Search

Objectives To characterize a clinical Klebsiella pneumoniae isolate from China co-harbouring tet(X4), blaOXA-181 and the aerobactin operon on an IncFIBk-FII-X3-ColKP3 hybrid plasmid. Methods A tigecycline-resistant strain was recovered from the intestinal sample of a patient. It was subjected to antimicrobial susceptibility testing, conjugation assay, virulence testing, WGS, bioinformatics analysis, plasmid stability testing and fitness cost testing. Results The strain K. pneumoniae T877 was resistant to tigecycline, intermediate to piperacillin/tazobactam and ertapenem, and positive for tet(X), blaOXA-181 and the virulence-associated operon iutAiucABCD, which were located on the same plasmid, named pKPT877-hybrid. It was 99.96% identical to the IncFIBk-FII plasmid pSCH6109-Vir (accession number CP050860) from K. pneumoniae strain SCH6109 at 96% coverage with the absence of a 50 kb region on pKPT877-hybrid; this region was highly homologous to the 51 kb IncX3-ColKP3-type, blaOXA-181-carrying plasmid pOXA181-191773 (accession number CP080367). Plasmid pKPT877-hybrid was conjugatively transferable to the ST11 K. pneumoniae strains FJ8 and KP04. pKPT877-hybrid did not have a significant impact on the fitness cost and could be maintained stably in T877. Conclusions We report for the first time (to the best of our knowledge) the co-transfer of last-line antibiotic resistance determinants [tet(X4) and blaOXA-181] and the aerobactin operon (iutAiucABCD) by a mobile IncFIBk-FII-X3-ColKP3 hybrid plasmid, which can be stably maintained in K. pneumoniae strains, even in the absence of antibiotic selective pressure. Once the plasmid transfers to a K. pneumoniae with porin deficiency, the strain might have high levels of resistance to carbapenems and tigecycline, which are the last line of defence against infections. Heightened and continuous efforts are needed to control its dissemination.

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.

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.

Findings described in this study constitute concrete evidence that it is possible to significantly enhance the antimicrobial activity of colistin by using an antifungal drug, econazole, as a colistin adjuvant. We showed that this drug combination can kill not only multidrug-resistant A. baumannii but also the tolerant subpopulation of such strains known as persisters, which may cause chronic and recurrent infections in clinical settings.

A plasmid that harbored the virulence factors highly like those of the virulence plasmid commonly found in clinical hypervirulent Klebsiella pneumoniae strains was detected in a foodborne Escherichia coli strain EC1108 and designated p1108-IncFIB. This virulent-like plasmid was found to be common in E. coli from various sources. To understand the contribution of this plasmid to the virulence of E. coli, plasmid p1108-IncFIB in strain EC1108 was first cured to generate strain EC1108-PC. The virulence plasmid p15WZ-82_Vir in Klebsiella pneumoniae strain 15WZ-82 was then transmitted to EC1108-PC to produce the transconjugant, EC1108-PC-TC to assess the contribution of this virulence plasmid to the virulence level of E. coli. During the process of conjugation, p15WZ-82_Vir was found to be evolved into p15WZ-82_int, which underwent homologous recombination with a plasmid encoding a carbapenemase gene

With the development of multidrug resistance in

The epidemiological features of the newly emerged carbapenem-resistant hypervirulent

Colistin is one of the few antibiotics that exhibit bactericidal effect on carbapenemase-producing

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.

Increased usage of daptomycin to treat infections caused by Gram-positive bacterial pathogens has resulted in emergence of resistant mutants. In a search for more effective daptomycin analogues through medicinal chemistry studies, we found that methylation at the nonproteinogenic amino acid kynurenine in daptomycin could result in significant enhancement of antibacterial activity. Termed "kynomycin," this new antibiotic exhibits higher antibacterial activity than daptomycin and is able to eradicate methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) strains, including daptomycin-resistant strains. The improved antimicrobial activity of kynomycin was demonstrated in in vitro time-killing assay, in vivo wax worm model, and different mouse infection models. The increased antibacterial activity, improved pharmacokinetics, and lower cytotoxicity of kynomycin, compared to daptomycin, showed the promise of the future design and development of next-generation daptomycin-based antibiotics.

