Search

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]

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 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]

BoNT/F7, one of the seven subtypes of botulinum neurotoxin type F (F1 to F7), is the second-most divergent subtype of this group. Despite sharing >60% identity with BoNT/F1 at both holotoxin and enzymatic domain levels, it requires an N-terminal extended peptide substrate for efficient substrate cleavage, suggesting its unique substrate recognition and specificity mechanism. Substrate mapping and saturation mutagenesis analysis revealed that VAMP2 (20–65) was likely a minimally effective substrate for LC/F7 (light chain of BoNT/F7), and in addition, LC/F7 recognized VAMP2 in a unique way, which differed significantly from that of LC/F1, although both of them share similar substrate binding and hydrolysis mode. LC/F7 utilizes distinct pockets for specific substrate binding and recognition in particular for the B1, B2 and S2 sites recognitions. Our findings provide insights into the distinct substrate recognition features of BoNT subtypes and useful information for therapy development for BoNT/F. [ABSTRACT FROM AUTHOR]

The erm (T) gene encodes the 23 s rRNA methyltransferase and confers erythromycin resistance in Gram-positive bacteria, while has rarely been identified in Gram-negative bacteria. In this study, we identified a small IncQ1 plasmid of 6135 bp harboring the erm (T) gene in a clinical K. pneumoniae strain and confirmed the role of the erm (T) gene in mediating azithromycin resistance. This plasmid was found to be generated by incorporating the erm (T) gene from mobile elements into an IncQ1 plasmid. Our data indicated the spread of the erm (T) gene from Gram-positive bacteria to Gram-negative bacteria and the clonal spread of the ST11-KL47 type K. pneumoniae strains carrying this plasmid. Generation of this kind of multi-host plasmid will promote the dissemination of the erm (T) gene among Gram-negative bacteria and result in failures of azithromycin in treating bacterial infections. [ABSTRACT FROM AUTHOR]

Hypervirulent Klebsiella pneumoniae (HvKP), which causes highly fatal infections, is a new threat to human health. In an attempt to investigate the underlying mechanisms of resistance to neutrophil-mediated killing and hence expression of high-level virulence by HvKP, we tested the binding affinity of HvKP strains to various types of human cells. Our data showed that HvKP exhibited weaker binding to both lung epithelial cells, intestinal Caco-2 cells and macrophages when compared to the classic, non-hypervirulent strains (cKP). Consistently, transconjugants that have acquired a rmpA or rmpA2-bearing plasmid were found to exhibit decreased adhesion to various types of human cells, and hence higher survival rate upon exposure to neutrophil cells. We further found that over production of hypermucoviscosity (HMV), but not capsular polysaccharide (CPS), contributed to the reduced binding and phagocytosis. The effect of hypermucoviscosity on enhancing HvKP virulence was further shown in human serum survival assays and animal experiments. Findings in this study therefore confirmed that rmpA/A2-mediated hypermucoviscosity in HvKP plays a key role in the pathogenesis of this organism through conferring the ability to evade neutrophil binding and phagocytosis. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae has an exceptional ability to acquire exogenous resistance-encoding and hypervirulence-encoding genetic elements. In this review we trace the key evolutionary routes of plasmids involved in the dissemination of such elements; we observed diverse, but convergent, evolutionary paths that eventually led to the emergence of conjugative plasmids which simultaneously encode carbapenem resistance and hypervirulence. One important evolutionary feature of these plasmids is that they contain a wide range of transposable elements that enable them to undergo frequent genetic transposition, resulting in plasmid fusion and presumably better adaptation of the plasmid to the bacterial host. Identifying the key molecular markers of resistance and virulence-bearing conjugative plasmids allows improved tracking and control of the life-threatening carbapenem-resistant and hypervirulent strains of K. pneumoniae. K. pneumoniae has become one of the most important bacterial pathogens; it causes a high mortality rate in patients. K. pneumoniae has an exceptional ability to acquire exogenous resistance-encoding and hypervirulence-encoding genetic elements, and it has evolved rapidly in the past decade. In one evolutionary route, classical K. pneumoniae strains acquired different types of bla KPC -encoding plasmid and evolved to become carbapenem-resistant K. pneumoniae (CRKP). In another evolutionary route, classical K. pneumoniae strains have become hypervirulent K. pneumoniae (hvKP) through the acquisition of pLVPK-like virulence plasmids. One important evolutionary feature of these plasmids is that they contain a wide range of transposable elements that enable them to undergo frequent genetic transposition, resulting in plasmid fusion and presumably better adaptation of the plasmid to the bacterial host. In recent years, convergence of these two evolutionary paths has been observed in K. pneumoniae , resulting in the emergence of carbapenem-resistant and hypervirulent K. pneumoniae (CR-hvKP) due to plasmid recombination and fusion processes. Identifying the key molecular markers of resistance and virulence-bearing conjugative plasmids allows improved tracking and control of the life-threatening carbapenem-resistant and hypervirulent strains of K. pneumoniae. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the genetic features of the blaVIM-1 gene first detected in a cephalosporin-resistant Vibrio alginolyticus isolate, Vb1978.Methods: The MICs of V. alginolyticus strain Vb1978 were determined, and the β-lactamases produced were screened and analysed using conjugation, S1-PFGE and Southern blotting. The complete sequence of the blaVIM-1-encoding plasmid was also obtained using the Illumina and MinION sequencing platforms.Results: V. alginolyticus strain Vb1978, isolated from a retail shrimp sample, was resistant to cephalosporins and exhibited reduced susceptibility to carbapenems. A novel blaVIM-1-carrying conjugative plasmid, designated pVb1978, was identified in this strain. Plasmid pVb1978 had 50 001 bp and comprised 59 predicted coding sequences (CDSs). The plasmid backbone of pVb1978 was homologous to those of IncP-type plasmids, while its replication region was structurally similar to non-IncP plasmids. The blaVIM-1 gene was found to be carried by the class 1 integron In70 and associated with a defective Tn402-like transposon.Conclusions: A novel blaVIM-1-carrying conjugative plasmid, pVb1978, was reported for the first time in V. alginolyticus, which warrants further investigation in view of its potential pathogenicity towards humans and widespread occurrence in the environment. [ABSTRACT FROM AUTHOR]

