Horizontal Transfer Mechanism Of Antimicrobial Resistance Plasmid Of ESBL-Producing Enterobacteriaceae Isolated From Water Environments In Mouse Intestinal Tract

The rational use of antibiotics helps control infectious diseases of humans and animals.Abuse and overuse of antibiotics in clinical practice has contributed to drug resistant bacteria“superbugs”.Extended-spectrum beta-lactamases(ESBLs)can hydrolyse penicillins,cephalosporins,and aztreonam but not cephamycins(e.g.,cefoxitin,cefotetan)or carbapenems,and they can be inhibited by clavulanic acid,sulbactam,and tazobactam.The global epidemic ESBL mainly includes SHV,TEM,and CTX-M types.The CTX-M type has been recognized as the predominant beta-lactamase.ESBLs are predominantly found among Enterobacteriaceae,which are inhabitants of intestinal flora and important pathogens in nosocomial and community settings.It is believed that drug resistant bacteria from humans and animals could be discharged into water bodies and their resistance genes would further spread among the bacterial community in water bodies.Water environments are considered as important reservoirs for resistance genes.More importantly,once ESBL-producing Enterobacteriaceae enters the intestine of humans and animals via contaminated food or drinking water,these bacteria could lead to the spread of resistance genes and to serious infections.The ESBLs are plasmid-encoded enzymes,and the plasmids have the potential to transfer between Enterobacteriaceae,leading to the concurrence of super virulent or resistance bacteria,which play an important role in evolution and transfer of drug-resistant gene.However,the mechanism of horizontal plasmid transfer remains to be elucidated.To learn the prevalence and characteristics of ESBL-producing Enterobacteriaceae from different water environments,and horizontal transfer mechanism of antibiotic-resistant plasmid in mice intestine,we conducted the following research.1.Prevalence and characteristics of extended-spectrum beta-lactamase(ESBL)-producing Enterobacteriaceae isolated from rural well water in Tai'an,China,2014To analyze prevalence and characterization of ESBL-producing Enterobacteriaceae from rural well waters in Tai'an,China.A total of 10 isolates expressing an ESBL phenotype,including 9 and 1 Klebsiella pneumoniae was obtained from 4(4 %)out of the 100 sampled wells.ESBL genotype revealed that 9 expressed CTX-M-15 and 1 produced CTX-M-27.Five out of 8 ESBL-producing E.coli expressing CTX-M-15 belonged to ST10,which are mostly detected from human feces in China.Importantly,the only strain of CTX-M-27-producing E.coli belonged to multi-locus sequence type B2:131(ST131),which may be related with severe infection in humans and animals.2.Multidrug resistance found in extended-spectrum beta-lactamase-producing Enterobacteriaceae from rural water reservoirs in Guantao,ChinaThis study aimed to isolate and characterize ESBL-producing Enterobacteriaceae in rural water reservoirs in Guantao,China.ESBL-producing Enterobacteriaceae were found in5(16.7%)of 30 sampled rural water reservoirs.Sixty-six individual isolates expressing an ESBL phenotype were obtained in the present study.Species identification showed that 42 representatives of Escherichia coli,17 Klebsiella pneumoniae,4 Raoultella planticola,and 3Enterobacter cloacae.Twenty isolates contained a single bla gene,including CTX-M(17strains),TEM(2 strains),and SHV(1 strain).Forty-six isolates contained more than one type of beta-lactamase genes.ESBL-producing Enterobacteriaceae isolated in this study were all multidrug resistant.These findings indicated that the serious contamination of ESBL-producing Enterobacteriaceae in rural water reservoirs existed in Guantao,China.3.Antibiotic Resistance and Integrons of Extended-Spectrum ?