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Abstract Objectives The clinical decision-making regarding choosing surgery alone (SA) or surgery followed by postoperative adjuvant chemotherapy (SPOCT) in esophageal squamous cell carcinoma (ESCC) remains controversial. We aim to propose a pre-therapy PET/CT image-based deep learning approach to improve the survival benefit and clinical management of ESCC patients. Methods This retrospective multicenter study included 837 ESCC patients from three institutions. Prognostic biomarkers integrating six networks were developed to build an ESCC prognosis (ESCCPro) model and predict the survival probability of ESCC patients treated with SA and SPOCT. Patients who did not undergo surgical resection were in a control group. Overall survival (OS) was the primary end-point event. The expected improvement in survival prognosis with the application of ESCCPro to assign treatment protocols was estimated by comparing the survival of patients in each subgroup. Seven clinicians with varying experience evaluated how ESCCPro performed in assisting clinical decision-making. Results In this retrospective multicenter study, patients receiving SA had a median OS 9.2 months longer than controls. No significant differences in survival were found between SA patients with predicted poor outcomes and the controls (p > 0.05). It was estimated that if ESCCPro was used to determine SA and SPOCT eligibility, the median OS in the ESCCPro-recommended SA group and SPOCT group would have been 15.3 months and 24.9 months longer, respectively. In addition, ESCCPro also significantly improved prognosis accuracy, certainty, and the efficiency of clinical experts. Conclusion ESCCPro assistance improved the survival benefit of ESCC patients and the clinical decision-making among the two treatment approaches. Critical relevance statement The ESCCPro model for treatment decision-making is promising to improve overall survival in ESCC patients undergoing surgical resection and patients undergoing surgery followed by postoperative adjuvant chemotherapy. Key Points ESCC is associated with a poor prognosis and unclear ideal treatments. ESCCPro predicts the survival of patients with ESCC and the expected benefit from SA. ESCCPro improves clinicians’ stratification of patients’ prognoses. Graphical Abstract

ABSTRACT: IncX4 plasmids are one of the most epidemiologically successful vehicles for mcr-1 spread. Here we found that the IncX4 plasmids carried two different replication proteins encoded by genes pir-1 and pir-2, respectively, but mcr-1 was only carried by IncX4 plasmid encoding pir-1. The copy number of pir-2 encoding plasmids (3.15 ± 0.9 copies) are higher than that of pir-1 encoding plasmids (0.85 ± 0.5 copies). When mcr-1 was cloned into IncX4 plasmid encoding pir-2, the higher copy number of these plasmids resulted in increased expression of mcr-1 and a greater fitness burden on their host cells. However, these plasmids exhibited a lower rate of invasion into the bacterial population compared with mcr-1-positive plasmids encoding the pir-1 gene. These findings collectively explain the absence of mcr-1 in all IncX4 plasmids encoding pir-2. Our results further confirmed that low-copy numbers are important for the spread of mcr-1 plasmid from the perspective of natural evolution.

ABSTRACT The appearance and prevalence of multidrug-resistance (MDR) Gram-negative bacteria (GNB) have limited our antibiotic capacity to control bacterial infections. The clinical efficacy of colistin (COL), considered as the “last resort” for treating GNB infections, has been severely hindered by its increased use as well as the emergence and prevalence of mobile colistin resistance (MCR)-mediated acquired drug resistance. Identifying promising compounds to restore antibiotic activity is becoming an effective strategy to alleviate the crisis of increasing MDR. We first demonstrated that the combination of berberine (BBR) and EDTA substantially restored COL sensitivity against COL-resistant Salmonella and Escherichia coli. Molecular docking indicated that BBR can interact with MCR-1 and the efflux pump system AcrAB-TolC, and BBR combined with EDTA downregulated the expression level of mcr-1 and tolC. Mechanically, BBR combined with EDTA could increase bacterial membrane damage, inhibit the function of multidrug efflux pump, and promote oxidative damage, thereby boosting the action of COL. In addition, transcriptome analysis found that the combination of BBR and EDTA can accelerate the tricarboxylic acid cycle, inhibit cationic antimicrobial peptide (CAMP) resistance, and attenuate Salmonella virulence. Notably, the combination of BBR and EDTA with COL significantly reduced the bacterial load in the liver and spleen of a mice model infected with Salmonella. Our findings revealed that BBR and EDTA can be used as adjuvants collectively with COL to synergistically reverse the COL resistance of bacteria.IMPORTANCEColistin is last-resort antibiotic used to treat serious clinical infections caused by MDR bacterial pathogens. The recent emergence of transferable plasmid-mediated COL resistance gene mcr-1 has raised the specter of a rapid worldwide spread of COL resistance. Coupled with the fact of barren antibiotic development pipeline nowadays, a critical approach is to revitalize existing antibiotics using antibiotic adjuvants. Our research showed that berberine combined with EDTA effectively reversed COL resistance both in vivo and in vitro through multiple modes of action. The discovery of berberine in combination with EDTA as a new and safe COL adjuvant provides a therapeutic regimen for combating Gram-negative bacteria infections. Our findings provide a potential therapeutic option using existing antibiotics in combination with antibiotic adjuvants and address the prevalent infections caused by MDR Gram-negative pathogens worldwide.

ABSTRACT Polymyxin is used as a last resort antibiotics for infections caused by multi-drug resistant (MDR) Gram-negative bacteria and is often combined with other antibiotics to improve clinical effectiveness. However, the synergism of colistin and other antibiotics remains obscure. Here, we revealed a notable synergy between colistin and flavomycin, which was traditionally used as an animal growth promoter and has limited activity against Gram-negative bacteria, using checkerboard assay and time–kill curve analyses. The importance of membrane penetration induced by colistin was assessed by examining the intracellular accumulation of flavomycin and its antimicrobial impact on Escherichia coli (E. coli) strains with truncated lipopolysaccharides. Besides, a mutation in the flavomycin binding site was created to confirm its role in the observed synergy. This synergy is manifested as an augmented penetration of the E. coli outer membrane by colistin, leading to increased intracellular accumulation of flavomycin and enhanced cell killing thereafter. The observed synergy was dependent on the antimicrobial activity of flavomycin, as mutation of its binding site abolished the synergy. In vivo studies confirmed the efficacy of colistin combined with flavomycin against MDR E. coli infections. This study is the first to demonstrate the synergistic effect between colistin and flavomycin, shedding light on their respective roles in this synergism. Therefore, we propose flavomycin as an adjuvant to enhance the potency of colistin against MDR Gram-negative bacteria.IMPORTANCEColistin is a critical antibiotic in combating multi-drug resistant Gram-negative bacteria, but the emergence of mobilized colistin resistance (mcr) undermines its effectiveness. Previous studies have found that colistin can synergy with various drugs; however, its exact mechanisms with hydrophobic drugs are still unrevealed. Generally, the membrane destruction of colistin is thought to be the essential trigger for its interactions with its partner drugs. Here, we use clustered regularly interspaced palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) for specifically mutating the binding site of one hydrophobic drug (flavomycin) and show that antimicrobial activity of flavomycin is critical for the synergy. Our results first give the evidence that the synergy is set off by colistin's membrane destruction and operated the final antimicrobial function by its partner drugs.

