Clostridium perfringens is a significant zoonotic foodborne pathogen. To systematically assess the potential risks associated with food-producing animals as a reservoir of C. perfringens in the early stages of the food production chain, we conducted whole-genome sequencing (WGS) and phenotypic analysis of 91 clinical C. perfringens isolates collected from pigs, chickens, cows, ducks, and geese across different regions of China. The results revealed that the isolates harbored a rich repertoire of toxin genes, with 71.43% (65/91) carrying greater than or equal to 10 toxin genes. Besides the classic type A, type C, which causes animal enterotoxemia, was most prevalent in pigs (45.76%). Notably, the and necrotic B-like (NetB) toxin, typically associated with avian necrotic enteritis, was also detected in isolates from cows and geese, suggesting potential cross-host transmission of toxin types. Antimicrobial susceptibility testing revealed a severe resistance situation, particularly among porcine isolates, which showed the highest resistance rates to clindamycin, penicillin, and tetracycline, with widespread multidrug resistance (MDR). Genomic analysis further identified 14 types of antimicrobial resistance (AMR) genes. The tetracycline resistance gene tetA(P) had an extremely high carriage rate of 94.51%, and AMR genes were most enriched in porcine isolates. Multilocus sequence typing (MLST) identified 59 sequence types (STs), 42 of which were newly discovered, demonstrating high genetic diversity. Major clonal complexes (CCs) showed certain host and geographic clustering. Furthermore, while the restriction-modification (RM) system was present in all isolates, the distribution of other defense systems like CRISPR-Cas was strain-specific. This study revealed that C. perfringens from Chinese food-producing animals is characterized by high virulence, extensive antimicrobial resistance, and high genetic diversity. It highlighted that pigs may serve as a crucial reservoir and evolutionary hub for virulent MDR isolates, posing a continuous threat to food safety and public health, and underscored the necessity for enhanced monitoring at the farm level.

Spatiotemporal coordination of peptidoglycan (PG) synthesis with the cell cycle is fundamental to bacterial life, yet how the rate-limiting step of PG synthesis is dynamically coupled to this process remains poorly understood. Here, we uncover that the conserved STK/STP-IreB module controls MurZ in Streptococcus suis through a phosphorylation-tunable liquid-liquid phase separation (LLPS) switch. Non-phosphorylated IreB dimers assemble into a septal ring that binds and inhibits MurZ. STK-mediated phosphorylation drives dimer-to-oligomer transition and LLPS, forming a biomolecular condensate, thereby relieving MurZ inhibition. Time-lapse imaging reveals that the septal ring and the condensate dynamically interconvert with the cell cycle. Thus, IreB couples the rate-limiting step of PG synthesis with the cell cycle through reversible interconversion between an inhibitory ring and a phase-separated reservoir, revealing a phosphorylation-tunable LLPS switch as a paradigm for spatiotemporal regulation of cellular biosynthesis.

BACKGROUND:The emergence and spread of hypervirulent Klebsiella pneumoniae (hvKp) poses a serious and growing challenge to public health worldwide. However, the knowledge regarding hvKp of animal origin remains very limited. In this study, we characterized a colistin-resistant hvKp isolate obtained from healthy chicken faeces and gained insight into the molecular basis of hypervirulence and colistin resistance. METHODS:Antimicrobial susceptibility testing was conducted by broth microdilution. The hypermucoviscous phenotype was determined by string tests. Whole-genome sequencing (WGS) was performed using a combination of Illumina NovaSeq/Oxford Nanopore PromethION platforms. Sequence alignment and complement assays were used to identify the mutations conferring colistin resistance. The virulence was investigated using both Galleria mellonella larvae and mice infection models. RESULTS:This strain KP20 displayed a hypermucoviscous phenotype and strong biofilm-forming capacity. WGS revealed that strain KP20 carried a pLVPK-like virulence plasmid with a novel replicon profile (IncFIB/RepB), harbouring key hypervirulence-associated genes, including iucABCD-iutA, iroBCDN, peg-344 and rmpA/rmpA2. Moreover, a plasmid harbouring the trimethoprim resistance gene dfrA50, pKP20-2, was identified as a member of the phage-like plasmids: genetic elements that function both as phages and plasmids. Sequence analysis and functional confirmation suggested that mutations in the crrB gene contributed to the colistin resistance observed in this strain. The virulence of KP20 was confirmed in the animal infection models. CONCLUSIONS:In this study, a high-risk ST412-K57 colistin-resistant hvKp of animal origin was identified and characterized. According to the One Health concept, our findings highlight the critical need for implementing enhanced molecular surveillance of hvKp in animals.

