Molecular Epidemiology of Enterococcal Bacteremia in Australia

Molecular Epidemiology of Enterococcal Bacteremia in Australia

Geoffrey W.Coombs,a,b Julie C.Pearson,a,b Denise A.Daley,b Tam Le,b Owen J.Robinson,a,b Thomas Gottlieb,c

Benjamin P.Howden,d,e Paul D.R.Johnson,d Catherine M.Bennett,f Timothy P.Stinear,d John D.Turnidge,g,h for the Australian Group on Antimicrobial Resistance

Australian Collaborating Centre for Enterococcus and Staphylococcus Species(ACCESS)Typing and Research,School of Biomedical Sciences,Curtin University,Perth, Western Australia,Australia a;Department of Microbiology and Infectious Diseases,PathWest Laboratory Medicine-WA,Royal Perth Hospital,Perth,Western Australia, Australia b;Department of Microbiology and Infectious Diseases,Concord Hospital,Concord,New South Wales,Australia c;Microbiology Department,Austin Health, Heidelberg,Victoria,Australia d;Department of Microbiology and Immunology,University of Melbourne,Melbourne,Victoria,Australia e;Population Health,Deakin University,Melbourne,Australia f;Department of Microbiology and Infectious Diseases,SA Pathology at Women’s and Children’s Hospital,South Australia,Australia g; Departments of Pathology,Paediatrics,and Molecular and Biomedical Science,University of Adelaide,South Australia,Australia h

Enterococci are a major cause of health care-associated infections and account for approximately10%of all bacteremias glob-ally.The aim of this study was to determine the proportion of enterococcal bacteremia isolates in Australia that are antimicro-bial resistant,with particular emphasis on susceptibility to ampicillin and the glycopeptides,and to characterize the molecular epidemiology of the Enterococcus faecalis and Enterococcus faecium isolates.From1January to31December2011,1,079unique episodes of bacteremia were investigated,of which95.8%were caused by either E.faecalis(61.0%)or E.faecium(34.8%).The majority of bacteremias were health care associated,and approximately one-third were polymicrobial.Ampicillin resistance was detected in90.4%of E.faecium isolates but was not detected in E.faecalis isolates.Vancomycin nonsusceptibility was reported in0.6%and36.5%of E.faecalis and E.faecium isolates,respectively.Unlike Europe and the United States,where vancomycin resistance in E.faecium is predominately due to the acquisition of the vanA operon,98.4%of E.faecium isolates harboring van genes carried the vanB operon,and16.1%of the vanB E.faecium isolates had vancomycin MICs at or below the susceptible breakpoint of the CLSI.Although molecular typing identi?ed126E.faecalis pulsed-?eld gel electrophoresis pulsotypes,>50% belonged to two pulsotypes that were isolated across Australia.E.faecium consisted of73pulsotypes from which43multilocus sequence types were identi?ed.Almost90%of the E.faecium isolates were identi?ed as CC17clones,of which approximately half were characterized as ST203,which was isolated Australia-wide.In conclusion,the Australian Enterococcal Sepsis Outcome Programme(AESOP)study has shown that although they are polyclonal,enterococcal bacteremias in Australia are frequently caused by ampicillin-resistant vanB E.faecium.

E nterococci,which were initially believed to be harmless inhab-

itants of the gastrointestinal tract?ora,have emerged as a ma-jor cause of health care-associated infections(1).Globally,they are now thought to account for approximately10%of all bacter-emias(2),and in North America and Europe are the fourth and ?fth leading cause of sepsis,respectively(3).

In the1970s,health care-associated enterococcal infections were associated with the introduction of third-generation cepha-losporins and were primarily due to Enterococcus faecalis(4). However,following the increased use of vancomycin and broad-spectrum antibiotics,signi?cant increases in the numbers of in-fections caused by the more frequently resistant E.faecium were reported in Europe and in North America(5,6).Preceded by an increase in infections and outbreaks caused by ampicillin-resis-tant E.faecium,clinically signi?cant isolates of vancomycin-resis-tant enterococci(VRE)were subsequently detected in the United Kingdom(7)and Europe(8)and shortly after in the United States (9).By the early1990s,VRE had become the second most com-mon nosocomial pathogen in the United States(9)and was en-demic in many North American hospitals(10).Vancomycin re-sistance in E.faecium bacteremia isolates ranges from5to35%in Europe(see www.earss.rivm.nl)to60%in North America(11). The predominant VRE genotype in these two regions is vanA(12). Although the origins of vanA in these settings are unclear,the past use of the glycopeptide avoparcin as a growth promoter in animal husbandry has been proposed as a contributing factor in Europe (13).

In Australia,the?rst reported VRE was a vanA E.faecium from a liver transplant recipient in1995(14).Since this time,however, the vast majority of VRE have been E.faecium harboring the vanB operon(15).Although prevalence or incidence rates of VRE in Australian hospitals are not routinely collected,several reported studies have shown a signi?cant increase in the number of patients infected or colonized with vanB E.faecium(16–18).In the2010 Australian Group on Antimicrobial Resistance(AGAR)period prevalence study of key resistances in clinical isolates of Enterococ-cus species,vancomycin nonsusceptibility occurred in36.5%of E. faecium isolates,of which98%were vanB VRE(see http://www https://www.wendangku.net/doc/1514406727.html,/surveys).

In2011,AGAR commenced the Australian Enterococcal Sepsis Outcome Programme(AESOP).In this study,we determined the proportion of bacteremia isolates of Enterococcus species demon-strating antimicrobial resistance,with particular emphasis on sus-Received

22November2013Returned for modi?cation19December2013

Accepted27December2013

Published ahead of print3January2014

Editor:W.M.Dunne,Jr.

Address correspondence to Geoffrey W.Coombs,

geoffrey.coombs@https://www.wendangku.net/doc/1514406727.html,.au.

Copyright?2014,American Society for Microbiology.All Rights Reserved.

doi:10.1128/JCM.03286-13

March2014Volume52Number3Journal of Clinical Microbiology p.897–https://www.wendangku.net/doc/1514406727.html,897

ceptibility to ampicillin and the glycopeptides and the molecular epidemiology of E.faecalis and E.faecium.

MATERIALS AND METHODS

The Australian Group on Antimicrobial Resistance(AGAR)is a network of laboratories located across Australia which provide microbiology ser-vices to over80%of the country’s tertiary acute care hospitals.AGAR has been conducting national resistance surveillance for more than25years (see https://www.wendangku.net/doc/1514406727.html,),and for the last decade AGAR has been funded by the Australian Department of Health.

Twenty-nine laboratories from all six states,the Australian Capital Territory(ACT),and the Northern Territory(NT)participated in the inaugural2011AGAR Australian Enterococcal Sepsis Outcome Program (AESOP).

From1January to31December2011,the29AGAR laboratories col-lected all enterococcal species isolated from blood cultures.Enterococci with the same species and antimicrobial susceptibility pro?les isolated from a patient’s blood culture within14days of the?rst positive blood culture were excluded.A new enterococcal sepsis episode in the same patient was recorded if it was con?rmed by a further culture of blood taken more than14days after the initial positive culture.

A web-based data entry system was constructed to enable the collec-tion of real-time data into a common database.To ensure patient ano-nymity,but to allow follow-up of discrepant results with each participat-ing site,a record identi?er unique to the participating laboratory was used.

Each episode of bacteremia was designated health care associated if any one of the following criteria applied(19,20),(i)the?rst positive blood culture(s)in an episode of infection were collected?48h after hospital admission,(ii)the patient resided in a nursing home or long-term-care facility within the year preceding the positive blood culture(s), (iii)the patient had a previous hospital admission for?2days within the year preceding the positive blood culture(s),or(iv)the patient was receiv-ing hemodialysis.If the patient did not meet the above criteria and the blood culture was taken before or within48h of hospital admission,then the episode was designated community associated(20).

Participating laboratories identi?ed isolates to the species level by one of the following methods:API20S(bioMérieux),API ID32Strep(bio-Mérieux),Vitek(bioMérieux),Phoenix(BD),Vitek-MS(bioMérieux), matrix-assisted laser desorption ionization(MALDI)Biotyper(Bruker Daltonics),PCR,or conventional biochemical tests.

Antimicrobial susceptibility testing at the contributing site was per-formed according to each laboratory’s routine standardized methodology (Clinical and Laboratory Standards[CLSI]-based disc diffusion,agar di-lution,Vitek2,or Phoenix).Ampicillin and vancomycin susceptibilities were tested by all laboratories.In addition810(75.1%)isolates were tested against linezolid and991(91.8%)and258(23.9%)screened for high-level gentamicin and streptomycin resistance,respectively.For tests performed by the participating institution,CLSI breakpoints were utilized for inter-pretation(21).Isolates with an intermediate or resistant category were classi?ed as nonsusceptible.

Quality control was conducted as part of the routine laboratory prac-tice in all29ISO15189-accredited laboratories.

