR method was used for the analysis of Salmonella isolates in a chicken farm. This PCR system successfully discriminated S. Gallinarum and S. Pullorum from other different Salmonella serovars. The PCR results were confirmed by the conventional serotyping method. The newly established multiplex PCR is a simple, accurate, and cost-effective method for the timely identification and differentiation of S. Pullorum and S. Gallinarum.

ABSTRACT: Salmonella is the most important foodborne pathogen in poultry production systems and can infect humans via consumption of contaminated food. Ducks, an important waterfowl widely raised in China, are also a vehicle that transmits Salmonella through the food supply chain. In this study, 701 samples were collected from each production stage of the duck production chain. Salmonella was isolated and identified, and the isolates were tested for drug sensitivity and molecular typing based on whole genome sequencing (WGS) to explore the prevalence of Salmonella in the duck production chain. Altogether, a total of 180 Salmonella isolates (25.7%) were obtained from the duck production chain, 82 (35.7%) isolates were from hatchery samples, followed by 64 (29.2%) from market samples, 17 (23.6%) from farm samples, and 17 (9.4%) from slaughterhouse samples. All isolates were divided into 9 serotypes, among which S. Typhimurium, S. Anatum, and S. Enteritidis were the dominant serotypes. The S. Typhimurium was distributed in various production stages in the duck production chain. Among the 16 antibiotics, selected 60 isolates were only resistant to NAL, indicating that resistance of Salmonella in the duck production chain was low. WGS phylogenetic relationship results based on core-genome SNPs showed that S. Typhimurium can spread across geographic regions and along between different stages of the duck production chain, eventually reaching the market where it is a potential threat to consumer health. This study explored the prevalence of Salmonella in the duck production chain which will provide data support for proposing some interventions to control Salmonella.

This study aimed to investigate the prevalence and diversity of extended-spectrum β-lactamases (ESBL)-producing Escherichia coli isolates from healthy individuals in a community and to elucidate their dissemination mechanism. Cefotaxime-resistant E. coli were isolated from 95 samples of healthy persons from one community in Yangzhou, China, and were tested for minimal inhibitory concentrations of 14 antimicrobial agents. The isolates were subjected to whole genome sequencing by Illumina Hiseq or PacBio single-molecule real-time sequencing. A total of 30 cefotaxime-resistant E. coli isolates were obtained, carrying blaCTX-M (n=29) or blaDHA (n=1), of which the blaCTX-M-55 (n=19) was the most predominant genotype. One novel blaCTX-M variant blaCTX-M-252 was identified. Thirteen CTX-M-55-producing E. coli isolates belonged to ST8369 from nasal (n=12) or faecal (n=1) samples shared the identical cgMLST type, resistance profiles, resistance genes, plasmid replicons, and a 5,053-bp blaCTX-M-55 structure ΔIS26-ΔISEcp1-blaCTX-M-55-Δorf477-ΔTn2. The blaCTX-M-55 gene was located on IncHI2/ST3 plasmid in E. coli ST8369. The lengths of blaCTX-M/blaDHA-carrying contigs in the remaining 17 E. coli strains ranged from 1,663 to 382,836 bp, located on chromosome (n=4) or plasmids (n=5); the location of the other eight contigs could not be determined due to incomplete assembly. The blaCTX-M was associated with ISEcp1 as previously reported. Nasal colonization of CTX-M-55-producing ST8369 E. coli strains has occurred among healthy individuals in one community. There is a potential risk of antimicrobial resistance dissemination between humans within one community through close contact or environment via aerosols or dust. Therefore, surveillance of nasal carriage of blaCTX-M in communities is warranted to further monitor the spread of the antimicrobial resistance genes in China.