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 ISApl1. 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.

Non-typical Salmonella is a primary cause of food-borne diseases and considered as a major public health concern worldwide(1, 2).….

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

Ciprofloxacin resistance in Salmonella has been increasingly reported due to the emergence and dissemination of multiple Plasmid-Mediated Quinolone Resistance (PMQR) determinants, which are mainly located in non-conjugative plasmids or chromosome. In this study, we aimed to depict the molecular mechanisms underlying the rare phenomenon of horizontal transfer of ciprofloxacin resistance phenotype in Salmonella by conjugation experiments, S1-PFGE and complete plasmid sequencing. Two types of non-conjugative plasmids, namely an IncX1 type carrying a qnrS1 gene, and an IncH1 plasmid carrying the oqxAB-qnrS gene, both ciprofloxacin resistance determinants in Salmonella, were recovered from two Salmonella strains. Importantly, these non-conjugative plasmids could be fused with a novel Incl1 type conjugative helper plasmid, which could target insertion sequence (IS) elements located in the non-conjugative, ciprofloxacin-resistance-encoding plasmid through replicative transcription, eventually forming a hybrid conjugative plasmid transmissible among members of Enterobacteriaceae. Since our data showed that such conjugative helper plasmids are commonly detectable among clinical Salmonella strains, particularly S. Typhimurium, fusion events leading to generation and enhanced dissemination of conjugative ciprofloxacin resistance-encoding plasmids in Salmonella are expected to result in a sharp increase in the incidence of resistance to fluoroquinolone, the key choice for treating life-threatening Salmonella infections, thereby posing a serious public health threat.

A total of 55 cefotaxime-resistant Escherichia coli isolates were obtained from retail meat products purchased in Shenzhen, China, during the period November 2012 to May 2013. Thirty-seven of these 55 isolates were found to harbor a bla CTX-M gene, with the bla CTX-M-1 group being the most common type. bla CMY-2 was detected in 16 isolates, alone or in combination with other extended-spectrum β-lactamase (ESBL) determinants. Importantly, the fosA3 gene, which encodes fosfomycin resistance, was detected in 12 isolates, with several being found to reside in the conjugative plasmid that harbored the bla CTX-M gene. The insertion sequence IS 26 was observed upstream of some of the bla CTX-M-55 and fosA3 genes. Conjugation experiments showed that bla CTX-M genes from 15 isolates were transferrable, with Inc I1 and Inc FII being the most prevalent replicons. High clonal diversity was observed among the bla CTX-M producers, suggesting that horizontal transfer of the bla CTX-M genes among E. coli strains in retail meats is a common event and that such strains may constitute an important reservoir of bla CTX-M genes, which may be readily disseminated to other potential human pathogens.

The human gut microbiome is the presumed site in which the emergence and evolution of antibiotic-resistant organisms constantly take place. To delineate the genetic basis of resistance formation in gut microbiome strains, we investigated the changes in the subpopulation structure of Escherichia coli in rat intestine before and after antimicrobial treatment. We observed that antibiotic treatment was selected for an originally minor subpopulation E. coli carrying the biofilm-forming genetic locus pgaABCD and the toxin-antitoxin system HipAB. Such strains possessed dramatically enhanced ability to withstand the detrimental effects of antibiotics, becoming a dominant subspecies upon antibiotic treatment and eventually evolving into resistant mutants. In contrast, E. coli strains that did not carry pgaABCD and HipAB were eradicated upon antibiotic treatment. Our findings, therefore, suggested that genes encoding biofilm-forming ability played an important role in conferring specific gut E. coli strains the ability to evolve into resistant strains upon a prolonged antibiotic treatment, and that such strains may therefore be considered bacterial antibiotic resistance progenitor cells in the gut microbiome.