ABSTRACTAntibiotic resistance could rapidly emerge from acquiring the mobile antibiotic resistance genes, which are commonly evolved from an intrinsic gene. The emergence of the plasmid-borne mobilized efflux pump gene cluster tmexCD1-toprJ1 renders the last-resort antibiotic tigecycline ineffective, although its evolutionary mechanism remains unclear. In this study, we investigate the regulatory mechanisms of the progenitor NfxB-MexCD-OprJ, a chromosomally encoded operon that does not mediate antibiotic resistance in the wild-type version, and its homologs, TNfxB1-TMexCD1-TOprJ1 mediating high-level tigecycline resistance, and TNfxB3-TMexCD3-TOprJ1. Mechanistic studies demonstrated that in nfxB-mexCD-oprJ, MexCD expression was under a weaker promoter, PmexC and inhibited by a strong repressor NfxB. For tmexCD1-toprJ1, TMexCD1 was highly expressed owing to the presence of a strong promoter, PtmexC1, and an inactive suppressor, TNfxB1, with a T39R mutation that rendered it unable to bind to promoter DNA. In tnfxB3-tmexCD3-toprJ1b, TMexCD3 expression was intermediate because of the local regulator TNfxB3, which binds to two inverted repeat sequences of PtmexC. Additionally, TNfxB3 exhibited lower protein expression and weaker DNA binding affinity than its ancestor NfxB, together with their promoter activities difference explaining the different expression levels of tmexCD-toprJ homologs. Distinct fitness burdens on these homologs-carrying bacteria were observed due to the corresponding expression level, which might be associated with their global prevalence. In summary, our data depict the mechanisms underlying the evolution and dissemination of an important mobile antibiotic resistance gene from an intrinsic chromosomal gene.IMPORTANCEAs antibiotic resistance seriously challenges global health, tigecycline is one of the few effective drugs in the pipeline against infections caused by multidrug-resistant pathogens. Our previous work identified a novel tigecycline resistance efflux pump gene cluster tmexCD1-toprJ1 in animals and humans, together with its various variants, a rising clinical concern. Herein, this study focused on how the local regulation modes of tmexCD1-toprJ1 evolved to a highly expressed efflux pump. Through comparative analysis between three tnfxB-tmexCD-toprJ homologs and their progenitor nfxB-mexCD-oprJ, modes, we demonstrated the evolutionary dynamics from a chromosomal silent gene to an active state. We found the de-repression of the local regulator and an increase of the promoter activity work together to promote a high production of drug efflux machines and enhance multidrug resistance. Our findings revealed that TMexCD1-TOprJ1 adopts a distinct evolutionary path to achieve higher multidrug resistance, urgently needing tight surveillance.

Abstract Background The use of neoadjuvant immunotherapy plus chemotherapy has revolutionized the management of esophageal squamous cell carcinoma (ESCC) patients. Nevertheless, patients who would maximally benefit from these therapies have not been identified. Methods We collected postoperative specimens from 103 ESCC patients, of which 66 patients comprised a retrospective cohort and 37 comprised a prospective cohort. Patient specimens were subjected to applied multi‐omics analysis to uncover the mechanistic basis for patient responsiveness to cancer immunotherapy. The tumor microenvironment characteristics of these patient specimens was explored and identified by multiplex immunofluorescence and immunohistochemistry. Results Results demonstrated high COL19A1 expression to be a novel biomarker for successful immunotherapy (COL19A1high, odds ratio [95% confidence interval]: 0.31 [0.10–0.97], p = 0.044). Compared with COL19A1low patients, COL19A1high patients benefited more from neoadjuvant immunotherapy (p

The New Delhi Metallo-β-lactamase (NDM) producing Enterobacterales has been detected from diverse sources but has rarely been reported in retail eggs. In this study, 144 eggshell and 96 egg content samples were collected in 2022 from Guangdong province and were screened for NDM-producing strains. Four Escherichia coli strains (ST3014, ST10, ST1485, and ST14747) recovered from two (1.39%, 2 of 144) eggshells and two (2.08%, 2 of 96) egg content samples were identified as blaNDM−5-positive strains. Oxford Nanopore MinION sequencing and conjugation assays revealed that the blaNDM−5 gene was carried by IncX3 (n = 1), IncI1 (n = 1), and IncHI2 (n = 2). The IncI1-plasmid-carrying blaNDM−5 displayed high homology with one plasmid pEC6563-NDM5 from the human clinic, while the IncHI2 plasmid harboring blaNDM−5 shared highly similar structures with plasmids of animal origin. To the best of our knowledge, this is the first report on the identification of blaNDM−5-positive bacteria in retail eggs. NDM-producing E. coli could be transmitted to humans by the consumption of eggs or direct contact, which could pose a potential threat to human health.

ABSTRACT To comprehensively understand the formation and evolution of IncF33 plasmids, a global collection of whole-genome sequences of 863 strains positive for IncF33 plasmid replicons was analyzed. The results showed that the IncF33 plasmids were mainly identified in Enterobacterales, of which Escherichia coli (86.44%) was the dominant host, followed by Salmonella (8.57%) and Klebsiella pneumoniae (3.48%). Salmonella ST11 and K. pneumoniae ST11 were common and mostly from humans. IncF33 plasmids were found worldwide but prevalent in Chinese farm animals, predominantly carrying antibiotic resistance genes such as bla CTX-M-55, fosA3, bla TEM-1B, rmtB, and floR. Comparative genomics analysis of 103 complete IncF33 plasmid sequences showed highly similar backbones except for 16 lacking partial backbone fragments. Variable regions were diverse, containing various antibiotic resistance genes, insertion sequences, or other plasmid fragments. They can be roughly divided into two sublines based on the production of different CTX-M enzymes. Similar IncF33 plasmids from different countries were identified. Some early IncF33 plasmids lacked part of their leader regions, which showed over 99% homology with early F2:A-:B- plasmids, indicating that leader regions of IncF33 likely came from F2:A-:B- plasmids. In addition, IncF33 plasmids cointegrating with other types of plasmids to form new cointegrate plasmids are increasing, making them more efficient in their dissemination and persistence in Enterobacterales, which could pose a significant threat to global public health. IMPORTANCE Plasmids that capture multiple antibiotic resistance genes are spreading widely, leading to the emergence and prevalence of multidrug-resistant bacteria. IncF33 plasmids are a newly emerged plasmid type highly prevalent in animal-source Enterobacterales in China, and they are important vectors for transmitting several clinically important antibiotic resistance genes. The study revealed that the IncF33 plasmid is mainly prevalent in China animal-derived Escherichia coli and has the potential for cointegration and intercontinental dissemination. Therefore, it is crucial to enhance surveillance and control measures to limit the spread of IncF33 plasmids and their associated antibiotic resistance genes.

