Towards a better understanding of antimicrobial resistance dissemination:what can be learnt from studying model conjugative plasmids?

Bacteria can evolve rapidly by acquiring new traits such as virulence,metabolic properties,and most importantly,antimicrobial resistance,through horizontal gene transfer(HGT).Multidrug resistance in bacteria,especially in Gram-negative organisms,has become a global public health threat often through the spread of mobile genetic elements.Conjugation represents a major form of HGT and involves the transfer of DNA from a donor bacterium to a recipient by direct contact.Conjugative plasmids,a major vehicle for the dissemination of antimicrobial resistance,are selfish elements capable of mediating their own transmission through conjugation.To spread to and survive in a new bacterial host,conjugative plasmids have evolved mechanisms to circumvent both host defense systems and compete with co-resident plasmids.Such mechanisms have mostly been studied in model plasmids such as the F plasmid,rather than in conjugative plasmids that confer antimicrobial resistance(AMR)in important human pathogens.A better understanding of these mechanisms is crucial for predicting the flow of antimicrobial resistance-conferring conjugative plasmids among bacterial populations and guiding the rational design of strategies to halt the spread of antimicrobial resistance.Here,we review mechanisms employed by conjugative plasmids that promote their transmission and establishment in Gram-negative bacteria,by following the life cycle of conjugative plasmids.

Construction of two selectable markers for integrative/conjugative plasmids in Flavobacterium columnare

Flavobacterium columnare, the etiological agent of colunmaris disease, is one of the most important and widespread bacterial pathogens of freshwater fish. In this study, we constructed two artificial selectable markers （chloramphenicol and spectinomycin resistance） for gene transfer in F. columnare. These two new artificial selectable markers, which were created by placing the chloramphenicol or spectinomycin resistance gene under the control of the native acs regulatory region of F. columnare, were functional in both F. columnare and Escherichia coli. The integrative/conjugative plasmids constructed by using these markers were introduced into F. columnare G4 via electroporation or conjugation. The integrated plasmid DNA was confirmed by Southern blotting and PCR analysis. These two markers can be employed in future investigations into gene deletion and the pathogenicity of virulence factors in F. columnare.

Analysis of Drug Resistance Spectra and Conjugative Plasmid Carrying Rates of P.aeruginosa and Acinetobacter

Antimicrobial susceptibility test was performed on 57 clinical isolates of P. aeruginosa and 36 clinical isolates of Acinetobacter with 11 antimicrobial agents including getamicin, amikacin, ciprofloxacin, ofloxacin, fleroxacin, piperacillin, cefotaxime, cefoperazone/sulbactam, ceftazidime, cefoperazone and doxycycline. Transferable drug resistance plasmid carrying rates of these clinical isolates were also studied. On the basis of the in vitro activities, 52.63%(30/57) of the isolated strains of P. aeruginosa were susceptible to antimicrobial agents selected (except doxycycline), 41.67%(15/36) of the isolated strains of Acinetobacter were susceptible to 11 antimicrobial agents. The sensitivity rate of P.aeruginosa and Acinetobacter to antimicrobial agents selected was 70% or greater to all except doxycycline. Furthermore, the sensitivity rate of P.aeruginosa to amikacin ciprofloxacin, ceftazidime, cefoperazone, cefoperazone/sulbactam, and that of Acinetobacter to cefoperazone/sulbactam, amikacin was more than 90%,among them amikacin, cefoperazone/sulbactam being the most effective. Plasmid analysis showed that 15.79%(9/57) P.aeruginosa strains and 13.89%(5/36) Acinetobacter strains carried plasmid. Conjugative plasmid carrying rates of P. aeruginosa strains and Acinetobacter strains were 7.02%(4/57), 13.89%(5/36), respectively. Conjugative plasmid didn′t play an important role in the formation and dissemination of drug resistance of P. aeruginosa and Acinetobacter.

Characteristics of colistin-resistant Escherichia coli from pig farms in Central China

The emergence and dissemination of colistin resistance in Enterobacterioceae mediated by plasmid-borne mcr genes in recent years now pose a threat to public health.In this study,we isolated and characterized colistin-resistant and for mcr-positive£coli from pig farms in Central China.Between 2018 and 2019,594 samples were collected and recovered 445 E.coli isolates.Among them,33 with colistin resistance phenotypes and 37 that were positive for mcr genes were identified,including 34 positive for mcr-1,one positive for mcr-3,and two positive for both mcr-1 and mcr-3.An insertion of nine bases("CTGGATACG")into mcr-7 in four mcr-positive isolates led to gene dysfunction,and therefore did not confer the colistin resistance phenotype.Antimicrobial susceptibility testing revealed that 37 mcr-positive isolates showed severe drug resistance profiles,as 50% of them were resistant to 20 types of antibiotics.Multilocus sequence typing revealed a heterogeneous group of sequence types in mcr-positive isolates,among which ST10(5/37),ST156(5/37),and 5T617(4/37)were the predominant types.Plasmid conjugation assays showed that mcr-carrying plasmids of 25 mcr-positive isolates were conjugated with£coli recipient,with conjugation frequencies ranging from 1.7 × 10^(-6) to 4.1 × 10^(-3) per recipient.Conjugation of these mcr genes conferred a colistin resistance phenotype upon the recipient bacterium.PCR typing of plasmids harbored in the 25 transconjugants determined six types of plasmid replicons,including lncX4(14/25),FrepB(4/25),Incl2(3/25),lncHI2(2/25),FIB(1/25),and Inch(1/25).This study contributes to the current understanding of antibiotic resistance and molecular characteristics of colistin-resistant£coli in pig farms.

Acquisition of a novel conjugative multidrug-resistant hypervirulent plasmid leads to hypervirulence in clinical carbapenem-resistant Klebsiella pneumoniae strains

The co-occurrence of plasmid-mediated multidrug resistance and hypervirulence in epidemic carbapenem-resistant Klebsiella pneumoniae has emerged as a global public health issue.In this study,an ST23 carbapenem-resistant hyper-virulent K.pneumoniae(CR-HvKP)strain VH1-2 was identified from cucumber in China and harbored a novel hybrid plasmid pVH1-2-VIR.The plasmid pVH1-2-VIR carrying both virulence and multidrug-resistance(MDR)genes was likely generated through the recombination of a virulence plasmid and an IncFIIK conjugative MDR plasmid in clinical ST2318622 isolated from a sputum sample.The plasmid pVH1-2-VIR exhibited the capacity for transfer to the clinical ST11 carbapenem-resistant K.pneumoniae(CRKP)strain via conjugation assay.Acquisition of pVH1-2-VIR plasmid directly converted a CRKP into CR-HvKP strain characterized by hypermucoviscosity,heightened virulence for Galleria mellonella larvae,and increased colonization ability in the mouse intestine.The emergence of such a hybrid plasmid may expedite the spread of CR-HvKP strains,posing a significant risk to human health.