-Lactamases-Producing Thermotolerant Coliforms(ESBL-TC)Isolated from the Huangqian Water Reservoir in Tai'an,ChinaTo investigate antibiotic resistance,bla gene types,and the presence of integrons(class 1,2,and 3)and gene cassettes in ESBL-TC isolated from the Huangqian Reservoir of Tai'an,China.A total of 96 non-duplicate ESBL-TC were obtained in this study and the ESBL genes included blaCTX-M-14(n=47),blaCTX-M-15(n=27),blaCTX-M-55(n=18),blaSHV-12(n=4),blaCTX-M-3(n=3),and blaCTX-M-123(n=1).Eighty three of the 96 ESBL-TC contained class 1 integrons(86.5%),and 2 isolates harbored class 2 integrons.The sizes of gene cassette regions within integrons were between 0.2 kb to 3.2 kb.Together,the Huangqian Reservoir is seriously contaminated by ESBL-TC and could become a potential reservoir for antibiotic resistance genes.4.High prevalence and risk factors of fecal carriage of CTX-M type extended-spectrum beta-lactamase-producing Enterobacteriaceae from healthy rural residents of Tai'an,ChinaTo understand the prevalence of CTX-M type ESBL-harboring Enterobacteriaceae and to analyze risk factors related with fecal carriage in healthy rural residents in Taian,China.A total of 620 stool samples were collected from rural residents.The ESBL-positive Enterobacteriaceae was screened using ChromID ESBL agar,and then further confirmed by double-disk diffusion.The CTX-M genes were determined using polymerase chain reaction.The risk factors associated with fecal carriage of CTX-M-positive isolates were analyzed using the standard statistic methods.458 isolates carrying CTX-M gene(458/620,73.9%)were obtained from different individuals,and the most dominant genotype was CTX-M-9group(303/458,66.2%).The dominant species were Escherichia coli(E.coli;403/458,88.0%)and Klebsiella pneumoniae(K.pneumoniae;26/458,5.7%)among the isolates carrying CTX-M genes.All the CTX-M producers were resistant to ampicillin,cefazolin,cefuroxime,and ceftriaxone,but were all susceptible to biapenem,imipenem,and meropenem.The results of multivariate logistic regression model identified the enrollment in formal education(OR 2.321;95% CI 1.302–3.768;P = 0.039),the hospitalization history within the last 6 months(OR 1.753;95% CI 1.127–2.584;P = 0.031)and the antibiotics use within the last 6 months(OR 1.892;95% CI 1.242–2.903;P = 0.034).The three variables were significantly associated with carriage of CTX-M ESBL producers(x2 = 21.21;df = 3;P <0.001).The prevalence of fecal carriage of CTX-M ESBL-producing Enterobacteriaceae among healthy rural humans in Tai'an was high,and the recent antibiotic use and hospitalization history may be the important contributors.5.Characterization of Extended-Spectrum ?-Lactamase-Producing Escherichia coli Isolates from Pigs and Farm WorkersFood-producing animals can serve as reservoirs for ESBL-producing Escherichia coli.The present study aimed to characterize and compare ESBL-carrying E.coli isolates from both pigs and farm workers.Rectal swabs were obtained from 60 pigs on four pig-fattening farms(15 samples per farm),and rectal swabs were taken from 40 farm workers on these farms(10 samples per farm).ESBL-carrying E.coli isolates from the workers and pigs were characterized by ESBL genotype,antibiotic susceptibility,enterobacterial repetitive intergenic consensus type,and multilocus sequence type.ESBL-producing E.coli was detected in 34 of60 pigs(56.7%),and 20.0%(8/40)of the farm workers were positive for ESBL-producing E.coli.More importantly,ESBL-producing E.coli isolates with the same ?-lactamase genes,antibiotic resistance profiles,enterobacterial repetitive intergenic consensus types,and multilocus sequence types were detected in both pigs and workers on the same pig farm.These findings were suggestive for transfer of ESBL-producing E.coli between animals andhumans.6.Sequence analysis of drug-resistant plasmid harboring ESBLs geneThe drug-resistant plasmid Tp from E.coli isolates water was sequenced completely and analysed concerning its genetic environment and distributing of antimicrobial resistance genes.The frequency of the horizontal gene transfer was also examined in vitro.The complete sequence of the plasmid was determined using a whole-genome shotgun approach.Tp was a circularly 94,495 bp resistance plasmid belonged to IncI1 incompatibility group.116 putative genes were identified according to the annotation of the finished sequence,including only two antimicrobial resistance genes blaCTX-M-15 and blaTEM-1.The backbone region of the plasmid is very similar to IncI1 plasmid R46,especially the conjugal transfer region and both including