Of the isolates obtained from the1,033Enterococcus faecium and En-terococcus faecalis sepsis episodes recorded by the participating laborato-ries,963(93.2%)isolates were referred to the Australian Centre for En-terococcus and Staphylococcus Species(ACCESS)Typing and Research for further susceptibility testing and molecular typing.Vancomycin and tei-coplanin MICs were performed by Etest(bioMérieux)according to the manufacturer’s instructions.Linezolid MICs were performed by Etest (bioMérieux)on linezolid-nonsusceptible isolates.Results were inter-preted using both CLSI standards(21)and those of the European Com-mittee on Antimicrobial Susceptibility Testing(EUCAST)(version3.1 [see https://www.wendangku.net/doc/1514406727.html,/clinical_breakpoints/]).Pulsed-?eld gel elec-trophoresis(PFGE)of SmaI-digested DNA agarose plugs was performed on all referred isolates as previously described(22).Banding patterns were examined visually,scanned with a Quantity One device(Bio-Rad Labo-ratories Pty,Ltd.),and digitally analyzed using FPQuest(Bio-Rad Labo-ratories Pty.,Ltd.).The Dice coef?cient and the unweighted pair group method with arithmetic mean were used with settings for tolerance and optimization of1.25%and0.5%,respectively.Isolates with up to a six-band difference were considered the same pulsotype(23).Multilocus se-quence typing(MLST)was performed as previously described on a rep-resentative isolate of each E.faecium PFGE pulsotype(24).If a PFGE pulsotype included vancomycin nonsusceptible and vancomycin suscep-tible E.faecium,MLST was performed on both phenotypes.The sequences were submitted to https://www.wendangku.net/doc/1514406727.html,/,where an allele pro?le was gen-erated and a sequence type(ST)assigned.Bu use of the eBURST V3 algorithm at the same website,STs were classi?ed into eBURST groups when a member of a group shared alleles at?5of the7loci.Double-locus variants(dlvs)of an ST were included within an eBURST group only if the linking single-locus variant(slv)was present.The ancestral ST of an eBURST group was de?ned as the ST that differed from the largest num-ber of other STs at only a single locus(i.e.,the ST that has the greatest number of slvs).STs that diverged by no more than two of the seven MLST loci of the ancestral ST were considered to belong to the same clonal complex(CC).

vanA and vanB PCR was performed on the963referred E.faecium and E.faecalis isolates either by the participating laboratory or by ACCESS Typing and Research.

Regular audits for data discrepancies and potential duplicate entries were conducted and resolved with each of the participating sites during collection.

A chi-square test for comparison of two proportions was performed and95%con?dence intervals were determined using MedCalc for Win-dows,version12.7(MedCalc Software,Ostend,Belgium).

Approval to conduct the prospective data collection was given by the research ethics committee associated with each participating laboratory.

RESULTS

From1January to31December2013,1,079unique episodes of enterococcal bacteremia were identi?ed by the AGAR laborato-

TABLE1Enterococcus faecalis and Enterococcus faecium susceptibility results

Antimicrobial agent E.faecalis E.faecium

No.tested No.(%)resistant(95%CI)a No.tested No.(%)resistant(95%CI)

Vancomycin6583(0.5)(0.1–1.4)b375137(36.5)(31.6–41.6)b Ampicillin6580375339(90.4)(87.0–93.2) Linezolid47211(2.3)(1.2–4.1)b3004(1.3)(0.4–3.3)b

High-level gentamicin607211(34.8)(31.0–38.7)340221(65.0)(59.7–70.1) High-level streptomycin17211(6.4)(3.2–11.2)7918(22.8)(14.1–33.69)

a As described in CLSI M100-A23(21).

b Nonsusceptible.

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ries,of which950(88.0%)were health care associated(95%CI, 85.9to89.9%)and359(33.3%)were polymicrobial(95%CI,30.5 to36.2%).Twenty-eight patients had more than one episode.

Although eight species of Enterococcus were identi?ed,95.8% were either E.faecalis(658isolates,61.0%)or E.faecium(375, 34.8%).Forty-?ve enterococci were identi?ed either as Enterococ-cus casseli?avus(15isolates),E.gallinarum(14),E.avium(8),E. raf?nosus(4),E.durans(2),or E.hirae(2).One isolate could not be identi?ed to the species level.

Overall,90.4%of E.faecium were ampicillin resistant(Table 1).Ampicillin resistance was not detected in E.faecalis.Vancomy-cin nonsusceptibility(CLSI)was reported by the participating lab-oratories in0.6%and36.5%of E.faecalis and E.faecium isolates, respectively.Of the137vancomycin-nonsusceptible E.faecium isolates,133(97.1%)were also ampicillin resistant.MICs were performed on14of the15isolates reported as linezolid nonsus-ceptible by the participating laboratory using CLSI interpretations (3resistant and11intermediate).Eight isolates had MICs of2mg/liter(susceptible)and six isolates had MICs of4mg/liter(in-termediate using CLSI interpretations and susceptible using EUCAST interpretations).High-level gentamicin resistance was reported by the participating laboratories in65.0%of E.faecium and34.8%of E.faecalis isolates.In contrast,22.8%of E.faecium and6.4%of E.faecalis isolates were high-level streptomycin resis-tant.

Of the963isolates referred to the Australian Centre for Entero-coccus and Staphylococcus Species(ACCESS)Typing and Re-search,622(64.6%)were E.faecalis and341(35.4%)were E.fae-cium.

Enterococcus faecalis.The vancomycin MICs for the622E. faecalis isolates ranged from0.25to?256mg/liter,with a mode of 2mg/liter(Fig.1).The three vancomycin nonsusceptible isolates (?4mg/liter,0.6%)harbored the vanB gene and had vancomycin MICs of16mg/liter,32mg/liter,and?256mg/liter.No vanA or vanB genes were detected in the vancomycin-susceptible isolates. The teicoplanin MICs ranged from0.064to2mg/liter,with

a

FIG1Enterococcus faecalis and Enterococcus faecium vancomycin

MICs.

FIG2Enterococcus faecalis and Enterococcus faecium teicoplanin MICs.

Enterococcal Bacteremia in Australia https://www.wendangku.net/doc/1514406727.html,899

mode of0.25mg/liter(Fig.2).The three vanB-containing isolates had teicoplanin MICs of0.125mg/liter(two isolates)and0.25 mg/liter(one isolate),i.e.,below the susceptible CLSI and EUCAST breakpoints.

By PFGE,618of the622E.faecalis isolates were classi?ed into 126pulsotypes,of which nine pulsotypes(Efs1to Efs9)had10or more isolates(Fig.3).Four isolates could not be typed by PFGE. Of the117pulsotypes that had?10isolates,66pulsotypes were represented by only one isolate.Geographically,the nine major pulsotypes were widely distributed,with the two predominant pulsotypes,Efs1(191isolates)and Efs2(103isolates),isolated across Australia.The three van B E.faecalis isolates were detected in pulsotype https://www.wendangku.net/doc/1514406727.html,pared to non-Efs1/Efs2bacteremia cases,a signi?cantly higher percentage of Efs1and Efs2bacteremia cases were health care associated(80.8%versus88.1%)(P?0.0171).

Enterococcus faecium.The vancomycin MICs for the341E. faecium isolates ranged from0.25to?256mg/liter,with a mode of1.0mg/liter(Fig.1).Of the234vancomycin-susceptible isolates (MIC?4mg/liter),20(8.5%)harbored the vanB gene and had vancomycin MICs that ranged from0.5mg/liter to4.0mg/liter (Fig.4).Of the107vancomycin-nonsusceptible isolates(MIC?4 mg/liter),two isolates harbored the vanA gene(vancomycin

MIC FIG3Distribution and proportion of Enterococcus faecalis(Efs)pulsotypes across

Australia.

FIG4MICs of vanA-,vanB-,and vanA/B-negative Enterococcus faecium.

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?256mg/liter)and104harbored the vanB gene(vancomycin MICs6to?256mg/liter)(Fig.4).One isolate with a vancomycin MIC of6mg/liter did not harbor vanA or vanB genes.The teico-planin MICs ranged from0.047to?256mg/liter,with a mode of 0.5mg/liter(Fig.2).The two vanA isolates had teicoplanin MICs of32and64mg/liter.Of the124vanB isolates,one was teicoplanin intermediate(MIC16mg/liter)and three were resistant(MIC ?16mg/liter)by CLSI criteria,and?ve were resistant by EUCAST criteria(MIC?2mg/liter).

