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Objectives An effective strategy for combating MDR Gram-negative pathogens can greatly reduce the cost and shorten the antibiotic development progress. Here, we investigated the synergistic activity of outer membrane disruptor SLAP-S25 in combination with hydrophobic antibiotics (LogP > 2, including novobiocin, erythromycin, clindamycin and rifampicin) against MDR Gram-negative pathogens. Methods Five representative Gram-negative bacteria were selected as model strains to analyse the synergistic combination of SLAP-S25 and hydrophobic antibiotics. Carbapenem-resistant hypervirulent Klebsiella pneumoniae CRHvKP4 was used to investigate the synergistic mechanism. The in vivo synergistically therapeutic activity of SLAP-S25 and hydrophobic antibiotics was measured in the mouse peritonitis/sepsis model infected with K. pneumoniae CRHvKP4. Results SLAP-S25 disrupted the outer membrane by removing LPS from Gram-negative bacteria, facilitating the entry of hydrophobic antibiotics to kill MDR Gram-negative pathogens. Moreover, the combination of SLAP-S25 and rifampicin exhibited promising therapeutic effects in the mouse infection model infected with K. pneumoniae CRHvKP4. Conclusions Our findings provide a potential therapeutic strategy to combine SLAP-S25 with hydrophobic antibiotics for combating MDR Gram-negative pathogens.

Luminogens with aggregation-induced emission (AIEgens) properties have numerous broad applications in fields of chemical and biological analyses due to their exceptional photostability, excellent signal reliability, high quantum yield, and large Stokes' shift. In particular, AIEgens also bring new blood for immunoassay. Since publication of the first 2004 paper, AIEgens-based immunoassays have received significant attention because of their high sensitivity, specificity, accuracy, and reliability. However, until now, there have been no comprehensive literature reviews focused on the evolving field of AIEgens-based immunoassays. Thus, we have extensively reviewed AIEgens-based immunoassays from their basic working principles to specific applications. We focus on several fundamental elements of AIEgens-based immunoassays, including the typical structures of AIEgens, emission mechanism of AIEgens probes, function of AIEgens in immunoassays, and platform of AIEgens-based immunoassays. Then, the representative applications of AIEgens-based immunoassays in food safety, medical diagnostics, and environmental monitoring are explored. Thus, proposals on how to further improve the AIEgens-based immunoassay performance are also discussed, as well as future challenges and perspectives, aiming to provide brief and valid guidelines for choosing suitable AIEgens-based immunoassays according to specific application requirements.

Chronic diabetic wounds are a growing threat globally. Many aspects contribute to its deterioration, including bacterial infection, unbalanced microenvironment, dysfunction of cell repair, etc. In this work, we designed a multipronged micelles-hydrogel platform loaded with curcumin and rifampicin (CRMs-hydrogel) for bacteria-infected chronic wound treatment. The curcumin- and rifampicin-loaded micelles (CRMs) exhibited both MMP9-responsive and epidermal growth factor receptor (EGFR)-targeting abilities. On the one hand, drugs could be released from micelles due to responsive disassembly by MMP9, a matrix metalloproteinase overexpressed in a chronic wound environment; on the other hand, CRMs showed specific targeting to EGFR on epithelial cells and fibroblasts and therefore increased intracellular drug delivery. The thermosensitive CRMs-hydrogel could form strong adhesion with the wound area and served as a suitable matrix for sustained release of CRMs directly at the wound bed, with excellent intracellular and extracellular bacterial elimination efficiency and wound healing promotion capability. We found that a single dose of CRMs-hydrogel achieved 99% antibacterial rate at the MRSA-infected diabetic wound, which effectively reduced inflammatory response and promoted the neovascularization and re-epithelialization process, with nearly half reduction of the skin barrier regeneration period. Collectively, our thermosensitive, MMP9-responsive, and targeted micelles-hydrogel nanoplatform is promising for chronic wound treatment.

