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Wan He,1,* Changbu Wu,2,3,* Guanping Chen,2– 4 Guili Zhang,2,3 Zihan Zhao,2,3 Shu’an Wen,2,3 Yuan Zhou,2,3 Xue Deng,2,3 Yu Feng,2,3 Lan-Lan Zhong,2,3 Guo-Bao Tian,2– 4 Min Dai1 1School of Laboratory Medicine, Chengdu Medical College, Chengdu, 610500, People’s Republic of China; 2Department of Immunology and Microbiology, Advanced Medical Technology Center, The First Affiliated Hospital, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, 510080, People’s Republic of China; 3Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou, 510080, People’s Republic of China; 4Department of Immunology, School of Medicine, Sun Yat-Sen University, Shenzhen, 518107, People’s Republic of China*These authors contributed equally to this workCorrespondence: Min Dai, School of Laboratory Medicine, Chengdu Medical College, Chengdu, Sichuan Province, People’s Republic of China, Tel +86 28-62739128, Email daimin1015@163.com Guo-Bao Tian, Department of Immunology and Microbiology, Advanced Medical Technology Center, The First Affiliated Hospital, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, People’s Republic of China, Tel/Fax +86 20 87335387, Email tiangb@mail.sysu.edu.cnBackground: The emergence and spread of hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is a potential epidemiological threat that needs to be monitored. However, the transmission and pathogenic characteristics of hv-CRKP in China remain unclear. We investigated the epidemiological characteristics of gut colonized hv-CRKP in a hospital in Guangdong Province, China.Methods: A total of 46 gut colonized hv-CRKP isolates were collected from Sun Yat-Sen Memorial Hospital (Guangzhou, China) from August 31st to December 31st, 2021. Minimum inhibitory concentrations (MICs) were obtained for 15 antibiotics for 46 hv-CRKP isolates. BALB/C mice infection model and mucoviscosity assay was used to evaluate the virulence of the isolates. The characteristics of genome, phylogenetic relationship and the structure of the plasmid of 46 gut colonized hv-CRKP isolates were compared with pathogenic isolates from GeneBank based on whole-genome data.Results: The hv-CRKP isolation rate of all gut colonized carbapenem-resistant Klebsiella pneumoniae was 17% (46/270), and the intestinal colonization rate of hv-CRKP was irrelevant to the sex, age, department of hospitalization, and history of antibiotic use of the host. The gut colonized hv-CRKP showed pandrug resistance and hypervirulence. The gut colonized hv-CRKP and pathogenic hv-CRKP prevalent in China were mainly ST11 hv-CRKP and had two major epidemic clades. The similarities in genomic characteristics between gut colonized hv-CRKP and pathogenic hv-CRKP were consistent. The gut colonized hv-CRKP carried an incomplete structure pK2044 virulence plasmid from hypervirulent K. pneumoniae NTUH-K2044 by analyzing the virulence plasmid structure.Conclusion: Our results suggest that the gut colonized ST11 hv-CRKP may serve as a reservoir for the clinical pathogenic ST11 HV-CRKP. It is necessary to further strengthen the monitoring of gut colonized hv-CRKP and research the potential mechanism of infection caused by gut colonized hv-CRKP.Keywords: K. pneumoniae, intestinal colonization, hypervirulent, genomic characterization, carbapenemase

Xiaoxue Liang,1,* Ping Chen,2,* Baoguo Deng,2,* Feng-Hui Sun,1,* Yongqiang Yang,3– 5 Yanxian Yang,3,4 Ruowen He,3,4 Mingyang Qin,2 Yiping Wu,3,4 Fan Yang,2 Guo-Bao Tian,3,4 Min Dai1 1School of Laboratory Medicine, Chengdu Medical College, Chengdu, Sichuan Province, People’s Republic of China; 2School of Basic Medical Science, Xinxiang Medical University, Xinxiang, Henan Province, People’s Republic of China; 3Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong Province, People’s Republic of China; 4Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou, Guangdong Province, People’s Republic of China; 5School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Guangzhou, Guangdong Province, People’s Republic of China*These authors contributed equally to this workCorrespondence: Min Dai, School of Laboratory Medicine, Chengdu Medical College, Chengdu, Sichuan Province, People’s Republic of China, Tel +86 28-62739128, Email daimin1015@163.comPurpose: To compare antimicrobial resistance, virulence, clinical characteristics, and risk factors between carbapenem-resistant K. pneumoniae (CRKP) and carbapenem-susceptible K. pneumoniae (CSKP) isolates from patients with bloodstream infections (BSIs) in China.Patients and Methods: The clinical data of 103 patients with K. pneumoniae BSI from 10 hospitals were retrospectively analyzed. The minimum inhibitory concentrations of 15 antibiotics against the bacteria were determined. A Galleria mellonella infection model was used to evaluate virulence of the isolates. Kaplan–Meier curves were calculated to evaluate the 28-day and in-hospital survival rates of the isolates. The risk factors for CRKP and CSKP infection and respective mortality rate were evaluated by univariate analysis, and independent risk factors were evaluated using the multivariate logistic regression model.Results: Our results indicated that CRKP isolates were more resistant to most tested antibiotics than CSKP isolates. The G. mellonella infection model was used to demonstrate that CRKP isolates were more virulent than CSKP isolates. We found that in-hospital deaths occurred in 39.3% (22/56) of patients with CRKP BSIs and were significantly higher than those in patients with CSKP infections (19.1%, 9/47). Patients infected with CRKP isolates had poorer outcomes than those infected with the CSKP strains. For in-hospital mortality of CRKP BSIs, the independent risk factors included carbapenem-resistant Enterobacterales bacteremia and length of hospitalization after the onset of BSI.Conclusion: Our findings confirm that CRKP isolates are more drug-resistant than CSKP isolates and are associated with poorer outcomes. To prevent CRKP infection, strict infection control strategies and active surveillance should be implemented in hospitals.Keywords: Klebsiella pneumoniae, bloodstream infections, virulence, antimicrobial resistance

Minmin Lin,1,* Yongqiang Yang,2– 4,* Yanxian Yang,2,3,* Guanping Chen,5 Ruowen He,2,3 Yiping Wu,2,3 Lan-Lan Zhong,2,3 Mohamed Abd El-Gawad El-Sayed Ahmed,2,3,6 Siyuan Feng,2,3 Cong Shen,2,3 Xin Wen,2,3 Jin Huang,1 Hongyu Li,7 Xiaobin Zheng,1 Guo-Bao Tian2,3 1Department of Respiratory Medicine, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, People’s Republic of China; 2Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, People’s Republic of China; 3Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou 510080, People’s Republic of China; 4School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Guangzhou 510006, People’s Republic of China; 5Sun Yat-sen University School of Medicine, Guangzhou 510006, People’s Republic of China; 6Department of Microbiology and Immunology, Faculty of Pharmaceutical Sciences and Drug Manufacturing, Misr University for Science and Technology (MUST), Cairo, 6th of October City, Egypt; 7Department of Laboratory, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, People’s Republic of China*These authors contributed equally to this workCorrespondence: Guo-Bao Tian; Xiaobin Zheng Email tiangb@mail.sysu.edu.cn; zhxbin@mail.sysu.edu.cnBackground: Bloodstream infection (BSI) caused by carbapenem-resistant Enterobacteriaceae are potentially life-threatening related to poorer outcomes. Colistin is considered one of the last-resort treatments against human infections caused by multidrug-resistant (MDR) Gram-negative bacteria. Therefore, emergence of strains from the blood that co-harboring mcr and carbapenem resistance genes were considered as a serious problem.Purpose: In this study, two mcr-9-harboring MDR Enterobacter cloacae isolates BSI034 and BSI072 recovered from BSI patients were identified, one of which co-harbored mcr-9 and blaNDM-1. The genetic characteristics of the MDR plasmid needed to be clarified.Methods: S1-PFGE and Southern blotting were conducted to determine the location of mcr-9. Whole-genome sequencing was performed to obtain the complete genome and plasmid sequences. The resistome and virulence genes of the strains, accompanied by the genetic characteristics of mcr-9- and blaNDM-1-harboring plasmids, were analyzed.Results: Whole-genome sequencing showed that BSI034 harbored mcr-9-carrying IncHI2-type pBSI034-MCR9 and blaNDM-1-carrying IncX3-type pBSI034-NDM1. The 278,517 bp pBSI034-MCR9 carried mcr-9 along with the other 19 resistance genes. mcr-9 was flanked by IS 903B (1057 bp) and IS 26 (820 bp) in the same orientation. In addition to resistance genes, strain BSI034 also carried a chromosome-located Yersinia high-pathogenicity island, which harbored genes of yersiniabactin biosynthesis operon ybtSXQPAUTE, irp1/2, and fyuA.Conclusion: We described the complete genome and mcr-9/blaNDM-1-co-harboring plasmid of E. cloacae from a BSI patient. Notable differences were observed within mosaic modules between pBSI034-MCR9 and other mcr-9-harboring plasmids due to extensive recombination via horizontal gene transfer.Keywords: MDR, mcr-9, colistin, virulence genes