Background: The mcr-1 gene has been widely reported in both bacterial chromosomes and plasmids, while its stability in these genetic materials is not well understood.Objectives: Our aim was to characterize the stability and dynamics of Tn6330 elements in both a plasmid and the chromosome in a single bacterial population.Methods: Plasmid-borne and chromosomal Tn6330 were characterized by PCR, conjugation, S1-PFGE, stability assay, single-molecule long-read sequencing and bioinformatics analysis.Results: Tn6330 was simultaneously detected in both a plasmid and the chromosome of a clinical Escherichia coli strain. The plasmid was found to comprise the IncFIB replicon and a phage-like replicon, as well as two integrons that harboured various mobile elements and resistance genes including mcr-1, floR, blaTEM-1b and strAB. Both plasmid-borne and chromosomal Tn6330 transposons could be re-organized into a circular intermediate that played a role in transmission of the mcr-1 gene. Tn6330 was found to be very stable in both the plasmid and chromosome after 30 passages of 12 h with or without colistin selective pressure. The decayed structure of Tn6330 in the genuine single DNA molecules of bacterial populations, although occurring at a very low frequency, could be detected for the first time, in which Tn6330 was degraded into a single ISApl1 element.Conclusions: Long-read sequencing technology is a good tool to study the evolution and stability of genetic elements in bacteria. The ultrastability of an mcr-1-encoding element in a bacterial plasmid and chromosome renders it unlikely to be eradicated quickly by the reduced use of colistin, and factors leading to the frequent demise of Tn6330 warrant further studies. [ABSTRACT FROM AUTHOR]

Objectives: To characterize a plasmid in a K1 hypervirulent Klebsiella pneumoniae (HvKP) strain encoding both hypervirulence and carbapenem resistance phenotypes.Methods: Plasmids from HvKP strain KP70-2 were subjected to whole-plasmid sequencing using both the Illumina NextSeq 500 sequencing platform and Nanopore MinION sequencer platforms.Results: A hybrid virulence- and resistance-encoding plasmid of 240 kb, harbouring both the virulence gene rmpA2 and the carbapenemase gene blaKPC-2, was recovered from a clinical HvKP strain. Designated pKP70-2, the plasmid was found to be almost structurally identical to various known hypervirulence-encoding plasmids harboured by other HvKP strains, except for an extra MDR-encoding region located within the genetically conserved plasmid backbone. This MDR region was flanked by two copies of IS26 in the same orientation, one at each end and linked to an external 8 bp (CTAAAATT) product of target site duplications, suggesting that an insertion event was responsible for the integration of the MDR region into the virulence plasmid. The MDR region was also found to harbour mobile elements that in turn contain the antibiotic resistance genes dfrA14 and blaKPC-2.Conclusions: Based on the genetic composition of pKP70-2, we postulate that the multiple insertion elements that it harbours were responsible for mediating the plasmid recombination events that underlie continuous emergence and genetic adaptation of novel resistance- and virulence-encoding mobile elements in K. pneumoniae. [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]

Salmonella Corvallis ST1541 has recently emerged as a globally disseminated pathogenic strain that often causes severe food-borne infections. Unlike most pandemic serotypes of Salmonella, the ST1541 strains harbored ColRNA1 plasmids that contain qnr-like determinants known to be responsible for the increasing incidence of ciprofloxacin-resistant food-borne Salmonella infections. In this study, we conducted a genomic analysis of a global collection of 388 S. Corvallis ST1541 strains collected within a twenty-year period. We investigated the genetic characteristics of plasmid-mediated quinolone resistance (PMQR) plasmids harbored by these S. Corvallis strains, established a minimum spanning tree (MST) to determine the temporal and spatial distribution of the top 10 MST clusters, inferred a time-phylogenies for the major sub-lineages and traced the routes of international dissemination of this serotype strains. Bayesian algorithm predicted that UK might be the origin of S. Corvallis strains currently prevalent in various countries. This idea is supported by the observation of the emergence of intercontinental-disseminated clonal strains and extensive transmission of the extensive-drug resistance (XDR)-encoding plasmid pSA663. This study therefore provides valuable insight into the evolution of globally transmitted S. Corvallis strains and suggests a need to strengthen cooperation between different countries to control the dissemination of these drug-resistant bacteria. [ABSTRACT FROM AUTHOR]

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]