By PFGE,the341E.faecium isolates were classi?ed into73 pulsotypes from which43multilocus STs were identi?ed(Table 2).Three of the seven housekeeping genes in PFGE pulsotype Efm56could not be ampli?ed using the recommended MLST primers and therefore a ST could not be assigned.By eBURST,28

TABLE2Molecular epidemiology of health care-and community-associated Enterococcus faecium isolates

Group CC ST MLST allelic pro?le Pulsotypes identi?ed by PFGE(no.of isolates)Total no.(%)

of isolates

No.of

isolates

with vanA

gene

No.of

isolates

with vanB

gene

No.of health

care-associated

isolates

No.of

community-

associated

isolates

11720315-1-1-1-1-20-1Efm1(77),Efm2(48),Efm6(15),Efm12(4),

Efm14(3),Efm15(3),Efm16(3),Efm17

(2),Efm35(1),Efm41(1),Efm50(1),

Efm69(1)

159(46.6)791536

171-1-1-1-1-1-1Efm5(26),Efm9(4),Efm10(4),Efm11(4),

Efm18(2),Efm19(2),Efm36(1),Efm46

(1),Efm49(1),Efm54(1),Efm57(1)

47(13.8)447

34115-5-1-1-1-1-1Efm3(37),Efm32(1)38(11.1)13338

5554-1-1-1-1-1-1Efm4(34)34(10.0)2304

2521-5-1-1-1-1-1Efm7(10),Efm38(1)11(3.2)111

809-1-1-1-12-1-1Efm8(5),Efm28(1)6(1.8)126

2021-1-1-1-1-7-1Efm48(1)1(0.3)1

1179-1-1-1-1-1-1Efm72(1)1(0.3)1

2331-1-1-1-1-20-1Efm37(1)1(0.3)1 UD41415-5-1-1-1-20-1Efm23(1),Efm64(1)2(0.6)211

UD19215-1-1-1-1-7-1Efm24(1)1(0.3)1

UD26615-1-6-6-1-7-1Efm20(2)2(0.6)2

UD86915-1-1-6-1-7-1Efm55(1)1(0.3)1

222222-3-1-2-1-1-1Efm13(3),Efm40(1)4(1.2)4

6823-3-2-2-1-1-1Efm29(1)1(0.3)1

5332-3-1-14-1-1-1Efm31(1)1(0.3)1

323-3-1-2-1-1-1Efm63(1)1(0.3)1

219-3-1-2-1-1-1Efm71(1)1(0.3)1

317836113-8-8-8-6-51-6Efm26(1)1(0.3)1 9413-8-8-8-6-10-6Efm30(1),Efm61(1)2(0.6)11

60413-8-8-23-6-27-6Efm60(1)1(0.3)1

6013-8-8-8-11-10-6Efm62(1)1(0.3)1

17813-8-8-8-6-27-6Efm67(1)1(0.3)1

4UD b187-1-1-1-5-1-1Efm21(1),Efm22(1),Efm45(1)3(0.9)3 UD2737-3-1-1-5-7-1Efm53(1)1(0.3)1

UD2627-1-1-1-5-7-1Efm51(1)1(0.3)1

5UD67425-8-14-22-10-73-6Efm47(1)1(0.3)1 UD64825-8-14-22-10-71-6Efm68(1)1(0.3)1

S a S197-1-1-1-1-11-1Efm27(1)1(0.3)1

S S542-9-6-6-1-11-1Efm25(1)1(0.3)1

S S2645-4-6-6-2-1-8Efm33(1)1(0.3)1

S S87025-8-14-22-10-19-11Efm34(1)1(0.3)1

S S5835-18-14-22-8-26-6Efm39(1)1(0.3)1

S S8673-7-3-73-1-1-1Efm42(1)1(0.3)1

S S6392-40-12-3-1-1-1Efm43(1)1(0.3)1

S S7985-13-18-17-8-19-6Efm44(1)1(0.3)1

S S8684-5-1-6-1-92-1Efm52(1)1(0.3)1

S S8634-59-1-1-1-20-1Efm58(1)1(0.3)11

S S479-2-1-11-1-14-5Efm59(1)1(0.3)1

S S8735-4-1-3-1-1-5Efm65(1)1(0.3)1

S S87136-21-9-17-10-59-6Efm66(1)1(0.3)1

S S87515-1-1-22-1-20-6Efm70(1)1(0.3)1

S S86613-8-58-72-6-27-6Efm73(1)1(0.3)1

NT c Efm56(1)1(0.3)1

a UD,unde?ned.

b S,singleton.

c NT,nontypeable using recommende

d Enterococcus faecium MLST primers.

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of the 43STs were grouped into ?ve eBURST groups (Fig.5).The remaining 15STs were classi?ed as singletons.eBURST group 1consisted of 13STs of which 9STs formed clonal complex (CC)17.The ancestral ST within CC17was identi?ed as ST17,from which seven single-locus variants (slvs)and the double locus vari-ant (dlv)ST203were identi?ed.ST203was considered a co-founder within eBURST group 1from which an additional four STs were linked.Overall,38pulsotypes were identi?ed in eBURST group 1,of which 12and 11pulsotypes were characterized as ST203and ST17,respectively.A further two CCs were character-ized,including CC22in eBURST group 2and CC178in eBURST group 3.An ancestral ST was not identi?ed in eBURST groups 4and 5.

Overall,304(89.1%)E.faecium isolates were grouped into eBURST group 1.Five STs,all found in CC17,had more than 10isolates (Fig.6).The two major STs,ST203(159isolates)and ST17(47isolates),were isolated across Australia.ST341(38isolates)and ST252(11isolates)were isolated only in the eastern regions of Australia,and ST555(34isolates)was isolated in the western and central regions.

Apart from ST863(pulsotype Efm58),isolates harboring a vanA or vanB gene were located in eBURST group 1.However,ST863,a ST203dlv,was not included in eBURST group 1be-cause no linking slv was identi?ed.van genes were identi?ed in the ?ve major STs and in ST80and ST414.Although only 8.5%(4/47)of ST17isolates harbored vanB ,vanB genes were iden-ti?ed in 50.6%(79/153)of ST203isolates,including seven of the 12ST203pulsotypes.All 48of the ST203Efm2isolates were vanB positive.

Overall,94.4%of E.faecium isolates were health care https://www.wendangku.net/doc/1514406727.html,pared to non-group 1E.faecium bacteremia,a signi?-cantly higher percentage of cases of group 1E.faecium bacteremia were health care associated,78.4%versus 96.4%(P ?0.0001).

DISCUSSION

Similar to the situation in the United States (25)and in Europe (26),in Australia enterococcal bacteremia,and notably bacter-emia caused by multidrug-resistant E.faecium ,has become a sig-ni?cant problem.In the AESOP 2011study,approximately one in three cases of enterococcal bacteremia was due to E.faecium ,of which 90.4%were ampicillin resistant and 36.5%were vancomy-cin nonsusceptible.However,unlike Europe and the United States,where vancomycin resistance in E.faecium is predomi-nately due to the acquisition of the vanA operon,almost all E.faecium blood culture isolates in Australia harboring van genes carried the vanB operon (98.4%).Twenty (16.1%)of the 124vanB E.faecium isolates had vancomycin MICs at or below the CLSI susceptible breakpoint (?4mg/liter)and would not have been identi?ed using routine phenotypic antimicrobial susceptibility methods.

Similar to the study performed by Pinholt and colleagues in Denmark (2),in our study the majority of bacteremia cases were health care associated,and approximately one-third of episodes were polymicrobial.

With the use of PFGE,both enterococcal species were shown to be very polyclonal,con?rming the enormous plasticity of the en-terococcal genome,which has been demonstrated to acquire ge-netic elements that can account for up to 25%of the genome (27

).

FIG 5eBURST-generated population snapshot of Enterococcus faecium sequence types (STs)isolated in the 2011Australian Enterococcus Sepsis Outcome Program (AESOP).Each ST is represented by a black dot.The numbers refer to a particular sequence type (ST).The size of each dot re?ects the number of isolates within a ST.The ancestral ST of a clonal complex is represented by a blue dot.The yellow-colored dot (ST203)is considered a subgroup cofounder.

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Journal of Clinical Microbiology

Although 126E.faecalis PFGE pulsotypes were identi?ed,47.6%of the isolates belonged to either Efs1or Efs2.Isolated across Aus-tralia,these two strains have had success in extending their geo-graphical range,which may be due to the acquisition of mobile genetic elements and single nucleotide polymorphisms that favor spread and persistence.However,this hypothesis will need to be con?rmed by whole-genome sequencing.Both ampicillin and vancomycin resistance in E.faecalis were uncommon.Only three Efs2isolates harbored van genes,and all were vanB .

The relative clinical importance of E.faecium bacteremia has increased with the emergence of resistance to antimicrobials such as ampicillin and vancomycin (28,29).In at least one Australian institution,VRE bacteremia has reportedly surpassed methicillin-resistant Staphylococcus aureus (MRSA)bacteremia (18).Glob-ally,hospital-derived E.faecium isolates have been shown to be part of a single clonal lineage,designated CC17after its presumed founder ST17,that has successfully adapted to hospital environ-ments (30).CC17is characteristically ampicillin and quinolone resistant,and subsequent acquisition of vanA -or vanB -contain-ing transposons by horizontal gene transfer in CC17clones has resulted in VRE with pandemic potential.In the AESOP 2011study,the ?ve major blood culture E faecium clones were all grouped into CC17.In addition,seven of the eight clones harbor-ing the vanA or vanB genes were also CC17clones.The eighth clone (ST863),although not a CC17clone,was an ST203dlv.ST203,a dlv of ST17,was ?rst described in Australia in 2010as the cause of a sustained outbreak of vanB E.faecium bacteremia in a hospital located on the eastern seaboard of Australia (18).Sub-sequent to this report,ST203has been identi?ed as a major vanB E.faecium clone in other Australian regions (31),Outside of Aus-tralia,ST203has also been reported in many Asian and European countries (https://www.wendangku.net/doc/1514406727.html,/),although isolates from

these countries almost always possess the vanA gene.In our study,46.6%of E.faecium isolates were characterized as ST203,and these were isolated Australia wide.Approximately 50%of ST203isolates carried the vanB operon,while none harbored vanA genes.Although 12PFGE ST203pulsotypes were identi?ed,almost 80%of isolates were classi?ed as either pulsotype Efm1or pulsotype Efm2.Why some clones within CC17dominate over other clones,and why within a clone some pulsotypes dominate over other pulsotypes,requires further genetic analysis.