Lan-Lan Zhong,1,2,* Qian Zhou,3,* Cui-Yan Tan,3,* Adam P Roberts,4,5 Mohamed Abd El-Gawad El-Sayed Ahmed,1–2,6 Guanping Chen,1,2 Min Dai,7 Fan Yang,8 Yong Xia,9 Kang Liao,10 Yingjian Liang,3 Yongqiang Yang,1,11 Siyuan Feng,1,2 Xiaobin Zheng,3,* Guo-Bao Tian1,2,*1Program in Pathobiology, The Fifth Affiliated Hospital, Zhongshan School of Medicine, Sun Yat-Sen University, Guangdong 519000, People’s Republic of China; 2Ministry of Education, Key Laboratory of Tropical Diseases Control, Sun Yat-sen University, Guangzhou 510080, People’s Republic of China; 3Department of Respiratory Medicine, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai 519000, People’s Republic of China; 4Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK; 5Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine, Liverpool L3 5QA, UK; 6Department of Microbiology and Immunology, Faculty of Pharmaceutical Sciences and Drug Manufacturing, Misr University for Science and Technology (MUST), Cairo, Egypt; 7School of Laboratory Medicine, Chengdu Medical College, Chengdu 610500, People’s Republic of China; 8Department of Microbiology, School of Basic Medical Science, Xinxiang Medical University, Xinxiang 453003, People’s Republic of China; 9Department of Clinical Laboratory Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou 510150, People’s Republic of China; 10Department of Clinical Laboratory, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510080, People’s Republic of China; 11School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University, Guangzhou 510006, People’s Republic of China*These authors contributed equally to this workPurpose: The discovery of the plasmid-mediated colistin resistance genes, mcr, revealed a mechanism of transmission of colistin resistance, which is a major, global public health concern especially among individuals infected with carbapenem-resistant Gram-negative bacteria. To monitor the spread and epidemiology of mcr genes, a convenient and reliable method to detect mcr genes in clinical isolates is needed, especially in the primary care institutions. This study aimed to establish a restriction endonuclease-based multiplex loop-mediated isothermal amplification (multi-LAMP) assay to detect mcr genes (mcr-1 to mcr-5) harbored by colistin-resistant bacteria.Methods: A triple-LAMP assay for mcr-1, mcr-3, and mcr-4 and a double-LAMP assay for mcr-2 and mcr-5 were established. The sensitivity and specificity of the LAMP reactions were determined via electrophoresis and visual detection.Results: The sensitivity of the LAMP assay was 10-fold greater than that of PCR, with high specificity among the screened primers. Specific mcr genes were distinguished in accordance with band numbers and the fragment length of the digested LAMP amplification products. Furthermore, the LAMP assay was confirmed as a rapid and reliable diagnostic technique upon application for clinical samples, and the results were consistent with those of conventional PCR assay.Conclusion: The multi-LAMP assay is a potentially promising method to detect mcr genes and will, if implemented, help prevent infections by drug-resistant bacteria in primary-care hospitals due to rapid and reliable surveillance. To our knowledge, this is the first study to report the application of LAMP to detect mcr-2 to mcr-5 genes and the first time that multi-LAMP has been applied to detect mcr genes.Keywords: mcr genes, colistin resistance, multi-LAMP, rapid detection, enzyme digestion

Fan Yang,1,* Cong Shen,2,3,* Xiaobin Zheng,4 Yan Liu,5 Mohamed Abd El-Gawad El-Sayed Ahmed,2,3,6 Zihan Zhao,2,3 Kang Liao,7 Yaling Shi,8 Xin Guo,2,3 Ruoxuan Zhong,2,3 Zhimin Xu,2,3 Guo-Bao Tian2,3 1Department of Microbiology, School of Basic Medical Science, Xinxiang Medical University, Xinxiang, China; 2Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China; 3Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou, China; 4Department of Respiratory Medicine, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China; 5Department of Laboratory, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China; 6Department of Microbiology and Immunology, Faculty of Pharmacy, Misr University for Science and Technology (MUST), Cairo, 6th of October City, Egypt; 7Department of Clinical Laboratory, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China; 8Department of Clinical Laboratory, Guangzhou Eighth People’s Hospital, Guangzhou Medical University, Guangzhou, China *These authors contributed equally to this work Background: As a result of the growing prevalence of the plasmid-mediated mobile colistin resistance gene mcr-1 among Gram-negative bacteria, the surveillance of mcr-1 has been globally applied. In our study, we aimed to shed light on the possibility of transmission of mcr-1-resistant isolates through market retail fruits. Methods and results: Herein, 133 different fruit surface samples were collected and screened for the different MCR variants (mcr-1 to mcr-8) using PCR and confirmed with sequencing. We identify for the first time mcr-1-carrying Escherichia coli and Klebsiella pneumoniae from market retail fruits in Guangzhou, China. Minimum inhibitory concentrations were detected by the broth microdilution method. Liquid mating was performed to check the transferability of the mcr-1 gene. Pulsed field gel electrophoresis analysis of S1 nuclease-digested DNA and Southern blotting were performed to check the location of the mcr-1 gene. Then, whole-genome sequencing and in silico multilocus sequence typing analysis were performed. Conclusion: We showed that E. coli GB110 can mediate the spreading of antibiotic resistance genes through the food chain, while K. pneumoniae GB015 was considered to be the progenitor of the most successful multidrug-resistant clone. Since fruits are usually consumed fresh, this may serve as a direct source of mcr-1-producing bacteria in humans that requires prompt surveillance and intervention to limit the spread of resistance. Keywords: colistin, mcr-1, Escherichia coli, Klebsiella pneumoniae, fruit

Yingjian Liang,1,* Changli Tu,1,* Cuiyan Tan,1 Mohamed Abd El-Gawad El-Sayed Ahmed,2,3,4 Min Dai,5 Yong Xia,6 Yan Liu,7 Lan-Lan Zhong,2,3 Cong Shen,2,3 Guanping Chen,2,3 Guo-Bao Tian,2,3 Jing Liu,1 Xiaobin Zheng1 1Department of Respiratory Medicine, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China; 2Department of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China; 3Key Laboratory of Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, China; 4Department of Microbiology and Immunology, Faculty of Pharmacy, Misr University for Science and Technology (MUST), 6th of October City, Egypt; 5School of Laboratory Medicine, Chengdu Medical College, Chengdu, China; 6Department of Clinical Laboratory Medicine, Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China; 7Clinical laboratory, The Fifth Affiliated Hospital of Sun Yat-Sen University, Zhuhai, China *These authors contributed equally to this work Purpose: The main objective of this study was to decipher the prevalence, antimicrobial resistance, major virulence genes and the molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolated from different clinical sources in southern China.Materials and methods: The present study was performed on 187 non-duplicate S. aureus clinical isolates collected from three tertiary hospitals in Guangdong Province, China, 2010–2016. Antimicrobial susceptibility testing was performed by the disk diffusion method and by measuring the minimum inhibitory concentration. Screening for resistance and virulence genes was performed. Clonal relatedness was determined using various molecular typing methods such as multilocus sequence typing, spa and staphylococcal chromosomal cassette mec (SCCmec) typing. Whole genome sequencing was performed for three selected isolates.Results: Out of 187 isolates, 103 (55%) were identified as MRSA. The highest prevalence rate was found among the skin and soft tissue infection (SSTI) samples (58/103), followed by sputum samples (25/103), blood stream infection samples (15/103) and others (5/103). Antimicrobial susceptibility results revealed high resistance rates for erythromycin (64.1%), clindamycin (48.5%), gentamicin (36.9%) and ciprofloxacin (33.98%). All isolates were susceptible to vancomycin. Resistance genes and mutation detected were as follows: aac(6’)-aph(2”) (24.3%), dfrG (10.7%), rpoB (21.4%), cfr (0%), fexA (1.94%), gyrA (35.92%), gyrB (0.97%), grlA (20.4%), grlB (10.68%), ermA (21.4%), ermB (18.44%), ermC (21.4%) and lnuA (18.44%). Profiling of virulence genes revealed the following: sea (11.7%), seb (21.4%), sec (0.97%), sed (0.97%), hla (86.41%), hlb (17.48%), hlg (10.68%), hld (53.4%), Tsst-1 (3.9%) and pvl (27.2%). Clonal relatedness showed that ST239-SCCmecA III-t37 clone was the most prevalent clone.Conclusion: Our study elucidated the prevalence, antibiotic resistance, pathogenicity and molecular characteristics of MRSA isolated from various clinical sources in Guangdong, China. We found that the infectious rate of MRSA was higher among SSTI than other sources. The most predominant genotype was ST239-SCCmecA III-t37 clone, indicating that ST239-t30 clone which was previously predominant had been replaced by a new clone. Keywords: MRSA, antibiotic resistance, resistance genes, virulence factors, molecular typing, whole genome sequencing

Siyuan Feng,1,2,* Cong Shen,1,2,* Hongtao Chen,3 Xiaobin Zheng,4 Yong Xia,5 Lan-Lan Zhong,1,2 Xi Huang,6 Xinwei Wu,7 Guo-Bao Tian1,2 1Department of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China; 2Key Laboratory of Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, China; 3Department of Laboratory, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China; 4Department of Respiratory Medicine, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China; 5Department of Clinical Laboratory Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China; 6Program of Pathobiology and Immunology, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, China; 7Department of Microbiology, Guangzhou Center for Disease Control and Prevention, Guangzhou, China *These authors contributed equally to this work Abstract: Colistin is increasingly used as an antibiotic of last resort for treating carbapenem-resistant Enterobacteriaceae. Mobile colistin resistance gene mcr-1 has been increasingly reported in Enterobacteriaceae around the world. Of particular concern is the spread of mcr-1 into carbapenemase-producing Enterobacteriaceae, which results in highly drug-resistant strains that are potentially untreatable. Notably, such mcr-1-carrying isolates harboring carbapenemase genes have been reported in animals and patients with infection. Here, we report an Escherichia coli strain carrying co-transferable mcr-1-harboring IncX4 and blaNDM-5-harboring IncX3 plasmids, which was recovered in the context of fecal colonization. Keywords: Escherichia coli, MCR-1, NDM-5, co-production

Colistin (CT) is the last-resort of antibiotic against multidrug-resistance (MDR) Acinetobacter baumannii ( A. baumannii ) infection. However, colistin resistance is increasingly reported in A. baumannii isolates partially due to the global emergence and dissemination of plasmid-borne mobile colistin resistance ( mcr ) gene and is a threat to human health. Thus, available treatment strategies urgently required in the fight against colistin-resistant A. baumannii . Here, we showed that mcr confers damaged outer membrane (OM) permeability in A. baumannii , which could compromise the viability of A. baumannii . Consistently, A. baumannii with colistin resistance exhibits increased susceptibility to macromolecular antibiotics such as rifampicin (RIF) and erythromycin (ERY). Moreover, the combination therapy of colistin and rifampicin demonstrates efficacy against colistin-resistant A. baumannii , regardless of the presence of mcr . Altogether, our data suggest that the synergy of colistin in combination with macromolecular hydrophobic antibiotics poses a promising therapeutic alternative for colistin-resistant A. baumannii ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Ma, Zhao, Zhou, Liang, Lv, Li, Wang, Xu, Liu, Tian, Feng and Xia.)