Klebsiella pneumoniae is an important zoonotic opportunistic pathogen. Of particular concern is the emergence of multidrug-resistant hypervirulent K. pneumoniae (MDR-hvKP), which represents a serious public health threat. Bacteriophage (phage) therapy has emerged as a promising solution to combat antibiotic-resistant K. pneumoniae infections. Here, we characterize a novel lytic phage, vB_Kp_H122, that specifically targets K57 capsular-type MDR-hvKP. vB_Kp_H122 exhibited the characteristic morphology of a siphovirus and demonstrated efficient infection kinetics, with an optimal multiplicity of infection (MOI) of 0.001 and a latent period of ~5 min. It also demonstrated considerable stability across a pH range of 4-11 and at temperatures from 4°C to 50°C, as well as potent activity against K. pneumoniae biofilms. The phage has a linear double-stranded DNA genome of 46,077 bp with a G + C content of 47.61% and belongs to a novel species within the genus Roufvirus. Its genome contained no identifiable genes associated with lysogeny, virulence, or antibiotic resistance, supporting its therapeutic safety. In a mouse infection model, a single dose of vB_Kp_H122 at 2 × 106 PFU significantly reduced bacterial loads in organs, alleviated pathological damage, and provided complete protection against lethal challenge with K57 MDR-hvKP. These findings suggest that vB_Kp_H122 may have potential as an antibacterial candidate against K57 MDR-hvKP isolates.

Klebsiella pneumoniae is an important emerging pathogen leading to serious clinical outcomes as the multidrug-resistant (MDR) isolates arise. While K. pneumoniae is a well-known pathogen in human clinical settings, its risks in nonhuman hosts and potential for cross-species transmission remain poorly understood. In this study, we performed whole-genome sequencing on 112 K. pneumoniae isolates from four host sources in Northeast China to elucidate their phylogenetic relationships and characterize their antimicrobial resistance (AMR) and virulence profiles. Our analysis identified 53 distinct sequence types (STs), 38 K-locus (KL) types, and nine O-loci. Notably, several strains with identical ST-KL combinations were shared across different hosts: ST76-KL5 was detected in both human and dairy cow sources; ST412-KL57 was found in human and chicken; ST111-KL63 was present in both pig and dairy cow sources, suggesting potential interspecies transmission. Antimicrobial susceptibility testing (AST) showed that nearly all human- and pig-derived isolates were resistant to β-lactams, quinolones, co-trimoxazole, tetracyclines, and at least one aminoglycoside. In contrast, isolates from chickens and dairy cows exhibited lower resistance rates to these antibiotics. Overall, 88 of the 112 isolates (78.6%) were classified as MDR phenotype. Virulence gene screening indicated that 34 isolates carried the iucABCD/iutA aerobactin synthesis cluster, a key marker for hypervirulence. These iuc-positive strains fell into two distinct phylogenetic lineages, including iuc1 and iuc3. It is necessary to conduct the regular monitoring of the antibiotic resistance and hypervirulence of clinical K. pneumoniae from animals to prevent and control the transmission of K. pneumoniae along the food chain.

The spread of the transferable optrA gene poses an increasing threat to the clinical efficacy of oxazolidinones. Here, we characterized a novel optrA-carrying plasmid, pEfa-optrA-Arg, from a linezolid-resistant Enterococcus faecalis clinical isolate from Argentina. The 68,653-bp conjugative plasmid harbored optrA together with multiple antimicrobial resistance genes and showed high similarity to a plasmid previously identified in a bovine isolate from Switzerland. pEfa-optrA-Arg, or a closely related variant, was also detected in E. faecalis isolates from several Argentinian hospitals, highlighting the role of horizontal gene transfer in the spread of antimicrobial resistance across human and animal reservoirs within the One Health continuum.

