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The future of antibiotics: facing antibiotic resistance
The future of antibiotics : facing antibiotic resistance
Center for Adaptation Genetics and Drug Resistance, Tufts University School of Medicine, Boston, MA, USA -IiI I Mir,.,bio/ b!fcrt 2000: 6 (Supplemcm 3): 101-106
Antibiotic resi tance originates locally, but manifests globally. R.esistance frequencie~ can vary widely in different parts of the world, and even in hospitals in the same city. A good example of this point is demonstrated by the l1lultidrug resistant pneumococcus. There are wide variations in the percentage frequency of resistance to multiple antibiotics from different parts of the world as reported for the t 9905. Large differences exist among cities in the USA, Europe and Africa (Figure I). The approach to the problem depends on empowering local groups to examine the situation and find the solution appropriate for that locality. Resistance traits are not a recent microbia l invention to confront the antibiotics we are developing and using. They
have been around for the many nullennia of bactenal existence, dUling which time they have preslImably served other purposes. However, III any parncular envIronment whether it be a home, a city or a hospital - the applicaoon of an antibiotic will select resistance genes and propagate strains beating resistance to that antlbioric [1]. Resistance as a clinical problem is not inevitable. Just because an antibiotic is lIsed does not mean that a patiem will present with a drug-resistant infection. The reason we con£i:om the problem so often today stems from the extensIVe overuse of antibiotics, which encourage, the resistant strams to sUlvive. Environments teem ,vith large numbers of resistant bacteria. This preponderance of resistant strams dramatically
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Figure 1 Frequency of mullldrug resislanl S. pneumoniae In various countries in l he 1990s. (Dala organized from published studies Marshall and Levy. In preparation)
I 2000 Copyrigh l by Ihe European Soci ty of Clinical Microbiology and InfectiOUs Diseases, eMJ, 6 (Suppl . 3) , 101106
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Clinical Microbiology and Infection, Volume 6, Supplement 3, 2000
increases the chances that a patient's infection (which, after aJJ, derives from the environment) will be drug resistant. Another feature of resistance that underm.ines our efforts, but certainly aids the microbial world, is that resistance traits can be exchanged among aJJ types of bacteria - from Gramnegative bacteria to Gram-positive and vice versa, including MycobacteriulII. Unless it is a chromosomal mutation, a resistance determinant that appears in one organism will not stay solely in that organism. R esistances are often carried on transferable units of DNA called plasmids and transposons, which can go directly or indirectly from one bacterial genus to another. They can be transferred by bacteriophages and even through cell acquisition of naked chromosomal DNA (Figure 2) [2). Antibiotics produce major changes in microbial ecology, dismpting the equilibria among species and genera, and among resistant and susceptible strains (3). Tills effect stems from the large amount of antibiotics used. Tn tlle USA, an estimated 50 million pounds of antibiotics are produced each year. About half of them are used in humans, and the other half for plants and animals [4,5) . In humans, outpatient prescriptions in the USA number about 200 million annually. In animals, ,about 80% of the antibiotics used are for growth promotion. More subtle, and less well known, is antibiotic use in agriculture. An estimated 300 000 pound~ of antibiotics, called 'pesticides', are annuaJJy sprayed onto fruit trees [5). This latter use must have a large environmental impact per geographic unit. The end re;ult of all this antibiotic use is that the surviving bacteria are dmg resistant and can continue to transfer their plasmids and transposons to other bacteria. There is another feature of resistance that is problematic, if not paradoxical. Antibiotics are quite stable, and most of the mechanisms of resistance leave them untouched. Whether applied to human or animal systems or dispersed in the environment, antibiotics remain active and capable of
continuing the resistance selection process anywhere in the environment [6). Dmg inactivation could be an answer to this problem, but only a small number of resistance determ.inants have the dmg itself as their target (e.g. the ~-lactamases for penicillins and cephalosporins, acetyltransferases for chloramphenicol, different inactivating enzymes for aminoglycosides and macrolides). AU others, e.g. rifampicin, quinolones, trimethoprim, tetracycline, involve mechanisms that either act on the target of tlle dmg or keep the dmg out of the cell. The net result is that an active dmg remains in the environment where it continues to select for any strains that are resistant to it [6.7) .