Drug-resistant Mycobacterium tuberculosis is a significant cause of infectious disease morbidity and mortality for which new antimicrobials are urgently needed. Inhibitors of mycobacterial respiratory energy metabolism have emerged as promising next-generation antimicrobials, but a number of targets remain unexplored. Succinate dehydrogenase (SDH), a focal point in mycobacterial central carbon metabolism and respiratory energy production, is required for growth and survival in M. tuberculosis under a number of conditions, highlighting the potential of inhibitors targeting mycobacterial SDH enzymes. To advance SDH as a novel drug target in M. tuberculosis , we utilized a combination of biochemical screening and in-silico deep learning technologies to identify multiple chemical scaffolds capable of inhibiting mycobacterial SDH activity. Antimicrobial susceptibility assays show that lead inhibitors are bacteriostatic agents with activity against wild-type and drug-resistant strains of M. tuberculosis . Mode of action studies on lead compounds demonstrate that the specific inhibition of SDH activity dysregulates mycobacterial metabolism and respiration and results in the secretion of intracellular succinate. Interaction assays demonstrate that the chemical inhibition of SDH activity potentiates the activity of other bioenergetic inhibitors and prevents the emergence of resistance to a variety of drugs. Overall, this study shows that SDH inhibitors are promising next-generation antimicrobials against M. tuberculosis .

Bacterial infections, especially those caused by multidrug-resistant pathogens, pose a significant threat to public health. Vaccines are a crucial tool in fighting these infections; however, no clinically available vaccine exists for the most common bacterial infections, such as those caused by Pseudomonas aeruginosa. Herein, a multiantigenic antibacterial nanovaccine (AuNP@HMV@SPs) is reported to combat P. aeruginosa infections. This nanovaccine utilizes the hybrid membrane vesicles (HMVs) created by fusing macrophage membrane vesicles (MMVs) with bacterial outer membrane vesicles (OMVs). The HMVs mitigate the toxic effects of both OMVs and bacterial secreted toxins (SP) adsorbed on the surface of MMVs, while preserving their stimulating properties. Gold nanoparticles (AuNPs) are utilized as adjuvant to enhance immune response without comprising safety. The nanovaccine AuNP@HMV@SPs induces robust humoral and cellular immune responses, leading to destruction of bacterial cells and neutralization of their secreted toxins. In murine models of septicemia and pneumonia caused by P. aeruginosa, AuNP@HMV@SPs exhibits superior prophylactic efficacy compared to control groups including OMVs, or MMVs@SPs and HMV@SPs, achieving 100% survival in septicemia and > 99.9% reduction in lung bacterial load in pneumonia. This study highlights AuNP@HMV@SPs as a safe and effective antibacterial nanovaccine, targeting both bacteria and their secreted toxins, and offers a promising platform for developing multiantigenic antibacterial vaccines against multidrug-resistant pathogens., (© 2024 The Author(s). Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Introduction: Staphylococcus aureus is one of the chief pathogens that cause chronic and recurrent infections. Failure of the antibiotics to curb the infections contributes to relapse and is an important reason for the high mortality rate. Treatment failure may also be due to antibiotic tolerance. Accumulating evidence suggests that t the host immune environment plays an important role in inducing antibiotic tolerance of S. aureus , but research in this area has been limited., Methods: In this study,the minimum inhibitory concentration (MIC) of the antibiotics against S. aureus was determined using the standard broth microdilution method.The study evaluated whether itaconate induces antibiotic tolerance in S. aureus through an antibiotic bactericidal activity assay.The effect of itaconate on the growth of S. aureus was evaluated by monitoring the growth of S. aureus in medium supplemented with itaconate. Additionally, RNA sequencing and metabolomics analyses were used to determine transcriptional and metabolic changes in S. aureus when exposed to itaconate., Results and Discussion: According to the study,we found that the immune metabolite itaconate can induce tolerance in both methicillin-resistant and -susceptible S. aureus to aminoglycosides. When S. aureus was exposed to itaconate, its growth slowed down and transcriptomic and metabolomic alterations associated with decreased energy metabolism, including the tricarboxylate cycle, glycolysis, pyruvate metabolism, and arginine biosynthesis, were observed. These changes are associated with aminoglycoside tolerance. This study highlights the role of immune signaling metabolites in bacterial antibiotic tolerance and suggests new strategies to improve antibiotic treatment by modulating the host immune response and stimulating the metabolism of bacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhao, Xu, Chen, Yang, Guo, Dai, Tian and Qin.)

Background: The intensive use of antibiotics has resulted in strong natural selection for the evolution of antimicrobial resistance (AMR), but whether, and under what circumstances, the removal of antibiotics would result in a rapid reduction in AMR has been insufficiently explored. We aimed to test the hypothesis that in the simple, yet common, case of AMR conferred by a single gene, removing antibiotics would quickly reduce the prevalence of resistance if the AMR gene imposes a high fitness cost and costless resistance is extremely rare among its proximal mutants., Methods: In this genetic study, to test our hypothesis, we used the mcr-1 gene in Escherichia coli, which confers resistance to the last-resort antibiotic colistin, as a model. A high-throughput reverse genetics approach was used to evaluate mcr-1 variants for their fitness cost and resistance levels relative to a non-functional construct, by measuring relative growth rates in colistin-free media and at 2 μg/mL and 4 μg/mL colistin. We identified costless resistant mcr-1 mutants, and examined their properties within the context of the sequential organisation of mcr-1's functional domains as well as the evolutionary accessibility of these mutations. Finally, a simple population genetic model incorporating the measured fitness cost was constructed and tested against previously published real-world data of mcr-1 prevalence in colonised inpatients in China since the 2017 colistin ban in fodder additives., Findings: We estimated the relative growth rates of 14 742 mcr-1 E coli variants (including the wild type), 3449 of which were single-nucleotide mutants. E coli showed 73·8% less growth per 24 h when carrying wild-type mcr-1 compared with the non-functional construct. 6252 (42·4%) of 14 741 mcr-1 mutants showed colistin resistance accompanied by significant fitness costs, when grown under 4 μg/mL colistin selection. 43 (0·3%) mcr-1 mutants exhibited costless resistance, most of which contained multiple mutations. Among the 3449 single mutants of mcr-1, 3433 (99·5%) had a fitness cost when grown in colistin-free media, with a mean relative growth of 0·305 (SD 0·193) compared with the non-functional variant. 3059 (88·7%) and 1833 (53·1%) of 3449 single mutants outgrew the non-functional mcr-1 in the presence of 2 μg/mL and 4 μg/mL colistin, respectively. Single mutations that gave rise to costless mutants were rare in all three domains of mcr-1 (transmembrane domain, flexible linker, and catalytic domain), but the linker domain was enriched with cost-reducing and resistance-enhancing mutations and depleted with cost-increasing mutations. The population genetics model based on the experimental data accurately predicts the rapid decline in mcr-1 prevalence in real-world data., Interpretation: Many identified costless resistant variants that consist of multiple mutations are unlikely to evolve easily in nature. These findings for colistin and mcr-1 might be applicable to other cases in which AMR entails a substantial fitness cost that cannot be mitigated in proximal mutants., Funding: National Natural Science Foundation of China, and National Key Research and Development Program of China., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction: The rapid spread of plasmid-mediated tet(X4) conferring high tigecycline resistance poses a significant threat to public health. Escherichia coli as the most common pathogen which carries tet(X4) has been widely disseminated in China. Thus, comprehensive investigations are required to understand the mechanism of transmission of tet(X4) -positive E. coli ., Methods: In this study, a total of 775 nonduplicate samples were collected in Guangdong, China from 2019 to 2020. We screened for tet(X4) -positive E. coli by PCR amplification and species identification. Furthermore, we analyzed the phylogenetics and genetic context of tet(X4) -positive E. coli through whole-genome sequencing and long-reads sequencing., Results: Overall, 146 (18.84%) tet(X4) -positive E. coli were isolated, comprising 2 isolates from humans and 144 isolates from pigs. The majority of tet(X4) -positive E. coli exhibited resistance to multiple antibiotics but all of them were susceptible to amikacin and colistin. Phylogenetic analysis showed that ST877, ST871, and ST195 emerged as the predominant sequence types in tet(X4) -positive E. coli . Further analysis revealed various genetic environments associated with the horizontal transfer of tet(X4) . Notably, a 100-kbp large fragment insertion was discovered downstream of tet(X4) , containing a replicon and a 40-kbp gene cluster for the bacterial type IV secretion system., Discussion: The high colonization rate of tet(X4) -positive E. coli in animals suggests that colonization as a key factor in its dissemination to humans. Diverse genetic context may contribute to the transfer of tet(X4) . Our findings underline the urgent need for controlling the spread of plasmid-mediated tigecycline resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Shao, Wu, Li, He, Chen, Sun, Yang, Feng, Zhang, Yan, Dai, Tian and Zhong.)