Mycoplasma synoviae (M. synoviae) infection remains a major cause of economic losses in intensive poultry production worldwide. The increased incidence of infections by M. synoviae has facilitated the emergence of diverse field strains. Characterizing the biological features of currently circulating strains will provide data support for region-specific disease control. In this study, 11 M. synoviae field strains were isolated from Northern China between 2018 and 2023 and comprehensively analyzed their genetic genotypes, antimicrobial susceptibility profiles, and potential resistance-associated mutations. Additionally, four representative strains (3S, GJZ, JB913, and YFS) were evaluated for pathogenicity through four inoculation routes: intravenous, footpad, intranasal, and intratracheal administration. Multilocus sequence typing (MLST) assigned the 11 isolates to six distinct sequence types (STs), including four STs (ST34, ST93, ST151 and ST153) exclusively found in China and two previously unreported novel STs (ST273 and ST274). Antimicrobial susceptibility assays demonstrated that most strains had elevated minimum inhibitory concentrations (MICs) against enrofloxacin and florfenicol, whereas most strains showed low MICs to tilmicosin, tylvalosin, tylosin, oxytetracycline, doxycycline, lincomycin, and tiamulin. Analysis of quinolone resistance-determining regions (QRDRs) presented a strong association between high enrofloxacin MICs (MIC ≥ 2 µg/mL) and specific amino acid mutations, including ParC Thr85Ile and GyrB Ser417Asn substitutions, as well as a putative ParE Pro446Ser point mutation based on M. synoviae reference numbering. Pathogenicity assays showed that intravenous and footpad inoculation of the four tested strains consistently induced severe systemic pathological lesions, including footpad/joint swelling and keel bursa edema, while the YFS strain additionally caused airsacculitis. In contrast, intranasal and intratracheal inoculation only caused negligible or no observable tissue damage. Notably, the GJZ strain induced significant upregulation of pro-inflammatory cytokines (IL-6, IL-17 A) and immunomodulatory cytokines (IL-4, IFN-γ) following intranasal inoculation, without evidence of clinical disease. This immunogenic yet attenuated phenotype is comparable to that of the MS-H vaccine strain administered via eye drops, indicating that GJZ holds promising potential as a novel vaccine candidate. Overall, this study provides a basic characterization of M. synoviae field strains from Northern China and identifies a single strain with favorable characteristics for further vaccine development research.

Oxazolidinones (linezolid and tedizolid) are last-resort antibiotics used to treat severe infections caused by multidrug-resistant Gram-positive bacteria. Although linezolid is not approved for veterinary use, resistant bacteria—particularly enterococci—have increasingly been reported in food-producing animals worldwide. Slaughterhouses represent potential hotspots for the transmission of antimicrobial-resistant pathogens along the food chain. This study investigated the presence of oxazolidinone resistance genes and relevant genetic elements in florfenicol-resistant enterococci and staphylococci isolated from wastewater collected from an Italian swine slaughterhouse. In total, five enterococci, six staphylococci, and one Mammaliicoccus sciuri (previously known as Staphylococcus sciuri) isolate were recovered. PCR screening revealed that all staphylococci and M. sciuri carried the cfr gene, while enterococci were positive for either optrA or poxtA. Nine isolates were selected for whole genome sequencing. Enterococcus hirae EM2 carried poxtA on the novel pEhEM2-poxtA plasmid. In E. faecium ED1 and EF1, optrA was located on the novel pEfmED1-optrA plasmid. E. durans EF2 carried poxtA within a Tn6657 transposon integrated into a pEgFS4-2-like plasmid also containing tet(M), tet(L), and genes related to transposition and plasmid replication. In Staphylococcus simulans SN1 and SF3, cfr was found on the novel pSsSN1-cfr plasmid and on p12-02300 plasmid, respectively. In Staphylococcus cohnii SD1, cfr was located on the previously described plasmid unnamed1, while in S. cohnii SF2 it was inserted into a chromosomal Tn558-like transposon along with fexA. M. sciuri SN4 harboured cfr on a small plasmid showing high similarity to the pK8D55P-cfr plasmid previously found in an M. sciuri isolate from ducks. These results highlight the role of slaughterhouse wastewater as a potential reservoir of oxazolidinone resistance genes and emphasize the need for continued monitoring to limit the environmental spread of antimicrobial resistance from animal production systems.