This study aimed to investigate the prevalence and characterization of tet(X4) in Escherichia coli isolates from a pig farm in Shanghai, China, and to elucidate tet(X4) dissemination mechanism in this swine farm. Forty-nine (80.33%) E. coli strains were isolated from 61 samples from a pig farm and were screened for the presence of tet(X). Among them, six (12.24%) strains were positive for tet(X4) and exhibited resistance to tigecycline (MIC ≥ 16 mg/L). They were further sequenced by Illumina Hiseq. Six tet(X4)-positive strains belonged to ST761 with identical resistance genes, resistance profiles, plasmid replicons, and cgMLST type except that additional ColE10 plasmid was present in isolate SH21PTE35. Isolate SH21PTE31, as a representative ST761 E. coli strain, was further sequenced using Nanopore MinION. The tet(X4) in SH21PTE31 was located on IncFIA18/IncFIB(K)/IncX1 hybrid plasmid pYUSHP31-1, highly similar to other tet(X4)-carrying IncFIA18/IncFIB(K)/IncX1 plasmids from ST761 E. coli and other E. coli lineages in China. These IncFIA18/IncFIB(K)/IncX1 plasmids shared closely related multidrug resistance regions, and could reorganize, acquire or lose resistance modules mediated by mobile elements such as ISCR2 and IS26. Phylogenetic analysis were performed including all tet(X4)-positive isolates obtained in this pig farm combined with 43 tet(X4)-positive E. coli from pigs, cow, pork, wastewater, and patients with the same ST from NCBI. The 50 tet(X4)-carrying E. coli ST761 isolates from different areas in China shared a close phylogenetic relationship (0-49 SNPs). In conclusion, clonal transmission of tet(X4)-positive E. coli ST761 has occurred in this swine farm. E. coli ST761 has the potential to become a high-risk clone for tet(X4) dissemination in China.

Abstract Background Genotype S H9N2 viruses have become predominant in poultry in China since 2010. These viruses frequently donate their whole internal gene segments to other emerging influenza A subtypes such as the novel H7N9, H5N6, and H10N8 viruses. We recently reported that the PB2 and M genes of the genotype S H9N2 virus, which are derived from the G1-like virus, enhance the fitness of H5Nx and H7N9 avian influenza viruses in chickens and mice. However, whether the G1-like PB2 and M genes are preferentially incorporated into progeny virions during virus reassortment remains unclear; whether the G1-like PB2 and M genes from different subtypes are differentially incorporated into new virion progeny remains unknown. Results We conducted a reassortment experiment with the use of a H7N9 virus as the backbone and found that G1-like M/PB2 genes were preferentially incorporated in progeny virions over F/98-like M/PB2 genes. Importantly, the preference varied among G1-like M/PB2 genes of different subtypes. When competing with F/98-like M/PB2 genes during reassortment, both the M and PB2 genes from the H7N9 virus GD15 showed an advantage, whereas only the PB2 gene from the H9N2 virus CZ73 and the M gene from the H9N2 virus AH320 displayed the advantage. Conclusion Our findings highlight the preferential and variable advantages of H9N2-derived G1-like M and PB2 genes in incorporating them into H7N9 progeny virions over SH14-derived F/98-like M/PB2 genes.

Abstract Since 2014, clade 2.3.4.4 has become the dominant epidemic branch of the Asian lineage H5 subtype highly pathogenic avian influenza virus (HPAIV) in southern and eastern China, while the H5N6 subtype is the most prevalent. We have shown earlier that lack of glycosylation at position 158 of the hemagglutinin (HA) glycoprotein due to the T160A mutation is a key determinant of the dual receptor binding property of clade 2.3.4.4 H5NX subtypes. Our present study aims to explore other effects of this site among H5N6 viruses. Here we report that N-linked glycosylation at site 158 facilitated the assembly of virus-like particles and enhanced virus replication in A549, MDCK, and chicken embryonic fibroblast (CEF) cells. Consistently, the HA-glycosylated H5N6 virus induced higher levels of inflammatory factors and resulted in stronger pathogenicity in mice than the virus without glycosylation at site 158. However, H5N6 viruses without glycosylation at site 158 were more resistant to heat and bound host cells better than the HA-glycosylated viruses. H5N6 virus without glycosylation at this site triggered the host immune response mechanism to antagonize the viral infection, making viral pathogenicity milder and favoring virus spread. These findings highlight the importance of glycosylation at site 158 of HA for the pathogenicity of the H5N6 viruses.