ABSTRACT Conjugative plasmids play a vital role in bacterial evolution and promote the spread of antibiotic resistance. They usually cause fitness costs that diminish the growth rates of the host bacteria. Compensatory mutations are known as an effective evolutionary solution to reduce the fitness cost and improve plasmid persistence. However, whether the plasmid transmission by conjugation is sufficient to improve plasmid persistence is debated since it is an inherently costly process. Here, we experimentally evolved an unstable and costly mcr-1 plasmid pHNSHP24 under laboratory conditions and assessed the effects of plasmid cost and transmission on the plasmid maintenance by the plasmid population dynamics model and a plasmid invasion experiment designed to measure the plasmid’s ability to invade a plasmid-free bacterial population. The persistence of pHNSHP24 improved after 36 days evolution due to the plasmid-borne mutation A51G in the 5′UTR of gene traJ. This mutation largely increased the infectious transmission of the evolved plasmid, presumably by impairing the inhibitory effect of FinP on the expression of traJ. We showed that increased conjugation rate of the evolved plasmid could compensate for the plasmid loss. Furthermore, we determined that the evolved high transmissibility had little effect on the mcr-1-deficient ancestral plasmid, implying that high conjugation transfer is vital for maintaining the mcr-1-bearing plasmid. Altogether, our findings emphasized that, besides compensatory evolution that reduces fitness costs, the evolution of infectious transmission can improve the persistence of antibiotic-resistant plasmids, indicating that inhibition of the conjugation process could be useful to combat the spread of antibiotic-resistant plasmids. IMPORTANCE Conjugative plasmids play a key role in the spread of antibiotic resistance, and they are well-adapted to the host bacteria. However, the evolutionary adaptation of plasmid-bacteria associations is not well understood. In this study, we experimentally evolved an unstable colistin resistance (mcr-1) plasmid under laboratory conditions and found that increased conjugation rate was crucial for the persistence of this plasmid. Interestingly, the evolved conjugation was caused by a single-base mutation, which could rescue the unstable plasmid from extinction in bacterial populations. Our findings imply that inhibition of the conjugation process could be necessary for combating the persistence of antibiotic-resistance plasmids.

Context Lung cancer, the most common type of cancer, has a high mortality rate. Cucurbitacin B (CuB), a natural compound extracted from Cucurbitaceae plants, has antitumor effects. Objective We investigated the role of CuB on lung cancer and its potential mechanisms. Materials and methods A549 cells were treated with 0.1, 0.3, 0.6, and 0.9 μM CuB for 12, 24, and 48 h or untreated. Gene and protein levels were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Enzyme-linked immunosorbent assay (ELISA) detected inflammatory factors levels (TNF-α and IL-10). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), flow cytometry, and colony formation assays measured cell viability, apoptosis, and proliferation. The interaction between miR-let-7c and long non-coding RNA X inactive-specific transcript (XIST) or interleukin-6 (IL-6) was verified by dual-luciferase reporter assays. Results CuB treatment inhibited the proliferation of lung cancer cells and promoted cell apoptosis, and increased the expression of Bax and cleaved caspase3, decreased cyclin B1 and Bcl-2 expression. CuB suppressed XIST and IL-6 expression, and enhanced miR-let-7c expression. XIST silencing enhanced the inhibitory effect of CuB on cell proliferation and the promotion effect on apoptosis via upregulating miR-let-7c. Moreover, XIST targeted miR-let-7c to activate the IL-6/STAT axis. MiR-let-7c overexpression enhanced the regulatory effect of CuB on proliferation and apoptosis via suppressing the IL-6/STAT3 pathway. Discussion and conclusion CuB regulated cell proliferation and apoptosis by inhibiting the XIST/miR-let-7c/IL-6/STAT3 axis in lung cancer. These findings indicate CuB may have the possibility of clinical application in lung cancer treatment.

ABSTRACT Several variants of the plasmid-carried tigecycline resistance gene cluster, tmexCD-toprJ, have been identified. This study characterized another novel variant, tmexC6D6-toprJ1b, located on the chromosome of environmental-origin Pseudomonas mendocina. TMexC6D6-TOprJ1 mediates resistance to multiple drugs, including tigecycline. The promoter activity of tmexC6D6-toprJ1b and negative transcriptional repression by the upstream regulator tnfxB6 are crucial for the expression of tmexC6D6-toprJ1b. tmexC6D6-toprJ1b was found in the plasmids or chromosomes of different Pseudomonas species from six countries. Two genetic backgrounds, class 1 integrons and int-carrying integrase units, were found adjacent to the tmexC6D6-toprJ1b gene cluster and might mediate the transfer of this novel efflux pump gene cluster in Pseudomonas. Further phylogenetic analysis revealed Pseudomonas as the major reservoir of tmexCD-toprJ variants, warranting closer monitoring in the future. IMPORTANCE Tigecycline is one of the treatment options for serious infections caused by multidrug-resistant bacteria, and tigecycline resistance has gained extensive attention. The emergence of a transferable tigecycline resistance efflux pump gene cluster, tmexCD-toprJ, severely challenged the efficiency of tigecycline. In this study, we identified another novel tmexCD-toprJ variant, tmexC6D6-toprJ1b, which could confer resistance to multiple classes of antibiotics, including tigecycline. Although tmexC6D6-toprJ1b was found only in Pseudomonas species, tmexC6D6-toprJ1b might spread to Enterobacteriaceae hosts via mobile genetic elements resembling those of other tmexCD-toprJ variants, compromising the therapeutic strategies. Meanwhile, novel transferable tmexCD-toprJ variants are constantly emerging and mostly exist in Pseudomonas spp., indicating Pseudomonas as the important hidden reservoir and origin of tmexCD-toprJ variants. Continuous monitoring and investigations of tmexCD-toprJ are urgent to control its spread.

