Exogenous Alanine and or Glucose plus Kanamycin Kills Antibiotic-Resistant Bacteria

Article Exogenous Alanine and/or Glucose plus Kanamycin Kills Antibiotic-Resistant Bacteria

Graphical Abstract

Highlights

d Glucos

e and alanine abundances are suppressed in

kanamycin-resistant E.tarda

d Alanin

e or glucose,via the TCA cycle,restores bacterial

susceptibility to antibiotics

d NADH and proton motiv

e force increases,which stimulates

uptake of antibiotic

d A functional metabolomics-based strategy to kill bacteria is

developed Authors

Bo Peng,Yu-bin Su,...,Yao-mei Tian, Xuan-xian Peng

Correspondence

pxuanx@https://www.wendangku.net/doc/873601174.html,

In Brief

Peng et al.show that exogenous alanine and/or glucose restores susceptibility to antibiotics in antibiotic-resistant bacteria by increasing TCA?ux,NADH production, and proton motive force to enhance kanamycin uptake,both in vitro and in a mouse model for urinary tract

infection. Peng et al.,2015,Cell Metabolism21,249–261

February3,2015a2015Elsevier Inc.

https://www.wendangku.net/doc/873601174.html,/10.1016/j.cmet.2015.01.008

Cell Metabolism

Article

Exogenous Alanine and/or Glucose

plus Kanamycin Kills

Antibiotic-Resistant Bacteria

Bo Peng,1,2,3Yu-bin Su,1,3Hui Li,1,3Yi Han,1Chang Guo,1Yao-mei Tian,1and Xuan-xian Peng1,*

1Center for Proteomics and Metabolomics,State Key Laboratory of Biocontrol,School of Life Sciences,MOE Key Lab Aquat Food Safety, School of Life Sciences,Sun Yat-sen University,Guangzhou510275,People’s Republic of China

2Molecular Foundry,Lawrence Berkeley National Laboratory,Berkeley,CA94720-8197,USA

3Co-?rst author

*Correspondence:pxuanx@https://www.wendangku.net/doc/873601174.html,

https://www.wendangku.net/doc/873601174.html,/10.1016/j.cmet.2015.01.008

SUMMARY

Multidrug-resistant bacteria are an increasingly

serious threat to human and animal health.However,

novel drugs that can manage infections by multi-

drug-resistant bacteria have proved elusive.Here

we show that glucose and alanine abundances are

greatly suppressed in kanamycin-resistant Edward-

siella tarda by GC-MS-based metabolomics.Exoge-nous alanine or glucose restores susceptibility of

multidrug-resistant E.tarda to killing by kanamycin,

demonstrating an approach to killing multidrug-

resistant bacteria.The mechanism underlying this

approach is that exogenous glucose or alanine pro-

motes the TCA cycle by substrate activation,which

in turn increases production of NADH and proton

motive force and stimulates uptake of antibiotic.

Similar results are obtained with other Gram-nega-

tive bacteria(Vibrio parahaemolyticus,Klebsiella

pneumoniae,Pseudomonas aeruginosa)and Gram-positive bacterium(Staphylococcus aureus),and the results are also reproduced in a mouse model for urinary tract infection.This study establishes a functional metabolomics-based strategy to manage infection by antibiotic-resistant bacteria.

INTRODUCTION

Bacterial antibiotic resistance is a threat to human health and to the viability of animal and plant species in the human food chain. Because efforts to develop antimicrobials that kill multidrug-resistant bacteria have not been successful to date(Raju et al., 2012;Coates and Hu,2007),there is an urgent need and high demand for agents that can kill or prevent infection with these bacteria.Unfortunately,the existing arsenal of antibiotics no longer provides protection against infection with Gram-negative bacteria and other pathogenic bacterial species or subspecies (Piddock,2012;Carlet et al.,2012).It has been noted that antibi-otics tend to achieve a lower intracellular concentration in antibi-otic-resistant bacteria than in closely related antibiotic-sensitive strains,indicating either lower rate of drug uptake or higher rate of drug exported(or both)(Page`s et al.,2008;Alekshun and Levy,2007).To overcome this phenomenon,novel approaches must be developed to achieve an intracellular drug concentra-tion suf?ciently high that the intended biological effect is observed(Hancock et al.,2012;Lee and Collins,2012;Roemer and Boone,2013).

Some evidence suggests that agents that increase the trans-membrane proton motive force(PMF)stimulate uptake of amino-glycoside antibiotics(Allison et al.,2011;Wang et al.,2009).We propose that the metabolic state of a bacterium signi?cantly in-?uences its relative susceptibility to killing by antibiotic drugs, and that speci?c metabolic pro?les correlate with antibiotic resistance.Furthermore,reagents that revert the metabolome of an antibiotic-resistant strain to that of an antibiotic-sensitive strain could potentially revert the phenotype of antibiotic resis-tance.In particular,we propose that exogenous metabolites might have this effect,if they increase transmembrane PMF, converting drug-resistant bacteria to drug-sensitive bacteria. Edwardsiella tarda is represented in the normal gut?ora of humans and?sh,but it is also an opportunistic intracellular pathogen in humans,?sh,and other species(Haenen et al., 2013;Park et al.,2012).In humans,E.tarda can cause gastroen-teritis,colitis,dysentery-like disease,septicemia,and meningitis (Nelson et al.,2009;Kawai et al.,2011).Infants can become in-fected with E.tarda during birth(Mowbray et al.,2003),and it has been linked to devastating declines in?sh populations (Park et al.,2012;Liu et al.,2013).Multidrug-resistant E.tarda has been isolated from?sh and human(Wang et al.,2009;Yu et al.,2012;Kawai et al.,2011),presenting signi?cant challenge for its control.Here,we describe the metabolic state of antibi-otic-resistant E.tarda and demonstrate that exogenous alanine or glucose revert the bacterium to kanamycin susceptibility. RESULTS

Metabolic Pro?le and Potential Biomarkers of

Drug-Resistant E.tarda

To study antibiotic resistance in E.tarda,wild-type kanamycin-susceptible E.tarda LTB4(LTB4-S)were grown in media con-taining kanamycin.Single cells that survived and proliferated in the presence of the antibiotic were used to establish a homog-enous kanamycin-resistant cell line,and

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E.tarda LTB4(LTB4-R)were selected(Figure S1A).Metabolomic pro?lings of LTB4-S and LTB-R,both of which were cultured in medium without kanamycin,were analyzed by gas chromatog-raphy-mass spectrometry(GC-MS).Eight biological and two technical replicates were performed,covering63metabolites in each strain(Figure S1B).The Z scores for LTB4-S were à17.5to+13.8(Figure S1C).Forty-nine of63metabolites (77.8%)showed signi?cant difference(p<0.01)corresponding to a false discovery rate of3.2%,with19metabolites at lower abundance and29at higher abundance in LTB4-R.Unsuper-vised hierarchical clustering and Z scores were used to rank metabolites whose abundance differed signi?cantly in the two strains(Figures1A and1B).Independent component analysis identi?ed two principal components,IC01and IC02,where IC01discriminates between the two strains,and IC02discrimi-nates within each strain(Figure1C).Glucose abundance was suppressed to a greater extent in LTB4-R than all other metabo-lites tested and may have the most impact(Figures1B,1D and S1D).However,the most strongly impacted KEGG pathway is alanine,aspartate,and glutamate metabolism(Figure1E). Decreased abundance of alanine and glutamate was observed (Figure1B),with higher impact on alanine(Figures1D and S1D).The concentration of alanine and glucose modulates the concentration of NADH;consistent with this,NADH abundance was lower in LTB4-R than in LTB4-S(Figure S1E).These results prompted us to explore whether the suppressed levels of alanine and glucose are useful biomarkers of antibiotic resistance and whether the phenotype of LTB4-R cells changes in the presence of exogenous alanine and/or glucose.

Exogenous Metabolites Alter Susceptibility of

Drug-Resistant E.tarda to Kanamycin

An earlier study reported that exogenous glucose stimulates up-take of aminoglycoside antibiotics by E.coli persisters(Allison et al.,2011).Here,we tested whether two metabolites,alanine or/and glucose,suppressed in LTB4-R,can be used to kill drug-resistant E.tarda with kanamycin including LTB4-R and E.tarda EIB202.EIB202is a wild multidrug-resistant strain car-rying a43.7kbp conjugative plasmid that confers resistance to tetracycline,streptomycin,sulfonamide,and chloramphenicol (Figure S2A)(Wang et al.,2009).The two drug-resistant bacteria were co-incubated with alanine with or without glucose and chal-lenged with an antibiotic,because we predicted the two metab-olites might act in a synergistic manner,by virtue of their ability to stimulate amino acid and carbon metabolisms,respectively. As predicted,cell survival decreased with increasing dose of alanine(Figure2A),glucose(Figure2B),or alanine plus glucose (Figures2C and2D),when grown in the presence of kanamycin. Relative to LTB4-R cells grown in the presence of1,000m g kana-mycin alone,cell survival decreased101-,3,228-,or276,000-fold in the presence of40mM alanine,10mM glucose,or both (Figure2E);cell survival of LTB4-R and EIB202was201-,927-, and123,600-fold and527-,2,916-,and283,089-fold lower for cells grown in the presence of kanamycin plus alanine,glucose, or both,respectively(Figure S2B).

