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Background: Carbapenemase-producing Vibrio spp., which exhibit an XDR phenotype, have become increasingly prevalent and pose a severe threat to public health.Objectives: To investigate the genetic characteristics of NDM-1-producing Vibrio spp. isolates and the dissemination mechanisms of blaNDM-1 in Vibrio.Methods: A total of 1363 non-duplicate Vibrio spp. isolates collected from shrimp samples in China were subjected to antimicrobial susceptibility tests and screened for blaNDM-1. The blaNDM-1-positive isolates were further characterized by PFGE, MLST, conjugation and WGS using Illumina and Nanopore platforms. Plasmid stability and fitness cost were assessed using Escherichia coli J53, Klebsiella pneumoniae Kpt80 and Salmonella spp. SA2051 as recipient strains.Results: In total, 13 blaNDM-1-positive isolates were identified, all exhibiting MDR. WGS analysis revealed that the 13 blaNDM-1 genes were all associated with a derivative of Tn125. Plasmid analysis revealed that six blaNDM-1 genes were located in IncC plasmids and the other seven were carried by plasmids of two different novel types. Conjugation and plasmid stability assays showed that only the IncC plasmids could be transferred to all the recipient strains and could be stably maintained in the hosts.Conclusions: The emergence of the novel plasmids has contributed to the variable genetic contexts of blaNDM-1 in Vibrio spp. and IncC plasmids harbouring the blaNDM-1 gene could facilitate the spread of such genes between Vibrio spp. and other zoonotic pathogens, leading to a rapid dissemination of blaNDM-1 in bacterial pathogens worldwide. [ABSTRACT FROM AUTHOR]

Recently, many TetX variants such as Tet(X3~14) were reported to confer resistance to tigecycline which is a last-resort antibiotic used to treat infections caused by multidrug-resistant bacteria. In this study, we identified essential residues including 329, 339, 340, 350, and 351 in TetX variants that mediated the evolution of the tigecycline-inactive Tet(X2) enzyme to the active forms of Tet(X3) and Tet(X4). Based on their amino acid sequences and functional features, we classified TetX variants into TetX-A class, TetX-B class and TetX-C class. We further found that TetX-A class variants originated from Bacteroidetes, with some variants further evolving to TetX-C class and acquired by Enterobacteriaceae. On the other hand, our data showed that some variants genes belonging to TetX-A class evolved directly to TetX-B class, which was further transmitted to Acinetobacter spp. This new classification system may facilitate better clinical management of patients infected by TetX-producing strains. TetX variants such as Tet(X3~14) were reported to confer resistance to tigecycline which is a last-resort antibiotic used to treat infections caused by multidrug-resistant bacteria. Here, Cheng et al. propose a new classification system for TetX variants. [ABSTRACT FROM AUTHOR]

Objectives: Bacterial antibiotic tolerance is responsible for the recalcitrance of chronic infections. This study aims to investigate a potential drug that can effectively kill antibiotic-tolerant bacteria and evaluate the ability of this drug on the eradication of tolerant cells both in vitro and in vivo.Methods: The in vitro effect of econazole on eradicating starvation-induced tolerant bacterial populations was studied by testing the amount of survival bacteria in the presence of econazole combining conventional antibiotics. Proton motive force (PMF) was determined after econazole treatment by DiOC2(3). Finally, mouse infection models were used to detect the ability of econazole on killing the tolerant populations in vivo.Results: Econazole eradicated starvation-induced tolerant cells of various bacterial species within 24 or 96 h when used in combination with conventional antibiotics. Moreover, mouse survival rate drastically increased along with the decrease of in vivo bacterial count after treatment of infected mice with the econazole and ceftazidime combination for 72 h. PMF was found to have dissipated almost completely in econazole-treated cells.Conclusions: Econazole could act in combination with conventional antibiotics to effectively eradicate bacterial tolerant cells. The combined use of econazole and ceftazidime was shown to be effective for eradicating tolerant cells in a mouse infection model. The ability of econazole to eradicate tolerant cells was due to its ability to cause dissipation of bacterial transmembrane PMF. Econazole-mediated PMF disruption is a feasible strategy for the treatment of chronic and recurrent bacterial infections. [ABSTRACT FROM AUTHOR]