The sharp increase in infections associated with multidrug-resistant (MDR) bacterial pathogens that have seriously compromised traditional chemotherapeutics is becoming a devastating public health threat worldwide. Thus, there is an urgent unmet demand to pursue alternative intervention strategies to circumvent this problem. Advances in host-acting antibacterial compounds (HACs) have provided promising, emerging approaches to reduce or eliminate internalized bacteria. In this review we focus mainly on the interactions between host cells and facultative intracellular bacteria to explore potential targets for the discovery and development of HACs. Additionally, we summarize the targets of recently described HACs and their modes of action and pharmacological activities against diverse pathogenic bacteria-associated infections. This overview of HACs sheds light on alternative strategies to treat clinical infections associated with cytosolic bacteria and the development of novel antibacterial agents.

China Antimicrobial Resistance Surveillance Network for Pets (CARPet) was established in 2021 to monitor the resistance profiles of clinical bacterial pathogens from companion animals. From 2018 to 2021, we recovered and tested 4,541 isolates from dogs and cats across 25 Chinese provinces, with Escherichia coli (18.5%) and Staphylococcus pseudintermedius (17.8%) being the most predominant bacterial species. The Enterobacterales were highly susceptible to tigecycline, meropenem, colistin, and amikacin (70.3%–100.0%), but showed moderate resistance to ampicillin, ceftriaxone, doxycycline, florfenicol, levofloxacin, enrofloxacin, and trimethoprim-sulfamethoxazole (29.3%–56.7%). About 66.3% of Acinetobacter spp. were resistant to florfenicol, with relatively low resistance to another 11 antibiotics (1.2%–23.3%). The Pseudomonas spp. showed high susceptibility to colistin (91.7%) and meropenem (88.3%). The coagulase-positive Staphylococcus spp. showed higher resistance rates to most antimicrobial agents than coagulase-negative Staphylococcus isolates. However, over 90.0% of Staphylococcus spp. were susceptible to linezolid, daptomycin and rifampin, and no vancomycin-resistant isolates were detected. E. faecium isolates demonstrated higher resistance rates to most antimicrobial agents than E. faecalis isolates. Streptococcus spp. isolates showed low resistance to most antimicrobial agents except for doxycycline (78.2%) and azithromycin (68.8%). Overall, the tested clinical isolates showed high rates of resistance to commonly used antimicrobial agents in companion animals. Therefore, it is crucial to strengthen the monitoring of bacterial resistance in pets. By timely and effectively collecting, analyzing, and reporting antimicrobial resistance dynamics in pets, the CARPet network will become a powerful platform to provide scientific guidance for both pet medical care and public health.

China banned the use of colistin as animal growth promoter in April 2017. Herein, we report the prevalence of mcr-1 in the intestine of healthy humans and risk factors associated with mcr-1 carriage after the implementation of the ban.We recruited 719 healthy volunteers from Shenzhen City from 1 March 2018 to 31 December 2019 to investigate the prevalence of mcr-1 in human intestine, and undertook a case-control study to ascertain the risk factors associated with the mcr-1-positive population. A further comparative study was conducted to identify differences between genetic characteristics of mcr-1-positive and mcr-1-negative Escherichia coli.Overall, 56 (7.8%, 95% CI 5.9%-10.0%, n = 719) individual faecal samples were positive for mcr-1, and prevalence of mcr-1 among individuals in 2019 (2.4%, 95% CI 8.7%-15.0%, 7/294) was significantly lower than that in 2018 (11.5%, 95% CI 1.0%-4.8%, 49/425) (p 0.0001). After the colistin ban, animal-derived food (pork and chicken meat) was no longer a risk factor for mcr-1 carriage in human intestine, whereas a higher intake of fish and seafood (75 g/day) and whole grains (150 g/day) was associated with higher and lower risk of mcr-1 carriage, respectively (OR 2.175, 95% CI 1.047-4.517; OR 0.045, 95% CI 0.004-0.567). Compared with mcr-1-negative E. coli, the mcr-1-positive E. coli had different patterns of resistance genes and genetic heterogeneity.Our study implicates aquatic food as beeing associated with mcr-1 carriage in the healthy population, even after the ban on colistin. Dietary modification (e.g. whole grains) may help to combat mcr-1-positive bacterial colonization of the gut.