Similar experiments were performed with three other E.tarda wild strains,WY28,WY37,and ATCC15947,and three other LTB4-S-derived the beta-lactam ampicillin-,the quinolone balo-?oxacin-,and the tetracyclins tetracycline-resistant strains.Minimum inhibitory concentration of these strains is shown in Figures S2A and S2C,respectively.The results were similar to those observed with LTB4-R and EIB202(Figure S2D),suggest-ing a common mechanism in glucose and/or alanine-mediated drug susceptibility.A more detailed analysis of cell survival of EIB202was performed by titrating the metabolite concentration, counting the number of surviving cells over time,and generating a two-dimensional heat map(Figure S3A)since EIB202is a wild strain with multidrug resistance.The degree of synergy was calculated using the combination index(CI)algorithm of Chou and Talalay(Chou and Talalay,1981),revealing CI97values 0.066–0.197for kanamycin and alanine(Table S1A),0.053–0.192for kanamycin and glucose(Table S1B),and0.0442–0.106for alanine and glucose(Table S1C).Synergy was also analyzed using isobolograms,con?rming the above results. Optimal conditions to promote kanamycin sensitivity and the highest level of synergy are revealed in Figure S3A.The dose-reduction index(DRI)for kanamycin is presented in Tables S1D–S1F.At97%effective dose ED97,for kanamycin with alanine(1:8),glucose(1:2),or both,the DRI is93.6%,93.7%, or88.1%,respectively.Alanine and/or glucose also potentiated gentamicin,an aminoglycoside antibiotic to replace kanamycin, to eliminate EIB202(Figure S3B).However,only weak effect was detected when aminoglycoside antibiotic kanamycin was re-placed with the beta-lactam ampicillin,ceftazidime,or the quino-lone balo?oxacin(Figure S3B),which is similar to previously reported data(Allison et al.,2011).Replacement of alanine or glucose with threonine or maltose showed similar effects,but not isoleucine(Figure S3C).Correspondingly,lower PMF was detected in cells incubated with isoleucine than with threonine or maltose(Figure S3D).To exclude the possibility that the ef-fects of exogenous metabolite were associated with growth state,survival rate of EIB202was monitored in M9medium or M9/LB mixed medium supplemented with or without metabolic stimuli(glucose or alanine).As shown in Figure S3E,the only fac-tor that makes the survival rate different is the supplementation of metabolic stimuli together with kanamycin rather than meta-bolic stimuli alone(Figure S3E).

Treating Clinically Relevant Bacterial Bio?lms and Persisters

Infection with bacterial bio?lms and persisters are a signi?cant concern in the clinical environment(Cohen et al.,2013;Corona and Martinez,2013;Drenkard and Ausubel,2002);therefore, it would be clinically useful if bio?lms and persisters were sus-ceptible to killing by kanamycin in the presence of alanine and/ or glucose.To test this possibility,?sh-derived pathogenic E.tarda EIB202and human-derived pathogenic E.tarda ATCC15947were cultured in vitro as bio?lms in the presence of kanamycin without or with alanine and/or glucose.In this sys-tem,EIB202and ATCC15947bio?lm growth was inhibited372-and162-fold by kanamycin plus alanine and glucose(Figure2F), respectively.Pathogenic E.tarda EIB202and ATCC15947bio-?lms were also injected via catheter into the urinary tracts of mice,and in this in vivo system,kanamycin plus alanine and glucose also inhibited infection in the urinary tract and sup-pressed spread of infection to kidneys(Figures2G and2H).In all cases,cell killing required kanamycin and was more severe in the presence of alanine and glucose.

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Figure1.Metabolic Pro?ling and Bioinformatics Analysis of Drug-Sensitive—LTB4-S—and Drug-Resistant—LTB4-R—E.tarda Strains (A)Heat map showing relative abundance of metabolites(Wilcoxon p<0.01)in E.tarda LTB4-R and LTB4-S.Heat map scale(green to red:low to high abundance) is shown at bottom.

(B)Z scores(standard deviation from average)corresponding to data in(A).Each point represents one technical repeat in one metabolite.Black,LTB4-S;blue, LTB4-R.

(C)Independent component analysis(ICA)of E.tarda LTB4-S and LTB4-R.Each dot shows one technical replicate.

(D)Hierarchical clustering of decreased abundance of metabolites in E.tarda LTB4-R.

(E)Enriched pathways in E.tarda LTB4-R(p<0.01).

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Bacterial persisters are a small phenotypically different sub-population of specialized survivor cells found within bio?lms and planktonic bacterial populations.They have been shown to be highly tolerant to antimicrobials and have been reported to be the cause of persistent and dif?cult-to-treat infections (Co-hen et al.,2013).Here,the frequency of persisters of EIB202and ATCC15947decreased 2,320-and 1,373-fold in the presence of kanamycin plus alanine and glucose,respectively (Figure 2I).Other relevant human and/or ?sh pathogens,including Vibrio parahaemolyticus ,Klebsiella pneumonia ,methicillin-resistant Staphylococcus aureus (MRSA),and Pseudomonas aeruginosa ,are also susceptible to killing by kanamycin plus alanine and glucose (Figure S3F).These ?ndings suggest that kanamycin plus alanine and glucose could be useful for managing or pre-

venting infection with drug-resistant pathogens,many of which cause diseases in humans and other economically and/or envi-ronmentally important species.

Increased PMF Stimulates Uptake of Antibiotic

Glucose promotes uptake of aminoglycoside antibiotics by increasing PMF (Allison et al.,2011).A similar mechanism was observed here (Figures 3A–3C and S4A).Speci?cally,higher concentration of NADH,higher PMF,and higher kanamycin-induced cell death were detected in in E.tarda EIB202incubated with exogenous alanine and/or glucose,effects that abolished by carbonylcyanide m-chlorophenyl hydrazone (CCCP).To iden-tify PMF as the leading factor for glucose-and/or alanine-medi-ated bacterial killing,we validate this by four ways.First,

the

Figure 2.Effect of Exogenous Alanine and/or Glucose on Susceptibility of Antibiotic-Resistant E.tarda ,Bio?lms,and Persisters to Kanamycin

(A and B)LTB4-R or EIB202were incubated with increasing concentrations of alanine (A)or glucose (B)for 6hr in the presence of kanamycin.

(C and D)Synergic effects of alanine and glucose on bacterial survival.Experiments were performed similarly to (A)and (B),except for the presence of 10mM glucose (C)or 40mM alanine (D).

(E)Percent survival of LTB4-R in the presence or absence of metabolite(s)in the indicated kanamycin concentrations.

(F)Percent survival of EIB202and ATCC15947in bio?lms treated with 40m g and 30m g/ml kanamycin,respectively,in the presence of alanine and/or glucose as indicated.

(G)Mouse urinary tracts were catheterized and infected with EIB202and ATCC15947.Mice were treated with 3,000m g/kg kanamycin as indicated and 3g/kg alanine,1.5g/kg glucose,or both twice daily for 3days.

(H)Kidney biopsies were obtained,and cfu/g kidney tissue was measured for mice treated as indicated.

(I)Percent survival of EIB202and ATCC15947persisters in the presence or absence of alanine and/or glucose plus kanamycin.

Results are displayed as mean ±SEM,and signi?cant differences are identi?ed (*p <0.05,**p <0.01;which caused by dose gradient are not marked)as determined by Student’s t test.Three biological repeats were carried out.

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kanamycin-mediated killing was sensitive to pH,indicating that the killing is attributed to PMF(Figure S4B)(Allison et al., 2011).Second,the level of pyruvate rather than lactate was elevated with increased concentration of alanine and/or glucose. Therefore,the pyruvate/lactate ratio was increased in a dose-dependent manner(Figures3D and S4C).This suggests the pri-ority to generating pyruvate over lactate,which is consistent with the elevated NADH and PMF.Third,the expression of NuoI and NuoE,the two enzymes for NADH oxidation,and respiratory chain dehydrogenase activity,which contribute to cytochrome quinol oxidases activity,were increased by exogenous alanine and/or glucose(Figures3E and3F).Forth,three inhibitors—rote-none,antimycin A,and NaN3—of the aerobic respiratory chain partly abolished increased PMF and acquired sensitivity to kana-mycin(Figures3G–3I and S4D),in which rotenone inhibits the transfer of electrons from iron-sulfur centers in complex I to ubiquinone and antimycin A and NaN3are inhibitors of the ubiq-uinoloxidation and the oxidases,respectively.One noticeable observation is that glucose but not alanine stimulated PMF and partially reverted the phenotype of drug resistance in E.tarda un-der anaerobic condition(Figures3J and S4E),a result consistent with the fact that glucose supports anaerobic respiration. Alanine and/or glucose stimulate antibiotic uptake.Intracel-lular concentration of kanamycin was signi?cantly higher in E.tarda grown in the presence of alanine and/or glucose than in the absence of alanine and glucose(Figure3K).And lower kanamycin was detected in LTB4-R than in LTB4-S in medium with one-half MIC drug of LTB4-S,which may be owed to higher rate of drug exported and/or lower rate of drug uptake in the resistant strain.The absence of alanine and glucose led to 9.5ng/ml drug difference between LTB4-R and LTB4-R(36.5 versus46.0ng/ml),whereas alanine and/or glucose promote more65–123ng/ml and113–231ng/ml drug uptake in LTB4-R and LTB4-S,respectively,suggesting the increased drug uptake overcomes the effects by drug ef?ux pump(Figure3L).It is sup-ported by the events that no mutations of AcrAB-TolC were de-tected(data not shown),and alanine and/or glucose did not affect expression of TolC(Figure S4F).These results suggest that increased PMF contributes to the mechanism by which alanine and/or glucose stimulates uptake of kanamycin by E.tarda and decreases cell survival.