Streptococcus suis, an important zoonotic pathogen capable of transmission from pigs to humans, represents a critical threat to both public health and the global pork industry. The increasing prevalence of multidrug-resistant S. suis strains, coupled with their ability to form biofilms, has necessitated the development of alternative antimicrobial strategies. In this study, we characterized the therapeutic potential of Ply113, an endolysin derived from an Enterococcus faecium phage, against S. suis. Ply113 has shown potent bactericidal activity against S. suis in vitro, with rapid time-kill characteristics and broad-spectrum efficiency against clinically prevalent serotypes (2, 3, 4, 7, and 9). Transmission electron microscopy analysis revealed that Ply113 induced distinct morphological alterations in S. suis, including cell wall disintegration and cytoplasmic leakage. This endolysin exhibited anti-biofilm functionality, eradicating biofilms formed by clinical strain of S. suis in a concentration-dependent manner. In murine models of bacteremia, a single administration of Ply113 provided complete protection against lethal S. suis infection, significantly decreasing the bacterial burden in the liver and spleen and attenuating organ injury. Additionally, Ply113 has been shown to be safe for mice, with no adverse effects. Taken together, our findings indicate that Ply113 is a promising alternative antimicrobial agent for combating biofilm-related infections caused by S. suis.

OBJECTIVES:To identify and characterize the oxazolidinone resistance genes cfr and optrA from a methicillin-resistant Macrococcoides bohemicum strain of chicken origin. METHODS:The presence of mobile oxazolidinone resistance genes was detected by PCR. Antimicrobial susceptibility testing was conducted by broth microdilution. Transfer experiments were carried out to evaluate horizontal transferability of the plasmid. WGS was performed using a combination of Illumina NovaSeq/Oxford Nanopore PromethION platforms. RESULTS:The M. bohemicum strain HLJ23 exhibited an MDR phenotype and was positive for both cfr and optrA genes. WGS revealed that the genes cfr and optrA co-exist on the novel MDR plasmid pHLJ23-71kb. Although conjugation experiments were unsuccessful, plasmid pHLJ23-71kb could be transferred to Staphylococcus aureus RN4220 by electrotransformation. Genetic context analysis showed that the cfr and optrA together with another four antimicrobial resistance genes are located in an MDR region on plasmid pHLJ23-71kb. Sequence analysis suggested that this MDR region possibly originated from Mammaliicoccus or Staphylococcus spp. CONCLUSIONS:To the best of our knowledge, this study represents the first report of the oxazolidinone resistance genes cfr and optrA in the genus Macrococcoides. Furthermore, attention should be paid to the exchange of resistance determinants between members of the genera Staphylococcus, Mammaliicoccus and Macrococcoides.

The Salmonella pathogenicity islands (SPIs) play crucial roles in the progression of Salmonella infection. In this study, we constructed an improved λ Red homologous recombination system to prepare single and triple deletion mutants of 3 prominent SPIs (SPI-1, 2, and 3), aiming at the impact of deletion on morphology, carbon source metabolism, adhesion and invasion capacity, in vivo colonization, and immune efficacy in chicks. Our examination revealed that the surface of the single deletion mutants (SM6ΔSPI1, ΔSPI2, and ΔSPI3) exhibited a more rugged texture and appeared to be enveloped in a layer of transparent colloid, whereas the morphology of the triple deletion mutant (SM6ΔSPI1&2&3) remained unaltered when compared to the parent strain. The carbon metabolic spectrum of the SPI mutants underwent profound alterations, with a notable and statistically significant modification observed in 30 out of 95 carbon sources, primarily carbohydrates (17 out of 30). Furthermore, the adhesion capacity of the 4 mutants to Caco-2 cells was significantly reduced when compared to that of the parent strain. Moreover, the invasion capacity of mutants SM6ΔSPI1 and SM6ΔSPI1&2&3 exhibited a substantial decrease, while it was enhanced to varying degrees for SM6ΔSPI3 and SM6ΔSPI2. Importantly, none of the 4 mutants induced any clinical symptoms in the chicks. However, they did transiently colonize the spleen and liver. Notably, the SM6ΔSPI1&2&3 mutant was rapidly cleared from both the spleen and liver within 8 days post-infection and no notable pathological changes were observed in the organs. Additionally, when challenged, the mutants immunized groups displayed a significant increase in antibody levels and alterations in the CD3+CD4+ and CD3+CD8+ subpopulations, and the levels of IL-4 and IFN-γ cytokines in the SM6ΔSPI1&2&3 immunized chicken serum surpassed those of other groups. In summary, the successful construction of the 4 SPI mutants lays the groundwork for further exploration into the pathogenic (including metabolic) mechanisms of SPIs and the development of safe and effective live attenuated Salmonella vaccines or carriers.