colicin Ib gene.Plasmid Tp is a chimera by transposon Tn3-mediated ISEcp1-blaCTX-M-15 mobile element,and its transfer will lead to spread of ESBLs gene,which have important public health significance.7.Colonization of ESBL-producing E.coli from water source in the mouse intestineThe transfer of antimicrobial resistance genes from the water-borne ESBL-producing E.coli was assessed in an intestinal colonization animal model.The ability to colonize and transfer was tested under cefotaxime animicrobial treatments.The water isolate of E.coli colonized in the intestine of mice treated by antimicrobial at levels up to 108cfu/g.In mice without antimicrobial treatment,the strain quickly decreased to below the detection limit due to competitive exclusion by the indigenous mouse flora.Onset of antimicrobial treatment gave immediate rise to detectable levels of the strain in the feces of up to 108 cfu/g feces.The experiment clearly shows that the treatment selects resistant strains and gives advantages to colonize the gastrointestinal tract.Furthermore,transfer of plasmid was observed during colonization of the mouse intestine.The blaCTX-M and blaTEM genotypes were transferred to both an indigenous recipient in the in vivo setting.ESBL-producing E.coli from water is an excellent colonizer of the intestine and is extremely promiscuous with respect to the transferability of its numerous plasmids.Antimicrobial treatment enhances the selection of resistant strains and results in an increase in the resistance gene pool,which ultimately raises the risk of spreading resistance genes.8.Horizontal transfer of antimicrobial resistance plasmid of ESBL-producing E.coli from water in the mouse inflammation intestineTo develop the gut inflammation model.Using a mouse colitis model,we found that Salmonella-inflicted gut inflammation elicits parallel blooms of the donor(108-1011 cfu/g feces)and recipient E.coli(108-1010 cfu/g faeces).These blooms boosted conjugative transferof antibiotic-resistant plasmid Tp from donor strain to recipient strain,and the level of TCs was equal to that of the recipients.The transconjugation efficiency is closed ~100% in vivo and were significantly higher than in control group,suggesting that almost all recipients become TCs harboring plasmid by 5 dpi.In the normal gut experiment,due to the colonization resistance,the level of the donors,recipients detected significantly lower than gut inflammation mice.The level of transconjugants was further lower than that of the recipients.In conclusion,Inflammation elicits concomitant Enterobacteriaceae blooms,which can raise donor and receptor densities to values >100-fold above those typically encountered in the normal mammalian intestine.This fuels the reassortment of genetic material between different Enterobacteriaceae and suggests that infected patients might enhance the spread of plasmid-encoded antibiotic resistance-determinants(e.g.,extened-spectrum ?-lactamases).9.Molecular Mechanism of Horizontal Plasmid Transfer between ESBL-Producing E.coli Isolated from Water Origin in Mouse Intestinal TractA large number of microorganisms in mammalian gut can exchange genes with invading microbes mainly using horizontal plasmid transfer,leading to the concurrence of super virulent or resistance bacteria.However,the mechanism of horizontal plasmid transfer remains to be elucidated.Therefore,this study aimed to explore molecular mechanism of horizontal plasmid transfer between E.coli W121 from water source and resident E.coli.Based on the structure of TP and the plasmid from resident E.coli(EC615),EC615 harbored an 94 kb plasmid >99% identical to plasmid Tp from E.coli W121 strain.The only difference between the two plasmids was an inversion in the shufflon region encoding the variable pilus tip antigen.Using gene knockout technology,experiments verified that the plasmid had retained its ability to conjugate into recipient E.coli.Analysis of plasmid Tp again,the major contributor to horizontal plasmid transfer was a benefit-driven factor--colicin Ib.In summary,this study not only successfully developed a mouse colitis model,but also found the core contributor to the horizontal plasmid transfer.