Exogenous Metabolites Promote Metabolic Flux of the TCA Cycle

We propose that exogenous alanine or glucose acts in a manner to revert multidrug-resistant E.tarda EIB202,which contributes to elevation of NADH and PMF.To investigate this possibility, metabolic pro?les(63metabolites,5biological and2technical replicates)of EIB202were compared in the presence and absence of alanine,glucose,or alanine plus glucose.Data were analyzed as described above,and the results are pre-sented in Figures4,S5,and S6.The four groups are clearly separately using ICA(Figure4A).Based on Z score analysis, exogenous alanine,glucose,or alanine plus glucose signi?cantly altered the metabolic state(Figure S5A).Forty-eight,?fty-one, and forty-nine of the63metabolites(76.20%,80.95%,and 77.78%)had signi?cant change in abundance,corresponding to a false discovery rate of5.9%,8.3%,and16.3%(Figures S5B–S5D).Unsupervised hierarchical clustering and pathway enrichment analysis identi?ed16biological pathways(Figures S5E and S5F),out of which the?rst three impact values are listed in Figure4B.The largest effects were observed on decreased amino acid metabolism(glycine,serine,asparagine,valine,and proline)(Figure S6)and increased the TCA cycle(Figure4C). The synergy of alanine and glucose increased higher production of succinate,malate,and citrate,but not fumarate,than alanine or glucose alone(Figure4D).These results indicate that the metabolic?ux of the TCA cycle increases in the presence of alanine and/or glucose.

Recently developed NTFD(non-targeted tracer fate detection) is a powerful tool to provide information about relative?ux magnitudes into each metabolite pool by determining the mass isotopomer distribution for all labeled compounds(Hiller et al., 2011;Walther et al.,2012).To further demonstrate the promotion in the TCA cycle as a result of exogenous alanine and/or glucose, we performed13C3-alanine and13C6-glucose tracer experiments in EIB202by GC-MS(Table S2).As shown in Figures4C and S6B,13C3-alanine or13C6-glucose is converted by acetyle-CoA synthase to produce labeled acetyl-CoA,which provides an acetyl group to citrate,succinate,fumarate,malate,and aspar-tate to generate M2label in the initial cycle.In the second cycle, both labeled and unlabeled acetyl-CoA are entered into the TCA cycle and are added to the M2label to generate M3and M1label through oxidative decarboxylation,respectively.Since the unla-beled acetyl-CoA has exogenous and endogenous sources,M1 is higher than M3label.Similarly,the labeled acetyl-CoA adding to M3label would produce M4label.Since an equal amount of M2is required for the M3generation,(M1+M3)/(M2+M3)ratio of each pool represents relative?ux for that metabolite in the TCA cycle(y TCA/y ALA,y GLC,or y BOTH),where y TCA refers to the turnover of a particular metabolite pool and y ALA and/or y GLC re-fers to the?ux of alanine and/or glucose carbon atoms to the TCA cycle.M1label is lower in citrate than in succinate,fuma-rate,or malate pools,which is due to the oxidative decarboxyl-ation of a-ketoglutarate.Low M1-fumarate and-aspartate and high M1-malate as well as low M2-and high M4-malate in the 13C

3

-alanine tracer experiment suggest glyoxylic acid cycle may be involved in the?ux,which is supported by elevated fatty acid synthesis in the13C3-alanine(data not shown).Thus,the relative?ux for13C3-alanine in the malate of glyoxylic acid cycle can be represented by(M1+M3)/(M2+M4+M3)(y MALAT/y ALA) (Figure S6C).In addition,most of M2-labeled metabolites are from labeled acetyl-CoA and unlabeled oxaloacetate.Although oxaloacetate and glutamate are interconverted to each other via alanine aminotransferase,the detection of few labeled gluta-mate indicates that the labeled metabolites are not converted to glutamate.The enriched M2-aspartate alone in aspartate pool indicates that L-aspartate may contribute to M2regulation of the TCA cycle through fumarate and oxaloacetate(Figure4C). In summary,the tracer experiments suggest that the exogenous glucose and/or alanine are converted to acetyl-CoA and then?ux to the TCA cycle,which is regulated by glutamate,aspartate,and glyoxylic acid cycle.

Promotion of the TCA Cycle Is Required for Restoring Sensitivity to Kanamycin

To understand how the TCA cycle was activated by alanine and/ or glucose and also further validate the?nding that activation of

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Figure3.Effect of Exogenous Alanine and/or Glucose on NADH,PMF,and Intracellular Kanamycin

(A)Intracellular NADH concentration in EIB202in the presence of alanine and/or glucose as indicated.

(B)PMF in EIB202in the presence of alanine and/or glucose and effect of CCCP as indicated.

(C)Percent survival of EIB202in the presence or absence of CCCP and in the presence of alanine and/or glucose.

(D)The pyruvate/lactate ratio of EIB202in the presence of alanine and/or glucose.

(E)Western blot for detection of NuoL and NuoF expression in the presence of alanine and/or glucose.Protein loading amounts were normalized to SDS-PAGE gel.

(F)Activity of respiration chain dehydrogenase in the presence of alanine and/or glucose.

(G–I)Percent survival of EIB202in the presence or absence of increasing doses of rotenone(G),antimycin A(H),and NaN3(I)and in the presence of alanine and/or glucose.

(J)Percent survival of EIB202cultured under aerobic or anaerobic conditions in the presence of alanine and/or glucose as indicated.

(legend continued on next page)

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the TCA cycle is required for the potential,we evaluated expres-sion and activity of enzymes in the TCA cycles by western blot and enzyme inhibitors (Figure 5A).The expressions of succinyl-CoA synthetase b subunit (SucC)and citrate synthase (GltA)were increased with alanine and/or glucose dose (Figure 5B).Similarly,the activity of three key enzymes,citrate synthase (CS),isocitrate dehydrogenase (ICDH),and a -oxoglutarate de-

(K)Intracellular kanamycin concentration in EIB202and ATCC15947in the presence of alanine and/or glucose as indicated.(L)Comparison of intracellular kanamycin between LTB4-R and LTB4-S in the presence of alanine and/or glucose as indicated.

Results in (A)–(E)and (G)–(L)are displayed as mean ±SEM,and signi?cant differences are identi?ed (*p <0.05,**p <0.01;which caused by dose gradient are not marked)as determined by Student’s t test.Three biological repeats were carried out

(A)–(L).

Figure 4.Metabolic Pathway Analysis

(A)Independent component analysis of cells grown in the presence or absence of alanine and/or glucose.

(B)Enriched pathways in the presence of alanine and/or glucose.

(C)Elevated abundance of metabolites (in red)in the TCA cycle and mass isotopomer distributions for 13C-labeled alanine or glucose detected in a nontargeted manner (histogram)in the presence of alanine,glucose,or both.Elevation of glutamate is detected only in the presence of glucose and plus alanine.

(D)Scatter plot showing normalized abundance of four TCA cycle metabolites.Each dot shows a technical replicate.

*p <0.05and **p <0.01as determined by Student’s t test.

hydrogenase (OGD),of the TCA cycle were elevated in the presence of alanine and/or glucose (Figure 5C).We further validate this by using two inhibitors,bro-mopyruvate or malonate.Bromopyruvate is an active-site-directed inhibitor of the pyruvate decarboxylase (El),a compo-nent of the pyruvate dehydrogenase complex,which converts pyruvate to acetyl-CoA (Figure 5A).Malonate is a competitive inhibitor of succinate dehy-drogenase (Figure 5A).As shown in Fig-ure 5C,the activities of the three enzymes were elevated in the presence of alanine and/or glucose.Bromopyruvate inhibited the activities of CS and ICDH in the pres-ence of glucose and glucose plus alanine but not alanine https://www.wendangku.net/doc/873601174.html,paratively,the activity of OGD was inhibited at all cases,indicating it may play more critical roles than CS and ICDH.These enzymes were affected because the rate of inhibi-tion by bromopyruvate was signi?cantly increased in the presence of pyruvate (Lowe and Perham,1984;Brown et al.,1997),which caused less pyruvate and then acetyl-CoA to enter the TCA cycle and affect the activity of these enzymes.

Malonate and bromopyruvate inhibited NADH and PMF genera-tion promoted by alanine and/or glucose (Figures 5D and 5E).Furthermore,malonate rescued higher viability of EIB202than bromopyruvate (Figures 5F and 5G),which is consistent with the fact that pyruvate decarboxylase regulates concentration of pyruvate as a source of the TCA cycle,whereas succinate dehydrogenase plays a role in the TCA cycle and generation of

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Figure5.Effect of Exogenous Alanine and/or Glucose on Enzymes of the TCA Cycle

(A)Superimposed on metabolic pathways related to glucose,alanine,and the TCA cycle.PDH,pyruvate dehydrogenase;GltA,citrate synthase;ICDH,isocitrate dehydrogenase;OGD,oxoglutarate dehydrogenase complex;SucC,succinyl-CoA synthetase b subunit;SDH,succinate dehydrogenase.

(B)Western blot analysis for SucC and GltA expression in the increasing concentration of alanine and/or glucose.

(C)Activity of three key enzymes of the TCA cycle in the presence or absence of exogenous alanine and/or glucose and effect of bromopyruvate.