• Clinical ST367 K1 K. quasipneumoniae subsp. similipneumoniae simultaneously harbouring bla KPC-2 and a virulence plasmid. • Carbapenem-resistant K1 K. quasipneumoniae with pLVPK-like virulence plasmid showed lower virulence and serum resistance. • K. quasipneumoniae may become an important reservoir of virulence and antimicrobial resistance genes in clinical settings. The continuous emergence of carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKP) poses a great challenge to human health owing to the associated extremely high morbidity and mortality. Klebsiella quasipneumoniae is a newly described bacterial species that is often misidentified as K. pneumoniae. Clinical K. quasipneumoniae strains have been reported worldwide, among which multidrug-resistant lineages have become a severe health problem, while less has been understood on this important pathogen. In this study, we characterised three clinical carbapenem-resistant K. quasipneumoniae subsp. similipneumoniae isolates belonging to sequence type 367 (ST367) and capsular type K1 and containing several virulence genes, including salmochelin (iroBCDN), aerobactin (iucABCDiutA) and regulator of mucoid phenotype (rmpA / A2), as well as some resistance genes, including bla KPC-2 , bla TEM-1 , bla OKP-B-9 and oqxAB. These carbapenem-resistant K. quasipneumoniae subsp. similipneumoniae strains containing virulence genes exhibited a higher level of virulence and serum resistance than a classical K. pneumoniae strain, while their virulence levels were slightly lower compared with typical ST11 CR-hvKP and ST23 K1 hvKP strains. This study reports for the first time the genetic and virulence characterisation of clinical K. quasipneumoniae subsp. similipneumoniae strains that simultaneously contained bla KPC-2 and virulence genes, contributing to a better understanding of their resistance and pathogenicity as well as for epidemic surveillance worldwide. [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]

This study reports a comprehensive epidemiological and genetic analysis of V. cholerae strains, specifically non-O1/non-O139 serogroups, isolated from animal-derived food samples in Guangdong province from 2015 to 2019. A total of 21 V. cholerae strains were obtained, which exhibited high resistance rates for nalidixic acid (57.14 %, 12/21), ampicillin (33.33 %, 7/21), and ciprofloxacin (19.05 %, 4/21). The quinolone resistance-related gene, qnrVC , was prevalent in 80.95 % (17/21) of the isolates. Additionally, chromosomally mediated quinolone-resistance mutations, including mutations in GyrA at position 83 (S83I) and ParC at position 85 (S85L), were detected in 47.62 % of the isolates. The combination of target mutation and qnrVC genes was shown to mediate resistance or intermediate resistance to ciprofloxacin in V. cholerae. Furthermore, an IncC-type conjugative plasmid carrying thirteen antibiotic resistance genes, including genes conferring resistance to two clinically important antibiotics, cephalosporins and fluoroquinolones, was identified in the shrimp-derived strain Vc516. While none of our food isolates harbored the toxigenic CTX- and TCP-encoding genes, they did possess genes encoding toxins such as HlyA and Autoinducer-2. Notably, some V. cholerae strains from this study exhibited a close genetic relationship with clinical strains, suggesting their potential to cause human infections. Taken together, this study provides a comprehensive view of the epidemiological features and genetic basis of antimicrobial resistance and virulence potential of V. cholerae strains isolated from food in southern China, thereby advancing our understanding of this important pathogen. • Vibrio cholerae strains have been isolated from animal-derived food samples purchased in Guangdong province, China. • 80.95 % (17/21) of the V. cholerae strains possessed quinolone resistance-related gene qnrVC , and ten of them carried both gyrA (Ser83Ile) and parC (Ser85Leu) mutations. • An IncC-type conjugative plasmid carried genes conferring resistance to two clinically important antibiotics, cephalosporins and fluoroquinolones, was identified in the shrimp-derived strain Vc516. • Non-O1/non-O139 V. cholerae strains isolated from food sources in China exhibited a higher frequency of AMR genes compared to strains from other geographical regions. [ABSTRACT FROM AUTHOR]

The escalating prevalence of multidrug-resistant (MDR) bacteria pose a significant public health threat. Understanding the genomic features and deciphering the antibiotic resistance profiles of these pathogens is crucial for development of effective surveillance and treatment strategies. In this study, we employed the R10.4.1 nanopore sequencing technology, specifically through the use of the MinION platform, to analyze eight MDR bacterial strains originating from clinical, ecological and food sources. A single 72-hour sequencing run could yield approximately 12 million reads which covered a total of 34 gigabases (Gbp). The nanopore R10.4.1 data was processed using the Flye assembler, successfully assembling the genomes of eight bacterial strains and their 18 plasmids. Notably, the assemblies generated solely from R10.4.1 nanopore data closely matched those from next-generation sequencing data. Diverse antibiotic resistance patterns and specific resistance genes in the test strains were identified. Hospital strains that exhibited multidrug resistance were found to harbor various resistance genes that encode efflux pumps and extended-spectrum β-lactamases. Environmental and food sources were found to display resistance profiles in a species-specific manner. The composition of structurally complex plasmids in the test strains could also be revealed by analysis of nanopore long reads, which also suggested evidence of horizontal transfer of plasmids between different bacterial species. These findings provide valuable insights into the genetic characteristics of MDR bacteria and demonstrating the practicality of nanopore sequencing technology for detecting of resistance elements in bacterial pathogens. [ABSTRACT FROM AUTHOR]

Antibiotic resistance is a global health issue, with Klebsiella pneumoniae (KP) posing a particular threat due to its ability to acquire resistance to multiple drug classes rapidly. OXA-232 is a carbapenemase that confers resistance to carbapenems, a class of antibiotics often used as a last resort for treating severe bacterial infections. The study reports the earliest known identification of six OXA-232-producing KP strains that were isolated in Zhejiang, China, in 2008 and 2009 within a hospital, two years prior to the first reported identification of OXA-232 in France. The four KP strains carry the OXA-232 gene and exhibit hypervirulent loci, suggesting a broader temporal and geographical spread and integration of this resistance and virulence than previously recognized with implications for public health. Global analysis of all OXA-232-bearing KP strains revealed that OXA-232-encoding plasmids are conservative, while the strains were very diverse suggesting the plasmid mediated transmission of this carbapenemase genes. Importantly, a large proportion of the OXA-232-bearing KP strains also carried virulence plasmids, in particular the recent emergence of ST15 type of KP that carried both OXA-232-encoding plasmids and hypervirulent (hv) plasmids in China since 2019, highlighting the importance of the emergence of this type of KP strains in clinical setting. The early detection and investigations of OXA-232 in these strains warrants the retrospective studies to uncover the true timeline of antibiotic resistance spread, which could provide valuable insights for shaping future strategies to tackle the global health crisis. [ABSTRACT FROM AUTHOR]