Background: Tigecycline is a tetracycline derivative that constitutes one of the last-resort antibiotics used clinically to treat infections caused by both multiple drug-resistant (MDR) Gram-negative and Gram-positive bacteria. Resistance to this drug is often caused by chromosome-encoding mechanisms including over-expression of efflux pumps and ribosome protection. However, a number of variants of the flavin adenine dinucleotide (FAD)-dependent monooxygenase TetX, such as Tet(X4), emerged in recent years as conferring resistance to tigecycline in strains of Enterobacteriaceae, Acinetobacter sp., Pseudomonas sp., and Empedobacter sp. To date, mechanistic details underlying the improvement of catalytic activities of new TetX enzymes are not available. Results: In this study, we found that Tet(X4) exhibited higher affinity and catalytic efficiency toward tigecycline when compared to Tet(X2), resulting in the expression of phenotypic tigecycline resistance in E. coli strains bearing the tet(X4) gene. Comparison between the structures of Tet(X4) and Tet(X4)-tigecycline complex and those of Tet(X2) showed that they shared an identical FAD-binding site and that the FAD and tigecycline adopted similar conformation in the catalytic pocket. Although the amino acid changes in Tet(X4) are not pivotal residues for FAD binding and substrate recognition, such substitutions caused the refolding of several alpha helixes and beta sheets in the secondary structure of the substrate-binding domain of Tet(X4), resulting in the formation of a larger number of loops in the structure. These changes in turn render the substrate-binding domain of Tet(X4) more flexible and efficient in capturing substrate molecules, thereby improving catalytic efficiency. Conclusions: Our works provide a better understanding of the molecular recognition of tigecycline by the TetX enzymes; these findings can help guide the rational design of the next-generation tetracycline antibiotics that can resist inactivation of the TetX variants. [ABSTRACT FROM AUTHOR]

Recent evidence suggests that metabolic shutdown alone does not fully explain how bacteria exhibit phenotypic antibiotic tolerance. In an attempt to investigate the range of starvation-induced physiological responses underlying tolerance development, we found that active maintenance of the transmembrane proton motive force (PMF) is essential for prolonged expression of antibiotic tolerance in bacteria. Eradication of tolerant sub-population could be achieved by disruption of PMF using the ionophore CCCP, or through suppression of PMF maintenance mechanisms by simultaneous inhibition of the phage shock protein (Psp) response and electron transport chain (ETC) complex activities. We consider disruption of bacterial PMF a feasible strategy for treatment of chronic and recurrent bacterial infections. The proton motive force (PMF) is a critical electrochemical gradient involved in bacterial energy metabolism. Here, Miaomiao Wang, Edward Wai Chi Chan et al. demonstrate that the PMF plays a key role in starvation-induced antibiotic tolerance in E. coli, suggesting that PMF disruption could be a future strategy to treat bacterial infection. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the genomic and phenotypic characteristics of an MDR Empedobacter falsenii strain isolated from a Chinese patient, which was phenotypically resistant to all last-line antibiotics (carbapenems, colistin and tigecycline).Methods: Species identity was determined by MALDI-TOF MS analysis. The complete genome sequence of the isolate was determined by WGS and the genetic elements conferring antimicrobial resistance were determined. The origin of this strain was tracked by phylogenetic analysis.Results: The E. falsenii strain was genetically most closely related to an Empedobacter sp. strain isolated from the USA. Members of E. falsenii are speculated to be intrinsically resistant to colistin. The carbapenem resistance of this strain was conferred by a chromosomal blaEBR-2 variant gene. Phylogenetic analysis indicated that the gene encoding the EBR β-lactamase was widely distributed in Empedobacter spp. Tigecycline resistance was mediated by a tet(X) variant gene encoded by a non-conjugative and non-typeable plasmid.Conclusions: The MDR phenotype of the E. falsenii isolate was conferred by different mechanisms. Findings from us and others indicate that E. falsenii may serve as a reservoir for carbapenem and tigecycline resistance determinants. [ABSTRACT FROM AUTHOR]