ABSTRACT: Salmonella enterica serovar Pullorum (S. Pullorum) causes pullorum disease (PD), which is an acute systemic disease, in chickens, and leads to serious economic losses in many developing countries because of its high morbidity and mortality rate in young chicks. The live-attenuated vaccine is considered to be an effective measure to control the Salmonella infection. In addition, the DIVA (differentiation of infected and vaccinated animals) feature without the interference of serological monitoring of Salmonella infection is an important consideration in the development of the Salmonella vaccine. In this study, we evaluated the immunogenicity and protective efficacy of a S. Pullorum rough mutant S06004ΔspiCΔrfaH as a live attenuated DIVA vaccine candidate in chickens. The S06004ΔspiCΔrfaH exhibited a significant rough lipopolysaccharides (LPS) phenotype which was agglutinated with the acriflavine, not with the O9 mono antibody. Compared to the wild-type, 50% lethal dose (LD50) of the rough mutant increased 100-fold confirmed its attenuation. The mutant strain also showed a decreased bacterial colonization in the spleen and liver. The immunization with the mutant strain had no effect on the body weight and no tissue lesions were observed in the liver and spleen. The high level of the S. Pullorum-specific IgG titers in the serum indicated that significant humoral immune responses were induced in the immunization group. The cellular immune responses were also elicited from the analysis of lymphocyte proliferation and expression of cytokines in the spleen. In addition, the S06004ΔspiCΔrfaH immunized group exhibited a negative response for the serological test, while the wild-type S06004 infection group was strongly positive for the serological test showing a DIVA capability. The survival rates in the vaccinated chickens were 87% after intramuscular challenge with wild-type S. Pullorum, while the survival rates were 20% in the control groups. Overall, these results have demonstrated that the rough mutant S06004ΔspiCΔrfaH strain can be developed as an efficient live attenuated DIVA vaccine candidate to control the systemic S. Pullorum infection without the interference of salmonellosis monitoring program in poultry.

Abstract Background Salmonella Enteritidis (SE) is one of the major foodborne zoonotic pathogens of worldwide importance which can induce activation of NLRC4 and NLRP3 inflammasomes during infection. Given that the inflammasomes play an essential role in resisting bacterial infection, Salmonella has evolved various strategies to regulate activation of the inflammasome, most of which largely remain unclear. Results A transposon mutant library in SE strain C50336 was screened for the identification of the potential factors that regulate inflammasome activation. We found that T3SS-associated genes invC, prgH, and spaN were required for inflammasome activation in vitro. Interestingly, C50336 strains with deletion or overexpression of Dam were both defective in activation of caspase-1, secretion of IL-1β and phosphorylation of c-Jun N-terminal kinase (Jnk). Transcriptome sequencing (RNA-seq) results showed that most of the differentially expressed genes and enriched KEGG pathways between the C50336-VS-C50336Δdam and C50336-VS-C50336::dam groups overlapped, which includes multiple signaling pathways related to the inflammasome. C50336Δdam and C50336::dam were both found to be defective in suppressing the expression of several anti-inflammasome factors. Moreover, overexpression of Dam in macrophages by lentiviral infection could specifically enhance the activation of NLRP3 inflammasome independently via promoting the Jnk pathway. Conclusions These data indicated that Dam was essential for modulating inflammasome activation during SE infection, there were complex and dynamic interplays between Dam and the inflammasome under different conditions. New insights were provided about the battle between SE and host innate immunological mechanisms.

ABSTRACTCRISPR-based typing was performed to subtype isolates of S. Typhimurium and its monophasic variant Salmonella 4,[5],12:i:- from humans and animals between 2009 and 2017 in China. CRISPR typing classified all isolates into two lineages and four sub-lineages. All isolates from Lineage II and Lineage IB-1 were Salmonella Typhimurium. All of Salmonella 4,[5],12:i: – isolates were distributed in Lineage IA and Lineage IB-2, which all belonged to ST34 by MLST typing. Only Lineage IB-2 contained ST34 isolates from both Salmonella Typhimurium and Salmonella 4,[5],12:i:-. Among the isolates of ST34, TST4 was identified as the most common CRISPR type representing 86.5% of Salmonella 4,[5],12:i:- and 14.5 % of Salmonella Typhimurium mainly from pigs and humans. This study demonstrated that TST4-ST34 isolates were predominant in Salmonella 4,[5],12:i:-, and pig was the main reservoir for Salmonella 4,[5],12:i:- in China, which might have the potential to transmit to humans by pig production.