Colistin is the last-line antibiotic against Gram-negative pathogens. Here we identify an FDA-approved drug, Otilonium bromide (Ob), which restores the activity of colistin against colistin-resistant Gram-negative bacteria in vitro and in a mouse infection model. Ob also reduces the colistin dosage required for effective treatment of infections caused by colistin-susceptible bacteria, thereby reducing the toxicity of the drug regimen. Furthermore, Ob acts synergistically with colistin in eradicating multidrug-tolerant persisters of Gram-negative bacteria in vitro. Functional studies and microscopy assays confirm that the synergistic antimicrobial effect exhibited by the Ob and colistin involves permeabilizing the bacterial cell membrane, dissipating proton motive force and suppressing efflux pumps, resulting in membrane damages, cytosol leakage and eventually bacterial cell death. Our findings suggest that Ob is a colistin adjuvant which can restore the clinical value of colistin in combating life-threatening, multidrug resistant Gram-negative pathogens. The drug otilonium bromide restores the activity of colistin against colistinresistant Gram-negative bacteria in vitro and in a mouse infection model, suggesting that this combination may restore the value of colistin in treatment of antibiotic resistant disease. [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]

• Over 220 TEM variants observed to date originated from two major mutant clones. • One began with the Gly238Ser change and the other originated with the Arg164Ser substitution. • These two first-step mutants led to stepwise alteration in enzyme structure and activity. • Dissemination of strains producing TEM-1 variants underlies the markedly increased prevalence of β-lactam resistance. The TEM-1 β-lactamase can only cleave penicillin and the first-generation cephalosporins but it has evolved to become active against second-, third- and fourth-generation drugs. Through sequence analysis of natural TEM variants and those created by mutagenesis experiments, we described two distinct evolution routes of TEM-1 that has generated over 220 enzyme variants. One began with the Gly238Ser alteration and the other originated with the Arg164Ser substitution. Further acquisition of mutations in the background of each of these two first-step mutants led to stepwise alteration in enzyme structure and hence activity, eventually producing a wide range of enzyme variants whose substrate specificities cover cephalosporins of all generations. Dissemination of strains producing TEM-1 variants generated from these two evolution routes underlies the markedly increased prevalence of bacterial resistance to β-lactams in the past few decades. This study provides insights into the evolution of hydrolysing enzymes, in particular β-lactamases. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a global threat due to its high mortality in clinical patients. However, the specific mechanisms underlying this increased mortality remain unclear. The objective of this study is to investigate how the development of a resistance phenotype contributes to the significantly higher mortality associated with this pathogen. To achieve this, a collection of isogeneic strains was generated. The clinical carbapenem-susceptible K. pneumoniae (CSKP) strain HKU3 served as the control isolate, while HKU3-KPC was created through conjugation with a bla KPC-2 -bearing plasmid and served as clinical CRKP strain. Using a sepsis model, it was demonstrated that both HKU3 and HKU3-KPC exhibited similar levels of virulence. Flow cytometry, RNA-seq, and ELISA analysis were employed to assess immune cell response, M1 macrophage polarization, and cytokine storm induction, revealing that both strains elicited comparable types and levels of these immune responses. Subsequently, meropenem was utilized to treat K. pneumoniae infection, and it was found that meropenem effectively reduced bacterial load, inhibited M1 macrophage polarization, and suppressed serum cytokine production during HKU3 (CSKP) infection. However, these effects were not observed in the case of HKU3-KPC (CRKP) infection. These findings provide evidence that the high mortality associated with CRKP is attributed to its enhanced survival within the host during antibiotic treatment, resulting in a cytokine storm and subsequent host death. The development of an effective therapy for CRKP infections could significantly reduce the mortality caused by this pathogen. • KPC-bearing plasmids do not alter the virulence of CRKP. • CRKP induces polarization of M1 macrophages and triggers a cytokine storm. • Acquired antibiotic resistance may play a main role in the high in-hospital mortality associated with CRKP. [ABSTRACT FROM AUTHOR]

The tigecycline resistance gene tet (X4) has been widely reported in animals and animal products in some Asian countries including China in recent years but only sporadically detected in human. In this study, we investigated the prevalence and genetic features of tet (X4)-positive clinical E. coli strains. A total of 462 fecal samples were collected from patients in four hospitals located in four provinces in China in 2023. Nine tet (X4)-positive E. coli strains were isolated and subjected to characterization of their genetic and phenotypic features by performing antimicrobial susceptibility test, whole-genome sequencing, bioinformatic and phylogenetic analysis. The majority of the test strains were found to exhibit resistance to multiple antimicrobial agents including tigecycline but remained susceptible to colistin and meropenem. A total of seven different sequence types (STs) and an unknown ST type were identified among the nine tet (X4)-positive strains. Notably, the tet (X4) gene in six out of these nine tet (X4)-positive E. coli strains was located in a IncFIA-HI1A-HI1B hybrid plasmid, which was an tet (X4)-bearing epidemic plasmid responsible for dissemination of the tet (X4) gene in China. Furthermore, the tet (X4) gene in four out of nine tet (X4)-positive E. coli isolates could be successfully transferred to E. coli EC600 through conjugation. In conclusion, this study characterized the epidemic tet (X4)-bearing plasmids and tet (X4)-associated genetic environment in clinical E. coli strains, suggested the importance of continuous surveillance of such tet (X4)-bearing plasmids to control the increasingly widespread dissemination of tigecycline-resistant pathogens in clinical settings in China. [ABSTRACT FROM AUTHOR]