Objectives: To test whether antibiotics of different functional categories exhibit differential potential in promoting transmission of MDR-encoding plasmids among members of the gut microbiome.Methods: Rats inoculated with blaNDM-1-bearing Klebsiella pneumoniae were subjected to treatment with different types of antibiotics. The structural changes in the gastrointestinal (GI) tract microbiome were determined by 16S rRNA sequencing and analysis. In addition, the efficiency of transmission of blaNDM-1-bearing plasmids to different subtypes of GI tract Escherichia coli was also confirmed in vitro.Results: We showed that drugs that are commonly used to treat Gram-negative bacterial infections, such as ampicillin and amoxicillin, could enrich both carbapenem-resistant Enterobacteriaceae (CRE) and antibiotic-susceptible E. coli in the GI tract, thereby promoting transmission of the blaNDM-1-bearing plasmid in the gut microbiome. In contrast, meropenem was found to minimize the population of CRE in the gut microbiome, hence treatment with this drug exhibited drastically lower potential to promote transmission of the blaNDM-1-bearing plasmid to the recipient strains. We further showed that an increased population size of Proteobacteria due to a suppressive effect on Firmicutes is a key factor in enhancing the efficiency of transmission of the blaNDM-1-bearing plasmid and hence dissemination of carbapenem-resistant strains.Conclusions: This study depicted for the first time the effect of different antibiotics on the structure of the rat GI tract microbiome, which in turn determined the pattern and rate of transmission of the blaNDM-1-bearing plasmid. Such findings can help establish new guidelines for prudent antibiotic usage to minimize the chance of dissemination of mobile resistance elements among members of the GI tract microbiome. [ABSTRACT FROM AUTHOR]

Background: ISCR1 is an important mobile genetic element mediating the transfer of antibiotic resistance genes. Genetic diversity regarding distribution and copy numbers of ISCR1 within a bacterial population derived from an ancestral strain, which may reflect the degree of genetic plasticity conferred by such an element, has not been studied.Objectives: To investigate the plasmid heterogeneity in Salmonella conferred by ISCR1.Methods: Nanopore long-read and other sequencing technologies were used to resolve the structures harbouring different copies of ISCR1-qnrB6 from the perspective of single molecules.Results: Salmonella London Sa128 was positive for ISCR1-qnrB6 and harboured an MDR-encoding conjugative IncF plasmid, pSa128, containing a complex class 1 integron. The plasmid pSa128T from the transconjugant was larger compared with the original plasmid pSa128, presumably due to amplification of ISCR1-qnrB6. Single-molecule, long-read analysis indicated that both plasmids in the donor and transconjugant strains were in a heterogeneous state that contains variable numbers of ISCR1-qnrB6, with four and eight copies in single plasmids being the dominant types. This type of plasmid heterogeneity in populations of one strain can be regarded as an atypical plasmidome.Conclusions: This study highlights the importance of investigation of a single plasmid structure based on long-read sequencing technologies, with a focus on analysing the complex structures of the MDR region, which is expected to exhibit genetic polymorphism or plasmid heterogeneity in various MDR-encoding elements even among members of the same strain. The availability of a single-molecule sequencing technique represents a paradigm shift in the capability of performing population genetic analysis of antibiotic-resistant organisms. [ABSTRACT FROM AUTHOR]