The increasing threat to global health posed by antibiotic resistance remains a serious concern. This troublesome scenario has steered a need for the discovery and evaluation of novel antibacterial agents. Natural products are the main sources of antimicrobials used in clinical practice, serving as a rich reservoir for the discovery of new antibiotics. Pharmaceutical phenolics especially xanthones widely exist in the plant kingdom, and are important plant metabolites. They possess versatile biological activities, including antiviral, antibacterial, neurotrophic, and anticancer. In the present study, we focus on the antibacterial activities of phytoxanthones and summarize their structures and sources, categories and drug-likeness evaluations, and antibacterial activities. A total of 226 different plant xanthones are identified through the NETs screening, and most of them are distributed in Clusiaceae family. These phytoxanthones are divided into four groups according to the intrinsic structural properties, including the most common simple xanthones and the majority of biprenylated ones. Moreover, their physicochemical parameters are calculated and the structure–activity relationships are discussed as well. These results indicate that the biprenylated xanthone derivatives may be promising antibacterial candidates and that the natural products of plants may be a poorly understood repository for the discovery of novel antibacterial agents.

A rare antibody that is able to tolerate physio-chemical factors is preferred and highly demanded in diagnosis and therapy. Rabbit monoclonal antibodies (RmAbs) are distinguished owing to their high affinity and stability. However, the efficiency and availability of traditional methods for RmAb discovery are limited, particularly for small molecules. Here, we present an indirect competitive screening method in nanowells, named CSMN, for single rabbit antibody-secreting cells (ASCs) selection with 20.6 h and propose an efficient platform for RmAb production against small molecules within 5.8 days for the first time. Chloramphenicol (CAP) as an antibacterial agent poses a great threat to public health. We applied CSMN to select CAP-specific ASCs and produced one high-affinity RmAb, surprisingly showed extremely halophilic properties with an IC

Mobile colistin resistance (

Biopharmaceutics Classification System (BCS) class II and IV drugs exhibit low solubility and suffer a limitation in oral administration. Exosomes have attracted intensive attention in the efficient delivery of such compounds. However, low gastrointestinal stability and high production cost of exosomes hinder their development as drug carriers. Here, milk exosomes are functionalized with phosphatidylserine and are capable of improving the solubility of BCS class II and IV drugs, resulting in facilitating the oral delivery of the drugs. A natural flavonoid, α-mangostin, is loaded into exosomes (AExo) to enhance the antibacterial efficiency, demonstrated by clearing 99% of bacteria in macrophages. Furthermore, AExo exhibits high mucus penetrability and shows a significant therapeutic efficacy in two animal infection models. Collectively, this work expands the application of exosomes from bovine milk with simple operation and low cost, shedding light on the potential of milk exosomes in improving the solubility of drugs to enhance the efficacy of oral administration.

Fluorescence polarization immunoassay (FPIA) is a homogeneous and rapid analytical method that is suitable for high-throughput screening of large numbers of samples. However, FPIA typically suffers from lower sensitivity than the well-established enzyme-linked immunosorbent assay (ELISA), limiting its wide application as an analytical tool that can be run with trace levels of an analyte. Herein, a highly sensitive FPIA for detecting amantadine (AMD) in chicken is described. To achieve high sensitivity, nine chemical tracers of AMD that employ different fluoresceins, fluorescein derivatives, and haptens were synthesized and paired with four previously produced monoclonal antibodies (mAbs). The effect of the tracer structure on the sensitivity of FPIA was investigated and discussed. We found that the tracers with a linear and shorter bridge between adamantane and fluorescein generally provided higher sensitivity. After optimization, N'-(1-adamantyl) ethylenediamine (AEDA), an AMD structural analogue labeled with fluorescein isothiocyanate (FITC), achieved the lowest IC50 value (1.0 ng/ml) in the FPIA, which was comparable to that of the heterologous ELISA format that used the same mAb7G2. We also investigated the possible recognition mechanism of mAbs in terms of conformational and electronic aspects. The developed FPIA was applied to chicken to detect AMD residue, demonstrating a limit of detection (LOD) of 0.9 µg/kg with recoveries of 76.5-89.3% and coefficients of variation (CVs) below 14.5%. These results show that the proposed FPIA is an efficient, accurate, and convenient method for the rapid screening of AMD residues in chicken. PRACTICAL APPLICATION: The fluorescence polarization immunoassay (FPIA) was developed to determine and quantify amantadine (AMD) in chicken samples with high sensitivity. This homogeneous method avoids coating and washing steps and may provide high-throughput AMD screening in chicken in 10 min with high accuracy and precision. FPIA can be used as a monitoring tool and contribute significantly to the rapid detection of AMD in chicken.