• A new approach for dynamic analysis of population structure of large numbers of genomes was developed. • Global distribution patterns of different types of Klebsiella pneumoniae strains were depicted. • Carbapenem-resistance and hypervirulence convergence are spreading and stabilising. Klebsiella pneumoniae is an important clinical bacterial pathogen that has hypervirulent and multidrug-resistant variants. Uniform Manifold Approximation and Projection (UMAP) was used to cluster genomes of 16 797 K. pneumoniae strains collected, based on core genome distance, in over 100 countries during the period 1937 to 2021. A total of 60 high-density genetic clusters of strains representing the major epidemic strains were identified among these strains. Using UMAP bedding, the relationship between genetic cluster, capsular polysaccharide (KL) types and sequence type (ST) of the strains was clearly demonstrated, with some important STs, such as ST11 and ST258, found to contain multiple clusters. Strains within the same cluster often exhibited significant diverse features, such as originating from different areas and being isolated in different years, as well as carriage of different resistance and virulence genes. These data enable the routes of evolution of the globally prevalent K. pneumoniae strains to be traced. Alarmingly, carbapenem-resistant K. pneumoniae strains accounted for 51.7% of the test strains and worldwide transmission was observed. Carbapenem-resistant and hypervirulent K. pneumoniae strains are mainly reported in China; however, these strains are increasingly reported in other parts of the world. Also identified in this study were several key genetic loci that facilitate development of a new K. pneumoniae typing method to differentiate between high- and low-risk strains. In particular, the acrR, ompK35 and hha genes were predicted to play a key role in expression of the resistance and virulence phenotypes. [Display omitted] [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]

Carbapenem-resistant and hypervirulent K. pneumoniae (CR-HvKP) strains that have emerged recently have caused infections of extremely high mortality in various countries. In this study, we discovered a conjugative plasmid that encodes carbapenem resistance and hypervirulence in a clinical ST86 K2 CR-HvKP, namely 17ZR-91. The conjugative plasmid (p17ZR-91-Vir-KPC) was formed by fusion of a non-conjugative pLVPK-like plasmid and a conjugative blaKPC-2-bearing plasmid and is present dynamically with two other non-fusion plasmids. Conjugation of p17ZR-91-Vir-KPC to other K. pneumoniae enabled them to rapidly express the carbapenem resistance and hypervirulence phenotypes. More importantly, genome analysis provided direct evidence that p17ZR-91-Vir-KPC could be directly transmitted from K2 CR-HvKP strain, 17ZR-91, to ST11 clinical K. pneumoniae strains to convert them into ST11 CR-HvKP strains, which explains the evolutionary mechanisms of recently emerged ST11 CR-HvKP strains. Carbapenem-resistant and hypervirulent Klebsiella pneumoniae strains are emerging. Here Xie et al. show that these phenotypes are carried on a plasmid formed from the fusion of a virulence plasmid with a conjugative plasmid. [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 investigate the genetic features of five plasmids recovered from an NDM-5-producing clinical Escherichia coli strain, BJ114, and to characterize the plasmid recombination event that occurred during the conjugation process.Methods: The genetic profiles of the five plasmids were determined by PCR, conjugation, S1-PFGE, Southern hybridization and WGS analysis. Plasmid sequences were analysed with various bioinformatic tools.Results: Complete sequences of five plasmids were obtained. Two small plasmids, pBJ114-141 and pBJ114-46, were speculated to have recombined into a large fusion plasmid, pBJ114T-190. When conjugated to other E. coli strains, some of the fusion plasmids were able to be resolved into the original two single plasmids. A non-conjugative plasmid, pBJ114-96, exhibited a high degree of sequence identity with the phage P7-like plasmid as well as an mcr-1-bearing plasmid. Another plasmid, pBJ114-78, was found to contain multidrug resistance genes and various mobile elements.Conclusions: The fusion plasmid recoverable from the transconjugant was found to be generated as a result of a recombination event that occurred upon interaction between a blaNDM-5-carrying plasmid and another plasmid present in the parental strain. Such recombination events presumably play a potential role in the dissemination of the blaNDM genes among different plasmids and pathogenic bacterial strains. [ABSTRACT FROM AUTHOR]