(legend continued on next page)

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PMF.Our?ndings indicate that promotion of the TCA cycle by substrate activation is required for restoring bacterial sensitivity to kanamycin.

a-Oxoglutarate Promotes Activity of a-Oxoglutarate Dehydrogenase and Elevates Bacteria to Kanamycin Susceptibility

The above experiments demonstrate that OGD is especially crucial for the killing potential.We reason that the similar result is obtained when activity of OGD elevates.To test this idea,we used a-oxoglutarate,the substrate of OGD(Figure6A),to pro-mote the activity of the enzyme.Results showed that the enzyme activity elevated with increasing a-oxoglutarate dose(Figure6B). The viability of EIB202cells was approximately1,000-fold lower in the presence of1.6mg/ml kanamycine and5mM a-oxogluta-rate than in the presence of kanamycin without a-oxoglutarate or a-oxoglutarate without kanamycin(Figure6C).Exogenous a-ox-oglutarate increased intracellular NADH of EIB202,which was abolished by malonate(Figure6D).Elevated PMF was also de-tected in a dose-dependent manner(Figure6E).CCCP reduced PMF,abrogated the ability of exogenous a-oxoglutarate to in-crease PMF(Figure6F),and increased survival in the presence of kanamycin(Figure6G).The intracellular concentration of kanamycin was measured in EIB202cells in the presence and absence of a-oxoglutarate.Results suggest that a-oxoglutarate stimulates uptake of kanamycin(Figure6H),which is similar to the result obtained using alanine or glucose.These results indi-cate that increased activity of a-oxoglutarate dehydrogenase may promote bacterial susceptibility to kanamycin,which further supports the conclusion that promotion of the TCA cycle by sub-strate activation may potentiate action of kanamycin in elimi-nating antibiotic-resistant bacteria.

DISCUSSION

A strategy for overcoming bacterial resistance to antibiotics is described that bypasses the need for discovery or design of novel drugs or reagents.This strategy uses non-toxic com-pounds to modulate the metabolome of antibiotic-resistant bacteria,promote the TCA cycle,increase PMF,and stimulate transport of extracellular antibiotics through the bacterial cell wall/membrane into the intracellular environment.The?rst step in our study was to characterize the antibiotic-resistant metabo-lome and identify low-(or high-)abundance metabolites that correlate with antibiotic resistance.

In this study,we observe that susceptibility to antibiotics is restored when the suppressed metabolites are supplied in the growth media.A mechanism for this effect is proposed:namely, increased?ux through the TCA cycle,increased PMF,increased drug uptake,and increased killing(Figure7).Although the observed effects in resistant strains and proposed mechanism are similar to those of Allison et al.for persistent bacteria,the mechanism of the metabolic pathway for PMF production is still unknown(Allison et al.,2011).To expand this,we showed that NADH,speci?cally generated from TCA cycle(Figures4,5, and6),is the source of proton to generate PMF,which is attrib-uted to elevated activity of the three key metabolites in the TCA cycle by substrate activation(Figures5and6)and thereby is responsible for the increased killing.Association of the TCA cy-cle with antibiotic resistance has been reported(Kohanski et al., 2007,2008,2010;Lin et al.,2014).The present study indicates that we may regulate the TCA cycle to promote antibiotic uptake and then increase killing.Moreover,we not only test carbon sources as previously described but also con?rmed the capa-bility of nitrogen source and amino acids,in potentiating antibi-otic to kill bacteria effectively,which is never tested by others. The high-level synergy of two different types of metabolites may relate to their metabolic pathways.On one hand,alanine is metabolized to pyruvate as glucose dose.Pyruvate is con-verted to acetyl-coenzyme A,which is the major upstream input for the TCA cycle.On the other hand,alanine transfers the amino group to a-ketoglutarate by alanine aminotransferase and pro-duces pyruvate and L-glutamate.Glutamate is the only amino acid that undergoes rapid oxidative deamination by glutamate dehydrogenase,which uses NAD or NADP as a coenzyme to generate a-ketoglutarate(Sharkey et al.,2013).Thus,this syn-ergy may contribute to a-ketoglutarate regulation and elevated NADH production,which is supported by our tracer experiment that glutamate?uxes from glutamate to a-ketoglutarate one way, though the exact mechanism needs further investigation.

The presence of an effective penetration barrier in bacteria has been regarded as a major problem largely responsible for the lack of progress in the?eld(Lewis,2013).The present study demonstrates that alanine and/or glucose subvert the penetra-tion barrier and potentiate aminoglycoside antibiotic uptake to eliminate antibiotic-resistant strains.Indeed,the effects related to uptake lead to elevation of intracellular antibiotic by several-fold so that the high concentration of drug surmounts other bac-terial antibiotic-resistant mechanisms,including the multidrug ef?ux pump,and thus effectively exerts the antibacterial effect. In this study,we sequenced three genes,AcrA,AcrB,and TolC,whose products consist the major ef?ux pump conferring intrinsic resistance to antibiotics in many pathogens,to exclude the possible acquired gene mutations contributing to decreased intracellular kanamycin in drug-resistant bacteria.Although mu-tations in other unknown genes might be present,which requires further investigation,our approach still shows an effective way to combat antibiotic-resistant bacteria by promoting uptake of antibiotics.

The experimental system reported here demonstrates rever-sion of multidrug-resistant E.tarda to susceptibility to killing by kanamycin.In four multidrug-resistant wild E.tarda strains with or without an antibiotic-resistant plasmid and four LTB4-S-derived strains with different antibiotic-resistant mechanisms, exogenous alanine and/or glucose effectively restores suscepti-bility to kanamycin.We believe that our approach has general

(D and E)NADH(D)and PMF(E)in the presence or absence of alanine and/or glucose and effect of malonate or bromopyruvate.

(F and G)Percent survival of EIB202in the presence of kanamycin,alanine,and/or glucose and the increasing concentrations of bromopyruvate(F)or malonate(G).

Results in(C)–(G)are displayed as mean±SEM,and signi?cant differences are identi?ed(*p<0.05,**p<0.01;which caused by dose gradient are not marked)as determined by Student’s t test.Three biological repeats were carried out.

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relevance and may be useful in future for treating clinically relevant diseases caused by antibiotic-resistant bacteria.For example,gentamicin also kills E.tarda in the presence of alanine,and the effect is also observed in other bacterial strains including multi-drug-resistant strains V.parahaemolyticus ,K.peneumoniae ,P.aeruginosa ,and a superbug MRSA.The role of metabolites in antibiotic resistance has been reported previously (Vega et al.,2012,2013;Lee et al.,2010;Gusarov et al.,2009;Shatalin et al.,2011;Bernier et al.,2011),but the fact that exogenous metabo-lites restore susceptibility to antibiotics and the mechanism by which this occurs are important ?ndings.

In summary,we demonstrate that the bacterial susceptibility to antibiotics is strongly associated with metabolic states and that speci?c metabolic pro?les are correlating with certain antibiotic resistance.Therefore,chemicals that revert the metab-olomics pro?le of an antibiotic-resistant strain to that of an anti-biotic-sensitive strain could potentially revert the antibiotic resis-tance.Our study proposed an approach to identify metabolic modulator through investigation of metabolomics,by which crucial modulators can be used for therapeutic purpose.

EXPERIMENTAL PROCEDURES

Antibiotics,Chemicals,and Antibodies

All antibiotics were obtained from Shanghai Sangon Biological Engineering Technology &Services Co.Ltd.Antibiotics were ?ltered with a 0.22m m pore

size hydrophilic PVDF membrane.All other chemicals were purchased from Sigma Aldrich (Sigma).CCCP,rotenone,and antimycin A were dissolved in di-methylsulphoxide,and sodium azide was dissolved in water.All reagents were stored at à20 C.Antibodies were obtained from Chenxue Biological Corp.Bacterial Strains and Growth Conditions

All bacterial species in this study were obtained from the collection of our lab-oratory.LTB4-S-derived strains including LTB4-R were selected by sequential propagations in LB broth with respective antibiotics.The sensitivity to antibi-otics was evaluated by the minimum inhibitory concentration,which was determined by serial 2-fold dilution of antibiotics.All bacterial cultures were grown in LB medium from frozen stock in a shaker bath for 24hr at 30 C for V.parahaemolyticus and E.tarda strains,except for ATCC15947,and at 37 C for the other bacteria.

Metabolomic Pro?ling

Sample preparation and GC-MS analysis were carried out as previously described (Cheng et al.,2014).

Statistical Analysis and Sample Pattern Recognition

Initial peak detection and mass spectral deconvolution were performed using XCalibur software (Thermo Fisher,version 2.1).Metabolites were identi?ed using spectral matching and retention indexes from National Institute of Standards and Technology (NIST)library using the NIST MS search 2.0.After the removal of any known arti?cial peaks and merger of the same com-pounds,the internal standard allows normalization of the resulting data.The peak intensities were normalized to form a single matrix with Rt-m/z pairs (retention time-mass charge ratio pairs)for each ?le in the data set.This ?le was then used for further analysis as described

previously

Figure 6.Exogenous a -Ketoglutarate Elevates Susceptibility of E.tarda to Kanamycin

(A)The overall reaction accomplished by oxoglutarate dehydrogenase complex (OGDC).

(B)Effect of exogenous a -ketoglutarate on activity of a -oxoglutarate dehydrogenase of EIB202.

(C)EIB202were incubated in the presence of kanamycin and increasing concentrations of a -ketoglutarate for 6hr.(D)Intracellular NADH in EIB202in the presence or absence of a -ketoglutarate and/or malonate.(E)PMF of EIB202in the increasing concentration of a -ketoglutarate.