Background Mobile colistin-resistance gene mcr-1 is prevalent among various bacteria, hosts and countries, especially in the guts of humans and animals. However, the biological basis for mcr-1 colonization and transmission in the intestines is largely unknown. Methods We used mouse models to mimic exposure to mcr-1-positive Escherichia coli (MCRPEC, harboring an IncI2 plasmid positive for mcr-1) in human intestines without and with antibiotic pretreatment, respectively. We used cultivation and qPCR method to determine the presence and quantity of MCRPEC. We also used Fluorescence in situ hybridization (FISH) method to locate mcr-1-positive bacteria in situ and confocal laser scanning microscopy method to examine the interaction between MCRPEC and Caco-2 cell in vitro. Finally, we used mouse model to investigate the transmission of MCRPEC among individuals. Results We found two approaches of MCRPEC colonization in the mouse intestines after exposure. In mice with intestinal microbiota homeostasis, MCRPEC was transient in the large intestines, while mcr-1 was detected at lower abundances (10-4–10-5) for at least 21 days as relic DNA or mcr-1-positive uncommon bacteria (MCRPUB). In mice with intestinal microbiota dysbiosis, MCRPEC colonized the intestines directly with a high shedding load (~108 CFU/g feces) and high mcr-1 abundance (~10-2). The transmission model confirmed that both MCRPEC and MCRPUB could cryptically transfer among individuals and persist for long periods. Conclusions These results demonstrate two approaches of MCRPEC colonization and a long persistence of MCRPUB or an antetype in mouse intestines as well as their transmission among mice, which partially explains the widespread prevalence of mcr-1-positive IncI2 plasmid among hosts and its long persistence in the gut even without antibiotic pressure. Contaminated food enables exposure to pathogens that can colonize human intestines; thus, reducing MCRPEC/MCRPUB in livestock and animal-derived food and preventing MCRPEC/MCRPUB transmission in ecosystems under the "One Health" perspective are crucial.

Phallotoxins, toxic cyclopeptides found in wild poisonous mushrooms, are predominant causes of fatal food poisoning. For the early and rapid diagnosis mushroom toxin poisoning, a highly sensitive and robust monoclonal antibody (mAb) against phallotoxins was produced for the first time. The half-maximum inhibition concentration (IC50) values of the mAb-based indirect competitive ELISAs for phallacidin (PCD) and phalloidin (PHD) detection were 0.31 ng mL−1 and 0.35 ng mL−1, respectively. In response to the demand for rapid screening of the type of poisoning and accurate determination of the severity of poisoning, colloidal gold nanoparticle (GNP) and time-resolved fluorescent nanosphere (TRFN) based lateral flow assays (LFA) were developed. The GNP-LFA has a visual cut-off value of 3.0 ng mL−1 for phallotoxins in human urine sample. The TRFN-LFA provides a quantitative readout signal with detection limit of 0.1 ng mL−1 in human urine sample. In this study, urine samples without pretreatment were used directly for the LFA strip tests, and both two LFAs were able to accomplish analysis within 10 min. The results demonstrated that LFAs based on the newly produced, highly sensitive, and robust mAb were able to be used for both rapid qualitative screening of the type of poisoning and accurate quantitative determination of the severity of poisoning after accidental ingestion by patients of toxic mushrooms.

Plasmid-mediated colistin-resistance genes have been reported in human origin clinical samples worldwide which raises its threats to human infections. Notably, mcr-1, mcr-3, mcr-8, and mcr-10 have been reported isolated directly from clinical samples which creates more seriously threaten to human health than other mcr gene types. A multiplex polymerase chain reaction (Multi-PCR) protocol was developed to detect and genotype mobile colistin-resistance genes (mcr-1, mcr-3, mcr-8, mcr-10) in Enterobacteria for clinical laboratory purposes. We first designed four pairs of new primers for the amplification of mcr-1, mcr-3, mcr-8, and mcr-10 gene respectively to achieve stepwise separation of amplicons between 216 and 241 bp, and complete this Multi-PCR system with the assistance of another pair of universal primer. Among which the forward primers for mcr-8 and mcr-10 amplicons were identical. The protocol was validated by testing 11 clinical isolates of Escherichia coli and 3 clinical isolates of Klebsiella from human origin, each well characterized and prospectively validated. The Multi-PCR assay showed full concordance with whole-genome sequence data and displayed higher sensitivity and 100% specificity. The assay could detect all variants of the various mcr alleles described. The Multi-PCR assay successfully genotyped of mcr alleles described in one test.