Compared with plasmid-borne mcr-1 , the occurrence of chromosomally-encoded mcr-1 is rare although it has been reported in several cases. This study aimed to investigate the genetic features of chromosomally-encoded mcr-1 among Escherichia coli strains as well as the potential genetic basis governing mobilisation of mcr-1 in bacterial chromosomes. The genome sequences of 16 E. coli strains containing a chromosomal mcr-1 gene were obtained and analysed. Phylogenetic and whole-genome sequencing (WGS) analysis demonstrated that mcr-1 was associated with four major types of genetic arrangements, namely IS Apl1 – mcr1 – orf , Tn 6330 , complex Tn 6330 and ΔTn 6330 in chromosomes of genetically unrelated E. coli strains. The mcr-1 -carrying mobile elements were shown to insert into the AT-rich region, which was also the case for IS Apl1 . Analysis of complete E. coli genome sequences showed that there were multiple copies of IS Apl1 present in E. coli chromosomes that also carried mcr-1 , whilst all mcr-1 -negative chromosomes were absent of any copy of IS Apl1 , suggesting the strong association of IS Apl1 and mcr-1 . Insertion of IS Apl1 into E. coli chromosomes may be a prerequisite for the insertion of mcr-1 -carrying mobile elements. Insertion of mcr-1 into E. coli chromosomes would enable it to become intrinsically resistant, which is expected to become more prevalent. Policy on the prudent use of colistin both in veterinary and clinical settings should be imposed globally to further prevent dissemination of mcr-1 in E. coli and other bacterial pathogens. [ABSTRACT FROM AUTHOR]

Background: Hypervirulent Klebsiella pneumoniae strains often cause life-threatening community-acquired infections in young and healthy hosts, but are usually sensitive to antibiotics. In this study, we investigated a fatal outbreak of ventilator-associated pneumonia caused by a new emerging hypervirulent K pneumoniae strain.Methods: The outbreak occurred in the integrated intensive care unit of a new branch of the Second Affiliated Hospital of Zhejiang University (Hangzhou, China). We collected 21 carbapenem-resistant K pneumoniae strains from five patients and characterised these strains for their antimicrobial susceptibility, multilocus sequence types, and genetic relatedness using VITEK-2 compact system, multilocus sequence typing, and whole genome sequencing. We selected one representative isolate from each patient to establish the virulence potential using a human neutrophil assay and Galleria mellonella model and to establish the genetic basis of their hypervirulence phenotype.Findings: All five patients had undergone surgery for multiple trauma and subsequently received mechanical ventilation. The patients were aged 53-73 years and were admitted to the intensive care unit between late February and April, 2016. They all had severe pneumonia, carbapenem-resistant K pneumoniae infections, and poor responses to antibiotic treatment and died due to severe lung infection, multiorgan failure, or septic shock. All five representative carbapenem-resistant K pneumoniae strains belonged to the ST11 type, which is the most prevalent carbapenem-resistant K pneumoniae type in China, and originated from the same clone. The strains were positive on the string test, had survival of about 80% after 1 h incubation in human neutrophils, and killed 100% of wax moth larvae (G mellonella) inoculated with 1 × 106 colony-forming units of the specimens within 24 h, suggesting that they were hypervirulent K pneumoniae. Genomic analyses showed that the emergence of these ST11 carbapenem-resistant hypervirulent K pneumoniae strains was due to the acquisition of a roughly 170 kbp pLVPK-like virulence plasmid by classic ST11 carbapenem-resistant K pneumoniae strains. We also detected these strains in specimens collected in other regions of China.Interpretation: The ST11 carbapenem-resistant hypervirulent K pneumoniae strains pose a substantial threat to human health because they are simultaneously hypervirulent, multidrug resistant, and highly transmissible. Control measures should be implemented to prevent further dissemination of such organisms in the hospital setting and the community.Funding: Chinese National Key Basic Research and Development Program and Collaborative Research Fund of Hong Kong Research Grant Council. [ABSTRACT FROM AUTHOR]

Objectives: To characterize a novel virulence-resistance plasmid pSE380T carried by a Salmonella enterica serotype Enteritidis clinical strain SE380.Methods: The plasmid pSE380T was conjugated to Escherichia coli strain J53 and sequenced by PacBio RSII, followed by subsequent annotation and genetic analysis.Results: Sequence analysis of this plasmid revealed that the entire Salmonella Enteritidis-specific virulence plasmid, pSEN, had been incorporated into an IncHI2 MDR plasmid, which comprises the cephalosporin and fosfomycin resistance determinants blaCTX-M-14 and fosA3. Based on BLAST analysis and scrutiny of insertion footprints, the insertion event was found to involve a replicative transposition process mediated by IS26, an IS element frequently detected in various resistance plasmids. The resulting pSE380T plasmid also comprises backbone elements of IncHI2 and IncFIA plasmids, producing a rare fusion product that simultaneously encodes functional features of both, i.e. virulence, resistance and high transmissibility.Conclusions: This is a novel hybrid plasmid mediating MDR and virulence from a clinical Salmonella Enteritidis strain. This plasmid is likely to be transmissible amongst various serotypes of Salmonella and other Enterobacteriaceae species, rendering a wide range of bacterial pathogens resistant to cephalosporins and fosfomycin, and further enhancing their virulence potential. It will be important to monitor the spread and further evolution of this plasmid among the Enterobacteriaceae strains. [ABSTRACT FROM AUTHOR]

Objectives: To investigate a set of MDR conjugative plasmids found in Vibrio species and characterize the underlying evolution process.Methods: pAQU-type plasmids from Vibrio species were sequenced using both Illumina and PacBio platforms. Bioinformatics tools were utilized to analyse the typical MDR regions and core genes in the plasmids.Results: The nine pAQU-type plasmids ranged from ∼160 to 206 kb in size and were found to harbour as many as 111 core genes encoding conjugative, replication and maintenance functions. Eight plasmids were found to carry a typical MDR region, which contained various accessory and resistance genes, including ISCR1-blaPER-1-bearing complex class 1 integrons, ISCR2-floR, ISCR2-tet(D)-tetR-ISCR2, qnrVC6, a Tn10-like structure and others associated with mobile elements. Comparison between a plasmid without resistance genes and different MDR plasmids showed that integration of different mobile elements, such as IS26, ISCR1, ISCR2, IS10 and IS6100, into the plasmid backbone was the key mechanism by which foreign resistance genes were acquired during the evolution process.Conclusions: This study identified pAQU-type plasmids as emerging MDR conjugative plasmids among important pathogens from different origins in Asia. These findings suggest that aquatic bacteria constitute a major reservoir of resistance genes, which may be transmissible to other human pathogens during food production and processing. [ABSTRACT FROM AUTHOR]