Rifampicin is the most powerful first-line antibiotic for tuberculosis, which is caused by Mycobacterium tuberculosis. Although accumulating evidence from sequencing data of clinical M. tuberculosis isolates suggested that mutations in the rifampicin-resistance-determining region (RRDR) are strongly associated with rifampicin resistance, the comprehensive characterisation of RRDR polymorphisms that confer this resistance remains challenging. By incorporating I-SceI sites for I-SceI-based integrant removal and utilizing an L5 swap strategy, we efficiently replaced the integrated plasmid with alternative alleles, making mass allelic exchange feasible in mycobacteria. Using this method to establish a fitness-related gain-of function screen, we generated a mutant library that included all single-amino-acid mutations in the RRDR, and identified the important positions corresponding to some well-known rifampicin-resistance mutations (Q513, D516, S522, H525, R529, S531). We also detected a novel two-point mutation located in the RRDR confers a fitness advantage to M. smegmatis in the presence or absence of rifampicin. Our method provides a comprehensive insight into the growth phenotypes of RRDR mutants and should facilitate the development of anti-tuberculosis drugs., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Clostridioides difficile is a leading cause of healthcare-associated infections, causing billions of economic losses every year. Its symptoms range from mild diarrhea to life-threatening damage to the colon. Transmission and recurrence of C. difficile infection (CDI) are mediated by the metabolically dormant spores, while the virulence of C. difficile is mainly due to the two large clostridial toxins, TcdA and TcdB. Producing toxins or forming spores are two different strategies for C. difficile to cope with harsh environmental conditions. It is of great significance to understand the molecular mechanisms for C. difficile to skew to either of the cellular processes. Here, we summarize the current understanding of the regulation and connections between toxin production and sporulation in C. difficile and further discuss the potential solutions for yet-to-be-answered questions., (© 2024 The Authors. mLife published by John Wiley & Sons Australia, Ltd on behalf of Institute of Microbiology, Chinese Academy of Sciences.)

The continuous reduction of clinically available antibiotics has made it imperative to exploit more effective antimicrobial therapies, especially for difficult-to-treat Gram-negative pathogens. Herein, it is shown that the combination of an antimicrobial nanozyme with the clinically compatible basic amino acid L-arginine affords a potent treatment for infections with Gram-negative pathogens. In particular, the antimicrobial activity of the antimicrobial nanozyme is dramatically increased by ≈1000-fold after L-arginine stimulation. Specifically, the combination therapy enhances bacterial outer and inner membrane permeability and promotes intracellular reactive oxygen species (ROS) generation. Moreover, the metabolomic and transcriptomic results reveal that combination treatment leads to the increased ROS-mediated damage by inhibiting the tricarboxylic acid cycle and oxidative phosphorylation, thereby inducing an imbalance of the antioxidant and oxidant systems. Importantly, L-arginine dramatically significantly accelerates the healing of infected wounds in mouse models of multidrug-resistant peritonitis-sepsis and skin wound infection. Overall, this work demonstrates a novel synergistic antibacterial strategy by combining the antimicrobial nanozymes with L-arginine, which substantively facilitates the nanozyme-mediated killing of pathogens by promoting ROS production., (© 2023 Wiley-VCH GmbH.)

The emerging and global spread of a novel plasmid-mediated colistin resistance gene, mcr-1, threatens human health. Expression of the MCR-1 protein affects bacterial fitness and this cost correlates with lipid A perturbation. However, the exact molecular mechanism remains unclear. Here, we identified the MCR-1 M6 variant carrying two-point mutations that conferred co-resistance to β-lactam antibiotics. Compared to wild-type (WT) MCR-1, this variant caused severe disturbance in lipid A, resulting in up-regulation of L, D-transpeptidases (LDTs) pathway, which explains co-resistance to β-lactams. Moreover, we show that a lipid A loading pocket is localized at the linker domain of MCR-1 where these 2 mutations are located. This pocket governs colistin resistance and bacterial membrane permeability, and the mutated pocket in M6 enhances the binding affinity towards lipid A. Based on this new information, we also designed synthetic peptides derived from M6 that exhibit broad-spectrum antimicrobial activity, exposing a potential vulnerability that could be exploited for future antimicrobial drug design., Competing Interests: The authors declare no conflicts of interest., (Copyright: © 2023 Liang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Antibiotic colistin is the last line of defense against multidrug-resistant (MDR) Gram-negative bacterial infections. Emergence of colistin resistance in microbes is a critical challenge. Herein, curcumin is discovered, for the first time, to reverse the resistance phenotype of colistin-resistant bacteria via a checkerboard assay. For the co-delivery of curcumin and colistin, negatively charged poly(ethylene glycol)-functionalized liposomes encapsulating both drugs (Lipo-cc) are prepared. Killing kinetics and live/dead assays confirm the antibacterial activity of Lipo-cc against colistin-resistant bacteria, which is more potent than that of the free curcumin and colistin combination. Mechanistical studies reveal that Lipo-cc restores the affinity of colistin for the bacterial membrane and improves the uptake of curcumin, which leads to reduced efflux pump activity, achieving a synergistic effect of colistin and curcumin. At the effective antibacterial dose, Lipo-cc does not exhibit any toxicity. The therapeutic efficacy of Lipo-cc is further demonstrated in an intestinal bacterial infection model induced with colistin-resistant Escherichia coli. Lipo-cc reduces the bacterial burden with over 6-log reduction and alleviated inflammation caused by infection. Importantly, unlike colistin, Lipo-cc does not affect the homeostasis of the intestinal flora. Taken together, Lipo-cc successfully overcame colistin resistance, indicating its potential for the treatment of colistin-resistant bacterial infections., (© 2023 Wiley-VCH GmbH.)

Colistin is the last-resort antibiotic to treat multidrug-resistant (MDR) Gram-negative bacterial infections that are untreatable by other clinically available antibiotics. However, the recently merged plasmid-borne gene mobilized colistin resistance ( mcr ) leads to modification of the colistin target (i.e., bacterial membrane), greatly compromising the therapy outcome of colistin. To address this unmet clinical need, a nanocomplex (CMS-pEt_20 NP) of anionic prodrug colistin methanesulfonate (CMS) and guanidinium-functionalized cationic polymer pEt_20 is developed through facile self-assembly for co-delivering an antibiotic and antimicrobial polymer with membrane affinity to reverse colistin resistance. The CMS-pEt_20 NP formation enables reversal of colistin resistance and complete killing of clinically isolated mcr -positive colistin-resistant bacteria including MDR E. coli and K. pneumoniae , while monotreatment of polymer or antibiotic at equivalent doses exhibits no antibacterial activity. Mechanistic studies reveal that the CMS-pEt_20 NP enhanced the affinity of delivered CMS to the modified membrane of colistin-resistant bacteria, reviving the membrane lytic property of colistin. The increased membrane permeability caused by colistin in turn promotes an influx of pEt_20 to generate intracellular ROS stress, resulting in elimination of colistin-resistant bacteria. More importantly, a colistin-resistant mouse peritonitis-sepsis infection model demonstrates the excellent therapeutic efficacy of CMS-pEt_20 NP with 100% survival of the infected mouse. In addition, the nanocomplex is proven not toxic both in vitro and in vivo . Taken together, the self-assembled antibiotic-polymer nanocomplex with two complementary antibacterial mechanisms successfully reverses the colistin resistance phenotype in bacteria, and it can be a potential strategy to treat untreatable colistin-resistant MDR bacterial infections.

Objectives: Tigecycline is recognized as one of the last-line antibiotics to treat serious bacterial infection caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). The plasmid-borne gene tet (X4) mediates high resistance to tigecycline. However, the prevalence and genetic context of tet (X4) in K. pneumoniae from various sources are not fully understood. Here, we investigated the prevalence of tet (X4)-positive K. pneumoniae and characterized the genetic context of tet (X4)-bearing plasmids in K. pneumoniae isolates., Methods: Polymerase chain reaction (PCR) was used to detect the tet (X4) gene. The transferability of the tet (X4)-carrying plasmids was tested by conjugation assays. The Galleria mellonella infection model was used to test virulence of tet (X4)-positive strains. Whole-genome sequencing and genome-wide analysis were performed to identify the antimicrobial resistance and the virulence genes, and to clarify the genetic characteristics of the tet (X4)-positive isolates., Results: Among 921 samples, we identified two tet (X4)-positive K. pneumoniae strains collected from nasal swabs of two pigs (0.22%, 2/921). The two tet (X4)-positive isolates exhibited high minimum inhibitory concentrations to tigecycline (32-256 mg/L) and tetracycline (256 mg/L). The plasmids carrying the tet (X4) gene can transfer from the donor strain K. pneumoniae to the recipient strain Escherichia coli J53. Genetic analysis of the complete sequence of two tet (X4)-carrying plasmids pTKPN_3-186k-tetX4 and pTKPN_8-216k-tetX4 disclosed that the tet (X4) gene was flanked by delta IS CR2 and IS 1R , which may mediate the transmission of the tet (X4) gene., Conclusion: The prevalence of tet (X4)-positive K. pneumoniae among different sources was low. IS CR2 and IS 1R may contribute to the horizontal transfer of tet (X4) gene. Effective measures should be taken to prevent the transmission of tet (X4)-producing K. pneumoniae in humans or animals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yang, He, Wu, Qin, Chen, Feng, Zhao, Xu, Guo, Tian, Dai, Yan and Qin.)