ST17,the presumed founder of CC17,is thought to have suc-cessfully adapted to the hospital environment by the cumulative acquisition of resistance (ampicillin and vancomycin)and genes (esp Efm ,hyl Efm ,and fms )that result in a putative selective advan-tage (32).The acquisition of these genes has been followed by the genetic diversi?cation of ST17,resulting in many slvs and dlvs that presumably have acquired or lost genetic elements.This appears to have facilitated clones in gaining further selective advantages in hospital environments.

Importantly,the successful transmission of a genetically adapted vancomycin-resistant E.faecium clone in a hospital may not be attributable solely to suboptimal infection control prac-tices.Recently,de novo generation of vancomycin-resistant vanB E.faecium has been demonstrated to occur within patients,pre-sumably occurring in the normal colonic ?ora by acquisition of the vanB operon from anaerobic ?ora (33).The acquisition of a selective advantage by a VRE clone not necessarily lead to the long-term survival of that clone.In 2004,we described a large single-strain outbreak of vanB E.faecium across several hospitals in Western Australia (17).Characterized as CC17ST173(34),once eradicated from the hospital environment this strain has not subsequently been isolated in Western Australia,nor was it de-tected in the AESOP 2011bacteremia study.Furthermore,

failure

FIG 6Distribution and proportion of Enterococcus faecium (Efm)multilocus sequence types (ST)across Australia.

Enterococcal Bacteremia in Australia

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to isolate ST173during long-term follow-up screening of previ-ously colonized patients suggests the strain no longer had a selec-tive advantage once outside the hospital environment.Whole-genome sequencing of isolates from the AESOP2011study will be an important resource in our efforts to understand this species and to develop novel infection control strategies to prevent the emergence of this hospital superbug.

Our study had a number of limitations.Although achieving national coverage,the participating laboratories service only a mi-nority of the Australian hospitalized population.Further,MIC assays for vancomycin,teicoplanin,and linezolid were performed by a commercial gradient diffusion method,and not the standard reference broth microdilution method.

In conclusion,the AESOP2011study has shown that the ma-jority of Australian enterococcal bacteremias are health care asso-ciated,and though predominantly caused by E.faecalis,they are frequently caused by ampicillin-resistant vanB E.faecium.Molec-ular typing characterized over50%of E.faecalis isolates as two PFGE pulsotypes,and almost90%of E.faecium isolates as CC17 clones,of which approximately half were ST203.Further studies of the enterococcal genome will contribute to our understanding of the evolution of enterococci in the hospital environment and assist in preventing their nosocomial transmission. ACKNOWLEDGMENTS

We gratefully acknowledge Hui-leen Tan,Yung Ching Lee,and Lynne Wilson from the Department of Microbiology and Infectious Diseases, PathWest Laboratory Medicine-WA,Royal Perth Hospital;Frances O’Brien and Ka Yan Wong from the Australian Collaborating Centre for Enterococcus and Staphylococcus Species(ACCESS)Typing and Research, School of Biomedical Sciences,Curtin University;and the WA Genome Resource Centre,Department of Clinical Immunology and Biochemical Genetics,Royal Perth Hospital,for the molecular typing of enterococci.

This study was primarily funded by a grant from the Australian Gov-ernment Department of Health and Ageing.

Members of the AGAR in2011were,in the Australian Capital Terri-tory,Peter Collignon and Susan Bradbury(The Canberra Hospital);in New South Wales,Tom Gottlieb and Graham Robertson(Concord Hos-pital),Miriam Paul and Richard Jones(Douglass Hanly Moir Pathology), James Branley and Donna Barbaro(Nepean Hospital),George Kotsiou and Peter Huntington(Royal North Shore Hospital),Colin MacLeod and Bradley Watson(Royal Prince Alfred Hospital),Iain Gosbell and Anna-belle LeCordier(Liverpool Hospital),David Mitchell and Lee Thomas (Westmead Hospital);in the Northern Territory,Jann Hennessy and Rob Baird(Royal Darwin Hospital);in Queensland,Enzo Binotto and Bron-wyn Thomsett(Pathology Queensland Cairns Base Hospital),Graeme Nimmo and Narelle George(Pathology Queensland Central Laboratory), Petra Derrington and Sharon Dal-Cin(Pathology Queensland Gold Coast Hospital),Chris Coulter and Sonali Coulter(Pathology Queensland Prince Charles Hospital),Joan Faoagali and Joel Douglas(Pathology Queensland Princess Alexandra Hospital),Jenny Robson and Georgia Peachey(Sullivan Nicolaides Pathology);in South Australia,Kelly Pa-panoum and Nicholas Wells(SA Pathology[Flinders Medical Centre]), Morgyn Warner and Fleur Manno(SA Pathology[Royal Adelaide Hos-pital]),John Turnidge and Jan Bell(SA Pathology[Women’s and Chil-dren’s Hospital]);in Tasmania,Mhisti Rele and Kathy Wilcox(Launces-ton General Hospital),Louise Cooley and Rob Peterson(Royal Hobart Hospital);in Victoria,Denis Spelman and Michael Huysmans(The Alfred Hospital),Benjamin Howden and Peter Ward(Austin Hospital),Tony Korman and Despina Kotsanas(Monash Medical Centre),Suzanne Gar-land and Gena Gonis(Royal Women’s Hospital),Mary Jo Waters and Linda Joyce(St Vincent’s Hospital);in Western Australia,David McGechie and Rebecca Wake(PathWest Laboratory Medicine–WA Fremantle Hospital),Barbara Henderson and Ronan Murray(PathWest Laboratory Medicine-WA Queen Elizabeth II Hospital),Keryn Christian-sen,Denise Daley,and Geoffrey Coombs(PathWest Laboratory Medi-cine-WA Royal Perth Hospital),Victoria D’Abrera and Sindy Budalich(St John of God Pathology).

REFERENCES

1.Hidron AI,Edwards JR,Patel J,Horan TC,Sievert DM,Pollock DA,

Fridkin SK,National Healthcare Safety Network Team Participating National Healthcare Safety Network Facilities.2008.NHSN annual update:antimicrobial-resistant pathogens associated with healthcare-associated infections:annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Preven-tion,2006–2007.Infect.Control Hosp.Epidemiol.29:996–1011.http: //https://www.wendangku.net/doc/1514406727.html,/10.1086/591861.

2.Pinholt M,Ostergaard C,Arpi M,Bruun NE,Schonheyder HC,Gradel

KO,Sogaard M,Knudsen JD,for the Danish Collaborative Bacteraemia Network.2013.Incidence,clinical characteristics and30-day mortality of enterococcal bacteraemia in Denmark2006–2009:A population-based cohort study.Clin.Microbiol.Infect.https://www.wendangku.net/doc/1514406727.html,/10.1111/1469 -0691.12236.

3.Deshpande LM,Fritsche TR,Moet GJ,Biedenbach DJ,Jones RN.2007.

Antimicrobial resistance and molecular epidemiology of vancomycin-resistant enterococci from North America and Europe:a report from the SENTRY antimicrobial surveillance program.Diagn.Microbiol.Infect.

Dis.58:163–170.https://www.wendangku.net/doc/1514406727.html,/10.1016/j.diagmicrobio.2006.12.022.

4.Murray BE.1990.The life and times of the Enterococcus.Clin.Microbiol.

Rev.3:46–65.

5.Simonsen GS,Smabrekke L,Monnet DL,Sorensen TL,Moller JK,

Kristinsson KG,Lagerqvist-Widh A,Torell E,Digranes A,Harthug S, Sundsfjord A.2003.Prevalence of resistance to ampicillin,gentamicin and vancomycin in Enterococcus faecalis and Enterococcus faecium isolates from clinical specimens and use of antimicrobials in?ve Nordic hospitals.

J.Antimicrob.Chemother.51:323–331.https://www.wendangku.net/doc/1514406727.html,/10.1093/jac /dkg052.

6.Treitman AN,Yarnold PR,Warren J,Noskin GA.2005.Emerging

incidence of E nterococcus faecium among hospital isolates(1993to2002).

J.Clin.Microbiol.43:462–463.https://www.wendangku.net/doc/1514406727.html,/10.1128/JCM.43.1.462 -463.2005.

7.Uttley AH,Collins CH,Naidoo J,George RC.1988.Vancomycin-

resistant https://www.wendangku.net/doc/1514406727.html,ncet i:57–58.

8.Leclercq R,Derlot E,Duval J,Courvalin P.1988.Plasmid-mediated

resistance to vancomycin and teicoplanin in Enterococcus faecium.N.Engl.