Phenotypic resistance to fluoroquinolones due to mutational changes in the gyrA and parC genes is common among clinical Vibrio strains; the plasmid-mediated quinolone resistance (PMQR) qnrVC genes were also suggested to play a role in enhancing resistance development. This study investigated the prevalence of qnrVC genes in foodborne Vibrio strains collected in Shenzhen, China, during the period August 2015 and April 2017. A total of 1811 foodborne Vibrio strains were collected, mostly (73.8%) from shrimp samples and 20.2% of these strains were resistant to ciprofloxacin. Investigation of resistance mechanisms showed that mutations in the gyrA and parC genes were commonly associated with ciprofloxacin resistance. The presence of qnrVC genes was shown to enhance ciprofloxacin MIC in Vibrio strains and 69.7% of Vibrio strains that harbored target mutations also carried qnrVC genes, yet only 27.5% of the isolates not harboring such mutations carried the qnrVC genes. A total of 141 strains were found to carry the qnrVC alleles, with qnrVC5 and qnrVC1 being the most common types. Fourteen qnrVC variant genes that contained novel mutations were detectable, with 12 (85.7%) involving qnrVC5 -like alleles. For the first time, we found a variant that was likely formed by the recombination of qnrVC1 and qnrVC5. The genetic context of the qnrVC genes found in this study was highly variable, with most being accompanied by mobile genetic elements and other resistance genes. The increasing prevalence of qnrVC genes in Vibrio and its contribution on mediating the development of ciprofloxacin resistance need to be further investigated. [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]

Colistin resistance mediated by mcr-1 -bearing plasmids poses a new challenge to treatment of Salmonella infections. To probe the scale of the problem that colistin resistance mediated by mcr-1 plasmids among Salmonella , the prevalence of mcr-1 in foodborne Salmonella recovered from 2014 to 2017 in Shenzhen, China and genetic profile of mcr -1 positive isolates were investigated. All mcr-1 positives Salmonella strains were collected from food products, characterized by PCR and MALDI-TOF, and subjected to antimicrobial susceptibility testing, whole-genome sequencing, bioinformatics analysis, and conjugation. Twenty-eight mcr-1 -positive Salmonella strains were recovered from pork. The rate of recovery displayed an increasing trend and was often accompanied by multidrug resistance. Salmonella Typhimurium was the most prevalent serotypes. Comparative genomic analysis indicated that the mcr-1 gene was located on the transferable IncX4 plasmids, as well as the IncHI2 plasmids, in which the gene was associated with IS Apl1. All two types of plasmids were often detected in zoonotic pathogen. Transferable 251K mcr-1 -bearing IncHI2 type plasmids were frequently reported in human and food-producing animals, but this is first time to detect a certain number in food. These findings show that dissemination of these two types of plasmids is responsible for the increase in the prevalence of colistin resistance in Salmonella strains in recent years, leading to rapid emergence of MDR Salmonella upon acquisition of these two mcr-1 -bearing plasmids. Transmission of IncX4 and IncHI2 plasmids in Salmonella would cause huge public health concerns in controlling foodborne infections caused by Salmonella. • Genetic profiles of mcr-1 -positive Salmonella derived from foods were uncovered. • IncHI2/IncX4 plasmid harbored mcr-1 dominated colistin resistant pork-borne Salmonella. • Surveillance of antimicrobial resistance Salmonella in food is essential. [ABSTRACT FROM AUTHOR]

Azithromycin resistance in bacterial pathogens has increased worldwide, and Klebsiella pneumoniae (K. pneumoniae) carries a variety of azithromycin resistance encoding genes. Genomic DNA of K. pneumoniae strain 16HN-12 was subjected to whole-plasmid sequencing using both the 150-bp paired-end Illumina NextSeq 500 platform and the long-read Oxford Nanopore Technologies MinION platform. Transferability of the azithromycin-resistance plasmid and the virulence plasmid was assessed by performing the conjugation assay. This study identified an IncB/O/K/Z conjugative plasmid that harboured erm (B) and mph (A) genes from a clinical K. pneumoniae strain. The plasmid was readily able to conjugate to Escherichia coli (E. coli) strain J53 and Salmonella enterica subsp. enterica serovar Typhimurium strain PY1 and promoted phenotypic resistance to azithromycin. Furthermore, the virulence plasmid harboured by this K. pneumoniae strain could be conjugated to E. coli strain EC600 and K. pneumoniae strain WZ1-2 via the help of this resistance plasmid through formation of a fusion plasmid. The fusion process was generated by homologous recombination through a homologous region located in both the virulence plasmid and resistance plasmid. Generation of this kind of conjugative plasmid simultaneously carrying virulence and resistance determinants could accelerate dissemination of these determinants and generate bacterial pathogens encoding these phenotypes. These data provide more information about transmission of azithromycin resistance and virulence determinants and call for action to further investigate and prevent such an evolutionary trend. [ABSTRACT FROM AUTHOR]