Ceftriaxone and ciprofloxacin are the drugs of choice in treatment of invasive Salmonella infections. This study discovered a novel type of plasmid, pSa44-CIP-CRO, which was recovered from a S. London strain isolated from meat product and comprised genetic determinants that encoded resistance to both ciprofloxacin and ceftriaxone. This plasmid could be resolved into two daughter plasmids and co-exist with such daughter plasmids in a dynamic form in Salmonella; yet it was only present as a single plasmid in Escherichia coli. One daughter plasmid, pSa44-CRO, was found to carry the blaCTX-M-130 gene, which encodes resistance to ceftriaxone, whereas the other plasmid, pSa44-CIP, carried multiple PMQR genes such as qnrB6-aac(6’)-Ib-cr, which mediated resistance to ciprofloxacin. These two daughter plasmids could be integrated into one single plasmid through ISPa40 mediated homologous recombination. Mouse infection and treatment experiments showed that carriage of plasmid, pSa44-CIP-CRO by S. typhimurium led to the impairment of treatment by ciprofloxacin or cefitiofur, a veterinary drug with similar properties as ceftriaxone. In conclusion, dissemination of such conjugative plasmids impairs current choices of treatment for life-threatening Salmonella infection and hence constitutes a serious public health threat.

This study aimed to characterize novel conjugative plasmids that encode transferable ciprofloxacin resistance in Salmonella. In this study, 157 nonduplicated Salmonella isolates were recovered from food products, of which 55 were found to be resistant to ciprofloxacin. Interestingly, 37 of the 55 CiprSalmonella isolates (67%) did not harbor any mutations in the quinolone resistance-determining regions (QRDR). Six Salmonella isolates were shown to carry two novel types of conjugative plasmids that could transfer the ciprofloxacin resistance phenotype to Escherichia coli J53 (azithromycin resistant [Azir]). The first type of conjugative plasmid belonged to the ∼110-kb IncFIB-type conjugative plasmids carrying qnrB-bearing and aac(6′)-Ib-cr-bearing mobile elements. Transfer of the plasmid between E. coli and Salmonella could confer a ciprofloxacin MIC of 1 to 2 μg/ml. The second type of conjugative plasmid belonged to ∼240-kb IncH1/IncF plasmids carrying a single PMQR gene, qnrS. Importantly, this type of conjugative ciprofloxacin resistance plasmid could be detected in clinical Salmonella isolates. The dissemination of these conjugative plasmids that confer ciprofloxacin resistance poses serious challenges to public health and Salmonella infection control.

Vibrio parahaemolyticus is an important causative agent of gastroenteritis, with the consumption of contaminated seafood being the major transmission route. Resistance to penicillin is common among V. parahaemolyticus strains, whereas cephalosporin resistance remains rare. In an attempt to assess the current prevalence and characteristics of antibiotic resistance of this pathogen in common food samples, a total of 54 (17% of the total samples) V. parahaemolyticus strains were isolated from 318 meat and seafood samples purchased from supermarkets and wet markets in Shenzhen, China, in 2013. These isolates exhibited high-level resistance to ampicillin, yet they were mostly susceptible to other antimicrobials, except for two that were resistant to extended-spectrum cephalosporins. The β-lactamase gene bla PER-1 was detectable in one strain, V. parahaemolyticus V43, which was resistant to both third- and fourth-generation cephalosporins. Compared to other bla PER-1 -positive V. parahaemolyticus strains reported in our previous studies, strain V43 was found to harbor an ∼200-kb conjugative plasmid carrying genes that were different from the antimicrobial resistance genes reported from the previous studies. The β-lactamase gene bla CMY-2 was detectable for the first time in another V. parahaemolyticus isolate, V4, which was resistant to third-generation cephalosporins. This bla CMY-2 gene was shown to be located in an ∼150-kb IncA/C-type conjugative plasmid with a genetic structure consisting of traB-traV-traA -IS Ecp1-bla CMY-2 - blc-sugE-encR-orf1-orf2-orf3-orf4-dsbC-traC , which is identical to that of other IncA/C conjugative plasmids in Enterobacteriaceae , albeit with a different size. These findings indicate that the transmission of extended-spectrum-β-lactamase (ESBL) and AmpC β-lactamase genes via conjugative plasmids can mediate the development of extended-spectrum cephalosporin resistance in V. parahaemolyticus , thereby posing a potential threat to public health.