The bla NDM gene is playing an increasingly important role in infections caused by CR-KP, especially in children. However, systematic detection and bioinformatics analysis of NDM-KP in children's hospitals are lacking in China.

Mammalian cells act as reservoirs of internalized bacteria to circumvent extracellular antibacterial compounds, resulting in relapse and reinfection diseases. The intracellular persistence of

As a rapidly developing cell engineering technique, cell electrofusion has been increasingly applied in the field of hybridoma preparation in recent years. However, electrofusion is a certain degree of difficulty to completely replace the polyethylene glycol-mediated cell fusion. The key elements limiting electrofusion in the field of hybridoma preparation are practical complicated. This review summarizes the state of art of cell electrofusion in hybridoma preparation based on recent published literatures, mainly focusing on electrofusion instruments and their components, process control, cell treatment, and process characterization. The review provides new information and insightful commentary, critically important to the promotion of further electrofusion development in the field of hybridoma preparation.

Antibiotic resistance nowadays is spreading much faster than the introduction of new antibiotics into clinical practice. There is an urgent need for potential compounds to combat multidrug-resistant (MDR) bacteria. Marine fungi provide a promising source for chemical diversity with antibiotic-like molecules. To identify structurally distinct compounds that effectively eradicate MDR pathogens and to control the development of antibiotic resistance, we have reinvestigated equisetin, a previously reported meroterpenoid isolated from a marine sponge-derived fungus. Equisetin exerted efficient antibacterial activities against either MRSA or VRE without detectable high-level resistance. Meanwhile, equisetin, as an antibiotic adjuvant, restores colistin susceptibility to colistin-resistant bacteria toward diverse Gram-negative pathogens. Intriguingly, the low-level equisetin-resistant Staphylococcus aureus displayed collateral sensitivity to multiple classes of existing antibiotics with decreased capacity to produce biofilm. Lastly, equisetin showed efficacy with MRSA in three infected animal models. This work suggests that equisetin derived from marine natural products is a promising lead to overcome antibiotic resistance, providing new insight in future antibiotic discovery and development.

In response to the spread of colistin resistance gene mcr-1, China banned the use of colistin in livestock fodders. We used a time-series analysis of inpatient colonization data from 2011-2019 to accurately reveal the associated fluctuations of mcr-1 that occurred in inpatients in response to the ban.

Mobile colistin resistance gene mcr-1 and extended-spectrum β-lactamase gene blaCTX-M are highly prevalent in human – and pet-derived bacteria. Isolation of identical strains of mcr-1-positive Escherichia coli (MCRPEC) or blaCTX-M-positive E. coli (CTX-MPEC) from pets and humans highlighted the potential for co-colonization of antibiotic-resistant bacteria which can be a risk for dissemination of resistance genes. In this study, the prevalence of mcr-1 and blaCTX-M carriage from rectal swabs in 299 families (dogs and their owners) were 2.7 and 5.3%, respectively. We identified a significant association of mcr-1 carriage between dogs and their owners. Whilst antibiotic use in the previous three months was associated with blaCTX-M carriage in dogs. Only one instance of dog and owner carrying identical CTX-MPEC was observed. Although the prevalence of identical strains in one family is rare, the huge number of dog ownership worldwide suggest that this threat should not be underestimated.

What is already known about this topic? Salmonella causes acute and chronic diseases in food animals, and infected food animals are one of the most important source of human infection. What does this report contribute? The prevalence of Salmonella was 10.5% in chicken samples, 24.4% in pig, 23.3% in duck, and 29.4% in milk. Salmonella isolates were highly resistant to ampicillin (59.60%). What are the implications for public health practices? Data on Salmonella infections among food animals in China could help identify sources and factors related to the spread of Salmonella in food animals and food production chains.