• This study was a longitudinal, large-scale surveillance of mcr-9 and mcr-10 in Enterobacter cloacae complex in China. • Enterobacter cloacae complex strains may be important reservoirs of mcr-9. • mcr-9 often coexisted with carbapenemase genes and they had the potential for co-transfer. • The mcr-9 - and mcr-10 -positive strains exhibited extreme genetic diversity. • Plasmids played an important role in the transmission of mcr in Enterobacter cloacae complex. Enterobacter cloacae complex (ECC) is among the most common carbapenem-resistant Enterobacteriaceae (CRE) in China. The emergence of mcr has rendered CRE strains resistant to the last-line antibiotic colistin. This study investigated the prevalence of mcr-9 and mcr-10 in carbapenem-resistant ECC (CRECC) and carbapenem-susceptible ECC (CSECC) in China. The CRECC and CSECC strains were collected from different regions of China. Antimicrobial susceptibility tests, conjugation experiments, whole genome sequencing, bioinformatic analysis, and quantitative RT-PCR were performed to understand the mechanisms of resistance and transmission of mcr in ECC. A total of 534 ECC were collected, among which 57 (10.7%) and 23 (4.3%) were positive for mcr-9 and mcr-10 , respectively. The prevalence of mcr-9 in CRECC was significantly higher than that in CSECC (31.8% vs. 3.7%; P < 0.001), while the prevalence of mcr-10 in CRECC was significantly lower (0.8% vs. 5.5%; P < 0.05). Most mcr-9 -positive strains (n = 45, 78.9%) exhibited multidrug-resistant phenotype, and four (17.4%) of the mcr-10 -positive strains exhibited multi-drug resistance. Coexistence of mcr and carbapenemase genes was commonly observed, including 41 (71.9%) mcr-9 -positive strains and one (4.3%) mcr-10 -positive strain, and the possibility of co-transfer was confirmed by conjugation experiments. The mcr -positive ECC were highly diverse, while most mcr genes were plasmid-encoded, indicating the important role of plasmids in the transmission of mcr in ECC. Furthermore, the expression of mcr-9 was increased after induction by colistin. The widespread mcr genes and co-transfer with carbapenemase genes among ECC strains pose an urgent threat to public health. [ABSTRACT FROM AUTHOR]

We report the discovery of a promising NDM-1 inhibitor, ebselen, through a cell-based screening approach. Enzymatic kinetic study and ESI-MS analysis suggested that ebselen could bind to NDM-1 by forming a S–Se bond with the Cys221 residue at the active site, thereby exhibiting a new inhibition mechanism with broad spectrum inhibitory potential. [ABSTRACT FROM AUTHOR]

Class D β-lactamase OXA-48 is widely distributed among Gram-negative bacteria and is an important determinant of resistance to the last-resort carbapenems. Nevertheless, the detailed mechanism by which this β-lactamase hydrolyzes its substrates remains poorly understood. In this study, the complex structures of OXA-48 and various β-lactams were modeled and the potential active site residues that may interact with various β-lactams were identified and characterized to elucidate their roles in OXA-48 substrate recognition. Four residues, namely S70, K73, S118, and K208 were found to be essential for OXA-48 to undergo catalytic hydrolysis of various penicillins and carbapenems both in vivo and in vitro. T209 was found to be important for hydrolysis of imipenem, whereas R250 played a major role in hydrolyzing ampicillin, imipenem, and meropenem most likely by forming a H-bond or salt-bridge between the side chain of these two residues and the carboxylate oxygen ions of the substrates. Analysis of the effect of substitution of alanine in two residues, W105 and L158, revealed their roles in mediating the activity of OXA-48. Our data show that these residues most likely undergo hydrophobic interaction with the R groups and the core structure of the β-lactam ring in penicillins and the carbapenems, respectively. Unlike OXA-58, mass spectrometry suggested a loss of the C6-hydroxyethyl group during hydrolysis of meropenem by OXA-48, which has never been demonstrated in Class D carbapenemases. Findings in this study provide comprehensive knowledge of the mechanism of the substrate recognition and catalysis of OXA-type β-lactamases. [ABSTRACT FROM AUTHOR]

Due to frequent outbreaks of sprouts-associated foodborne illness, sprouts have been considered as a "high risk" food. In this study, the antimicrobial peptide zp37 (GIKAKIIIKIKK-NH 2), which possesses a helical structure, was found to exhibit a minimum inhibitory concentration (MIC) value of 16 μM on E. coli O157:H7, effectively killing such strain in vitro and reducing its population size by 94.7% in sprouts upon storage for seven days. The effect of zp37 on cell membrane was investigated, with results showing that zp37 caused membrane depolarization and weak membrane permeabilization, which in turn resulted in cellular deformation observable by scanning electron microscope (SEM). Peptide zp37 was labeled by FITC to track the cellular location of zp37. Fluorescence microscopy studies showed that a larger amount of FITC-zp37 entered the bacterial cells when exposed to a higher concentration of the labeled peptide. Confocal microscopy showed that FITC-zp37 was detectable in cell membrane and cytoplasm. Upon entering the cytoplasm, zp37 was found to bind to the DNA of E. coli O157:H7, causing DNA aggregation and precipitation. Moreover, in vitro (HEK293 cell) and in vivo (Galleria mellonella) assays demonstrated that zp37 exhibited low toxicity. • Peptide zp37 had a MIC value of 16 μM on E. coli O157:H7 with low toxicity. • In sprouts, zp37 treatment reduced 94.7% E. coli O157:H7 upon storage for 7 days. • zp37 caused membrane depolarization and weak membrane permeabilization. • zp37 bound to the DNA of E. coli O157:H7, causing DNA aggregation and precipitation.. [ABSTRACT FROM AUTHOR]