(F)PMF in EIB202in the presence or absence of a -ketoglutarate and/or CCCP.

(G)Percent survival of EIB202in the presence or absence of CCCP,kanamycin,and/or a -ketoglutarate.

(H)Intracellular kanamycin in EIB202in the presence or absence of a -ketoglutarate.Results are displayed as mean ±SEM,and signi?cant differences are identi?ed (*p <0.05,**p <0.01;which caused by dose gradient are not marked)as determined by Student’s t test.Three biological repeats were carried out.

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(Sreekumar et al.,2009).Z score analysis scaled each metabolite according to a reference distribution.

Multivariate statistical analysis included independent component analysis (ICA)(http://metagenealyse.mpimp-golm.mpg.de/).ICA was used to discrim-inate sample patterns,identify the metabolites associated with resistance,and minimize the interindividual variation’s in?uence.Principal component analysis (PCA)was applied to reduce the high dimension of the data set.GraphPad Prism v5.01was used to draw the histogram and the scatter plot.We analyzed the differential metabolites to their respective biochemical pathways as out-lined in the Kyoto Encyclopedia of Genes and Genomes (KEGG,release 41.1,http://www.genome.jp/kegg )and MetPA (http://metpa.metabolomics.ca/MetPA/faces/Home.jsp ),which provides impact factors as an index.Antibiotic Bactericidal Assays

Antibiotic bactericidal assay was carried out as previously described (Allison et al.,2011).In brief,a single bacterial colony was grown in 50ml LB broth in 250ml ?asks for 24hr at 30 C.After centrifugation at 8,0003rpm for 5min,samples were washed twice with 30ml sterile saline and re-suspended in M9minimal media supplemented with 10mM acetate,1mM MgSO 4,and 100m M CaCl 2to 0.2at OD 600.Reaction samples were added to metabolites and antibiotic and incubated at 30 C for 6hr.The use of M9medium offers us the advantage to test the effects of different metabolites on antibiotic-resis-tant strains in a reliable way,by which potential confounding factors would not be present as in more complex medium.In all experiments with CCCP,sam-ples were pre-treated with the proton ionophore for 5min before adding anti-biotic.After incubation,100m l aliquot samples were periodically removed,serially diluted,and plated (10m l aliquots)onto LB agar plates.The plates were cultured at 30 C or 37 C for 18–22hr depending on bacteria species.Only those dilutions yielding 20–200colonies were enumerated to calculate colony-forming units (cfu).Percent survival was determined by dividing the cfu obtained from a treated sample by the cfu obtained from control.Isotope Tracer Experiment

The effects of nine 13C-labeled tracers (six glucose and three alanine)on ?ux estimation precision were investigated as previously described (Hiller et al.,2010).In brief,EIB202was cultured using a 1:1mixture (total 20mM alanine and/or 10mM glucose)of unlabeled substrate and [U-13C 3]alanine and/or [U-13C 6]glucose.Three biological replicates were performed for each growth condition.GC-MS was performed using an Agilent 7890A GC equipped with a 30m DB-35MS capillary column connected to an Agilent 5975C MS oper-

ating under electron impact (EI)ionization.The effectiveness of each tracer was gauged using the software downloaded from the internet (Hiller et al.,2013).The software provides the mass isotopomer distribution (MID)data of all labeled compounds detected in the GC-MS https://www.wendangku.net/doc/873601174.html,beled compounds were identi?ed from the MID data,which were used for estimation of metabolic ?uxes.The estimation was carried out by the tracing of labeled atoms present in an externally supplied compound as it is metabolized.Both the spectrum obtained from the labeled chromatogram and the spectrum obtained from the unlabeled chromatogram were normalized by their total signal.The detailed analysis is described in Figure S6.

Anaerobic Experiments

E.tarda EIB202cells were re-suspended in M9medium with an OD 600of 0.2.The re-suspended cells were mixed with kanamycin plus alanine and/or glucose and incubated in an anaerobic jar (MART Microbiology)at 30 C for 6hr.The cultures were spotted onto LB agar plates to determine cfu/ml as described above.

Measurement of Membrane Potential

The membrane potential was examined via BacLight Bacterial Membrane Po-tential Kit (Life Technologies)as previously described (Allison et al.,2011).NADH Measurements

E.tarda culture was diluted to OD 600of 0.2and incubated with metabolite(s)at 30 C for 6hr.Cell pellets were washed and re-suspended with NADH extrac-tion buffer.Heat extracts were performed at 60 C for 5min.Buffer and NAD extraction buffer were added to neutralize the extracts.The cells were vor-texed and spun down at 14,000rpm for 5min.Supernatant was collected for the EnzyChrom NAD/NADH Assay Kit (BioAssay Systems).

Measurement of Intracellular Kanamycin

Bacterial cells were incubated with kanamycin or kanamycin plus metabolite(s)at 30 C for 6hr.The cells were washed and re-suspended in sterile saline and adjusted to OD 600of 1.0.Each aliquot of 1ml sample was sonicated for 3min and centrifuged to remove insoluble matters.Kanamycin in supernatants was quanti?ed with Kanamycin ELISA Rapid Diagnostic Kit (Beijing Clover Tech-nology Group Inc.).Luminescence intensity was measured on a PerkinElmer LS55Fluorescence Spectrophotometer (PerkinElmer).A quartz cuvette with an optical path length of 10mm was selected with excitation and emission at 287and 450nm,

respectively.

Figure 7.Proposed Mechanism for Cell Killing in the Presence of Alanine and/or Glucose plus Kanamycin

Alanine and/or glucose promote the TCA cycle by substrate activation and lead to elevated concentration of NADH.The NADH is oxidized through electron transport chain to generate PMF.The PMF facilitates aminoglycoside uptake,thereby elevating intracellular antibiotic concentration and effectively killing antibiotic-resistant bacteria.

Cell Metabolism 21,249–261,February 3,2015a2015Elsevier Inc.259

Bio?lm Assay and Mouse Infection Assay

Bio?lm-formation culture was determined as previously described(Allison et al.,2011).In brief,6mm PE50catheters(0.58mm30.96mm)were inocu-lated in1ml fresh LB and10m l cultured stationary phase bacteria,then incu-bated aerobically for24hr at30 C.The medium was changed every24hr for a total of72hr.The PE50catheters were washed?ve times with1ml of sterile saline to remove loosely adherent cells and planktonic cells.For in vitro exper-iments,catheters with1.563106cfu as bio?lm were used for kill bacteria directly.For in vivo experiments,the bio?lm-coated tubing with1.563106 cfu was surgically implanted in the urinary tract of female Balb/c mice(4weeks, weighing20–25g).Forty-eight hours after surgery,mice were randomly divided into seven groups,eight mice for each group,including intraperitoneal injection with saline,glucose(1.5g/kg),alanine(3g/kg),kanamycin(3mg/kg), glucose(1.5g/kg)plus kanamycin(3mg/kg),alanine(3g/kg)plus kanamycin (3mg/kg),or glucose(1.5g/kg)plus alanine(3g/kg)plus kanamycin(3mg/kg) for3days(twice per day).After the treatment,mice were observed for an addi-tional24hr.Finally,the catheter tubings were removed and bacteria were collected in1ml sterile saline to determine cfu/ml.Meanwhile,kidneys were obtained to determine bacterial load(cfu/g).Mice were provided by the Center of Experiment Animal of Sun Yat-sen University.Animal protocols were approved by the Committee for the Use and Care of Laboratory Animals, Sun Yat-sen University.

Western Blot Analysis

Bacterial protein samples were resolved on10%SDS-PAGE and transferred to nitrocellular membranes(GE Healthcare Life Sciences).The membranes were incubated with1:1,000of the primary rabbit antibodies anti-NouF, anti-TolC,anti-SucC,and anti-GltA or the primary mouse antibody anti-NouI,followed by corresponding goat anti-rabbit or rabbit anti-mouse second-ary antibodies conjugated with horseradish peroxidase.Band intensities were detected by using a gel documentation system,LAS-3000(Fuji?lm Medical Systems).

Measurement of Enzyme Activity

Citrate synthase,isocitrate dehydrogenase,and a-ketoglutarate dehydro-genase were measured by citrate synthase,isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase activity Colorimetric Assay Kits (Genmed Scienti?cs Inc.).In brief,EIB202cultures were collected,washed, re-suspended in lysate(from the assay kits),and disrupted by sonic oscil-lation.Following centrifugation,supernatant was transferred to a new tube, and a Bradford assay was used to detect protein concentrations.Respira-tory chain dehydrogenase activity was detected as previously described(Li et al.,2010).

Pyruvate and Lactate Concentration Detection

Pyruvate and lactate concentrations were detected through Pyruvate and Lactate Detection Kits,respectively(Nanjing Jiancheng Bioengineering Insti-tute).In brief,EIB202cultures were collected,washed,re-suspended with saline solution,and adjusted to OD600of1.0.Aliquots of1ml samples were sonicated for3min,and the resulting supernatant was used for detection of pyruvate and lactate.

SUPPLEMENTAL INFORMATION

Supplemental Information includes six?gures and two tables and can be found with this article online at https://www.wendangku.net/doc/873601174.html,/10.1016/j.cmet.2015.01.008.