J.Med.319:157–161.https://www.wendangku.net/doc/1514406727.html,/10.1056/NEJM198807213190307.

9.Frieden TR,Munsiff SS,Low DE,Willey BM,Williams G,Faur Y,

Eisner W,Warren S,Kreiswirth B.1993.Emergence of vancomycin-resistant enterococci in New York https://www.wendangku.net/doc/1514406727.html,ncet342:76–79.http://dx.doi .org/10.1016/0140-6736(93)91285-T.

10.Murray BE.2000.Vancomycin-resistant enterococcal infections.N.Engl.

J.Med.342:710–721.https://www.wendangku.net/doc/1514406727.html,/10.1056/NEJM200003093421007.

11.Bearman GM,Wenzel RP.2005.Bacteremias:a leading cause of death.

Arch.Med.Res.36:646–659.https://www.wendangku.net/doc/1514406727.html,/10.1016/j.arcmed.2005.02 .005.

12.Cetinkaya Y,Falk P,Mayhall CG.2000.Vancomycin-resistant entero-

cocci.Clin.Microbiol.Rev.13:686–707.https://www.wendangku.net/doc/1514406727.html,/10.1128/CMR .13.4.686-707.2000.

13.Klare I,Heier H,Claus H,Reissbrodt R,Witte W.1995.vanA-mediated

high-level glycopeptide resistance in Enterococcus faecium from animal husbandry.FEMS Microbiol.Lett.125:165–171.https://www.wendangku.net/doc/1514406727.html,/10 .1111/j.1574-6968.1995.tb07353.x.

14.Kamarulzaman A,Tosolini FA,Boquest AL,Geddes JE,Richards MJ.

1995.Vancomycin resistant Enterococcus faecium in a liver transplant re-cipient.Aust.N.Z.J.Med.25:560.

15.Bell J,Turnidge J,Coombs G,O’Brien F.1998.Emergence and epide-

miology of vancomycin-resistant enterococci in https://www.wendangku.net/doc/1514406727.html,mun.Dis.

Intell.22:249–252.

16.Cooper E,Paull A,O’Reilly M.2002.Characteristics of a large cluster of

vancomycin-resistant enterococci in an Australian hospital.Infect.Con-trol Hosp.Epidemiol.23:151–153.https://www.wendangku.net/doc/1514406727.html,/10.1086/502027. 17.Christiansen KJ,Tibbett PA,Beresford W,Pearman JW,Lee RC,

Coombs GW,Kay ID,O’Brien FG,Palladino S,Douglas CR,Mont-gomery PD,Orrell T,Peterson AM,Kosaras FP,Flexman JP,Heath

Coombs et al.

https://www.wendangku.net/doc/1514406727.html, Journal of Clinical Microbiology

CH,McCullough CA.2004.Eradication of a large outbreak of a single strain of vanB vancomycin-resistant Enterococcus faecium at a major Aus-tralian teaching hospital.Infect.Control Hosp.Epidemiol.25:384–390.

https://www.wendangku.net/doc/1514406727.html,/10.1086/502410.

18.Johnson PD,Ballard SA,Grabsch EA,Stinear TP,Seemann T,Young

HL,Grayson ML,Howden BP.2010.A sustained hospital outbreak of vancomycin-resistant Enterococcus faecium bacteremia due to emergence of vanB E.faecium sequence type203.J.Infect.Dis.202:1278–1286.http: //https://www.wendangku.net/doc/1514406727.html,/10.1086/656319.

19.Klevens RM,Morrison MA,Nadle J,Petit S,Gershman K,Ray S,

Harrison LH,Lyn?eld R,Dumyati G,Townes JM,Craig AS,Zell ER, Fosheim GE,McDougal LK,Carey RB,Fridkin SK,Active Bacterial Core surveillance(ABCs)Investigators MRSA.2007.Invasive methicillin-resistant Staphylococcus aureus infections in the United States.JAMA298: 1763–1771.https://www.wendangku.net/doc/1514406727.html,/10.1001/jama.298.15.1763.

20.Klevens RM,Morrison MA,Fridkin SK,Reingold A,Petit S,Gershman K,

Ray S,Harrison LH,Lyn?eld R,Dumyati G,Townes JM,Craig AS, Fosheim G,McDougal LK,Tenover https://www.wendangku.net/doc/1514406727.html,munity-associated me-thicillin-resistant Staphylococcus aureus and healthcare risk factors.Emerg.

Infect.Dis.12:1991–1993.https://www.wendangku.net/doc/1514406727.html,/10.3201/eid1212.060505. 21.Clinical and Laboratory Standards Institute..2012.Performance stan-

dards for antimicrobial susceptibility testing;22nd informational supple-ment.CLSI M100-S22.Clinical and Laboratory Standards Institute, Wayne,PA.

22.Kulski JK,Wilson RD,Bending R,Grubb W.1998.Antibiotic resistance

and genomic analysis of enterococci in an intensive care unit and general wards.Pathol.30:68–72.https://www.wendangku.net/doc/1514406727.html,/10.1080/00313029800169705.

23.Tenover FC,Arbeit RD,Goering RV,Mickelsen PA,Murray BE,

Persing DH,Swaminathan B.1995.Interpreting chromosomal DNA restriction patterns produced by pulsed-?eld gel electrophoresis:criteria for bacterial strain typing.J.Clin.Microbiol.33:2233–2239.

24.Homan WL,Tribe D,Poznanski S,Li M,Hogg G,Spalburg E,Van

Embden JD,Willems RJ.2002.Multilocus sequence typing scheme for Enterococcus faecium.J.Clin.Microbiol.40:1963–1971.https://www.wendangku.net/doc/1514406727.html, /10.1128/JCM.40.6.1963-1971.2002.

25.Wisplinghoff H,Bischoff T,Tallent SM,Seifert H,Wenzel RP,Edmond

MB.2004.Nosocomial bloodstream infections in US hospitals:analysis of 24,179cases from a prospective nationwide surveillance study.Clin.In-fect.Dis.39:309–317.https://www.wendangku.net/doc/1514406727.html,/10.1086/421946.26.de Kraker ME,Jarlier V,Monen JC,Heuer OE,van de Sande N,

Grundmann H.2013.The changing epidemiology of bacteraemias in Europe:trends from the European Antimicrobial Resistance Surveillance System.Clin.Microbiol.Infect.19:860–868.https://www.wendangku.net/doc/1514406727.html,/10.1111 /1469-0691.12028.

27.Arias CA,Murray BE.2012.The rise of the Enterococcus:beyond vanco-

mycin resistance.Nat.Rev.Microbiol.10:266–278.https://www.wendangku.net/doc/1514406727.html,/10 .1038/nrmicro2761.

28.Iwen PC,Kelly DM,Linder J,Hinrichs SH,Dominguez EA,Rupp ME,

Patil KD.1997.Change in prevalence and antibiotic resistance of Entero-coccus species isolated from blood cultures over an8-year period.Antimi-crob.Agents Chemother.41:494–495.

29.Cheah AL,Spelman T,Liew D,Peel T,Howden BP,Spelman D,

Grayson ML,Nation RL,Kong DC.2013.Enterococcal bacteraemia: factors in?uencing mortality,length of stay and costs of hospitalization.

Clin.Microbiol.Infect.19:E181–E189.https://www.wendangku.net/doc/1514406727.html,/10.1111/1469 -0691.12132.

30.Willems RJ,Top J,van Santen M,Robinson DA,Coque TM,Baquero

F,Grundmann H,Bonten MJ.2005.Global spread of vancomycin-resistant Enterococcus faecium from distinct nosocomial genetic complex.

Emerg.Infect.Dis.11:821–828.https://www.wendangku.net/doc/1514406727.html,/10.3201/1106.041204.

31.Kamolvit W,Sidjabat HE,Nimmo GR,Anuj SN,Bergh H,Richardson

LJ,Paterson DL.2013.Predominance of VREfm ST203subgroup in Queensland.Pathol.45:99.https://www.wendangku.net/doc/1514406727.html,/10.1097/PAT.0b013e32835 b68d2.

32.Top J,Willems R,Bonten M.2008.Emergence of CC17Enterococcus

faecium:from commensal to hospital-adapted pathogen.FEMS immunol Med.Microbiol.52:297–308.https://www.wendangku.net/doc/1514406727.html,/10.1111/j.1574-695X.20

08.00383.x.

33.Howden BP,Holt KE,Lam MM,Seemann T,Ballard S,Coombs GW,

Tong SY,Grayson ML,Johnson PD,Stinear TP.2013.Genomic insights to control the emergence of vancomycin-resistant enterococci.mBio 4:eoo412–13.https://www.wendangku.net/doc/1514406727.html,/10.1128/mBio.00412-13.

34.Freitas AR,Tedim AP,Novais C,Ruiz-Garbajosa P,Werner G,

Laverde-Gomez JA,Canton R,Peixe L,Baquero F,Coque TM.2010.

Global spread of the hyl(Efm)colonization-virulence gene in mega-plasmids of the Enterococcus faecium CC17polyclonal subcluster.Anti-microb.Agents Chemother.54:2660–2665.https://www.wendangku.net/doc/1514406727.html,/10.1128 /AAC.00134-10.