T-2 toxin is one of the most toxic and common trichothecene mycotoxins, and can cause various cardiovascular diseases. In this review, we summarized the current knowledge-base and challenges as it relates to T-2 toxin related cardiotoxicity. The molecular mechanisms and potential treatment approaches were also discussed. Pathologically, T-2 toxin-induced cardiac toxicity is characterized by cell injury and death in cardiomyocyte, increased capillary permeability, necrosis of cardiomyocyte, hemorrhage, and the infiltration of inflammatory cells in the heart. T-2 toxin exposure can cause cardiac fibrosis and finally lead to cardiac dysfunction. Mechanistically, T-2 toxin exposure-induced cardiac damage involves the production of ROS, mitochondrial dysfunction, peroxisome proliferator-activated receptor-gamma (PPAR-γ) signaling pathway, endoplasmic reticulum (ER stress), transforming growth factor beta 1 (TGF-β1)/smad family member 2/3 (Smad2/3) signaling pathway, and autophagy and inflammatory responses. Antioxidant supplementation (e.g., catalase, vitamin C, and selenium), induction of autophagy (e.g., rapamycin), blockade of inflammatory signaling (e.g., methylprednisolone) or treatment with PPAR-γ agonists (e.g., pioglitazone) may provide protective effects against these detrimental cardiac effects caused by T-2 toxin. We believe that our review provides new insights in understanding T-2 toxin exposure-induced cardiotoxicity and fuels effective prevention and treatment strategies against this important food-borne toxin-induced health problems.

Rare antibody that is able to tolerate physio-chemical factors is preferred and highly demanded in diagnosis and therapy. Rabbit monoclonal antibodies (RmAbs) are distinguished owing to their high affinity and stability. However, the efficiency and availability of traditional methods for RmAb discovery are limited, especially for small molecules. Here, we present an indirect competitive screening method in nanowells, named CSMN, for single rabbit antibody secreting cells (ASCs) selection with 20.6 h and proposed an efficient platform for RmAb production against small molecule with 5.8 days for the first time. Chloramphenicol (CAP) as an antibacterial agent has the great threats for public health. We applied the CSMN to select CAP-specific ASCs and produced one high affinity RmAb, surprisingly showing extremely halophilic properties with an IC50of 0.08 ng mL-1in saturated salt solution which has as yet not been shown by other antibodies. Molecular dynamic simulation showed that the negatively charged surface improved the stability of the RmAb structure with additional disulfide bonds compared with mouse antibody. Moreover, the reduced solvent accessible surface area of the binding pocket increased the interactions of RmAb with CAP in a saturated salt solution. Furthermore, the RmAb was used to develop an immunoassay for the detection of CAP in real biological samples with simple pretreatment, shorter assay time, and higher sensitivity. The results demonstrated that the practical and efficient CSMN is suitable for rare RmAb discovery against small molecules.

The pursuit of the limit between dimensionalities is a scientific goal with high applicability. Sandwich immunoassay, usually based on two antibodies binding two epitopes, is one of the most popular mainstay tools in both academic and industrial fields. Herein, we determined and evaluated the minimum distance of two epitopes in sandwich immunoassays for small molecules. Briefly, nine model analytes comprising two hapten epitopes, i.e., melamine (MEL) and p-nitroaniline (NIA), were designed by increasing the linear chain linkers brick by brick. Two groups of monoclonal antibodies (mAbs) were produced with different recognition properties toward MEL and NIA using 12 new haptens with different spacer arms. The results indicated that two epitopes of the analyte with a distance of only 2.4 Å could be simultaneously bound by two mAbs, which is the known limit of epitope distance in sandwich immunoassays thus far. We further found that an epitope distance of below 8.8 Å for the analyte generally induces noticeable steric hindrance of antibodies, preventing a sandwich immunoassay with high probability. These observations were investigated and evaluated by molecular docking, molecular dynamics, and surface plasmon resonance and using model and real analytes. Altogether, we determined the minimum distance of two epitopes and explored the molecular mechanism of the antibody–analyte–antibody ternary complex in sandwich immunoassays, providing a theoretical basis for hapten design, antibody discovery and development, and sandwich immunoassay establishment for small molecules.