The inappropriate use of cephalosporins lead to the occurrence and global spread of bacteria resistant to these antimicrobials. In this study, we isolated four Escherichia albertii strains from broilers in eastern China. The antimicrobial susceptibility and genomic characterization of these E. albertii isolates were determined. Our results revealed that these four E. albertii isolates exhibited resistance to tetracyclines, chloramphenicol, beta-lactams, aminoglycosides, polymyxin B, sulfonamides, quinolones, and other antimicrobials. Among them, EA04 isolate was multidrug resistant and harbored extended-spectrum beta-lactamases (ESBL) genes blaCTX-M and blaTEM. Whole genome sequencing and core-genome multilocus sequence typing (cgMLST) based on all ST4638 E. albertii for EA04 inferred highly probable epidemiological links between selected human isolates. Additionally, the ESBL genes blaTEM-141 and blaCTX-M-55 were coexistent in an approximately 75 kb IncFII plasmid pEA04.2 in EA04. Comparative analysis indicated that genes blaTEM-141 and blaCTX-M-55 were located in IS15-blaCTX-M-55-wbuC-blaTEM-141-IS26 region, which similar structures were identified in various bacteria. Furthermore, the plasmid pEA04.2 could be transferable to E. coli EC600 and lead to the resistance to third-generation cephalosporins. These results suggested that chicken potentially serve as a reservoir for multidrug resistant E. albertii, which increases the risk of horizontal transfer of antimicrobial resistance between humans, animals and environment.

A novel polyvalent broad-spectrum phage, vB_EcoM_XAM237 (XAM237), was isolated from pig farm sewage. It can simultaneously lyse multiple strains of pathogenic Escherichia coli (E. coli), demonstrating a broad host range. When the enteropathogenic E. coli (EPEC) strain E711 was used as the host bacterium, the phage XAM237 exhibited a short latent period, high stability at different temperatures and pH values and good tolerance to chloroform. Moreover, phage XAM237 can efficiently adsorb and lyse host bacteria in vitro. Whole-genome sequencing revealed that XAM237 is a double-stranded DNA (dsDNA) phage consisting of 170 541 bp with a G + C content of 35%. Phylogenetic analysis confirmed that XAM237 belongs to the family Straboviridae, genus Tequatrovirus. In addition, the genome of XAM237 did not contain genes related to lysogenicity, virulence or antimicrobial resistance. The effects of phage XAM237 in treating EPEC infections in vivo were evaluated in a mouse model. Phage XAM237 was able to reduce the number of colonized aEPEC strain E711 in the small intestine, liver, spleen, and kidney. This study suggested that phage XAM237 may be a promising candidate biologic agent for controlling pathogenic E. coli infections.

>Highlights● A novel conjugative plasmid pHJ90-cfr carrying the multiresistance gene cfr was characterized in Proteus vulgaris.● A new IS5-family member, ISPmi4, was identified for the first time.● Both plasmids and ICEs were vital mobile genetic elements for horizontal transmission of cfr gene in Proteus species.

