Horizontal transfer of vanA between probiotic Enterococcus faecium and Enterococcus faecalis in fermented soybean meal and in digestive tract of growing pigs

Background: The aim of this study was to investigate the intergeneric transfer of vancomycin resistance gene vanA between probiotic enterococci in the fermentation progress of soybean meal and in the digestive tract of growing pigs.One vanA genotype vancomycin resistant E.faecium strain,Efm4,and one chloramphenicol-resistant E.faecalis strain,Efs2,were isolated from twenty-nine probiotic basis feed material/additive samples.For in vitro conjugation,Efm4 and Efs2 were used as starter to ferment soybean meal.For in vivo conjugation,thirty growing pigs were randomly assigned to five groups(n = 6),treated with a basic diet,or supplemented with 10% fermented soybean meal,1% Efm4,5% Efs2 or a combination of 1% Efm4 + 5% Efs2 for 7 d,respectively.Fecal samples of pigs in each group were collected daily for the isolation and dynamic analysis of Efm4,Efs2 and transconjugants.The sequence types(STs) of Efm4,Efs2 and transconjugants were analyzed by multilocus sequence typing(MLST).The vanA harboring plasmid in Efm4 and transconjugants was analyzed by S1-pulsed field gel electrophoresis(PFGE)and further verified by multiple alignments.Results: The results showed that,in FSBM,transconjugants were detected 1 h after the fermentation,with a conjugation frequency of ~ 10^-3 transconjugants/recipient.Transconjugants proliferated with Efm4 and Efs2 in the first 8 h and maintained steadily for 10 d till the end of the experiment.Additionally,in vivo experiment showed that transcojugants were recovered in one of six pigs in both FSBM and Efm4 + Efs2 groups,with conjugation frequency of ~ 10^-5 and ~ 10^-4,respectively.MLST revealed the ST of Efm4,Efs2 and transconjugants was ST1014,ST69 and ST69,respectively.S1-PFGE confirmed the existence of the vanA-harboring,142,988-bp plasmid,which was also a multi-drug resistant plasmid containing Tn1546-like transposon.Conclusions: The findings revealed the potential safety hazard existing in the commercial probiotic enterococci in China,because the horizontal transfer from farm to fork could potentially pose a safety risk to the public.

Pooled Plasmid Sequencing Reveals the Relationship Between Mobile Genetic Elements and Antimicrobial Resistance Genes in Clinically Isolated Klebsiella pneumoniae

Plasmids remain important microbial components mediating the horizontal gene transfer(HGT)and dissemination of antimicrobial resistance.To systematically explore the relationship between mobile genetic elements(MGEs)and antimicrobial resistance genes(ARGs),a novel strategy using single-molecule real-time(SMRT)sequencing was developed.This approach was applied to pooled conjugative plasmids from clinically isolated multidrug-resistant(MDR)Klebsiella pneumoniae from a tertiary referral hospital over a 9-month period.The conjugative plasmid pool was obtained from transconjugants that acquired antimicrobial resistance after plasmid conjugation with 53 clinical isolates.The plasmid pool was then subjected to SMRT sequencing,and 82 assembled plasmid fragments were obtained.In total,124 ARGs(responsible for resistance to b-lactam,fluoroquinolone,and aminoglycoside,among others)and 317 MGEs[including transposons(Tns),insertion sequences(ISs),and integrons]were derived from these fragments.Most of these ARGs were linked to MGEs,allowing for the establishment of a relationship network between MGEs and/or ARGs that can be used to describe the dissemination of resistance by mobile elements.Key elements involved in resistance transposition were identified,including IS26,Tn3,IS903 B,ISEcp1,and ISKpn19.As the most predominant IS in the network,a typical IS26-mediated multi-copy composite transposition event was illustrated by tracing its flanking 8-bp target site duplications(TSDs).The landscape of the pooled plasmid sequences highlights the diversity and complexity of the relationship between MGEs and ARGs,underpinning the clinical value of dominant HGT profiles.

Transconjugation and genotyping of the plasmid-mediated AmpC β-lactamase and extended-spectrum β-lactamase genes in Klebsiella pneumoniae

Bsckgroud AmpC β-lactamases and extended-spectrum β-lactamases （ESBLs） are becoming predominant causes of resistance to third and forth-generation cephalosporins in Klebsiella pneumoniae （K. pneumoniae）. It is very difficult to treat infectious diseases caused by multidrug-resistant K. pneumoniae. The purpose of the present study was to investigate transconjugation and characteristics of β-lactamase genes in K. pneumoniae producing AmpC β-lactamases and ESBLs. Methods AmpC β-lactamases were detected by three-dimension test and ESBLs by disc confirmatory test. Minimum inhibitory concentrations （MICs） were determined by agar dilution. Transfer of resistance to EC600 （Rifr） was attempted by conjugation in broth and screened on agar containing cefotaxime （2 μg/ml） plus rifampin （1024 μg/ml）. The genes encoding AmpC or ESBLs and their transconjugants were detected by PCR and verified by DNA sequencing. Results The resistant rates to ampicillin and piperacillin were 100% in 18 isolates of K. pneumoniae. However, imipenem was still of great bactericidal activity on K. pneumoniae, and its MIC50 was 0.5 μg/mL. Eleven β-lactamase genes, including TEM-1, TEM-11, SHV-13, SHV-28, CTX-M-9, CTX-M-22, CTX-M-55, OXA-1, LEN, OKP-6 and DHA-1, were found from 18 isolates. And at least one β-lactamase gene occurred in each isolate. To our surprise, there were six β-lactamase genes in the CZ04 strain. Among 18 isolates of K. pneumoniae, the partial resistant genes in 8 isolates were conjugated successfully, which had 100% homological sequence with donors by sequence analysis. Compared with donors, 8 transconjugants had attained resistance to most β-lactams, including ampicillin, piperacillin, cefoxitin, cefotaxime and aztreonam, or even amikacin and gentamicin. Conclusions R plasmids can be easily transferred between the resistant and sensitive negative bacilli. It is very difficult to block and prevent the spread of antimicrobial resistance. So more attention should be paid to reducing the frequency, times and dosage of antimicrobials, especially third or fourth cephalosporins.