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.

The recently discovered colistin resistance-encoding element, mcr-1, adds to the list of mobile resistance genes whose products rapidly erode the antimicrobial efficacy of not only the commonly used antibiotics, but also the last line agents of carbapenems and colistin. The relative prevalence of mcr-1-bearing strains in various ecological niches including 1,371 food samples, 480 animal faecal samples, 150 human faecal samples and 34 water samples was surveyed using a novel in-house method. Bacteria bearing mcr-1 were commonly detected in water (71% of samples), animal faeces (51%), food products (36%), and exhibited stable carriage in 28% of human subjects surveyed. Such strains, which exhibited variable antibiotic susceptibility profiles, belonged to various Enterobacteriaceae species, with Escherichia coli being the most dominant in each specimen type. The mcr-1 gene was detectable in the chromosome as well as plasmids of various sizes. Among these, two conjugative plasmids of sizes ca 33 and ca 60 kb were found to be the key vectors that mediated mcr-1 transmission in organisms residing in various ecological niches. The high mcr-1 carriage rate in humans found in this study highlights the importance of continued vigilance, careful antibiotic stewardship, and the development of new antimicrobials.

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 ISApl1-mcr1-orf, Tn6330, complex Tn6330 and ΔTn6330 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 ISApl1. Analysis of complete E. coli genome sequences showed that there were multiple copies of ISApl1 present in E. coli chromosomes that also carried mcr-1, whilst all mcr-1-negative chromosomes were absent of any copy of ISApl1, suggesting the strong association of ISApl1 and mcr-1. Insertion of ISApl1 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.

This study surveyed the prevalence of mcr-1 in extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli strains of food origin in China and identified strains that carried mcr-1 , fosA3 , and ESBL genes, which were carried in various plasmids. The mcr-1 and ESBL genes could be cotransferred by one or more types of plasmids. The presence of these multidrug-resistant E. coli strains in food products might pose a huge threat to public health.

This study aims to investigate the prevalence and transmission dynamics of the bla NDM-1 gene in animal Escherichia coli strains. Two IncFII bla NDM-1 -encoding plasmids with only minor structural variation in the MDR region, pHNEC46-NDM and pHNEC55-NDM, were found to be responsible for the transmission of bla NDM-1 in these strains. The bla NDM-1 gene can be incorporated into plasmids and stably inherited in animal-borne E. coli strains that can be maintained in animal gut microflora even without carbapenem selection pressure.

Fluoroquinolone resistance in Salmonella has become increasingly prevalent in recent years. To probe the molecular basis of this phenomenon, the genetic and phenotypic features of fluoroquinolone resistant Salmonella strains isolated from food samples were characterized. Among the 82 Salmonella strains tested, resistance rate of the three front line antibiotics of ceftriaxone, ciprofloxacin and azithromycin was 10%, 39% and 25% respectively, which is significantly higher than that reported in other countries. Ciprofloxacin resistant strains typically exhibited cross-resistance to multiple antibiotics including ceftriaxone, primarily due to the presence of multiple PMQR genes and the blaCTX-M-65, blaCTX-M-55blaCMY-2 and blaCMY-72 elements. The prevalence rate of the oqxAB and aac(6’)-Ib-cr genes were 91% and 75% respectively, followed by qnrS (66%), qnrB (16%) and qnrD (3%). The most common PMQR combination observable was aac(6’)-Ib-cr-oqxAB-qnrS2, which accounted for 50% of the ciprofloxacin resistant strains. Interestingly, such isolates contained either no target mutations or only a single gyrA mutation. Conjugation and hybridization experiments suggested that most PMQR genes were located either in the chromosome or a non-transferrable plasmid. To summarize, findings in this work suggested that PMQRs greatly facilitate development of fluoroquinolone resistance in Salmonella by abolishing the requirement of target gene mutations.