Enterococcal Bacteremia in Australia

https://www.wendangku.net/doc/1514406727.html,905

细胞线粒体毒性的检测(Molecular Devices)

如何借助于具有SpectraMax MinMax300 细胞成像系统的SpectraMax i3多功能微孔板检测平台进行细胞线粒体毒性的检测 简介 优势 Application Highlight 评价待选新药对细胞线粒体产生的毒性作用一直被认为是药物筛选过程中的关键步骤，目前可轻松通过不同颜色的荧光染料来标记细胞线粒体后来检测其活性的变化，整个检测过程中通常也需要结合细胞核复染色法，例如使用DAPI染料，这样就可以在图像中轻松识别出所有细胞。可以利用成像系统的一个荧光通道识别被染核的细胞，而细胞线粒体活性检测可以通过特定的线粒体染料标记后，来分析第二个荧光通道中每个标记后细胞的荧光信号强度值。然而，细胞核复染色过程会增加一系列额外的操作步骤，使得实验过程变得繁琐、拖延检测时间。来自于Molecule Devices公司的StainFree ?技术(无标记分析技术)可以解放研究人员，使其无需借助于繁琐的细胞核复染色法来识别细胞，可以直接利用SpectraMax ? MiniMax ? 300 细胞成像系统的透射光通道来直接识别出单个细胞。此篇应用文献重点介绍如何利用SpectraMax ? i3多功能微孔板检测平台的细胞成像功能来检测细胞线粒体毒性，缬氨霉素，一种离子型抗生素，可对PC12细胞线粒体功能造成损伤，使用MitoTracker Deep Red FM来标记线粒体后对整个过程进行检测，然后分别使用StainFree方法和细胞核荧光染料标记法对细胞计数，SoftMax Pro软件分析比较两组结果。SpectraMax MiniMax 300细胞成像系统和SoftMax Pro软件组合后，可以帮助研究人员获取图像信息和进行数据分析，准确、快速的给出潜在化合物对细胞线粒体毒性的浓度效应学曲线。 基于细胞检测试验方法的设置 使用含有2.5%胎牛血清、15%的马血清和1%青霉素/链霉素的F-12K培养基来培养PC12(大鼠肾上腺嗜铬瘤细胞)细胞，在黑色底通的96孔板中每孔加入100ul配好的培养基，细胞的密度为10,000/孔，培养过夜。隔天，细胞孔内加入缬氨霉素，其浓度范围是1μM至1nM并按12浓度梯度关系进行稀释，然后将处理后的微孔板放入37度二氧化碳培养箱中培养24小时。完成孵育处理后，将混有化合物的培养基去除，随后加入含有100nM 的MitoTracker Deep Red FM(MTDR)染料的新鲜培养基。此试剂可以标记活细胞中的线粒体，可以利用MinMax 细胞成像系统的远红外荧光通道检测其在远红外波段下的荧光强度值。 细胞孵育30分钟后，去除混有染料的培养基，使用4%多聚甲醛固定细胞，固定后的细胞核使用EarlyTox Dead Green 染料来标记(Molecular Devices cat. #R8216)。虽然固定后是无需使用StainFree方法来分析细胞数目、形态的，但是此目的是比较这次实验中所有细胞在不同通道下获得数据的一致性。利用MinMax 细胞成像系统的透射光通道对细胞进行成像，获取细胞数目和分析其形态变化，利用其红色荧光通道检测其线粒体活性。同样利用系统的绿色荧光通道对染核细胞进行计数分析后与使用透射光通道下StainFree方法计数的细胞进行比较。 使用SoftMax Pro软件，设置两种不同的成像分析方法，第一种分析，单个细胞的识别是通过StainFree方法，可在透射光通道下识别单个细胞而无需利用细胞核荧光染料分子进行标记。为识别每个细胞，在软件中将测量的线粒体的红色荧光信号以一定形式参数输出。 ???StainFree技术无需利用细胞核复染方法就可以对细胞进行成像分析 利用SoftMax Pro软件所具有的细胞成像分析功能，可以检测出基于每一个细胞线粒体荧光信号强度值可更快的获得细胞线粒体毒性检测结果

The neutral theory of molecular evolution分子进化的中性理论

The neutral theory of molecular evolution Introduction I didn’t make a big deal of it in what we just went over,but in deriving the Jukes-Cantor equation I used the phrase“substitution rate”instead of the phrase“mutation rate.”As a preface to what is about to follow,let me explain the di?erence. ?Mutation rate refers to the rate at which changes are incorporated into a nucleotide sequence during the process of replication,i.e.,the probability that an allele di?ers from the copy of that in its parent from which it was derived.Mutation rate refers to the rate at which mutations arise. ?An allele substitution occurs when a newly arisen allele is incorporated into a popula-tion,e.g.,when a newly arisen allele becomes?xed in a population.Substitution rate refers to the rate at which allele substitutions occur. Mutation rates and substitution rates are obviously related related—substitutions can’t happen unless mutations occur,after all—,but it’s important to remember that they refer to di?erent processes. Early empirical observations By the early1960s amino acid sequences of hemoglobins and cytochrome c for many mam-mals had been determined.When the sequences were compared,investigators began to notice that the number of amino acid di?erences between di?erent pairs of mammals seemed to be roughly proportional to the time since they had diverged from one another,as inferred from the fossil record.Zuckerkandl and Pauling[8]proposed the molecular clock hypothesis to explain these results.Speci?cally,they proposed that there was a constant rate of amino acid substitution over time.Sarich and Wilson[6,7]used the molecular clock hypothesis to propose that humans and apes diverged approximately5million years ago.While that c 2001-2012Kent E.Holsinger

检测细胞活性或毒性(Molecular Devices)

Application Highlight 在SpectraMax MiniMax 300细胞成像系统上应用 EarlyTox细胞完整性试剂 盒检测细胞活性或毒性 图1. EarlyTox细胞完整性试剂盒检测原理 Live Red Dye Dead Green Dye Live Cell Dead Cell EarlyTox ?细胞完整性试剂盒由一系列易于识别活细胞和死细胞的优化试剂组成。可用于检测不同处理对细胞活性的影响，也可评估不同机制产生的细胞毒性，包括凋亡和坏死。该试剂盒设计工作于多种细胞类型，贴壁和悬浮均可。简单的操作流程和优异的试剂表现使其能应用到高通量扫描上。试剂盒中使用了两种结合DNA的荧光染料，一种为细胞膜可穿透的活细胞红色染料，一种为细胞膜不可穿透的死细胞绿色染料。红色染料可进入所有细胞的细胞核，细胞膜是否完整对其无影响，并可被激发产生亮红色荧光信号。绿色染料只能进入死细胞并产生亮绿色荧光，此类细胞的细胞膜已不完整(图1)。因此，活细胞和死细胞能通过成像系统拍摄到的荧光信号被轻易区分和识别到。 SpectraMax ? MiniMax ? 300细胞成像系统和SoftMax ? Pro软件为EarlyTox细胞完整性试剂盒的应用提供了成像和数据分析的整套硬件软件工具。使用仪器的双通道荧光成像功能以及软件的单细胞分析功能，可在5分钟内分析一块96孔板细胞活性。 方法 HeLa细胞以5000个/孔的密度接种到黑壁底透384孔板，培养24小时使其贴壁并生长，然后孔中加入具细胞毒性的药物。药物处理24小时候，按照试剂盒说明书加入染料标记后检测细胞活性。操作方法应尽量均一，以减少移除培养基、冲洗步骤造成的细胞损失和结果不准确性。可选的遮蔽试剂能用于减少培养基或待测药物产生的背景值。 特点 ? ? ? 信号强，缩短曝光时间，成像速度更快 适用于各种细胞类型，应用广泛拍照和分析一体完成，流程简化 通过试剂盒中两种不同特性的染料区分出活细胞和死细胞。红色染料进入所有细胞并将其细胞核标记为红色，另外绿色染料能进入具有不完整细胞膜的细胞中，并将其细胞核标记为绿色。只带红色荧光的细胞被成像和识别为活细胞，同时那些既发绿光又发红光的细胞被识别为死细胞。