Background: P. aeruginosa, a highly virulent Gram-negative bacterium, can cause severe nosocomial infections, and it has developed resistance against most antibiotics. New therapeutic strategies are urgently needed to treat such bacterial infection and reduce its toxicity caused by endotoxin (lipopolysaccharide, LPS). Neutrophils have been proven to be able to target inflammation site and neutrophil membrane receptors such as Toll-like receptor-4 (TLR4) and CD14, and exhibit specific affinity to LPS. However, antibacterial delivery system based on the unique properties of neutrophils has not been reported., Methods: A neutrophil-inspired antibacterial delivery system for targeted photothermal treatment, stimuli-responsive antibiotic release and endotoxin neutralization is reported in this study. Specifically, the photothermal reagent indocyanine green (ICG) and antibiotic rifampicin (RIF) are co-loaded into poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP-ICG/RIF), followed by coating with neutrophil membrane to obtain antibacterial delivery system (NM-NP-ICG/RIF). The inflammation targeting properties, synergistic antibacterial activity of photothermal therapy and antibiotic treatment, and endotoxin neutralization have been studied in vitro. A P. aeruginosa-induced murine skin abscess infection model has been used to evaluate the therapeutic efficacy of the NM-NP-ICG/RIF., Results: Once irradiated by near-infrared lasers, the heat generated by NP-ICG/RIF triggers the release of RIF and ICG, resulting in a synergistic chemo-photothermal antibacterial effect against P. aeruginosa (~ 99.99% killing efficiency in 5 min). After coating with neutrophil-like cell membrane vesicles (NMVs), the nanoparticles (NM-NP-ICG/RIF) specifically bind to inflammatory vascular endothelial cells in infectious site, endowing the nanoparticles with an infection microenvironment targeting function to enhance retention time. Importantly, it is discovered for the first time that NMVs-coated nanoparticles are able to neutralize endotoxins. The P. aeruginosa murine skin abscess infection model further demonstrates the in vivo therapeutic efficacy of NM-NP-ICG/RIF., Conclusion: The neutrophil-inspired antibacterial delivery system (NM-NP-ICG/RIF) is capable of targeting infection microenvironment, neutralizing endotoxin, and eradicating bacteria through a synergistic effect of photothermal therapy and antibiotic treatment. This drug delivery system made from FDA-approved compounds provides a promising approach to fighting against hard-to-treat bacterial infections., (© 2023. The Author(s).)

The global dissemination of the mobilized colistin resistance gene, mcr-1 , threatens human health. Recent studies by our group and others have shown that the withdrawal of colistin as a feed additive dramatically reduced the prevalence of mcr-1 . Although it is accepted that the rapid reduction in mcr-1 prevalence may have resulted, to some extent, from the toxic effects of MCR-1, the detailed mechanism remains unclear. Here, we found that MCR-1 damaged the outer membrane (OM) permeability in Escherichia coli and Klebsiella pneumonia and that this event was associated with MCR-1-mediated cell shrinkage and death during the stationary phase. Notably, the capacity of MCR-1-expressing cells for recovery from the stationary phase under improved conditions was reduced in a time-dependent manner. We also showed that mutations in the potential lipid-A-binding pocket of MCR-1, but not in the catalytic domain, restored OM permeability and cell viability. During the stationary phase, PbgA, a sensor of periplasmic lipid-A and LpxC production that performed the first step in lipid-A synthesis, was reduced after MCR-1 expression, suggesting that MCR-1 disrupted lipid homeostasis. Consistent with this, the overexpression of LpxC completely reversed the MCR-1-induced OM permeability defect. We propose that MCR-1 causes lipid remodelling that results in an OM permeability defect, thus compromising the viability of Gram-negative bacteria. These findings extended our understanding of the effect of MCR-1 on bacterial physiology and provided a potential strategy for eliminating drug-resistant bacteria.

Clostridioides difficile ( C. difficile ) is a Gram-positive, spore-forming, toxin-producing, obligate anaerobic bacterium. C. difficile infection (CDI) is the leading cause of healthcare-associated infective diarrhoea. The infection is mediated by the spore, a metabolically inactive form of C. difficile . The spore coat acts as a physical barrier to defend against chemical insults from hosts and natural environments. The composition of spore coat has already been revealed; therefore, the interactive networks of spore coat proteins and the dynamic process of coat assembly are the keys to design strategies to control and cure CDI. This review gives a brief discussion of the signal processing and transcriptional regulation of C. difficile sporulation initiation. Following the discussion, the spore formation is also introduced. Finally, this review mainly focuses on the spore coat assembly, a poorly understood process in C. difficile , and important proteins that have been studied.

The oral and upper respiratory tracts are closely linked anatomically and physiologically with the lower respiratory tract and lungs, and the influence of oral and upper respiratory microbes on the lung microbiota is increasingly being recognized. However, the ecological process and individual heterogeneity of the oral and upper respiratory tract microbes shaping the lung microbiota remain unclear owing to the lack of controlled analyses with sufficient sample sizes. Here, the microbiomes of saliva, nasal cavity, oropharyngeal area, and bronchoalveolar lavage samples are profiled and the shaping process of multisource microbes on the lung microbiota is measured. It is found that oral and nasal microbial inputs jointly shape the lung microbiota by occupying different ecological niches. It is also observed that the spread of oral microbes to the lungs is heterogeneous, with more oral microbes entering the lungs being associated with decreased lung function and increased lung proinflammatory cytokines. These results depict the external shaping process of lung microbiota and indicate the great value of oral samples, such as saliva, in monitoring and assessing lung microbiota status in clinical settings., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

A sharp increase in multidrug-resistant tuberculosis (MDR-TB) threatens human health. Spontaneous mutation in essential gene confers an ability of Mycobacterium tuberculosis resistance to anti-TB drugs. However, conventional laboratory strategies for identification and prediction of the mutations in this slowly growing species remain challenging. Here, by combining XCas9 nickase and the error-prone DNA polymerase A from M . tuberculosis , we constructed a CRISPR-guided DNA polymerase system, CAMPER, for effective site-directed mutagenesis of drug-target genes in mycobacteria. CAMPER was able to generate mutagenesis of all nucleotides at user-defined loci, and its bidirectional mutagenesis at nick sites allowed editing windows with lengths up to 80 nucleotides. Mutagenesis of drug-targeted genes in Mycobacterium smegmatis and M . tuberculosis with this system significantly increased the fraction of the antibiotic-resistant bacterial population to a level approximately 60- to 120-fold higher than that in unedited cells. Moreover, this strategy could facilitate the discovery of the mutation conferring antibiotic resistance and enable a rapid verification of the growth phenotype-mutation genotype association. Our data demonstrate that CAMPER facilitates targeted mutagenesis of genomic loci and thus may be useful for broad functions such as resistance prediction and development of novel TB therapies., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Staphylococcus haemolyticus is an opportunistic pathogen associated with hospital-acquired infections. However, the genetic diversity of S. haemolyticus among the patients and the hospital environment is largely unknown. Here, we isolated 311 S. haemolyticus strains from different sampling sites of patients and hospital environment. Genomic analysis showed that ST42 is an emerging clone widely disseminated in the hospital. S. haemolyticus ST42 strains exhibited decreased susceptibilities for multiple antibiotics compared with other STs and carried significantly more antibiotic resistance genes (ARGs). Furthermore, ST42 strains harbored more virulence genes per isolate than in other STs, and the capsular biosynthesis genes cap DEFG were more prevalent in ST42 strains. Using the Galleria mellonella infection model, we demonstrated that ST42 strains are highly virulent compared with non-ST42 strains. Taken together, our data identified an emerging ST42 clone of S. haemolyticus with aggregated ARGs and virulence determinants in the hospital, representing a significant health threat in terms of both disease and treatment. IMPORTANCE S. haemolyticus is an emerging opportunistic pathogen with a high burden of antimicrobial resistance. We performed molecular epidemiological analysis of S. haemolyticus that was isolated from a hospital, and found that the phylogenetic lineages are diverse accompanied by a dominant epidemic clonal lineage ST42. We demonstrated that S. haemolyticus ST42 strains have been disseminated among patients and the hospital environment. The data provide mechanistic insight and indicate that S. haemolyticus ST42 strains are multidrug-resistance and virulent clones via accumulating more ARGs and virulence genes.