Oxazolidinones are highly effective antimicrobial agents for the treatment of serious infections caused by Gram-positive organisms, including methicillinresistant Staphylococcus aureus（MRSA）, vancomycinresistant enterococci（VRE）, multidrug-resistant（MDR） pneumococci and MDR mycobacteria（Brenciani et al. 2022）. However, the emergence and prevalence of acquired oxazolidinone resistance genes, including cfr, optrA and poxtA, presents a great challenge for public health（Schwarz et al. 2021）.

Oxazolidinones are potent antimicrobial agents used to treat human infections caused by multidrug-resistant Gram-positive bacteria. The growing resistance to oxazolidinones poses a significant threat to public health. In August 2021, a linezolid-resistant Enterococcus faecium BN83 was isolated from a raw milk sample of cow in Inner Mongolia, China. This isolate exhibited a multidrug resistance phenotype and was resistant to most of drugs tested including linezolid and tedizolid. PCR detection showed that two mobile oxazolidinones resistance genes, optrA and poxtA, were present in this isolate. Whole genome sequencing analysis revealed that the genes optrA and poxtA were located on two different plasmids, designated as pBN83-1 and pBN83-2, belonging to RepA_N and Inc18 families respectively. Genetic context analysis suggested that optrA gene on plasmid pBN83-1 was located in transposon Tn6261 initially found in E. faecalis. Comprehensive analysis revealed that Tn6261 act as an important horizontal transmission vector for the spread of optrA in E. faecium. Additionally, poxtA-bearing pBN83-2 displayed high similarity to numerous plasmids from Enterococcus of different origin and pBN83-2-like plasmid represented a key mobile genetic element involved in movement of poxtA in enterococcal species. The presence of optrA- and poxtA-carrying E. faecium in raw bovine milk represents a public health concern and active surveillance is urgently warranted to investigate the prevalence of oxazolidinone resistance genes in animal-derived food products.

ABSTRACT Glaesserella parasuis is a commensal bacterial organism found in the upper respiratory tract of healthy pigs and the etiological agent of Glässer’s disease, which causes severe economic losses in the swine industry. This study aimed to better understand the epidemiological characteristics of this opportunistic pathogen. We investigated the prevalence and distribution of sequence types (STs), serovars, antimicrobial resistance genes (ARGs), and potential virulence factors (VFs) in 764 G . parasuis isolates collected from diseased and healthy pigs from 19 countries, including China. Multilocus sequence typing showed a high degree of variation with 334 STs, of which 93 were not previously recognized. Phylogenetic analysis revealed two major clades distinguished by isolation year, source, country, and serovar. The dominant serovars of G. parasuis were serovars 4 (19.50%), 7 (15.97%), 5/12 (13.87%), and 13 (12.30%). Serovar 7 gradually became one of the dominant serovars in G. parasuis with more VFs and fewer ARGs. Serovars 4 and 5/12 were the most frequent serovars in diseased pigs, whereas serovars 2, 8, and 11 were predominant in healthy pigs. Serovars 7 and 13 possessed more VFs than the other serovars. This study provides novel insights into the global prevalence and epidemiology of G. parasuis and valuable clues for further investigation into the pathogenicity of G. parasuis , which will facilitate the development of effective vaccines. IMPORTANCE Glaesserella parasuis is a clinically important gram-negative opportunistic pathogen, which causes serious financial losses in swine industry on a global scale. No vaccine is known that provides cross-protection against all 15 serovars; furthermore, the correlation between serovar and virulence is largely unknown. This study provides a large number of sequenced strains in 19 countries and compares the genomic diversity of G. parasuis between diseased and healthy pigs. We found a slight change in the dominant serovar of G. parasuis in the world, with serovar 7 gradually emerging as one of the predominant serovars. The observed higher average number of VFs in this particular serovar strain challenges the previously held notion that serovar 7 is non-virulent, indicating a more complex virulence landscape than previously understood. Our analysis indicating that six ARGs [ tet (B), sul2 , aph(3')-Ia , aph (6)-Id , bla ROB-1 , and aph(3'')-Ib ] are likely to be transmitted horizontally in their entirety. By analyzing VFs, we provided an improved understanding of the virulence of G. parasuis , and these key findings suggest that vaccine development will be challenging.