ABSTRACT During a 2020 routine epidemiological investigation of carbapenem-resistant Enterobacterales at a local food market in Guangzhou, China, two Escherichia coli ST410 isolates coproducing NDM-5 and OXA-181 were obtained from environmental samples. Antimicrobial susceptibility testing, whole-genome sequencing, and conjugation assays were applied to identify their resistance phenotypes, phylogenetic relatedness, and genetic characteristics. Phylogenetic analysis showed that the two isolates were clonally related with only one core-genome single-nucleotide polymorphism (SNP) difference and clustered into a branch with 87 E. coli ST410 isolates deposited in GenBank. These 89 ST410 isolates were closely related (≤51 SNPs), and most were from humans in Southeast Asian countries (n = 47). A Vietnamese clinical isolate collected in 2017 showed the strongest epidemiological link (seven SNPs) to the two ST410 isolates detected in this study. Complete-genome analysis revealed that the carbapenem resistance determinants blaNDM-5 and blaOXA-181 were located on an IncF1:A1:B49-IncQ1 plasmid and IncX3 plasmid, respectively. Conjugation experiments confirmed that the IncX3 plasmid was self-transmissible while the IncF1:A1:B49-IncQ1 plasmid was nonconjugative. BLASTn analysis indicated that the two plasmids showed high similarity to other blaNDM-5-bearing IncF1:A1:B49-IncQ1 and blaOXA-181-bearing IncX3 plasmids from other countries. Altogether, the high similarity of the core genomes and plasmids between the ST410 isolates found in this study and those human source isolates from foreign countries suggested the clonal spread of E. coli ST410 strains and horizontal transmission of blaOXA-181-bearing IncX3 plasmids across Southeast Asian countries. Stringent sanitary management of food markets is important to prevent the dissemination of high-risk clones to the public. IMPORTANCE This is the first report of an Escherichia coli ST410 clone that coproduces NDM-5 and OXA-181 in China. The high similarity of the core genomes and plasmids between the ST410 isolates characterized in this study and human source isolates from foreign countries strongly suggests that this ST410 lineage is an international high-risk clone, highlighting the need for continuous global surveillance of ST410 clones.

The catalytic asymmetric benzylations of prochiral nucleophiles are very limited. Here, the authors disclose an asymmetric α−benzylation of N-unprotected amino acids with benzyl alcohol derivatives by a chiral aldehyde-involved catalytic system.

Background: This study aimed to develop an artificial intelligence-based computer-aided diagnosis system (AI-CAD) emulating the diagnostic logic of radiologists for lymph node metastasis (LNM) in esophageal squamous cell carcinoma (ESCC) patients, which contributed to clinical treatment decision-making. Methods: A total of 689 ESCC patients with PET/CT images were enrolled from three hospitals and divided into a training cohort and two external validation cohorts. 452 CT images from three publicly available datasets were also included for pretraining the model. Anatomic information from CT images was first obtained automatically using a U-Net-based multi-organ segmentation model, and metabolic information from PET images was subsequently extracted using a gradient-based approach. AI-CAD was developed in the training cohort and externally validated in two validation cohorts. Results: The AI-CAD achieved an accuracy of 0.744 for predicting pathological LNM in the external cohort and a good agreement with a human expert in two external validation cohorts (kappa = 0.674 and 0.587, p

ObjectiveThe burden of both general and drug-resistant tuberculosis in rural areas is higher than that in urban areas in China. To characterize the genetic structure and transmission risk of Mycobacterium tuberculosis in rural China, we used whole genome sequencing to analyze clinical strains collected from patients in two counties of Yichang for three consecutive years.MethodsFrom 2018 to 2020, sputum samples were collected for cultures from patients with suspected tuberculosis in Yidu and Zigui county, and DNA was extracted from the positive strains for genome sequencing. The online SAM-TB platform was used to identify the genotypes and drug resistance-related mutations of each strain, establish a phylogenetic tree, and calculated the genetic distances between pairwise strains. Twelve single nucleotide polymorphisms (SNPs) were used as thresholds to identify transmission clusters. The risk of related factors was estimated by univariable and multivariable logistic regression.ResultsA total of 161 out of the collected 231 positive strains were enrolled for analysis, excluding non-tuberculous mycobacterium and duplicate strains from the same patient. These strains belonged to Lineage 2 (92, 57.1%) and Lineage 4 (69, 42.9%), respectively. A total of 49 (30.4%) strains were detected with known drug resistance-related mutations, including 6 (3.7%) multidrug-resistant-TB (MDR-TB) strains and 11 (6.8%) RIF-resistant INH-susceptible TB (Rr-TB) strains. Six of the MDR/Rr-TB (35.3%) were also resistant to fluoroquinolones, which made them pre-extensively drug-resistant TB (pre-XDR-TB). There were another seven strains with mono-resistance to fluoroquinolones and one strain with resistance to both INH and fluoroquinolones, making the overall rate of fluoroquinolones resistance 8.7% (14/161). A total of 50 strains (31.1%) were identified as transmission clusters. Patients under 45 years old (adjusted odds ratio 3.46 [95% confidential intervals 1.28–9.35]), treatment-naive patients (6.14 [1.39–27.07]) and patients infected by lineage 4 strains (2.22 [1.00–4.91]) had a higher risk of transmission.ConclusionThe drug resistance of tuberculosis in rural China, especially to the second-line drug fluoroquinolones, is relatively serious. The standardized treatment for patients and the clinical use of fluoroquinolones warrant attention. At the same time, the recent transmission risk of tuberculosis is high, and rapid diagnosis and treatment management at the primary care needs to be strengthened.

Exosomes isolated from mesenchymal stem cells (MSC) had shown beneficial effect on acute lung injury (ALI). However, the effective components in MSC-derived exosomes need further investigation. ALI mice model was established by lipopolysaccharide (LPS) injection. In vitro inflammatory model was established by LPS stimulation of MLE-12 cells. The cell proliferation was evaluated by EdU assay. TUNEL and Annexin V/PI were applied to evaluate the apoptosis of tissue and cell respectively. HE staining was performed to evaluate the lung injury. Transmission electronic microscope was used to observe isolated exosomes. Level of cytokines, MDA, KGF were determined by ELISA kit. Direct interaction of miR-132-3p and TRAF6 were verified by dual luciferase assay. The level of mRNA or proteins were determined by qRT-PCR or western blots respectively. TRAF6 was upregulated while miR-132-3p was downregulated in LPS-stimulated ALI model. MiR-132-3p negatively regulated TRAF6 by direct binding. MiR-132-3p potentiated proliferation and suppressed apoptosis of LPS-induced MLE-12 cells at least partly dependent on targeting TRAF6. Treatment of exosome alleviated the LPS-induced ALI in mice and LPS-induced inflammatory response in MLE-12 cells. Moreover, overexpression of miR-132-3p promoted the protective effect of exosomes in LPS-induced MLE-12 cells injury and LPS-induced ALI. Mechanically, it was suggested that miR-132-3p inactivated PI3K/Akt signalling via targeting TRAF6. In the present study, our results indicated that miR-132-3p mediated protective effect of MSC-derived exosomes on LPS-induced ALI. Exosomal miR-132-3p ameliorated LPS-induced ALI via targeting TRAF6 and inactivating PI3K/Akt signalling.