The rapid emergence of multidrug-resistant/extensively drug-resistant tuberculosis (TB) is responsible for treatment failure in patients with TB and significantly endangers global public health. Recently, bioenergetics has become a new paradigm for anti-TB drug discovery and is based on the link between bacterial ATP levels and drug efficacy. A better understanding of the role of ATP fluctuations during antibiotic treatment may provide insight into antibiotic-mediated killing of mycobacteria. Here, we employed an advanced single-fluorescence FRET (fluorescence resonance energy transfer)-based ATP biosensor, ATPser, for the stable and convenient detection of intracellular ATP fluctuations in mycobacteria. This strategy correlated closely with the results obtained from conventional luminescence ATP assays, indicating the reliability of the system for bioenergetics analysis in mycobacteria. Moreover, the reporter strains expressing ATPser displayed obvious ATP changes when subjected to different stresses, such as starvation and ATP depletion. Interestingly, we observed that different antibiotics induced fluctuations in cellular ATP levels in individual cells of various magnitudes, revealing a strong connection between ATP fluctuations and drug efficacy. Furthermore, drug combinations accelerated ATP perturbation, resulting in increased cell death. We concluded that ATPser enabled real-time measurement of ATP at the single-cell level in mycobacteria, and monitoring ATP dynamics in drug-treated bacteria may shed light on novel treatment strategies. IMPORTANCE Bioenergetics has emerged as a new paradigm for antituberculosis (anti-TB) drug discovery, and the cellular ATP level is the core indicator reflecting bacterial metabolic homeostasis. Although several bulk assays have been designed for the measurement of cellular ATP content, a more convenient strategy is required for real-time ATP measurement of single viable cells. In this study, by combining the ε-subunit of Bacillus subtilis F o F 1 -ATP synthase with a circularly permuted green fluorescent protein [(cp)GFP], we constructed a FRET-based single-fluorescence ATP sensor, ATPser, for real-time single-cell ATP detection among a mycobacterial population. Using the ATPser, we designed different drug combinations containing components that have similar/opposite effects on ATP alternation. Our results demonstrated that increased cellular ATP fluctuations were associated with depletion of mycobacterial viability, while counteracting ATP fluctuations weakened the killing effect of the drug regime. Thus, potentially efficient drug combinations can be considered based on their similar effects on mycobacterial ATP levels, and ATPser may be a useful tool to study mycobacterial bioenergetics and to guide drug regime design.

The antibiotic resistance crisis continues to threaten human health. Better predictions of the evolution of antibiotic resistance genes could contribute to the design of more sustainable treatment strategies. However, comprehensive prediction of antibiotic resistance gene evolution via laboratory approaches remains challenging. By combining site-specific integration and high-throughput sequencing, we quantified relative growth under the respective selection of cefotaxime or ceftazidime selection in ∼23,000 Escherichia coli MG1655 strains that each carried a unique, single-copy variant of the extended-spectrum β-lactamase gene blaCTX-M-14 at the chromosomal att HK022 site. Significant synergistic pleiotropy was observed within four subgenic regions, suggesting key regions for the evolution of resistance to both antibiotics. Moreover, we propose PEARP and PEARR, two deep-learning models with strong clinical correlations, for the prospective and retrospective prediction of blaCTX-M-14 evolution, respectively. Single to quintuple mutations of blaCTX-M-14 predicted to confer resistance by PEARP were significantly enriched among the clinical isolates harboring blaCTX-M-14 variants, and the PEARR scores matched the minimal inhibitory concentrations obtained for the 31 intermediates in all hypothetical trajectories. Altogether, we conclude that the measurement of local fitness landscape enables prediction of the evolutionary trajectories of antibiotic resistance genes, which could be useful for a broad range of clinical applications, from resistance prediction to designing novel treatment strategies., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Two tet (X4)-positive Enterobacter cloacae isolates TECL_1 and TECL_2 were isolated from pigs in China. S1-PFGE and Southern blotting showed that tet (X4) located on plasmids in the size of ∼290 kb and ∼190 kb in TECL_1 and TECL_2, respectively. Conjugation experiment demonstrated that the tet (X4)-harboring plasmid can transfer from the donor strain TECL_1 and TECL_2 to the recipient strain Escherichia coli J53, and the tigecycline resistance of transconjugants was increased by 128-fold and 64-fold compared with E. coli J53, respectively. We obtained the complete plasmid sequence of pTECL_2-190k-tetX4 (190,185 bp) from E. cloacae TECL_2 and found that the plasmid was a hybrid plasmid with replicon types of IncFIA, IncHI1A and IncHI1B. We further analyzed 85 tet (X4)-carrying plasmids in the public database and clarified that pTECL_2-190k-tetX4-like plasmid was widespread in multiple species of Enterobacteriaceae. IMPORTANCE We identified two tet (X4)-positive E. cloacae isolates, which has not been previously reported. We obtained the complete sequence of pTECL_2-190k-tetX4 and found that it was a hybrid plasmid with multiple replicon types, including IncFIA, IncHI1A and IncHI1B. By comparing all the known tet (X4)-carrying plasmids, we found that pTECL_2-190k-tetX4-like plasmid has been disseminated across various species in China. Our study expanded the identification of tet (X4)-positive species and emphasized that pTECL_2-190k-tetX4-like plasmid has spread widely in various species.

Clostridioides difficile, which causes life-threatening diarrheal disease, presents an urgent threat to health care systems. In this study, we present a retrospective genomic and epidemiological analysis of C. difficile in a large teaching hospital. First, we collected 894 nonduplicate fecal samples from patients during a whole year to elucidate the C. difficile molecular epidemiology. We then presented a detailed description of the population structure of C. difficile based on 270 isolates separated between 2015 and 2020 and clarified the genetic and phenotypic features by MIC and whole-genome sequencing. We observed a high carriage rate (19.4%, 173/894) of C. difficile among patients in this hospital. The population structure of C. difficile was diverse with a total of 36 distinct STs assigned. In total, 64.8% (175/270) of the isolates were toxigenic, including four CDT-positive (C. difficile transferase) isolates, and 50.4% (135/268) of the isolates were multidrug-resistant. Statistically, the rates of resistance to erythromycin, moxifloxacin, and rifaximin were higher for nontoxigenic isolates. Although no vancomycin-resistant isolates were detected, the MIC for vancomycin was higher for toxigenic isolates ( P < 0.01). The in-hospital transmission was observed, with 43.8% (110/251) of isolates being genetically linked to a prior case. However, no strong correlation was detected between the genetic linkage and epidemiological linkage. Asymptomatic colonized patients play the same role in nosocomial transmission as infected patients, raising the issue of routine screening of C. difficile on admission. This work provides an in-depth description of C. difficile in a hospital setting and paves the way for better surveillance and effective prevention of related diseases in China. IMPORTANCE Clostridioides difficile infections (CDI) are the leading cause of healthcare-associated diarrhea and are known to be resistant to multiple antibiotics. In the past decade, C. difficile has emerged rapidly and has spread globally, causing great concern among American and European countries. However, research on CDI remains limited in China. Here, we characterized the comprehensive spectrum of C. difficile by whole-genome sequencing (WGS) in a Chinese hospital, showing a high detection rate among patients, diverse genome characteristics, a high level of antibiotic resistance, and an unknown nosocomial transmission risk of C. difficile. During the study period, two C. difficile transferase (CDT)-positive isolates belonging to a new multilocus sequence type (ST820) were detected, which have caused serious clinical symptoms. This work describes C. difficile integrally and provides new insight into C. difficile surveillance based on WGS in China.

Background: Mobile colistin resistance like gene (mcr-like gene) is a new type of polymyxin resistance gene that can be horizontally transferred in the Enterobacteriaceae. This has brought great challenges to the treatment of multidrug-resistant Escherichia coli and K. pneumoniae., Results: K. pneumoniae 16BU137 and E. coli 17MR471 were isolated from the bus and subway handrails in Guangzhou, China. K. pneumoniae 19PDR22 and KP20191015 were isolated from patients with urinary tract infection and severe pneumonia in Anhui, China. Sequence analysis indicated that the mcr-1.1 gene was present on the chromosome of E. coli 17MR471, and the gene was in the gene cassette containing pap2 and two copies of ISApl1.The mcr-1.1 was found in the putative IncX4 type plasmid p16BU137&#95;mcr-1.1 of K. pneumoniae 16BU137, but ISApl1 was not found in its flanking sequence. Mcr-8 variants were found in the putative IncFIB/ IncFII plasmid pKP20191015&#95;mcr-8 of K. pneumoniae KP20191015 and flanked by ISEcl1 and ISKpn26., Conclusion: This study provides timely information on Enterobacteriaceae bacteria carrying mcr-like genes, and provides a reference for studying the spread of mcr-1 in China and globally., (© 2022. The Author(s).)

Background: Carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP) causes serious infections with significant morbidity and mortality. However, the epidemiology and transmission mechanisms of CR-hvKP and the corresponding carbapenem-resistant plasmids require further investigation. Herein, we have characterized an ST11 K. pneumoniae strain EBSI041 from the blood sample encoding both hypervirulence and carbapenem resistance phenotypes from a patient in Egypt., Results: K. pneumoniae strain EBSI041 showed multidrug-resistance phenotypes, where it was highly resistant to almost all tested antibiotics including carbapenems. And hypervirulence phenotypes of EBSI041 was confirmed by the model of Galleria mellonella infection. Whole-genome sequencing analysis showed that the hybrid plasmid pEBSI041-1 carried a set of virulence factors rmpA, rmpA2, iucABCD and iutA, and six resistance genes aph(3')-VI, armA, msr(E), mph(E), qnrS, and sul2. Besides, bla OXA-48 and bla SHV-12 were harboured in a novel conjugative IncL-type plasmid pEBSI041-2. The bla KPC-2 -carrying plasmid pEBSI041-3, a non-conjugative plasmid lacking the conjugative transfer genes, could be transferred with the help of pEBSI041-2, and the two plasmids could fuse into a new plasmid during co-transfer. Moreover, the emergence of the p16HN-263&#95;KPC-like plasmids is likely due to the integration of pEBSI041-3 and pEBSI041-4 via IS26-mediated rearrangement., Conclusion: To the best of our knowledge, this is the first report on the complete genome sequence of KPC-2- and OXA-48-coproducing hypervirulent K. pneumoniae from Egypt. These results give new insights into the adaptation and evolution of K. pneumoniae during nosocomial infections., (© 2021. The Author(s).)