Objectives: To analyse and compare mcr-1-bearing plasmids from animal Escherichia coli isolates, and to investigate potential mechanisms underlying dissemination of mcr-1.Methods: Ninety-seven ESBL-producing E. coli strains isolated from pig farms in China were screened for the mcr-1 gene. Fifteen mcr-1-positive strains were subjected to molecular characterization and bioinformatic analysis of the mcr-1-bearing plasmids that they harboured.Results: Three major types of mcr-1-bearing plasmids were recovered: IncX4 (∼33 kb), IncI2 (∼60 kb) and IncHI2 (∼216-280 kb), among which the IncX4 and IncI2 plasmids were found to harbour the mcr-1 gene only, whereas multiple resistance elements including blaCTX-M, blaCMY, blaTEM, fosA, qnrS, floR and oqxAB were detected, in various combinations, alongside mcr-1 in the IncHI2 plasmids. The profiles of mcr-1-bearing plasmids in the test strains were highly variable, with coexistence of two mcr-1-bearing plasmids being common. However, the MIC of colistin was not affected by the number of mcr-1-carrying plasmids harboured. Comparative analysis of the plasmids showed that they contained an mcr-1 gene cassette with varied structures (mcr-1-orf, ISApl1-mcr-1-orf and Tn6330), with the IncHI2 type being the most active in acquiring foreign resistance genes. A novel transposon, Tn6330, with the structure ISApl1-mcr-1-orf-ISApl1 was found to be the key element mediating translocation of mcr-1 into various plasmid backbones through formation of a circular intermediate.Conclusions: The mcr-1 gene can be disseminated via multiple mobile elements including Tn6330, its circular intermediate and plasmids harbouring such elements. It is often co-transmitted with other resistance determinants through IncHI2 plasmids. The functional mechanism of Tn6330, a typical composite transposon harbouring mcr-1, should be further investigated. [ABSTRACT FROM AUTHOR]

Emergence of tigecycline-resistance tet(X) gene orthologues rendered tigecycline ineffective as last-resort antibiotic. To understand the potential origin and transmission mechanisms of these genes, we survey the prevalence of tet(X) and its orthologues in 2997 clinical E. coli and K. pneumoniae isolates collected nationwide in China with results showing very low prevalence on these two types of strains, 0.32% and 0%, respectively. Further surveillance of tet(X) orthologues in 3692 different clinical Gram-negative bacterial strains collected during 1994–2019 in hospitals in Zhejiang province, China reveals 106 (2.7%) tet(X)-bearing strains with Flavobacteriaceae being the dominant (97/376, 25.8%) bacteria. In addition, tet(X)s are found to be predominantly located on the chromosomes of Flavobacteriaceae and share similar GC-content as Flavobacteriaceae. It also further evolves into different orthologues and transmits among different species. Data from this work suggest that Flavobacteriaceae could be the potential ancestral source of the tigecycline resistance gene tet(X). Emergence of tigecycline-resistance tet(X) genes is of concern. Here, the authors determine tet(X) prevalence in more than 6,000 clinical Gram-negative bacterial isolates collected between 1994 to 2019 in hospitals in China and suggest that Flavobacteriaceae could be the potential ancestral source of the tigecycline resistance genes. [ABSTRACT FROM AUTHOR]

Multidrug resistant (MDR) Escherichia coli poses great threat to human and animal health. To investigate the genetic characteristics of conjugative plasmids that encode ciprofloxacin, carbapenem and colistin resistance in a MDR E. coli strain (974) isolated from pork samples in China, the E. coli strain 974 was subjected to conjugation study, S1-PFGE, Southern hybridization and was sequenced by Illumina Nextseq 500 and MinION Platforms. The ciprofloxacin-resistance-encoding plasmid, p974-IncF, was found to harbor multiple mobile elements that in turn contain genes resistant to β-lactams, quinolone, florephenicol, tetracycline, aminoglycosides, sulfonamides, streptomycin, trimethoprim and chloramphenicol. The backbone of bla NDM-1 -bearing plasmid, p974-NDM, was found consist of a 17.3kb resistance region and a 56.7kb of transfer region. However, the classical IS CR1 mobile element and class I integron were missing in plasmid p974-NDM. Resembling the backbone of other IncI2 plasmids, the mcr-1 -bearing plasmid, p974-MCR, contained genes that encode replication function, plasmid stability, proteins related to type IV secretion complex, as well as two pilus-encoding genes, which are conserved among all IncI2 plasmids. Enhanced surveillance efforts and limitation of usage of antibiotics are urgently warranted to control the dissemination of these resistance determinants. • Escherichia coli strain 974 was resistant to multiple antibiotics. • Plasmid p974-IncF harbored four representative genes resistant to ciprofloxacin. • Classical IS CR1 mobile element and class I integron were missing in plasmid p974-NDM. • Plasmid p974-MCR harbored a gene resistance to colistin but lacked IS Apl1 element. [ABSTRACT FROM AUTHOR]