Hybridoma technology is widely used for monoclonal antibody (mAb) discovery, whereas the generation and identification of single hybridomas by the limiting dilution method (LDM) are tedious, inefficient, and time- and cost-consuming, especially for hapten molecules. Here, we describe a single transgenic hybridoma selection method (STHSM) that employs a transgenic Sp2/0 with an artificial and stable on-cell-surface anchor. The anchor was designed by combining the truncated variant transmembrane domain of EGFR with a biotin acceptor peptide AVI-tag, which was stably integrated into the genome of Sp2/0 via a piggyBac transposon. To ensure the subsequent precise selection of the hybridoma, the number of on-cell-surface anchors of the transfected Sp2/0 for fusion with immunized splenocytes was further normalized by flow cytometry at the single cell level. Then the single antigen-specific transgenic hybridomas were precisely identified and automatically selected using a CellenONE platform based on the fluorescence assay of the on-cell-surface anchor with the corresponding secreted antigen-specific mAb. The STHSM produced 579 single chloramphenicol (CAP)-specific transgenic hybridomas with a positive rate of 62.7% in 10 plates within 2 h by one-step selection, while only 12 single CAP-specific hybridomas with a positive rate of 6.3% in 40 plates required at least 32 days using the LDM with multiple subcloning steps. The best affinity of mAbs from the STHSM was more than 2-fold higher than that of those from the LDM, and this was mainly due to the preaffinity selection based on the on-cell-surface anchors and more interactions between the mAb and CAP. Then the mAbs from the STHSM and LDM were used to develop an immunoassay for CAP in spiked and natural biological samples. The method displayed satisfactory sensitivity, accuracy, and precision, demonstrating that the STHSM we developed is a versatile, practical, and efficient method for mAb discovery.

Public health interventions to control the recent emergence of plasmid-mediated tigecycline resistance genes rely on a comprehensive understanding of its epidemiology and distribution over a wide range of geographical scales. Here we analysed an Escherichia coli collection isolated from pigs and chickens in China in 2018, and ascertained that the tet(X4) gene was not present at high prevalence across China, but was highly endemic in northwestern China. Genomic analysis of tet(X4)-positive E. coli demonstrated a recent and regional dissemination of tet(X4) among various clonal backgrounds and plasmids in northwestern China, whereas a parallel epidemic coincided with the independent acquisition of tet(X4) in E. coli from the remaining provinces. The high genetic similarity of tet(X4)-positive E. coli and human commensal E. coli suggests the possibility of its spreading into humans. Our study provides a systematic analysis of the current epidemiology of tet(X4) and identifies priorities for optimising timely intervention strategies., Sun et al. analyse E. coli strains from pigs and chickens in Chinese farms and slaughterhouses, and reveal that while the tet(X4) gene is not ubiquitous across China, it is highly endemic in northwestern China. Genomic analysis of these strains shows recent and independent acquisitions of plasmids in across regions.

The rapid dissemination of life-threatening multidrug-resistant bacterial pathogens calls for the development of new antibacterial agents and alternative strategies. The virulence factor secreted by bacteria plays a crucial role in the sophisticated processes during infections. Inspired by the unique capacity of many bacteria inducing clotting of plasma to initiate colonization, we propose a programmable antibiotic delivery system for precision therapy using methicillin-resistant S. aureus (MRSA) as a model. Coagulase utilized by MRSA to directly cleave fibrinogen into fibrin, is an ideal target not only for tracking bacterial status but for triggering the collapse of fibrinogen functionalized porous microspheres. Subsequently, staphylokinase, another virulence factor of MRSA, catalyzed hydrolysis of fibrin to further release the encapsulated antibiotics from microspheres. Our sequential triggered-release system exhibits high selectivity to distinguish live or dead MRSA from other pathogenic bacteria. Furthermore, such programmable microspheres clear 99% MRSA in 4 h, and show increased efficiency in a wound healing model in rats. Our study provides a programmable drug delivery system to precisely target bacterial pathogens using their intrinsic enzymatic cascades. This programmable platform with reduced selective stress of antibiotics on microbiota sheds light on the potential therapy for future clinical applications.

Colistin resistance has attracted substantial attention after colistin was considered as a last-resort drug for the treatment of infections caused by carbapenem-resistant and/or multidrug-resistant (MDR) Gram-negative bacteria in clinical settings. However, with the discovery of highly mobile colistin resistance (mcr) genes, colistin resistance has become an increasingly urgent issue worldwide. Despite many reviews, which summarized the prevalence, mechanisms, and structures of these genes in bacteria of human and animal origin, studies on the prevalence of mobile colistin resistance genes in aquaculture and their transmission between animals and humans remain scarce. Herein, we review recent reports on the prevalence of colistin resistance genes in animals, especially wildlife and aquaculture, and their possibility of transmission to humans via the food chain. This review also gives some insights into the routine surveillance, changing policy and replacement of polymyxins by polymyxin derivatives, molecular inhibitors, and traditional Chinese medicine to tackle colistin resistance.