ABSTRACT The appearance and prevalence of novel plasmid-encoded tigecycline resistance efflux pump gene clusters tmexC1D1-toprJ1 and tmexC2D2-toprJ2 in Enterobacteriaceae have raised a threat to public health. Here, another tigecycline resistance gene cluster, tmexC2D2.2-toprJ2, was identified in two Aeromonas isolates recovered from fish meat and vegetables. Cloning confirmed the expression of tmexC2D2.2-toprJ2 mediated the resistance to tigecycline and decreased susceptibility to tetracyclines and cephalosporins in both Escherichia coli and Aeromonas. In an Aeromonas veronii strain, four copies of tmexC2D2.2-toprJ2 were located on the chromosome. Further analysis revealed that tmexC2D2.2-toprJ2 has been detected in the chromosomes of A. veronii, Aeromonas hydrophila, and Aeromonas caviae with one to four copies due to the insertion of a potential integrative transferable unit. The occurrence of multiple copies of chromosomal tmexC2D2.2-toprJ2 may act as a sink for this tigecycline resistance gene cluster, which requires continuous monitoring. IMPORTANCE Tigecycline is regarded as one of the few effective drugs against multidrug-resistant bacterial infection. However, mobile tigecycline resistance efflux pump gene clusters such as tmexC1D1-toprJ1 and its variants have been identified in both animal- and human-origin Enterobacteriaceae. In this study, we first found another efflux pump gene cluster, tmexC2D2.2-toprJ2, in the Aeromonas chromosome. This gene cluster could mediate tigecycline resistance and decrease susceptibility to tetracyclines and cephalosporins in the Aeromonas host strain. Meanwhile, tmexC2D2.2-toprJ2 was detected with multiple copies in Aeromonas spp. This multidrug resistance efflux pump gene cluster with multiple copy numbers might stably exist in Aeromonas and serve as a reservoir for tmexCD2-toprJ2, facilitating its persistent presence and spread.

Abstract Bone marrow-derived mesenchymal stem cells (BM-MSCs), the common progenitor cells of adipocytes and osteoblasts, have been recognized as the key mediator during bone formation. Herein, our study aim to investigate molecular mechanisms underlying circular RNA (circRNA) AFF4 (circ_AFF4)-regulated BM-MSCs osteogenesis. BM-MSCs were characterized by FACS, ARS, and ALP staining. Expression patterns of circ_AFF4, miR-135a-5p, FNDC5/Irisin, SMAD1/5, and osteogenesis markers, including ALP, BMP4, RUNX2, Spp1, and Colla1 were detected by qRT-PCR, western blot, or immunofluorescence staining, respectively. Interactions between circ_AFF4 and miR-135a-5p, FNDC5, and miR-135a-5p were analyzed using web tools including TargetScan, miRanda, and miRDB, and further confirmed by luciferase reporter assay and RNA pull-down. Complex formation between Irisin and Integrin αV was verified by Co-immunoprecipitation. To further verify the functional role of circ_AFF4 in vivo during bone formation, we conducted animal experiments harboring circ_AFF4 knockdown, and born samples were evaluated by immunohistochemistry, hematoxylin and eosin, and Masson staining. Circ_AFF4 was upregulated upon osteogenic differentiation induction in BM-MSCs, and miR-135a-5p expression declined as differentiation proceeds. Circ_AFF4 knockdown significantly inhibited osteogenesis potential in BM-MSCs. Circ_AFF4 stimulated FNDC5/Irisin expression through complementary binding to its downstream target molecule miR-135a-5p. Irisin formed an intermolecular complex with Integrin αV and activated the SMAD1/5 pathway during osteogenic differentiation. Our work revealed that circ_AFF4, acting as a sponge of miR-135a-5p, triggers the promotion of FNDC5/Irisin via activating the SMAD1/5 pathway to induce osteogenic differentiation in BM-MSCs. These findings gained a deeper insight into the circRNA-miRNA regulatory system in the bone marrow microenvironment and may improve our understanding of bone formation-related diseases at physiological and pathological levels.

Hemifacial spasm (HFS) and temporomandibular joint (TMJ) pain are common facial diseases which cause depression, anxiety, insomnia, and poor quality of life. However, currently there are still no effective therapies to treat HFS and TMJ. Electroacupuncture (EA) has advantages of safety, rapid work, easy operation and convenience. Here, we reported a case of a 50-year-old woman who presented with irregular spasm of eyelids and facial muscles on the left side, and TMJ pain on the right side. The patient had been treated with carbamazepine (20mg per day) and alternative therapies for a year, but still not much improvement in the symptoms. The scores of the Jankovic Rating Scale (JRS), global rating scale (GRS), and visual analog scale (VAS) were 7, 60, and 7 points, respectively. The EMG test showed that the spastic side had higher R1 amplitude, longer R2 duration, and larger R2 area than the non-spasmodic side, and the occurrence rate of the lateral spread responses (LSR) in the Orbicularis oris and the Orbicularis oculi muscle was 60% and 40%, respectively. We considered this patient had left HFS and right TMJ pain. EA was successfully undertaken for two periods over 30 weeks. After EA, JRS and VAS were reduced sharply, and the symptoms of HFS were stable without recurrence. However, the frequency of the lower eyelid increased gradually during the 6-month follow-up. These findings reveal that EA with the frequency of 2 Hz and intensity of ~ 1–2 mA may be a benefit for alleviating symptoms of HFS and TMJ pain without adverse reaction. The potential mechanisms of EA in HFS and TMJ pain co-morbidity involve brain stem mechanism and DNIC mechanism for distal acupuncture and segmental mechanism for local acupuncture analgesia.

ABSTRACT Antimicrobial resistance in Morganella morganii is increasing in recent years, which is mainly introduced via extra genetic and mobile elements. The aim of our study is to analyze the multidrug resistance (MDR) and characterize the mobile genetic elements (MGEs) in M. morganii isolates. Here, we report the characteristic of a pathogenic M. morganii isolate containing multidrug resistance genes that are mainly carried by a novel transposon Tn7376 and a genomic island. Sequence analysis suggested that the Tn7376 could be generated through homologous recombination between two different IS26-bounded translocatable units (TUs), namely, module A (IS26-Hp-IS26-mph(A)-mrx(A)-mphR-IS6100-chrA-sul1-qacEΔ1) and module B (ISCR1-sul1-qacEΔ1-cmlA1-aadA1-aadB-intI1-IS26), and the genomic island named MMGI-4 might derive from a partial structure of different original genomic islands that also carried IS26-mediated TUs. Notably, a 2,518-bp sequence linked to the module A and B contains a 570-bp dfrA24 gene. To the best of our knowledge, this is the first report of the novel Tn7376 possessing a complex class 1 integron that carried an infrequent gene dfrA24 in M. morganii. IMPORTANCE Mobile genetic elements (MGEs), especially for IS26-bounded translocatable units, may act as a reservoir for a variety of antimicrobial resistance genes in clinically important pathogenic bacteria. We expounded this significant genetic characteristic by investigating a representative M. morganii isolate containing multidrug resistance genes, including the infrequent dfrA24. Our study suggested that these acquired resistance genes were mainly driven by IS26-flanked important MGEs, such as the novel Tn7376 and the MMGI-4. We demonstrated that IS26-related MGEs contributed to the emergence of the extra gene dfrA24 in M. morganii through some potential genetic events like recombination, transposition, and integration. Therefore, it is of importance to investigate persistently the prevalence these MEGs in the clinical pathogens to provide risk assessment of emergence and development of novel resistance genes.