The phenotypic consequence of a genetic mutation depends on many factors including the expression level of a gene. However, a comprehensive quantification of this expression effect is still lacking, as is a further general mechanistic understanding of the effect. Here, we measured the fitness effect of almost all (>97.5%) single-nucleotide mutations in GFP, an exogenous gene with no physiological function, and URA3, a conditionally essential gene. Both genes were driven by two promoters whose expression levels differed by around tenfold. The resulting fitness landscapes revealed that the fitness effects of at least 42% of all single-nucleotide mutations within the genes were expression dependent. Although only a small fraction of variation in fitness effects among different mutations can be explained by biophysical properties of the protein and messenger RNA of the gene, our analyses revealed that the avoidance of stochastic molecular errors generally underlies the expression dependency of mutational effects and suggested protein misfolding as the most important type of molecular error among those examined. Our results therefore directly explained the slower evolution of highly expressed genes and highlighted cytotoxicity due to stochastic molecular errors as a non-negligible component for understanding the phenotypic consequence of mutations., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Type I and type II CRISPR-Cas systems are employed to evade host immunity by targeting interference of bacteria's own genes. Although Mycobacterium tuberculosis ( M. tuberculosis ), the causative agent of tuberculosis, possesses integrated type III-A CRISPR-Cas system, its role in mycobacteria remains obscure. Here, we observed that seven cas genes ( csm2 ∼ 5 , cas10 , cas6 ) were upregulated in Mycobacterium bovis BCG under oxidative stress treatment, indicating the role of type III-A CRISPR-Cas system in oxidative stress. To explore the functional role of type III-A CRISPR-Cas system, TCC ( T ype III-A C RISPR -C as system, including cas6 , cas10 , and csm2 - 6 ) mutant was generated. Deletion of TCC results in increased sensitivity in response to hydrogen peroxide and reduced cell envelope integrity. Analysis of RNA-seq dataset revealed that TCC impacted on the oxidation-reduction process and the composition of cell wall which is essential for mycobacterial envelop integrity. Moreover, disrupting TCC led to poor intracellular survival in vivo and in vitro . Finally, we showed for the first time that TCC contributed to the regulation of regulatory T cell population, supporting a role of TCC in modulating host immunity. Our finding reveals the important role of TCC in cell envelop homeostasis. Our work also highlights type III-A CRISPR-Cas system as an important factor for intracellular survival and host immunoregulation in mycobacteria, thus may be a potential target for therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yang, Xu, Liang, Liang, Li, Lin, Dai, Zhou, Li, Chen, Zhao, Tian and Feng.)

Bloodstream infections (BSIs) caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are potentially life-threatening and an urgent threat to public health. The present study aims to clarify the characteristics of carbapenemase-encoding and virulent plasmids, and their interactions with the host bacterium. A total of 425 Kp isolates were collected from the blood of BSI patients from nine Chinese hospitals, between 2005 and 2019. Integrated epidemiological and genomic data showed that ST11 and ST307 Kp isolates were associated with nosocomial outbreak and transmission. Comparative analysis of 147 Kp genomes and 39 completely assembled chromosomes revealed extensive interruption of acrR by IS Kpn26 in all Kp carbapenemase-2 (KPC-2)-producing ST11 Kp isolates, leading to activation of the AcrAB-Tolc multidrug efflux pump and a subsequent reduction in susceptibility to the last-resort antibiotic tigecycline and six other antibiotics. We described 29 KPC-2 plasmids showing diverse structures, two virulence plasmids in two KPC-2-producing Kp , and two novel multidrug-resistant (MDR)-virulent plasmids. This study revealed a multifactorial impact of KPC-2 plasmid on Kp , which may be associated with nosocomial dissemination of MDR isolates.

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.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and analysis of the plasmids associated with carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) in Egypt have not been presented. Therefore, we attempted to decipher the plasmid sequences that are responsible for transferring the determinants of carbapenem resistance, particularly bla NDM-1 and bla KPC-2 Out of 34 K. pneumoniae isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383 and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed by Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as a CR-HvKP strain: it harbored four plasmids, namely, pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes bla NDM-1 and bla KPC-2 were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including the regulator of the mucoid phenotype ( rmpA ), the regulator of mucoid phenotype 2 ( rmpA2 ), and aerobactin ( iucABCD and iutA ). Thus, we set out in this study to analyze in depth the genetic basis of the pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We report a high-risk clone ST11 KL47 serotype of a CR-HvKP strain isolated from the blood of a 60-year-old hospitalized female patient from the intensive care unit (ICU) in a tertiary care hospital in Egypt, which showed the cohabitation of a novel hybrid plasmid coharboring the bla NDM-1 and virulence genes and a bla KPC-2 -carrying plasmid. IMPORTANCE CRKP has been registered in the critical priority tier by the World Health Organization and has become a significant menace to public health. The emergence of CR-HvKP is of great concern in terms of both disease and treatment. In-depth analysis of the carbapenemase-encoding and virulence plasmids may provide insight into ongoing recombination and evolution of virulence and multidrug resistance in K. pneumoniae Thus, this study serves to alert contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals., (Copyright © 2021 Ahmed et al.)

Background: Previous studies reported the prevalence of mcr-1 among clinical infected Salmonella isolates in China. However, the transmission dynamics of mcr-1 in different ecological niches were not well investigated. Our objective is to exhibit the transmission dynamics of mcr-1 in Salmonella., Methods: 598 Salmonella isolates were recovered from ten hospitals; besides 936 pig faces and 167 pork samples were collected from January 2015 to December 2017 in Guangzhou, China. PCR and sequencing were used to identify mcr-1-positive Salmonella. Antimicrobial susceptibility testing was performed with 16 antimicrobials. Conjugation, S1-PFGE, and Southern blot were used to determine the transferability and location of mcr-1. Whole-genome sequencing was used to investigate pangenome, phylogeny, plasmid, and transposon., Results: Eleven mcr-1-positive Salmonella isolates were identified from patients with infectious diarrhea. Five pig fecal samples and three pork samples contained mcr-1-positive Salmonella isolates. All isolates were multi-drug resistant. The mcr-1 genes were located on ∼210-250 kb IncHI2-pST3 plasmids, and 12 mcr-1 genes were transferable. All isolates were assigned to ST34 or its genetically closed STs. The distribution of the core-genome network was significantly correlated with source distributions. The accessory genes-based network demonstrated that the diverse clonal complexes could share highly similar accessory genomes., Conclusions: The prevalence of mcr-1-positive Salmonella among different sources was low. Clonal transmission could not be the main reason for the expansion of mcr-1-positive Salmonella, but be attributed to the horizontal transfer of IncHI2-pST3 plasmid. Continuous surveillance on Salmonella should be performed to investigate the response of colistin banning in food-producing animals by mcr-1-positive Salmonella populations., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Genome scale mutagenesis identifies many genes required for mycobacterial infectivity and survival, but their contributions and mechanisms of action within the host are poorly understood. Using CRISPR interference, we created a knockdown of ppe31 Mm gene in Mycobacterium marinum ( M. marinum ), which reduced the resistance to acid medium. To further explore the function of PPE31, the ppe31 mutant strain was generated in M. marinum and Mycobacterium tuberculosis ( M. tuberculosis ), respectively. Macrophages infected with the ppe31 Mm mutant strain caused a reduced inflammatory mediator expressions. In addition, macrophages infected with M. marinum Δ ppe31 Mm had decreased host cell death dependent on JNK signaling. Consistent with these results, deletion of ppe31 Mtb from M. tuberculosis increased the sensitivity to acid medium and reduced cell death in macrophages. Furthermore, we demonstrate that both ppe31 mutants from M. marinum and M. tuberculosis resulted in reduced survival in macrophages, and the survivability of M. marinum was deceased in zebrafish due to loss of ppe31 Mm . Our findings confirm that PPE31 as a virulence associated factor that modulates innate immune responses to mycobacterial infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Feng, Hong, Zhang, Li, Tian, Zhou and Huang.)

Two multidrug-resistant (MDR) mcr-1 -harboring Klebsiella pneumoniae isolates from patients with urinary tract infections and one MDR Klebsiella quasipneumoniae isolate from a patient with bloodstream infection were identified to carry tmexCD1-toprJ1 The addition of the efflux pump inhibitor reduced the tigecycline MIC against all three isolates by 8- to 16-fold. pKQBSI104-1 was transferred from K. quasipneumoniae to Escherichia coli J53 via conjugation. The tmexCD1-toprJ1 -carrying plasmids pKP15ZE495-1 (102,569 bp) and pKQBSI104-1 (121,996 bp) were completely sequenced and analyzed., (Copyright © 2021 American Society for Microbiology.)

Increasing antibiotic resistance in multidrug-resistant (MDR) Gram-negative bacteria (MDR-GNB) presents significant health problems worldwide, since the vital available and effective antibiotics, including; broad-spectrum penicillins, fluoroquinolones, aminoglycosides, and β-lactams, such as; carbapenems, monobactam, and cephalosporins; often fail to fight MDR Gram-negative pathogens as well as the absence of new antibiotics that can defeat these "superbugs". All of these has prompted the reconsideration of old drugs such as polymyxins that were reckoned too toxic for clinical use. Only two polymyxins, polymyxin E (colistin) and polymyxin B, are currently commercially available. Colistin has re-emerged as a last-hope treatment in the mid-1990s against MDR Gram-negative pathogens due to the development of extensively drug-resistant GNB. Unfortunately, rapid global resistance towards colistin has emerged following its resurgence. Different mechanisms of colistin resistance have been characterized, including intrinsic, mutational, and transferable mechanisms.In this review, we intend to discuss the progress over the last two decades in understanding the alternative colistin mechanisms of action and different strategies used by bacteria to develop resistance against colistin, besides providing an update about what is previously recognized and what is novel concerning colistin resistance.