分子原子原胞晶体显示软件Visual Molecular Dynamics 使用说明

简介： 这个教程为新用户介绍了VMD的用法。老用户也可以用本教程进一步熟悉程序的应用，以更好地利用VMD。本教程是针对VMD 1.8.3设计的，需要约3个小时来完成。 本教程新增的内容可用三个独立的单元讲解。第一个单元主要内容是分子图形表现方法 基础，还会介绍制作形象逼真的图像要了解的知识。另外的两个单元是针对高级用户，介 绍了VMD的脚本。尽管非技术性用户可以略去脚本的阅读，但是我们鼓励每个人都去试一 试着读一下，因为它会提供一些有力而易用的工具，这些工具是简单的图形用户界面所无法 提供的。 本教程以一种有趣的小蛋白质泛素的研究为例来说明VMD的应用。在本文中，一些资 料是在小框中出现的。这些小框中包括教程的补充内容，例如泛素扮演的生物学角色，使用VMD的一些提示和捷径等等。 如果你有对本教程的评论和问题，请发邮件至tutorial-l@https://www.wendangku.net/doc/1514406727.html,。邮件列表可以在https://www.wendangku.net/doc/1514406727.html,/Training/Tutorials/mailing list/tutorial-l/.中找到。 泛素本教程会用VMD来显示泛素。泛素是一个由76个氨基酸组成 的小蛋白质，在所有的真核生物中普遍存在。在所有真核生物蛋白 质中，泛素是最为保守的蛋白质之一（在昆虫，鱼，牛和人中，前 74个氨基酸是完全一样的）。它已被证明存在于细胞核、细胞质和 细胞表面。它首要的功能是介导蛋白质降解，在降解过程中，作为 细胞内蛋白水解酶识别的标志。 需要的程序： 以下是本教程中需要的程序 VMD: 可以从https://www.wendangku.net/doc/1514406727.html,/Research/vmd/下载（在所有平台上均可使用）。 绘图程序：要观看从VMD输出的图像，需要专门的程序。VMD有一个内置的绘图程序， 也可以应用外部程序。应用什么程序是由你的操作系统决定的。例如： – Unix/Linux: xmgrace, http://plasma-gate.weizmann.ac.il/Grace/ – Windows: Excel, https://www.wendangku.net/doc/1514406727.html,/en-us/FX010*********.aspx (需要购买) – Mac/Multiple Platforms: Mathematica, https://www.wendangku.net/doc/1514406727.html,/ (需要购买); gnuplot, https://www.wendangku.net/doc/1514406727.html,/(免费下载) 现在开始学习VMD 你可以在VMD-tutorial-files目录里找到本教程的文件。如图1所示的VMD-tutorial-files的 文件和目录

第六章 分子动力学模拟 Molecular Dynamics

第六章 分子动力学模拟 Molecular Dynamics –MD 6.1引言 分子动力学模拟方法是在牛顿力学的理论框架下，根据体系内分子之间的相互作用势，获得每个原子随时间运动的轨迹，通过系综平均，可以得到感兴趣的与结构和动力学性质有关的物理量，如：平均原子坐标，平均能量、平均温度及原子运动的自相关函数等。这些物理量是通过对每个原子的运动轨迹，即微观量求平均而得到的宏观量，因此可以与实验观测量进行比较。 用计算机模拟方法在向空间采样方法有两种： （1） 随机采样 MC （2） 确定性方法MD 以上讲过的MC （Monte Carlo ）采样方法就是随机方法，与随机方法不同，确定性方法是按照动力学规律使系统在相空间运动。分子动力学模型就是一种确定性方法。它的基本出发点是从一个完全确定的物理模型出发，通过解牛顿运动方程而得到原子运动的轨迹。我们感兴趣的可测量的客观物理量可以通过相空间的采样求系综平均而得到。在多态历经假设成立的情况下，系综平均与长时间平均是相同的。 ? ∞ →∞==τ τ τ0 1 ))(),((lim dt t p t q A A A 系综 其中q,p 为t 的函数。A 表示系综平均，∞A 表示无穷长时间平均。因模拟时间总是有限的。对耦分子体系，当模拟时间大于分子的弛豫时间时，有限观测时间可以变成为无穷长的。 当弛豫模拟〉τt ，模拟t 可认为∞，因物理上的∞是不可能的。 6.2基本原理 1.动力学方程 基本动力学方程包括在经典力学（CM ）框架下的牛顿方程和在量子动力学（QM ）框架下的薛定谔方程。在常温下，经典的牛顿方程对研究生物分子体系的结构和动力学性质已经足够了，因为这时体系的量子效应并不十分重要。但是，对研究包含隧道效应的反应时间问题时，量子效应十分明显，这时就必须用QM 方程来模拟体系的量子动力学性质。

高通量筛选神经毒性(Molecular Devices)

高通量筛选神经毒性 优点 完全自动化的图像获取与分析 快速得到每个细胞的多表型参数 实时监测活细胞的毒性，可持续几分钟至几天 神经系统对许多毒性化合物以及自然环境中生成的有害物质非常敏感。在疾病发生过程中，神经毒性可以对大脑或者外周神经系统造成短暂或持续性的损伤，例如脊髓损伤，中风，创伤性脑损伤等。同时这种神经毒性也是造成很多神经退行性疾病诸如阿尔兹海默和帕金森的主要诱因。 诱导人多功能干细胞的高通量成像技术可以用于筛查候选药物或者环境污染物的神经营养，保护功能或者神经毒性大小。本篇说明会阐述如何结合iPSCs，分子仪器和软件自动对诱导多功能干细胞进行终点法及活细胞的神经毒性筛选。 以iPSC为基础的毒性筛查 诱导多功能干细胞技术对我们研究神经元毒性是十分有用的，iPSCs可以大批量的展示成熟神经元的功能。如果将这种独特的生物学相关的细胞类型和高通量成像分析结合在一起，我们就可以快速的筛选出在引起神经元毒性中起主导作用的化合物并大大减少临床前研究和相关动物实验的成本。此篇中用到的是Cellular Dynamics International公司完全分化的人神经细胞。 量化完整的神经细胞网 高通量分析是一种定量化的分析方法，其可以测量神经突触长度表征被测物的阳性或阴性作用。iPSC衍生的神经元细胞可以在3-5天内于96或者384孔板中形成神经元网络，然后用毒性化合物刺激48-72小时。神经网被?-tubulin标记后，可利用ImageXpress? Micro 宽敞成像系统获取图像。和抗抗体我们可以实现神经元网络的可视化。使用MetaXpress? 图像处理软件中Neurite Outgrowth模块和AcuityXpress? 高内涵数据分析软件对相关图像和实验数据进行分析。 无论是否进行了细胞核复染色，Neurite Outgrowth模块都能找到神经细胞胞体，并根据这些荧光标记的神经细胞轴突将之标定为阳性细胞。神经细胞网络具有几个重要的特征参数包括轴突数目，轴突长度，每个细胞或者区域的分支数。我们可以针对每个孔的相关数据和细胞表型进行计算和统计。

细胞活力和细胞毒性的评价(Molecular Devices)

优势：利用SpectraMax i3x多功能微孔板读板机所具有的超灵敏化学发光检测功能进行细胞活力和细胞毒性的评价 简介 方法 准备试剂 ? 仪器具有超高灵敏度化学发光检测功能，最低至10个细胞/每孔；? 微孔板读板高度自动优化设置，可提高检测信号强度? 软件预置模板可以更快分析出检测结果SpectraMax i3x是Molecular Devices公 司最新推出的一款多功能微孔板读板机， 可利用仪器所具有的化学发光检测功能， 进行细胞活力和细胞毒性相应检测。仪器 可灵敏、快速检测出培养基中活细胞的数 目和经相应处理后细胞毒性情况。 Promega公司推出的CellTiter-Glo试剂是 利用了萤火虫荧光素酶反应体系中需要 ATP参与才能使其发光的特点，化学发光 信号强弱取决于培养基中ATP含量的高 低，也就是依赖于其中活细胞数目的多 少。来自于BioVision公司基于生物化学发 光原理的细胞毒性检测试剂盒，目的是检 测腺苷酸激酶(AK)的含量，AK为一种存在 于所有细胞中的常见蛋白，当破坏了细胞 膜完整性后其会释放至培养基中，AK可转 化ADP至ATP，所以可以利用类似方式进 行化学发光检测。 材料 ? CellTiter-Glo Luminescent Cell Viability Assay (Promega P/N G7570) ? Bioluminescence Cytotoxicity Assay Kit (BioVision P/N K312-500) ? HeLa 细胞(ATCC P/N CCL-2) ? 黑色底透 96孔细胞培养板 (Corning P/N 3904) ? 白色96孔细胞培养板(Corning P/N 3917) ? SpectraMax i3x多功能微孔板读板机使用前预先将CellTiter-Glo缓冲液和底物解冻并且平衡其至室温，将CellTiter-Glo 缓冲液加至含有CellTiter-Glo底物的棕色小瓶中，按照试剂盒说明书提示，将试剂轻轻反复颠倒进行混匀。对于生物化学发光细胞毒性检测试剂盒，其中包含了1瓶AK检测试剂，此试剂使用前预先将1.1ml AK试剂缓冲液加入其中并轻轻混匀，需在室温环境下平衡15分钟后使用。这个10X 的AK试剂的储存液需要稀释后才能成为试剂缓冲液。细胞数目与化学发光信号关系Hela 细胞培养在含有10%胎牛血清和双抗的MEM培养基中，细胞经胰酶消化后，使其悬浮于培养基中进行计数。处理后的细胞铺在96孔板中，经细胞培养基稀释后其密度从50,000细胞每孔/100ul至10个细胞每孔/100ul。如果将细胞铺在384孔板时，稀释后密度从12,500细胞每孔/25ul至6个细胞每孔/25ul。对照孔中仅需加入细胞培养基用于检测其背景的化学发光信号值。细胞试验过程中，精确的细胞数目可用于生成标准曲线，将不同体积的CellTiter-Glo试剂加入相应的孔板中，如100ul(96孔板)或25ul(384孔板)。将微孔板放置于 振荡器上轻混2分后，室温条件下孵育10分 钟，等化学发光信号值稳定后再进行检 测。