Chiral aldehyde catalysis is a useful strategy in the catalytic asymmetric α-functionalization of amino methyl compounds but these reaction types are limited. Here, the authors report two chiral aldehyde-catalysed diastereodivergent reactions, namely, a 1,6-conjugate addition and a bio-inspired Mannich reaction.

ABSTRACT The emergence of the plasmid-borne colistin resistance gene mcr-1 threatens public health. IncX4-type plasmids are one of the most epidemiologically successful vehicles for spreading mcr-1 worldwide. Since MCR-1 is known for imposing a fitness cost to its host bacterium, the successful spread of mcr-1-bearing plasmids might be linked to high conjugation frequency, which would enhance the maintenance of the plasmid in the host without antibiotic selection. However, the mechanism of IncX4 plasmid conjugation remains unclear. In this study, we used high-density transposon mutagenesis to identify factors required for IncX4 plasmid transfer. Eighteen essential transfer genes were identified, including five with annotations unrelated to conjugation. Cappable-seq, transcriptome sequencing (RNA-seq), electrophoretic mobility shift assay, and β-galactosidase assay confirmed that a novel transcriptional regulator gene, pixR, directly regulates the transfer of IncX4 plasmids by binding the promoter of 13 essential transfer genes to increase their transcription. PixR is not active under nonmating conditions, while the expression of the pixR, pilX3-4, and pilX11 genes increased 3- to 6-fold upon contact with recipient Escherichia coli C600. Plasmid invasion and coculture competition assays revealed the essentiality of pixR for spreading and persistence of mcr-1-bearing IncX4 plasmids in bacterial populations. Effective conjugation is crucial for alleviating the fitness cost exerted by mcr-1 carriage. The existence of the IncX4-specific pixR gene increases plasmid transmissibility while promoting the invasion and persistence of mcr-1-bearing plasmids in bacterial populations, which helps explain their global prevalence. IMPORTANCE The spread of clinically relevant antibiotic resistance genes is often linked to the dissemination of epidemic plasmids. However, the underlying molecular mechanisms contributing to the successful spread of epidemic plasmids remain unclear. In this report, we shine a light on the transfer activation of IncX4 plasmids. We show how conjugation promotes the invasion and persistence of IncX4 plasmids within a bacterial population. The dissection of the regulatory network of conjugation helps explain the rapid spread of epidemic plasmids in nature. It also reveals potential targets for the development of conjugation inhibitors.

ABSTRACT Streptococcus suis strain 1112S was isolated from a diseased pig in a feedlot from Henan, China, in 2019. The isolate harbored a linezolid resistance gene optrA. WGS data revealed that the optrA gene was associated with a single copy ETAf ISS1S, in tandem with erm(B) and tet(O), located in a novel 72,587 bp integrative and conjugative element (ICE). Notably, this novel element, designated ICESsu1112S, also carried a novel bacitracin resistance locus. ICESsu1112S could be excised from chromosome and transferred to the recipient strain S. suis P1/7 with a frequency of 5.9 × 10−6 transconjugants per donor cell. This study provided the first description of the coexistence of optrA and a novel bacitracin locus on a multiple antibiotic resistant ICE and highlighted that ICE were major vehicle and contribute to the potential transfer of clinically relevant antibiotic resistance genes. IMPORTANCE Antimicrobial resistance (AMR) caused by the imprudent use of antimicrobials has become a global problem, which poses a serious threat to treatment of S. suis infection in pigs and humans. Importantly, AMR genes can horizontally spread among commensal organisms and pathogenic microbiota, thereby accelerating the dissemination of AMR determinants. These transfers are mainly mediated by mobile genetic elements, including ICEs. In S. suis, ICEs are the major vehicles that contribute to the natural transfers of AMR genes among different bacterial pathogens. However, ICEs that carry optrA and bacitracin resistance locus are rarely investigated in S. suis isolates. Here, we investigated a S. suis isolate carrying an optrA and a novel bacitracin resistance locus, which were co-located on a novel multiple antibiotic resistant ICESsu1112S. Our study suggests that more research is needed to access the real significance of ICEs that horizontally spread clinical important resistance genes.

A recently modern stochastic optimization algorithm has been developed by observing the life of slime mold physarum polycephalum in nature. The algorithm is called the slime mold algorithm (SMA) with an excellent exploratory capacity and exploitation inclination. Still, slipping into optimal local is easy to happen and slowly converges speed while dealing with complicated problems. This article proposes a new process of improving SMA (namely ISMA) by adapting the weight coefficient and cooperating the reverse learning strategy in the expression of agents updating locations to enhance the algorithm's optimization performance. Many selected benchmark functions and the optimal operation of cascade reservoirs are applied to evaluate the performance of the proposed algorithm. Comparisons of the proposed approach's results with the various algorithms under the case situations show that the recommended solution produces better performance than the different competing algorithms.

Background: Asthma, a common chronic inflammatory disease, is treated with corticosteroid in most cases, but corticosteroid resistance in severe asthma patients seriously impairs the therapeutic effects. LncRNA-CASC7 inhibits cell proliferation and enhances drug sensitivity, but the molecular mechanisms of corticosteroid resistance in severe asthma are still unknown. Methods: Airway smooth muscle cells (ASMCs) from healthy and severe asthmatic subjects were used in this study. The expression of CASC7 and miR-21 were modified by transfection with the pcDNA3.1-CASC7, miR-21 mimics and inhibitor. MTT assay was conducted to measure cell proliferation. ELISA assay was used to determine the secretion of CCL5, CCL11 and IL-6. The phosphorylation of glucocorticoid receptor (GR) and the PI3K/AKT signaling were assessed by western blotting assays. qRT-PCR was used to analyze the expression of CASC7, miR-21 and PTEN. Dual-luciferase reporter assay was used to assess the interaction among CASC7, miR-21 and PTEN. Results: Compared with AMSCs from severe asthma patients, dexamethasone inhibited cytokines (CCL5, CCL11 and IL-6) and promoted the phosphorylation of GR more significantly in normal AMSCs. CASC7 expression was suppressed while miR-21 expression and AKT activity were promoted in ASMCs from severe asthma patients. CASC7 promoted PTEN expression via directly inhibiting miR-21 expression. Overexpression of CASC7 suppressed the PI3K/AKT signaling pathway and promoted the inhibition effects of dexamethasone on cell proliferation and cytokines secretion via targeting miR-21. Conclusion: CASC7 increased corticosteroid sensitivity by inhibiting the PI3K/AKT signaling pathway via targeting miR-21, which provided a promising potential target for designing novel therapeutic strategy for severe asthma. Keywords: Asthma, lncRNA-CASC7, miR-21, PI3K/AKT signaling pathway, Corticosteroid insensitivity

Colistin is a last-line antibiotic against Gram-negative pathogens. However, the emergence of colistin resistance has substantially reduced the clinical effectiveness of colistin. In this study, synergy between colistin and capric acid was examined against twenty-one Gram-negative bacterial isolates (four colistin-susceptible and seventeen colistin-resistant). Checkerboard assays showed a synergistic effect against all colistin-resistant strains [(FICI, fractional inhibitory concentration index) = 0.02–0.38] and two colistin-susceptible strains. Time–kill assays confirmed the combination was synergistic. We suggest that the combination of colistin and capric acid is a promising therapeutic strategy against Gram-negative colistin-resistant strains.