AUTHOR CONTRIBUTIONS

X.X.P.,B.P.,and Y.B.S.wrote the manuscript.X.X.P.,B.P.,and H.L.concep-tualized and designed the project.X.X.P.,B.P.,H.L.,and Y.B.S.interpreted the data.X.X.P.,Y.B.S.,H.L.,and C.G.performed data analysis.Y.B.S.,B.P.,H.L., Y.H.,and Y.M.T.performed experiments.

ACKNOWLEDGMENTS

This work was sponsored by grants from the‘‘973’’project(2012CB114406), China Ocean Mineral Resources Research and Development Association (DY125-15-T-07),NSFC projects(41276145,31272702),and Guangdong Pro-vincial Science and technology projects(2012A031100004).

Received:August12,2014

Revised:November2,2014

Accepted:January13,2015

Published:February3,2015

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农村小学英语教学存在的问题与对策

农村小学英语教学存在的问题与对策目前农村孩子上学的趋势是家庭条件稍好的都到城镇去就读，主要原因是农村教学质量没城镇好，尤其是农村的英语教学更是导致农村学生数量减少的最主要原因。作者在本镇教学期间发现了一些农村学校英语教学中存在的问题并结合自己多年的教学经验就这些问题给出了一些相应的建议，希望对农村从事英语教学的同行有所帮助。 一、英语师资水平普遍较低 在日常教学中，大多数教师用的是老方法，教师管教，学生管背，其基本教学模式仍为“open your books-listen to me-read after me-recite”.这种教法的直接后果是学生只会死记硬背，而运用语言进行口头交流的能力得不到提高。造成这种情况的原因是：教师思想不解放，自身素质不高。 农村学校学生英语差的最主要原因是教师，大部分教师都不是正规学校毕业本身业务能力就很差，学生刚开始接触英语，就被教师引入歧途，使学生对英语产生了畏难情绪并且失去兴趣，应号召一些有爱心、能吃苦、业务能力强的正规院校的毕业生去农村学校任教。如果教师问题不解决，一切教学改革都是空谈。 教师作为素质教育的实施者，应积极主动地成为素质教育的参与者，而不是消极，被动的旁观者，要彻底地解放思想，打破应试教育的束缚，同时要提高自身的素质。农村中小学的英语

教师在听，说等方面相对较薄弱，就无力去提高学生的运用能力。因此各级主管部门应把农村中小学的英语教师的培训工作当做 一项工程抓，切实抓紧，抓落实。教师在不断的系统培训，学习过程中，主动地接受新的英语教学理论和教学方法，借此不断提高业务水平适应素质教育的需要。同时政府应号召一些有爱心、能吃苦、业务能力强的正规院校的毕业生去农村学校任教。 二、农村学生外语学习的心理和行为偏差 在农村初中英语教学中发现约有25%-30%的学生无法适应这一阶段的外语学习，他们一开始对英语怀有好奇心，也满怀希望学好英语。然而，随着字母阶段的学习结束，他们的好奇心也随之而去，对英语产生厌学情绪，对接踵而来的困难一筹莫展，渐渐地挫伤了他们的积极性，有相当数量的学生开始怀疑自己是不是学习外语的材料，动摇了学习外语的决心，也丧失了自信心，产生了明显的心理抑制障碍，由此两极分化日趋严重，听说读写能力明显差于城镇学生。 那么出现农村学生这种心理和行为偏差的原因何在？ （一）目的不明确，盲目好奇 农村学生因为地域和环境等因素见识狭窄，开始时，对应用感到新鲜，带着好奇开始学英语，但是又没有目的。随着困难的日渐增多，好奇心也随之而去，日积月累，厌烦或反感取而代之。 （二）缺乏动机，学习时冷时热

and_的用法【超全】

一、并列连词and所连接的分句在语义上的含义 1.表示在动作或事物的前后，一般译为“然后、随即”。 She came in and sat down. 她走了进来，随后就坐了下来。 I pulled the trigger and the gun went off. 我扣动扳机，枪随即响了。 2.表示递进关系，一般可译为“甚至、何况、还”等。 Friction is not always undesirable and can be very useful. 摩擦并不总是不好的，它甚至可能是有用的。 It was not easy to carry such a heavy load,and during the dog day. 提那样的重物已经是够困难的了，何况是在三伏天。 3.表示意义上的转折或让步，可译为“虽然、尽管、但、却”等。 He tried hard and (yet) he failed to get the job. 他努力尝试，却（仍然）没能得到那个工作。 You are not lazy,and still you are an idler. 你并不懒，但多少有点游手好闲。 4.表示对照、对比关系，可译为“而、却”。 She bought me cakes,and I helped her do her homework. 她买蛋糕给我吃，而我帮她做作业。 It can be difficult when you don’t think something is important and someone else does. 如果你并不认为某事重要而某人却认为重要，这时情况就可能不大好办。 5.表示对前面所述事项的结论或看法，and后可加分句或名词组，and一般不译出。 You gave him a piece of your mind,and a very good thing too. 你率直地批评他，这是件好事。 They disliked Joe——and that’s not surprising. 他们不喜欢乔，这并不让人惊讶。 6.表示因果关系，当前面的部分表示原因时，and=so that，译为“因此、从而、所以”；也有将表示原因的部分放在and后的，这时and=because，直接译为“因为”。 Sound is carried by air,and without air there can be no sound. 声音靠空气传播，因此没有空气也就没有声音。 Aluminum is used as the engineering material for planes and spaceships and it is both light and tough. 铝用作制造飞机和宇宙飞船的工程材料，因为铝质轻且韧性好。 7.引起一个插入句，用于中断语气，以便表示意见、看法。这时，and引起的句子中的主语多为第一人称，and引起的句子与句子其余部分用逗号隔开。

浅谈农村小学英语自然拼读法

浅谈农村小学英语自然拼读法 肇庆市怀集县马宁中心小学林明娴 摘要：著名英语教育专家胡春洞教授说过：“语音是存在的物质基础，英语语音教学是整体教学发展的起点，也是教学的第一关，并始终影响着以后的其它教学。”如何提高语音教学的成效，让学生听得懂、说得出、用得好呢?对于小学英语教学来说，课堂是教学的主阵地，帮助学生形成有效的英语语音学习策略是每位英语教师的职责，也是新课程标准所制定的学习目标之一。 关键词：语言环境专业素质方法 著名英语教育专家胡春洞教授说过：“语音是存在的物质基础，英语语音教学是整体教学发展的起点，也是教学的第一关，并始终影响着以后的其它教学。”自然规范的语音、语调将为有效的口语交际打下良好的基础.在小学英语教学中，教师应特别重视语音教学。小学阶段语音方面的教学目标是：培养学生具有按照规律自然拼读，记忆单词的意识，使学生逐步做到“见词能读，听音能写”。 我在小学英语教学实践中发现，语音教学的效果不尽如人意。一些学生没有学好语音，不会诵读，也就难以朗读单词和句子。学生学习英语出现两极分化、掉队，其中一个重要原因就是学生未学好语音，没有过好语音关。因为学生语音未学好，发音不准，听音能力差，不仅学不会口语，甚至连听课、记单词、读句子都有困难，从而对英语学习不感兴趣，产生畏难情绪，甚至放弃学习，丧失学习英语的兴趣和信心。如何提高语音教学的成效，让学生听得懂、说得出、用得好呢?对于小学英语教学来说，课堂是教学的主阵地，帮助学生形成有效的英语语音学习策略是每位英语教师的职责，也是新课程标准所制定的学习目标之一。以下是我在课堂教学中对自然拼读法的一些见解。 一、创造良好的语音环境，强化口语练习 我们发现：语音好的学生，听说读的能力都很强；语音差的学生，听说读往往障碍重重。所以语音是英语教学入门阶段的重点。要想学生学好语音，就必须要让他们开口说。在我们这里，课堂上很少听到男生的声音，一直以来英语学得好的都是女生，这是因为女生在课堂上大胆开口说、读，男生就怕读错，同学取笑。久而久之，让男生开口说就很难了，所以男生的成绩一直没有得到提高。那么创造良好的语音环境，强化口语练习就很有必要。首先坚

Or和and的用法

Or和and的用法 一、连词or主要用法分述如下： 1、用在选择疑问句中连结被选择的对象，意为“或者，还是”。例如： Is he a doctor or a teacher? 他是医生还是教师？ Did you do your homework or watch TV last night?你昨晚做作业还是看电视了？Are they singing or reading English? 他们是在唱歌还是在读英语？ 下列两个疑问句中的并列成份由于使用了不同的连词，因而句式有所不同。试比较： A、Does he like milk and bread? 他喜欢牛奶或者面包吗？ B、Does he like milk or bread? 他喜欢牛奶还是面包？ 分析：A 句中使用了连词and,是一般疑问句，对其作肯定或否定回答应用：Yes,he does.No,he doesn`t.B句中使用了并列连词or,因而是选择疑问句，对其回答不用“yes”或“no”，而应根据实际情况直接选择回答：He likes milk.或：He likes bread. 2、用于否定句中连结并列成分，表示“和，与”之意。例如： There isn`t any air or water on the moon.月球上既没有空气也没有水。 The baby is too young. He can`t speak or walk.那婴儿太小，他不会说话，也不会走路。 He hasn`t got any brothers or sisters.他没有兄弟和姐妹。 肯定句中并列连词应用and，在把含有and的肯定句改为否定句时，莫忘把连词and改为or。例如： The students sang and danced in the park yesterday. →The students didn`t sing or dance in the park yesterday. 3、用于句型“祈使句+or+陈述句”中，表示在以祈使句为条件下的相反假设，意为“否则，要不然”。例如： Work hard,or you will fall behind.你要努力学习，否则会落后。 Get up early tomorrow,or you will miss the early train.你明天要早点起身，要不然就赶不上早班火车了。 Don`t jump the queue,or other peopoe will not be pleased.别插队，否则别人会不高兴的。 可以把这类句型中的祈使句换为一个条件句（注意改写时应去掉连词or）。例如：Hurry up,or you will be late for the meeting.→If you don`t hurry up,you will be late for the meeting. 4、用于“either…or…”结构中，意为“不是……就是……”，“要么……要么……”。连结的并列成份可在句中作主语、表语、谓语、宾语等。（注意：连结并列成份作主语时，谓语动词通常与or后的部分保持一致。）例如： Either she or I am right.不是她对就是我对。（连接主语） The shoes in the shop were either too big or too small for me. 店里的鞋对我来说不是太大就是太小 He either does his homework or watches TV on Sundays.他星期天要么做作业，要么看电视。（ We play either football or basketball in the afternoon.下午我们不是踢足球就是打篮球。 5、用于连结并列成份，表示不确切、模糊的陈述。例如： This story happened five or six years ago. 这个故事发生在四、五年前。