Objectives To report a novel tigecycline resistance gene, tet(X6), and its variants in four bacterial species isolated from chickens and pigs in China. Methods WGS was conducted to identify the suspected resistance genes in the tigecycline-resistant Myroides phaeus 18QD1AZ29W. Functional cloning, homology modelling and molecular docking were performed to compare the function with other Tet(X) variants. Retrospective screening for tet(X6) was conducted for 80 isolates in our WGS data collection, and all genomic environments of tet(X6)-positive isolates were analysed. Results The tigecycline-resistant M. phaeus 18QD1AZ29W isolated from a pig farm in Shandong in 2018 was positive for tet(X2) and a novel tet(X) gene, designated tet(X6). Tet(X6) could increase the MICs of all tested tetracyclines/glycylcyclines for Escherichia coli only 2- to 4-fold, which was possibly due to a lower tetracycline binding capacity of Tet(X6) compared with that of other Tet(X) variants. Retrospective screening showed that seven other isolates (7/80, 8.8%), comprising four Proteus spp. and three Acinetobacter spp. from chickens and pigs in Shandong and Guangdong, were positive for three different variants of tet(X6). The analysis of the genomic environment revealed that two tet(X6)-positive isolates from M. phaeus and Proteus cibarius, respectively, contained ISCR2, which may play a role in tet(X6) transmission. Conclusions This study identified a novel type of tigecycline resistance gene, tet(X6), in Myroides, Acinetobacter and Proteus from chickens and swine. Tet(X6) conferred lower tetracycline/glycylcycline MICs than other Tet(X) variants, and ISCR2 may play a role in the transmission of tet(X6).

Campylobacter spp. are the leading cause of human gastroenteritis worldwide. RE-CmeABC is a newly identified resistance-enhancing multidrug efflux pump of Campylobacter spp. (C. spp.) that confers high-level resistance to fluoroquinolones, phenicols, macrolides, and tetracyclines (TETs), all of which are critical drugs in both human and veterinary medicine. In this study, we analyzed the presence and antimicrobial susceptibility of RE-cmeABC-positive Campylobacter isolates of food-animal origin from three representative regions (Shandong, Shanghai, and Guangdong) in China over three successive years, from 2014 to 2016. A total of 1088 Campylobacter isolates (931 C. coli and 157 C. jejuni) were recovered from the RE-cmeABC screening. We detected 122 (11.2%) RE-cmeABC-positive isolates of chicken origin, including 111 (70.7%) C. jejuni and 11 (1.2%) C. coli. This multidrug efflux pump is more prevalent among C. jejuni than C. coli. The level of resistance was significantly different in 111 RE-cmeABC-positive C. jejuni versus 46 RE-cmeABC-negative C. jejuni for florfenicol, clindamycin, and erythromycin (P

As antibodies are the main biological binder for hazards in food samples, their performance directly determines the sensitivity, specificity, and reproducibility of the developed immunoassay. The overwhelmingly used mammalian-derived antibodies usually suffer from complicated preparation, high cost, frequent bleeding of animals, and sometimes low titer and affinity. Chicken yolk antibody (IgY) has recently attracted considerable attention in the bioanalytical field owing to its advantages in productivity, animal welfare, comparable affinity, and high specificity. However, a broad understanding of the application of IgY-based immunoassay for the detection of chemical and biological hazards in food samples remains limited. Here, we briefly summarized the diversity, structure, and production of IgY including polyclonal and monoclonal formats. Then, a comprehensive overview of the principles, designs, and applications of IgY-based immunoassays for these hazards was reviewed and discussed, including food-borne pathogens, food allergens, veterinary drugs, pesticides, toxins, endocrine disrupting chemicals, etc. Thus, the trend of IgY-based immunoassays is expected, and more IgY types, higher sensitivity, and diversification of recognition-to-signal manners are necessary in the future.