To characterize the formation mechanism and characteristics of two cointegrate plasmids in Salmonella enterica serotype Enteritidis strain S13, plasmids from strain S13 and three corresponding transconjugants were subjected to whole genome sequencing and analyzed using bioinformatics tools. The traits of two fusion plasmids in transconjugants were characterized by stability and conjugation experiments. Sequence analysis indicated that strain S13 contained four plasmids, including mcr-1-bearing pS13-1, blaCTX–M–55-carrying pS13-2, tet(M)-bearing pS13-3, and floR-carrying pS13-4. IncN1-F33:A–:B– plasmid pS13-2, respectively, fused with IncFI:A–:B– plasmid pS13-3 and IncX1 plasmid pS13-4, which generated two cointegrate plasmids, designated pS13D and pS13F, which involved in two intermolecular replicative mechanisms mediated by IS26 and the novel transposon Tn6952 (ΔTnAS3-IS26-ΔISEcp1-ramA-ΔIS26-ΔTnAS1), respectively. This is the first report of the fusion of the IncN1-F33:A–:B– plasmid and IncFI:A–:B– plasmid mediated by IS26, and with IncX1 plasmid mediated by Tn6952. The formation and evolution of cointegrate plasmids could expand the resistance and host spectrum of fusion plasmids.

Plasmid-mediated colistin resistance gene mcr-1 generally confers low-level resistance. The purpose of this study was to investigate the impact of mcr-1 on the development of high-level colistin resistance (HLCR) in Klebsiella pneumoniae and Escherichia coli. In this study, mcr-1-negative K. pneumoniae and E. coli strains and their corresponding mcr-1-positive transformants were used to generate HLCR mutants via multiple passages in the presence of increasing concentrations of colistin. We found that for K. pneumoniae, HLCR mutants with minimum inhibitory concentrations (MICs) of colistin from 64 to 1,024 mg/L were generated. Colistin MICs increased 256- to 4,096-fold for mcr-1-negative K. pneumoniae strains but only 16- to 256-fold for the mcr-1-harboring transformants. For E. coli, colistin MICs increased 4- to 64-folds, but only 2- to 16-fold for their mcr-1-harboring transformants. Notably, mcr-1 improved the survival rates of both E. coli and K. pneumoniae strains when challenged with relatively high concentrations of colistin. In HLCR K. pneumoniae mutants, amino acid alterations predominately occurred in crrB, followed by phoQ, crrA, pmrB, mgrB, and phoP, while in E. coli mutants, genetic alterations were mostly occurred in pmrB and phoQ. Additionally, growth rate analyses showed that the coexistence of mcr-1 and chromosomal mutations imposed a fitness burden on HLCR mutants of K. pneumoniae. In conclusion, HLCR was more likely to occur in K. pneumoniae strains than E. coli strains when exposed to colistin. The mcr-1 gene could improve the survival rates of strains of both bacterial species but could not facilitate the evolution of high-level colistin resistance.

Abstract The mechanical and wear properties of CFRP laminate were investigated using a method of cycling low velocity impact, to study the trend and mechanism of impact resistance of the CFRP laminate under repeated impact during its service process. The interface responses of CFRP laminate under different impact kinetic energy during the cycling impact process were studied were studied experimentally, such as impact contact duration, deformation and energy absorption. The worn surface morphologies were observed through optical microscopy and a 3-D surface profiler and the cross-sectional morphologies were observed through SEM to investigate the mechanism of impact material damage. Based on a single-degree-of-freedom damping vibration model, the normal contact stiffness and contact damping of the material in different wear stages were calculated. It shows the failure process of CFRP laminate damaged by accumulated absorption energy under the cycling impact of different initial kinetic energy. The results indicate that the stiffness and damping coefficients will change at different impact velocities or cycle numbers. The damage mechanism of CFRP laminates under cycling low kinetic energy is delamination. After repeated experiments, it was found that there was a threshold value for the accumulated absorption energy before the failure of the CFRP laminate.

Background/Aims: The TLR/MyD88/NF-κB signaling pathway has been successfully used to treat renal interstitial fibrosis (RIF). However, the exact therapeutic mechanism is still unknown. Here, we assessed the therapeutic efficacy of TJ-M2010-2, a small molecular compound that inhibits MyD88 homodimerization, in RIF induced by ischemia reperfusion injury (IRI). Methods: In vivo, RIF was induced in mice by IRI, and the mice were prophylactically treated with TJ-M2010-2. In vitro, HK-2 cells were incubated with TGF-β1 to induce EMT, and the cells were pretreated with TJ-M2010-2. Results: We found that, compared with the IRI group, the TJ-M2010-2 group showed marked attenuation of RIF and renal function injury; decreased expression of TGF-β1, α-SMA, vimentin, MMP2 and MMP9; and increased E-cadherin expression. Furthermore, TGF-β1-induced EMT was blocked by TJ-M2010-2 in HK-2 cells, as evidenced by blocked morphologic transformation, restored E-cadherin expression and inhibited α-SMA expression. In addition, compared to the TGF-β1 group, the TJ-M2010-2 group showed profound inhibition of the expression of TRAF6, p65 and Snail and upregulation of the expression of IκBα. Conclusion: This MyD88 inhibitor may be a potential therapeutic agent to ameliorate RIF.

Rapid dissemination of the plasmid-born polymyxin resistance gene mcr-1 poses a critical medical challenge. MCR-1 expression is tightly controlled and imposes a fitness cost on the bacteria. We used growth studies and metabolomics to examine growth and metabolic changes within E. coli TOP10 at 8 and 24 h in response to different levels of expression of mcr-1. Induction of mcr-1 greatly increased expression at 8 h and markedly reduced bacterial growth; membrane disruption and cell lysis were evident at this time. At 24 h, the expression of mcr-1 dramatically declined with restored growth and membrane integrity, indicating regulation of mcr-1 expression in bacteria to maintain membrane homeostasis. Intermediates of peptide and lipid biosynthesis were the most commonly affected metabolites when mcr-1 was overexpressed in E. coli. Cell wall biosynthesis was dramatically affected with the accumulation of lipids including fatty acids, glycerophospholipids and lysophosphatidylethanolamines, especially at 8 h. In contrast, levels of intermediate metabolites of peptides, amino sugars, carbohydrates and nucleotide metabolism and secondary metabolites significantly decreased. Moreover, the over-expression of mcr-1 resulted in a prolonged reduction in intermediates associated with pentose phosphate pathway and pantothenate and CoA biosynthesis. These findings indicate that over-expression of mcr-1 results in global metabolic perturbations that mainly involve disruption to the bacterial membrane, pentose phosphate pathway as well as pantothenate and CoA biosynthesis.