Aeromonas dhakensis is an important ubiquitous Gram-negative and freshwater bacterium detected in different reservoirs. It can cause invasive diseases in humans. Herein, we report the first case in Mainland China of a fulminant death of a 29-year-old man as a result of a new, unexpected association between septicemic A. dhakensis and hepatitis B viral infection (HBV). Herein, the patient died from multiple organ failure 5 d postadmission after the ingestion of Snakehead Fish meal. The isolated bacterium was initially misidentified as Aeromonas hydrophila using VITEK-2, while whole-genome sequencing (WGS) revealed that the isolate is A. dhakensis . WGS revealed the occurrence of three antimicrobial genes of resistance: imiH , cphA2 , and bla OXA-12 ; besides, major virulence factors were detected. In silico , multilocus sequence typing (MLST) showed that our A. dhakensis 17FW001 belonged to a novel sequence type (ST557). A comparative genomic analysis of our isolate with nine selected Aeromonas species was done, which elucidated the pathogenicity of our A. dhakensis . In conclusion, we reported for the first time the association between A. dhakensis and HBV in Mainland China. We revealed that septicemic A. dhakensis could result in severe adverse clinical outcomes that end up with unexpected fulminant death especially when it is accompanied with HBV and sheds light on the virulence of A. dhakensis and the high rate of its misdiagnosis that requires to urgently consider screening of all cases of A. dhakensis for HBV in the future. Besides, caution should be taken while dealing with snakeheads which act as a vector for A. dhakensis .

The emergence and transmission of the mobile colistin resistance gene (mcr-1) threatened the extensive use of polymyxin antimicrobials. Accumulated evidence showed that the banning of colistin additive in livestock feed efficiently reduce mcr-1 prevalence, not only in animals but also in humans and environments. However, our previous study has revealed that a small proportion of Escherichia coli could continually carry chromosomally-encoded mcr-1. The chromosomally-encoded events, indicated the existence of stabilized heritage of mcr-1 and revealed a potential threat in the antimicrobial stewardship interventions, are yet to be investigated. In this study, we systematically investigated the genetic basis of chromosomally-encoded mcr-1 in prevalence and potential mechanisms of lineage, plasmid, insertion sequence, and phage. Our results demonstrated that the emergence of chromosomally-encoded mcr-1 could originate from multiple mechanisms, but mainly derived through the recombination of ISApl1/Tn6330. We reported a specific transmission mechanism, which is a phage-like region without lysogenic components, could associate with the emergence and stabilization of chromosomally-encoded mcr-1. These results highlighted the potential origin and risks of chromosomally-encoded mcr-1, which could be a heritable repository and thrive again when confronted with new selective pressures. To the best of our knowledge, this is the first study to systematically reveal the genomic basis of chromosomally-encoded mcr-1, and report a specific transmission pattern involved in phage-like region. Overall, we demonstrate the origin mechanisms and risks of chromosomally-encoded mcr-1. It highlights the need of public attention on chromosome-encoded mcr-1 to prevent from its reemergence.

Background: Bloodstream infection (BSI) caused by carbapenem-resistant Enterobacteriaceae are potentially life-threatening related to poorer outcomes. Colistin is considered one of the last-resort treatments against human infections caused by multidrug-resistant (MDR) Gram-negative bacteria. Therefore, emergence of strains from the blood that co-harboring mcr and carbapenem resistance genes were considered as a serious problem., Purpose: In this study, two mcr-9 -harboring MDR Enterobacter cloacae isolates BSI034 and BSI072 recovered from BSI patients were identified, one of which co-harbored mcr-9 and bla NDM-1. The genetic characteristics of the MDR plasmid needed to be clarified., Methods: S1-PFGE and Southern blotting were conducted to determine the location of mcr-9 . Whole-genome sequencing was performed to obtain the complete genome and plasmid sequences. The resistome and virulence genes of the strains, accompanied by the genetic characteristics of mcr-9 - and bla NDM-1 -harboring plasmids, were analyzed., Results: Whole-genome sequencing showed that BSI034 harbored mcr-9 -carrying IncHI2-type pBSI034-MCR9 and bla NDM-1 -carrying IncX3-type pBSI034-NDM1. The 278,517 bp pBSI034-MCR9 carried mcr-9 along with the other 19 resistance genes. mcr-9 was flanked by IS 903B (1057 bp) and IS 26 (820 bp) in the same orientation. In addition to resistance genes, strain BSI034 also carried a chromosome-located Yersinia high-pathogenicity island, which harbored genes of yersiniabactin biosynthesis operon ybtSXQPAUTE, irp1/2 , and fyuA ., Conclusion: We described the complete genome and mcr-9 / bla NDM-1 -co-harboring plasmid of E. cloacae from a BSI patient. Notable differences were observed within mosaic modules between pBSI034-MCR9 and other mcr-9 -harboring plasmids due to extensive recombination via horizontal gene transfer., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Lin et al.)

Background: The global dissemination of colistin resistance encoded by mcr-1 has been attributed to extensive use of colistin in livestock, threatening colistin efficacy in medicine. The emergence of mcr-1 in common pathogens, such as Escherichia coli, is of particular concern. China banned the use of colistin in animal feed from May 1, 2017. We investigated subsequent changes in mcr-1 prevalence in animals, humans, food, and the environment, and the genomic epidemiology of mcr-1-positive E coli (MCRPEC)., Methods: Sampling was done before (October to December, 2016) and after (October to December, 2017, and 2018, respectively) the colistin ban. 3675 non-duplicate pig faecal samples were collected from 14 provinces (66 farms) in China to measure intervention-related changes in mcr-1 prevalence. 15 193 samples were collected from pigs, healthy human volunteers, patients colonised or infected with Enterobacteriaceae who were admitted to hospital, food and the environment in Guangzhou, to characterise source-specific mcr-1 prevalence and the wider ecological effect of the ban. From these samples, 688 MCRPEC were analysed with whole genome sequencing, plasmid conjugation, and S1 pulsed-field gel electrophoresis with Southern blots to characterise associated genomic changes., Findings: After the ban, mcr-1 prevalence decreased significantly in national pig farms, from 308 (45%) of 684 samples in 2016 to 274 (19%) of 1416 samples in 2018 (p<0·0001). A similar decrease occurred in samples from most sources in Guangzhou (959 [19%] of 5003 samples in 2016; 238 [5%] of 4489 samples in 2018; p<0·0001). The population structure of MCRPEC was diverse (23 sequence clusters); sequence type 10 clonal complex isolates were predominant (247 [36%] of 688). MCRPEC causing infection in patients admitted to hospital were genetically more distinct and appeared less affected by the ban. mcr-1 was predominantly found on plasmids (632 [92%] of 688). Common mcr-1 plasmid types included IncX4, IncI2, and IncHI2 (502 [76%] of 656); significant increases in IncI2-associated mcr-1 and a distinct lineage of mcr-1-associated IncHI2 were observed post ban. Changes in the frequency of mcr-1-associated flanking sequences (ISApl1-negative MCRPEC), 63 core genome single nucleotide polymorphisms, and 30 accessory genes were also significantly different after the ban (Benjamini-Hochberg-adjusted p<0·05), consistent with rapid genetic adaptation in response to changing selection pressures., Interpretation: A rapid, ecosystem-wide, decline in mcr-1 was observed after the use of colistin in animal feed was banned, with associated genetic changes in MCRPEC. Withdrawal of antimicrobials from animal feed should be an important One Health measure contributing to the wider control of antimicrobial resistance globally., Funding: National Natural Science Foundation of China., (Copyright © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

There is growing evidence that GreA aids adaptation to stressful environments in various bacteria. However, the functions of GreA among mycobacteria remain obscure. Here, we report on cellular consequences following deletion of greA gene in Mycobacterium spp. The greA mutant strain (Δ greA ) was generated in Mycobacterium smegmatis , Mycobacterium tuberculosis (MTB) H37Ra, and M. tuberculosis H37Rv. Deletion of greA results in growth retardation and poor survival in response to adverse stress, besides rendering M. tuberculosis more susceptible to vancomycin and rifampicin. By using RNA-seq, we observe that disrupting greA results in the differential regulation of 195 genes in M. smegmatis with 167 being negatively regulated. Among these, KEGG pathways significantly enriched for differentially regulated genes included tryptophan metabolism, starch and sucrose metabolism, and carotenoid biosynthesis, supporting a role of GreA in the metabolic regulation of mycobacteria. Moreover, like Escherichia coli GreA, M. smegmatis GreA exhibits a series of conservative features, and the anti-backtracking activity of C-terminal domain is indispensable for the expression of glgX , a gene was down-regulated in the RNA-seq data. Interestingly, the decrease in the expression of glgX by CRISPR interference, resulted in reduced growth. Finally, intracellular fitness significantly declines due to loss of greA . Our data indicates that GreA is an important factor for the survival and resistance establishment in Mycobacterium spp. This study provides new insight into GreA as a potential target in multi-drug resistant TB treatment., (Copyright © 2020 Feng, Liu, Liang, El-Sayed Ahmed, Zhao, Shen, Roberts, Liang, Liao, Zhong, Guo, Yang, Wen, Chen and Tian.)