如何构建小学英语高效课堂模式

如何构建小学英语高效课堂模式 课堂教学是实施素质教育的主要阵地，实践水平和创新精神的培养，应该首先从课堂教学上予以突破。而提升课堂教学效率就成为当前的首要任务。所谓"高效课堂"就是用尽可能少的时间获取最大教学效益的教学活动。只有高效的课堂才能实现学生素质的全面发展，并为其持续发展打好基础。我们该从哪些方面去构建高效的课堂，从而全面提升小学英语课堂教学效率呢？我在课堂教学中尝试着从以下几方面构建高效课堂，取得了较好的效果，现提出与各位同仁共勉。 “良好的开端就是成功的一半”。 在导入新课时我们要根据教学任务和内容，采取符合学生年龄和心理需求的方式，这样能够有效地吸引学生的注意力，尽快引导学生进入学习状态。如在教doll, car, plane, boat, balloon 等单词时，我们可用猜一猜的游戏来导入：我举起一个书包对学生说：“圣诞节马上就要到了，圣诞老人给你们准备了好多的礼物，你们猜一猜都有什么礼物。”这时学生被调足了胃口，他们会迫不及待地猜："pencil, pen, ruler, crayon"等，教师再从书包里拿出物品一一展示给学生，这不但复习了旧知，也自然地导入新词，再如在学习apple, banana, orange, pear, peach 等词时，我们就可用实物导入。 所以说良好的开端能很好地调动学生的学习热情，吸引学生的注意力，促使学生较快地投入到英语学习中去。这个环节可采

用的形式有很多：英文儿歌、英语歌曲、游戏、绕口令等等。效果比较好的是动口、动手、动脑“三动合一”的活动，根据Asher 的TPR理论，这样的活动通过肢体语言的配合使用，能一下子抓住学生的注意力，降低学习英语的难度，有利于协助学生消除紧张心理，从而协助他们更好地理解并学习英语。 二、激发学生兴趣为基点 动力和源泉。小学英语是入门教学，应该把培养学生学习英语的兴趣放在首位。所以，教师可在每节课前唱英语歌曲，说歌谣，用富于感染力的音乐激发情趣，让学生以饱满的愉快的情绪进入学习英语的状态；能够用图片，优美的体态语、简笔画、照片、多媒体课件实行直观的教学活动；能够选用学生们熟悉的内容或游戏，使学生的兴趣点达到高潮 三、教学方法多样，生动活泼，激发学生的学习兴趣 兼收并蓄、集各家所长，采用综合的教学路子。情景法、视听法、直接法、结构法、功能法（交际法）、全身反应法、沉浸法、折衷法、综合法等，只要有助于实现教学目标都能够采纳。 创设情景、相互交流、激励情意，启发学习动机，树立学习信心。教师要充分利用教科书中的课文创设栩栩如生的情景，如打电话、购物、生日聚会、野餐、旅游、问路、看病等，为学生提供使用英语实行交流的机会。每当孩子们进入角色，成功地做成一件事，他们便情不自禁，喜形于色，所以兴趣倍增、信心增强，动机和情意受到了很大的激励。

and的用法及含义

and的用法及含义 And是英语中一个普通的连词，然而and并非只作并列连词用，它还具有一些不太常见的表达方式和意义，应根据上下文的特殊环境，作出判断才能准确理解其用法和意思。 1．and作并列连词，译为“和、并且”等，当连接三个以上的并列成分时，它放在最后一个成分之前，其余用逗号分开，例如： He bought a book and a pen．他买了一本书和一支笔。 Solid，liquid and gas are the three states of matter．固态、液态和气态是物质的三种状态。 2．名词+and+名词，若这种结构表示一个概念时，and+名词相当于介词with+名词，译为“附带、兼”的意思，例如： Noodle and egg is a kind of delicious food．（and egg=with egg）鸡蛋面是一种美味食物。 Whose is this watch and chain？（and chain＝with chain）这块带表链的手表是谁的？ 3．名词复数+and+同一名词的复数，强调连续或众多的含义。例如： There are photos and photos．照片一张接着一张。 They saw film hours and hours last week．上星期他们一小时接一小时地看电影。 4．形容词+and+形容词，这种结构形似并列，实际并非并列结构。例如： This roon is nice and warm（＝nicely warm）． The coffee is nice and hot（=thoroughly hot）． 5．用and连接动词的用法：and+动词作目的状语。动词go（come，stop等）＋and+动词，此时，and+动词相当于in order to+动词，例如： ①I'll go and bring back your boots，（go and bring back=go in order to bringback）我去把你的靴子拿来。 ②and+动词，起现在分词的作用，表示方式或伴随情况。例如： He sat and waited．（and waited=waiting）他坐着等。 ③and+同一动词表示动作长时间地“继续”或“重复”，例如：

并列连词“or”的八种最全用法

并列连词“or”的八种最全用法 英语中，表示选择关系的并列连词主要有or和either。。。or。其中or作为连词，也是有多种不同的含义和用法。小编为大家整理如下： 1、用于引出另一种可能性，表示“或者，还是”： Are you coming or not？你来还是不来？ 2、用于否定句，提出两种或多种事物时，表示“也不”： I don’t like tea or coffee。我不喜欢喝茶，也不喜欢喝咖啡。 3、用于警告，表示“否则”： Hurry up，or you will be late for school。快点，否则你上学要迟到了。 4、用于两个数字间，表示“大约”： There are seven or eight people in front of us。我们前面有七八个人。 5、用于引出解释性词语，表示“或者说”：

He likes geology，or the scien ce of the earth’s crust。他喜欢地质学，或者说地壳的科学。 6、用于引出对比概念： He was lying-or was he？他在说谎，还是没有说谎？ 7、or so 有“大约”的意思： It cost me $100 or so to buy this book。这本书大约花了我100美元。 8、or some---和some--- or other表示对人（somebody）、事（something）、地点（somewhere）不太确定： ---Who told you the story？这个故事是谁告诉你的？ ---somebody or other。I can’t remember。某个人吧，我记不得了。 另外，童鞋们要记住一些含or的固定结构： now or never 勿失良机more or less或多或少 believe it or not 信不信由你or better yet更好的是

农村小学英语的现状

农村小学英语的现状

农村小学英语的现状 摘要: 农村小学英语的开设，为农村孩子文化素养的提升给予了广阔的平台，缩短了与城市的差距。但在现实教学实践中，农村小学英语教学的情况不容乐观。本文从农村的特点及小学生英语学习实际情况出发，指出当前农村小学英语教学中存在的一些突出问题，提出了对其的反思与对策，为我们的老师和孩子解决困惑，力争让我们农村小学英语进一步有效、健康地发展。 关键词：小学英语困难与困惑反思与对策 （一）、英语学习环境的欠缺 小学英语重在说，在交流中感受语言的魅力。农村环境缺少英语交流的场所与氛围，学生在家里学习只是为了完成家庭书面作业，而真正在课堂上感受的机会不多。而农村的活动场地广，孩子回到家很容易成群结队地在水泥场上，广阔的田野里“疯”、“野”，却很少会在一起交流、讨论一下任何所学知识，操练一下英语会话，玩玩英语游戏。 （二）、家庭教育的短缺 “父母是孩子的第一任教师”，这一定程度上说明了家庭教育的重要性。但是农村的家长知识水平普遍偏低，对英语更是知之甚少。地区外出打工现象普遍，孩子大都由祖父母看管，对儿童开展英语家庭教育的可能性几乎为零。与城市相比，农村学校教育和家庭教育的脱节也加大了英语教育开展的难