化学发光法检测细胞活性(Molecular Devices)

介绍 在SpectraMax ? L和SpectraMax ? M5酶标仪上应用Promega CellTiter-Glo化学发光法 检测细胞活性 MD公司的SpectraMax ? L及SpectraMax ? M 5型号酶标仪结合P r o m e g a 公司的CellTiter-Glo化学发光细胞活性检测试剂盒能进行快速灵敏的活细胞数量测定。CellTiter-Glo检测法使用了萤光素酶，需要ATP使其反应发光。发光信号强度由ATP量决定，而ATP的多少取决于活细胞数目。SpectraMax ? L及M5酶标仪均能提供96孔和384孔化学发光检测功能。 方法 准备CellTiter-Glo试剂 使用前将CellTiter-Glo缓冲液和底物在室温中平置解冻。然后将缓冲液倒入装有底物的棕色瓶中，并按照CellTiter-Glo操作手册中指示，温和颠倒瓶子以混匀。细胞计数及产生发光信号 用含10%胎牛血清和L-谷氨酰胺的Ham ’s F12培养基培养CHO-K1细胞。胰酶消化后使细胞悬浮在培养基中，并进行细胞计数。用96孔板检测时，每孔中加入100ul细 作者 Cathy Olsen, Ph.D., MDS Analytical Technologies,1311 Orleans Drive, Sunnyvale, CA 94089.材料： 1.CellTiter-Glo化学发光细胞活性检测试剂盒(Promega公司，货号G7570)，包括CellTiter-Glo缓冲液和CellTiter-Glo底物(冻干粉) CHO-K1细胞(ATCC，货号CCL-61)白色96和384孔板(Greiner Lumitrac 200，货号655075和781075)SpectraMax ? L化学发光微孔板检测仪或SpectraMax ? M5多功能微孔板读板机 胞悬液，细胞浓度从50000个/孔稀释到24个/孔。384孔进行检测时，每孔中加入25ul悬液，细胞浓度从12500个/孔稀释到25个/孔。空白孔中加入不含细胞的培养基，用于检测背景光值。 为了确保CellTiter-Glo检测中细胞数量的准确性，细胞不能贴壁和生长，但能及时被检测到。每孔中加入100ul(96孔板)或25ul(384孔板)CellTiter-Glo试剂。然后将微孔板放在振荡器上温和振荡混匀2分钟，室温孵育10分钟，使其产生稳定的发光信号。 制作ATP标准曲线 ATP标准样品用培养基制备，浓度范围从100fmol/孔到1nmol/孔(96孔板)，或25fmol/孔到250pmol/孔(384孔板)，按上述同样体积加入微孔板中。空白对照孔加入不含ATP的培养基。适量CellTiter-Glo 试剂加入每孔，并按上述步骤孵育。仪器设置和样品分析 检测板用SpectraMax ? L和SpectraMax ? M5酶标仪进行读数，并使用SoftMax ? Pro 软件中预设参数的CellTiter-Glo模板。根据Promega推荐，检测时间设置为1秒。然后在SoftMax ? Pro软件上进行平均发光强度和标准偏差计算，细胞浓度稀释曲线和ATP标准曲线作图。

Molecular Shape

Molecular Shape While Lewis dot structures can tell us how the atoms in molecules are bonded to each other, they don’t tell us the shape of the molecule. In this section, we’ll discuss the methods for predicting molecular shape. The most important thing to remember when attempting to predict the shape of a molecule based on its chemical formula and the basic premises of the VSPER model is that the molecule will assume the shape that most minimizes electron pair repulsions. In attempting to minimize electron pair repulsions, two types of electron sets must be considered: electrons can exist in bonding pairs, which are involved in creating a single or multiple covalent bond, or nonbonding pairs, which are pairs of electrons that are not involved in a bond, but are localized to a single atom. The VSPER Model—Determining Molecular ShapeTotal number of single bonds, double bonds, and lone pairs on the central atomStructural pair geometryShape 2Linear

【专业英语】2-Molecular Weight

Molecular Weight The molecular weight of a polymer is of prime importance in the polymer’s synthesis and application. Chemists usually use the term molecular weight to describe the size of a molecule. The more accurate term is molar mass, usually in units of g mol-1. The interesting and useful mechanical properties that are uniquely associated with polymeric materials are a consequence of their high molecular weight. Most important mechanical properties depend on and vary considerably with molecular weight as seen in Fig. 1-3. 2 There is a minimum polymer molecular weight (A), usually a thousand or so, to produce any significant mechanical strength at all. Above A, strength increases rapidly with molecular weight until a critical point (B) is reached. Mechanical strength increases more slowly above B and eventually reaches a limiting value (C). The critical point B generally corresponds to the minimum molecular weight for a polymer to begin to exhibit sufficient strength to be useful. Most practical applications of polymers require higher molecular weights to obtain higher strengths. The minimum useful molecular weight (B), usually in the range 5000–10,000, differs for different polymers. The plot in Fig. 1-3 generally shifts to the right as the magnitude of the intermolecular forces decreases. Polymer chains with stronger intermolecular forces, for example, polyamides and polyesters, develop sufficient strength to be useful at lower molecular weights than polymers having weaker intermolecular forces, for example, polyethylene. Properties other than strength also show a significant dependence on molecular weight. However, most properties show different quantitative dependencies on molecular weight. Different polymer properties usually reach their optimum values at different molecular weights. Further, a few properties may increase with molecular weight to a maximum value and then decrease with further increase in molecular weight. An example is the ability to process polymers into useful articles and forms (e.g., film, sheet, pipe, fiber). Processability begins to decrease past some molecular weight as the viscosity becomes too high and melt flow too difficult. Thus the practical aspect of a polymerization requires one to carry out the process to obtain a compromise molecular weight—a molecular weight sufficiently high to obtain the required strength for a particular application without overly sacrificing other properties. Synthesizing the highest possible molecular weight is not necessarily the objective of a typical polymerization. Instead, one often aims to obtain a high but specified, compromise molecular weight. The utility of a polymerization is greatly reduced unless the process can be carried out to yield the specified molecular weight. The control of molecular weight is essential for the practical application of a polymerization process.

分子伴侣(molecular chaperones)

分子伴侣 (molecular chaperone) （2018年10月） 分子伴侣(molecular chaperone)是指细胞中某些蛋白质分子可以识别正在合成的多肽或部分折叠的多肽，并与多肽的某些部位相结合，从而协助蛋白质的正确折叠、组装、转运、降解错误折叠及抑制蛋白质聚集，维持正常的蛋白质稳态，本身并不参与最终产物的形成的一类分子。分子伴侣是生物体内普遍存在的一类蛋白质，广泛存在于原核生物和真核生物中。解螺旋 https://www.wendangku.net/doc/1514406727.html, 一、分子伴侣分类 1. 伴侣素家族(Charperonin，Cpn) Cpn家族具有独特的双层7-9元环状结构的寡聚蛋白，它们以依赖ATP方式促进体内正常和应急条件下蛋白质折叠。它又可以分为GroE1(HSP60)家族和Tris家族。GroE1伴侣蛋白ATP依赖性构象变化从而促进底物蛋白质的折叠[1]。GroE1在体内与一种辅助因子，如 E.coli中的GroEs，发挥协同作用。Tris家族没有类似的辅助因子。 2. 热休克蛋白70（HSP70）家族 热休克蛋白（HSPs）其表达受包括热休克、营养缺乏、缺氧、中毒等的不同应激诱导，能够防止蛋白的错误折叠和聚集，维持细胞内稳态[2]。 HSP70家族是进化史上最保守的蛋白质之一，家族成员包括四个：grp78、mtp70、hsc70及hsp70。HSP70同疏水的肽类有高亲和力，并且随着ATP的水解而增高。HSP70与多肽之间的可逆作用在蛋白质的折叠、转运、错误折叠多肽的降解及其调控过程中有着重要的作用。HSP70表达和转录激活主要通过转录激活热休克因子1（HSF1）的作用而迅速调节。RNA聚合酶II启动子近端停顿的转录，受HSP70基因表达的调控。Hsp70是蛋白质稳态的重要参与者，在蛋白质折叠，解聚和降解中具有重要作用。HSP70通过泛素-蛋白酶体系统以及不同的自噬途径（巨自噬，微自噬和分子伴侣介导的自噬（CMA）在底物降解中起重要作用，有助于蛋白质降解[3]。 3. 热休克蛋白90（HSP 90）家族 热休克蛋白90（Hsp90）蛋白家族的成员是高度保守的普遍存在的分子，其分子量约为90kDa。属于分子伴侣，可以促进从头合成或错误折叠的蛋白质的折叠，并抑制蛋白的聚集。HSP90蛋白参与必需的细胞过程和调节途径，如细胞凋亡，细胞周期控制，细胞活力，蛋白质折叠和降解以及信号传导事件。此外，它们通过激活抗原呈递细胞和树突细胞诱导适应性