ABSTRACT The plasmid-mediated fosfomycin resistance gene fosA3 has been detected in Enterobacterales from various sources but has rarely been reported in vegetables. In this study, the aim was to investigate the prevalence of and, subsequently, to characterize fosA3-positive Enterobacterales isolates from retail vegetables. Seventeen (7.3%) fosA3-carrying strains were identified from 233 extended-spectrum-β-lactamase-producing Enterobacterales isolates from vegetables. All 17 isolates, including six Escherichia coli, seven Klebsiella pneumoniae, two Raoultella ornithinolytica, and two Citrobacter freundii isolates, carried blaCTX-M. S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) and hybridization confirmed that the fosA3 genes in 16 isolates were located on plasmids ranging in size from ∼40 kb to ∼250 kb, except one located on chromosome of C. freundii. All the fosA3-carrying plasmids from 16 fosA3-positive isolates were successfully transferred into the recipient bacteria by transformation or conjugation. In agreement with data determined with isolates from food animals, the IncHI2/ST3 and IncN-F33:A-:B-/F33:A-:B plasmids were the main vectors of fosA3 in E. coli. Additionally, F24:A-:B1, IncFIIK-IncR, IncFIIS, IncR, and two untypeable plasmids were found for the first time to be vectors for fosA3 in Enterobacterales. The genetic contexts of fosA3 in 15 Enterobacterales isolates differed due to insertion and/or loss of molecular modules mediated by mobile elements. However, all fosA3 genes were flanked by IS26, as commonly observed in other fosA3-carrying plasmids. Here, we report a high rate of detection of fosA3 genes, mediated by multiple plasmid vectors, in ESBL-producing Enterobacterales from retail vegetables. FosA3-producing Enterobacterales could be transmitted to the human body by direct contact or consumption of vegetables, which might pose a potential threat to public health. IMPORTANCE This report provides important information on the transmission and epidemiology of fosA3 among Enterobacterales isolates from vegetables. The rate of occurrence of fosA3 in ESBL-producing Enterobacterales from retail vegetables is high, and fosA3 was found to be carried by diverse plasmids. Some novel genetic contexts of fosA3 and novel fosA3-carrying plasmids, including several plasmid types common in K. pneumoniae, were identified, increasing the number of known transfer vectors for the fosA3 gene and reflecting the complexity of fosA3 transmission in Enterobacterales. The capture of fosA3 by the resident plasmid of K. pneumoniae will accelerate the spread of fosA3 in K. pneumoniae, one of the most pathogenic species in clinical medicine. Considering the clinical importance of fosfomycin, and the fact that vegetables are directly consumed, the fosfomycin resistance genes present a risk of transmission to the human body through the food chain and thus pose a threat to public health.

The objective of this study was to explore the genetic and biological features of the tet(M)-harboring plasmid pTS14 in Salmonella enterica strain S14 isolated from a chicken fecal sample. Plasmid pTS14 was identified by conjugation, S1-pulsed-field gel electrophoresis (PFGE), Southern hybridization, and plasmid sequencing. The biological characteristics of pTS14 were assessed via stability, growth kinetics, and starvation survival experiments. Strain S14, belonging to ST3007, harbored a 119-kb tet(M)-bearing IncF2:A1:B1 conjugative plasmid pTS14. The plasmid pTS14 contained a novel transposon Tn6709 with the genetic structure IS26-tnpA1-tnpA2-Δorf13-LP-tet(M)-tnpX-ΔtnpR-IS26, and the resistance genes tet(B), tet(D), strAB, sul2, and blaTEM–1b. In addition, pTS14 was found to be highly stable in the recipient strain E. coli J53. The transconjugant TS14 exhibited a higher survival ratio than E. coli J53 under permanent starvation-induced stress. The tet(M)-bearing IncF2 epidemic plasmid lineage may accelerate the dissemination of tet(M) and other genes by coselection, which could constitute a potentially serious threat to clinical treatment regimens.

ABSTRACT Transporters belonging to the chromosomally encoded resistance-nodulation-division (RND) superfamily mediate multidrug resistance in Gram-negative bacteria. However, the cotransfer of large gene clusters encoding RND-type pumps from the chromosome to a plasmid appears infrequent, and no plasmid-mediated RND efflux pump gene cluster has yet been found to confer resistance to tigecycline. Here, we identified a novel RND efflux pump gene cluster, designated tmexCD1-toprJ1, on plasmids from five pandrug-resistant Klebsiella pneumoniae isolates of animal origin. TMexCD1-TOprJ1 increased (by 4- to 32-fold) the MICs of tetracyclines (including tigecycline and eravacycline), quinolones, cephalosporins, and aminoglycosides for K. pneumoniae, Escherichia coli, and Salmonella. TMexCD1-TOprJ1 is closely related (64.5% to 77.8% amino acid identity) to the MexCD-OprJ efflux pump encoded on the chromosome of Pseudomonas aeruginosa. In an IncFIA plasmid, pHNAH8I, the tmexCD1-toprJ1 gene cluster lies adjacent to two genes encoding site-specific integrases, which may have been responsible for its acquisition. Expression of TMexCD1-TOprJ1 in E. coli resulted in increased tigecycline efflux and in K. pneumoniae negated the efficacy of tigecycline in an in vivo infection model. Expression of TMexCD1-TOprJ1 reduced the growth of E. coli and Salmonella but not K. pneumoniae. tmexCD1-toprJ1-positive Enterobacteriaceae isolates were rare in humans (0.08%) but more common in chicken fecal (14.3%) and retail meat (3.4%) samples. Plasmid-borne tmexCD1-toprJ1-like gene clusters were identified in sequences in GenBank from Enterobacteriaceae and Pseudomonas strains from multiple continents. The possibility of further global dissemination of the tmexCD1-toprJ1 gene cluster and its analogues in Enterobacteriaceae via plasmids may be an important consideration for public health planning. IMPORTANCE In an era of increasing concerns about antimicrobial resistance, tigecycline is likely to have a critically important role in the treatment of carbapenem-resistant Enterobacteriaceae, the most problematic pathogens in human clinical settings—especially carbapenem-resistant K. pneumoniae. Here, we identified a new plasmid-borne RND-type tigecycline resistance determinant, TMexCD1-TOprJ1, which is widespread among K. pneumoniae isolates from food animals. tmexCD1-toprJ1 appears to have originated from the chromosome of a Pseudomonas species and may have been transferred onto plasmids by adjacent site-specific integrases. Although tmexCD1-toprJ1 still appears to be rare in human clinical isolates, considering the transferability of the tmexCD1-toprJ1 gene cluster and the broad substrate spectrum of TMexCD1-TOprJ1, further dissemination of this mobile tigecycline resistance determinant is possible. Therefore, from a “One Health” perspective, measures are urgently needed to monitor and control its further spread. The current low prevalence in human clinical isolates provides a precious time window to design and implement measures to tackle this.