度，有很多孩子的家长虽然知道英语非常重要，但不知道如何去指导孩子，也就是说缺乏在家教育孩子英语的能力。（三）、英语师资的匮乏 师资力量缺乏可以说是农村小学英语教学的一大软肋。当前农村学校严重缺少英语教师，工作量普遍过重，一般都要担任多个班和跨几个年级的教学，甚至兼教语文、数学等科目，每周担任20节课左右，这样花在教材钻研、教学设计、课堂管理、学生辅导的时间和精力也相对地减少了。况且现有的英语教师大部分是转岗培训出来的，甚至许多老师连转岗培训也不曾有，只是找一个英语基础略好的兼课罢了。 （四）、农村小学英语配套设施跟不上 农村小学英语从软件上得不到很好的满足，间接影响学生学习的效果。再者，从学校的硬件设施考虑，英语教学设备、设施、图书、音像、教具不能到位，教师上课什么都没有，仅靠一张嘴、一块黑板、一支粉笔，一些学校没有语言配套卡片、磁带、挂图、活动手册，更别说一些音像设备了。这样的条件与城市相比，简直是天堂与地狱。这直接影响了教学的效果、学习的态度及学习的热情。 多年的教学实践我深知：在农村，想要依靠学生的自觉,借助家长的帮助来拓展英语学习的时间和空间，那是很困难的。为此，我一直在苦苦思索：如何让英语融入学生的生活，让学生在真实的语言氛围中学习、使用英语？面对小学英语教

浅谈如何打造小学英语高效课堂

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小学英语教学论文：浅谈农村小学英语教学

浅谈农村小学英语教学 ————赖 一、小学开设英语课的背景要求 （一）、小学开设英语课是社会发展的需要 21世纪，知识经济的发展使人类进入了一个学习社会。科学技术发展迅速，国际竞争也日趋激烈，世界范围的综合国力的竞争，主要是科学技术的竞争，而现代科学技术的主要标志是信息技术。众所周知，当今国际上，英语已成为世界性的通用语言，学习英语已不仅是与国际发展接轨的需要更是我们学好科学知识的重要工具。而我们国家要想更好的对外开放跟上国际时代潮流，就首先应当使我国公民具备使用外语的基本技能。 如何才能更有效地提高外语的教学质量？为了解决这一社会需要，教育部决定，把小学开设英语课程作为21世纪初基础教育课程改革的重要内容，并就小学开设英语课程提出了具体指导意见，制订了《小学英语课程教学基本要求（试行）》。 （二）、小学开设英语课程有助于民族素质的提高 小学一、二年级的学生正处于7、8岁的少年时期。这个年龄段的孩子具有好奇、好动、爱表现、模仿等特点。他们喜欢新鲜事物，对陌生语言的好奇心能激起他们对外语的兴趣，他们喜欢引起别人的注意，希望得到老师的表扬，他们爱玩、爱唱、爱游戏、爱活动。处于这个年龄段的儿童学习外语时有许多成人甚至中学生所不具备的优越性。例如：模仿力、可塑性强等。因此，在小学开设英语课也可以充分发挥小学生学习语言的潜力，通过对外语的学习提高他们的思维、品德、文化、和心理素质。外语课可以使小学生了解其他国家和民族的优秀文化传统；可以更好地发展他们的思维，开阔视野，丰富他们的知识与经历；也可以提高他们的各种修养，形成国际竞争意识，使他们长大后更适应社会、经济、文化、科技的发展和满足以后国际交往的需要。 二、农村小学的特点 农村小学的生源主要来自于附近的一些行政村，与城里小孩相比会有一定的差别。因而农村小学生在学习英语时存在一定的困难。通过调查，主要可分为以下几个原因： 1、农村孩子比较胆小，羞于说英语。农村小学的学生土生土长，从小受周围特定环境与家庭教育的影响，与城市小孩相比，往往会显得更加胆小拘谨、不善言谈，缺少自信心理，又害怕会说错被老师批评，并且易落于机械的条文背诵的俗套，因而羞于说英语也不太敢说英语。 2、农村小学教学方法、手段落后于城市小学，在农村小学中还存在使用传统的英语教学方法的情况，而这种“满堂贯、英译汉”的教学方法，不仅使课堂气氛沉

or的用法归纳

or的用法归纳 你们知道or的用法吗?我们一起来学习学习吧，下面就和大家分享，来欣赏一下吧。 or的用法归纳 1. 表示选择，意为“或”“还是”： Is the radio off orWould you prefer tea or coffee? 你喜欢茶还是咖啡? Is he asleep or awake? 他睡着了还是醒着? Are you going to America by boat or by air? 你到美国是坐船还是坐飞机? You may go or stay, according as you decide. 是去是留由你自己决定。 Are you from North China or South China? 你是华北人还是华南人? You can come now or you can meet us there later. 你可以现在来，也可以稍晚和我们在那里碰头。

2. 表示一种否定的条件，意为“否则”： Come on, or we’ll be late. 快点，否则我们要迟到了。 Hurry up, or youll be late for school.赶快，否则你上学就要迟到了。 Dress warmly, or else you’ll catch cold. 穿暖和点，否则你会感冒的。 Be careful, or you’ll break that vase! 小心，否则你会把那花瓶打碎! Cross the road very carefully. Look both ways, or you might be knocked down. 过马路要非常小心，要看两边，不然会被车撞倒。 3. 可表示“要不就是”： He must be joking, or else he’s mad. 他一定在说笑话，要不就是疯了。 The book must be here, or else you’ve lost it. 这书一定在这儿，要不就是你丢失了。 4. 用于否定句中代替and。 He was not clever or good-looking. 他不聪明，也长得不好看。 比较：

小学英语教学模式

小学英语教学模式 【摘要】我们总结了小学英语课堂教学的基本模式——“六步”教学法。教无定法，但教必有法，贵在得法。此模式的探索实践旨在为广大小学英语教 师的课堂教学指明方向，从而全面提高小学英语课堂教学的质量。 【关键词】小学英语，高效课堂，教学模式 【正文】我们通过近两年的探索与实践，总结出小学英语课堂教学的基本模式与方法——“六步”教学法。即唱歌热身、温故知新、情景引入、新知呈 现、趣味操练、运用拓展。在本文中，我们以快乐英语第七册Unit2 Lesson8做为典型课例，进行教学模式的研讨。 一、唱歌热身 唱歌热身就是在上课前唱一首或几首英语歌曲，将学生无目的的动作变成音乐声中有规律的动作，将学生注意力迅速转移到课堂活动中来，促使学生做好上课的心理准备；通过优美的韵律，使学生的身心由疲劳变为放松，营造一种和谐、愉悦的学习气氛，让学生快速进入英语学习的氛围。在这一环节教师要注意以下几点： 1、围绕目标，唱歌热身。所选唱的歌曲或歌谣要生动活泼，贴近生活，尤其要紧密结合教材的内容，为本节课或本单元的目标服务。如：在学了五官之后唱Head ,Shoulers,Knees and Toes;学了颜色内容之后唱Colour Song ; 学了居室家俱后，唱My Bed Room;学了家庭成员之后，唱Father and Mother ;学了水果之后，唱An Apple a Day等等。以我校所授Unit2 Lesson8一课为例，在此环节教学，演唱What are you doing?歌曲，不仅复习巩固了已学的内容，也为本节课的内容进行了铺垫。 2、编排动作，师生同唱。我们建议教师根据歌曲内容编排动作，教师与学生互动互唱，营造了和谐的情感氛围，无形中拉进了师与生的距离，为本节课的学习做好了情感准备。 二、温故知新 本环节就是复习巩固已学过的知识，通过师生对话、chant, Brainstorm, TPR 活动、duty report、自我介绍、自由会话等各种形式，复习与新知识有关的旧知识，使学生具备必要的认知前提，保证新知学习的顺利进行；同时也可培养学生的口头表达能力及大胆表现自我的能力。建议教师要做到以下两点：

小学英语高效课堂教学模式

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and用法

一、and可以连接语法作用相同的词、短语或句子，表示并列或对称的关系，可翻译为“和”、“并”、“又”、“兼”等。 1. 连接两个并列主语。如： Millie and Amy go to the park every week. Millie和Amy每周去公园。 2. 连接两个并列谓语。如： You must take care of yourself and keep healthy.你必须照顾你自己并保持健康。 3. 连接两个简单句。如： They love playing football and we love playing football too.他们喜欢踢足球，我们也喜欢踢足球。 4. 如果连接两个或两个以上的词语，通常把and放在最后一个词语前面；为了强调，可在两者之间分别加上and；把词语连接起来时，通常把较短的词语放在前面。如： I like eggs, meat, rice, noodles and dumplings.我喜欢鸡蛋、肉、米饭、面条和饺子。 All that afternoon we jumped and sang and did all kinds of things.整个下午我们又唱又跳，做各种各样的事情。 但是有些用and连接的词语，顺序是固定的，不能随意改变。如：Men, women and children男人、妇女和儿童，fish and chips 炸鱼加炸土豆片。 二、表示动作的先后关系

And常用来连接两个动词或动词词组，后面一个动词所表示的动作比前面的动作发生得迟一点，可翻译为“然后”。如：Go along the street, and take the second turning on the right，you will find the cinema.沿着这条街走，然后在第二个路口向右拐，你就会发现电影院。 He got out of the lift and climbed the fifteenth floor on foot.他从电梯里走出来，然后步行爬上第十五层楼。 三、表示目的 在口语中，and常用在come，go，try等动词后连接另一个动词，表示目的，此时and相当于to，不需要翻译。如：Let us go and ask Miss Green.让我们去问格林小姐吧。Come and meet this family.来见见这家人吧。 四、表示因果关系 And连接两个动词或者两个分句，带有因果关系，此时and 相当于so，可以翻译为“便”、“于是”、“因而”、“结果”等。如：She could not find her mother and began to cry.她找不到妈妈，于是哭了起来。 It is a fine day today，and everyone is busy.今天是个好天气，因而人人都很忙。 五、表示条件和结果 在祈使句中，经常用and连接一个简单句，表示条件与结果的关系，相当于if引导的条件状语从句。它们在语法上是并

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