ANTIBIOTIC AVAILABILITY Several years ago, on a visit to Santiago, Chile, to give a lecture, I was placed in a hotel across the street from the local pharmacy. ) decided to go over to see what drugs were available. I did not even have to enter the store. There was a 'desquento', a sale, on antibiotics. In particular, there was a 30% savings on the 10caJJy made chloramphenicol sold over the counter. In tlle window display I could see ampicillin and tetracycline [21 . It is notable that over-the-counter availability of antibiotics in Chile is no longer permitted. However, the same easy access to dmgs occ urs in Mexico and other Latin and South American countries. In many developing cou ntries, antibiotics are sold over tlle counter like other commoditi es. Tn fact, even in the USA one can get antibiotics quite readily frOI11 physicians and other providers; while one nuy not necessarily be able to buy them over the co unter, one ca n demand them from physicians. Once obtained, they may be improperly used andlor stored for later use (2). Some time ago, we underwent the pleasures and ttials of raising chickens to evaluate the effect of antibiotic use as
bacterlopl1age
Figure 2 The genetics of antibiotic resis-
-- .....
"fre." DNA
tance transfer. Resistance genes can enter new hosts via plasm ids, phages or naked DNA. Once inside the cell, many resistance genes reside on transposons that can move from one DNA molecule to anoth er. (Adapted from Figure 4.3 in [2].)
<" 2000 Copyright by the European Society of Clinical Microbiology and InfectiOUs Diseases, eMI. 6 (Suppl. 3), 101 - 106
Levy Future of antibiotics
growth promoters. Starting with eggs, we raised 3()O chi ckens. H alf of these were treated with low dose oxytetracyclin e in their feed. Whi le studyi ng the mi crob io logy of the treated and control groups, it became rapidly clear that th e low, subth erapeuri c dose of tetracycline led to a dramatic change: resistant orgalllsms appeared in the chi ckens within 24 h of eating the antibiotic-containi ng feed [8] . What was perhaps more interesting was the finding that as we contin ucd th e chi ckens on the tetracycline-laced feed, Esrherirhia coli em erged
103
co nsidered as lacking a specific cellular target. Its use was previously reserved fo r hospitals. We examined this chemical fo r its abi lity to selec mutants of E. roli and succeeded in obtaining them. Resistance emerged by a mutation in the Jab! gene involved in fatty acid biosynthesis [J 1]. Tlus findmg led to a confirmation that the protein product of the gene, enoyl reductase, was the target of triclosan. Perhaps more important is the emcrgence of triclosan resIstance through activation of the /liar locus, whic h leads to multiple resistance to antibiotics
with resistance, not just to tetracycl in e, but to multipl e o th er
as well [1 2]. Overexpression of the
antibiotics including ampi ci llin, streptomycin and sulfonamides (Figure 3). Within 4-6 weeks, multidrug resistant E. roli
expression of the AcrAB dmg efflux protein, which can pump out pine oi l, triclosan, quaternary ammonium compounds,
were recovered from worke rs and their family members, but not trom their neighbors (8). H ere, then, IS an illustration of an
chlorhexidine and organic solvents in addition to antibiotics.
ecological effect. In the animals, we see a direct effect of the antibiotic on the kinds of bacteria associated with them. At the same time we also see that the immediate enviro nm ent is
IIlar
locus causes increased
Consequently, these antibacterials are affecting the bacterial world in a fashion similar to antibiotics. SIIlUlarly, multidrug efflux systems can and do provide triclosan resistance m
Pseudomollas [13] . The intrinsIC pumps are presumably the reason o;closan is not particularly active against tim organism.
being changed by the presence of an antibiotic - even de livered at subtherapeutic amounts. [n a couple of the fa1111 workers, we traced a biochemically marked 111ultidrugresistant E. coli in their feces to chickens on the fa1111 [9). Thus. bacteria emerging in one animal group can m ove to another. This phenomenon was also clearly demonstrated in a
We have to recognize that we have entered the IlUCrobla.l world - tlley are not visitors in ours. We cannot continue to
later study performed on the same £ann ri O]. As we look into the futurc, we see anoth er spectre arisi ng
'a ntimi crobe' battle. Bacteria will always be victorious - they
fr0111 the past decade: another form of antimic robia l activity
are mo re numerous and genetically more flexible - and they
has made its way through the USA, Europe and Asia. Under the guise of hygiene, the re is increased substitu tion of
perform important fennentation and degradation fi.mctions that are essential to life processes .
WHAT CAN WE DO?
try to steri li ze o ur world in an attempt to win some kind of
'antibacterial' chemicals for soap and water, added to all
We have to rea.lize that antibiotics are socIetal and
manner of cleansers. Moreover, mattresses, cutting boards,
ecological dmgs as well as therapeutics, and choo e our
utensIls and toys 3re among the many items impregnated with an antibacteria l to offer 'b uilt-in anti bacte ri al protection'.
actions accordingly. Treatment of the indIvidual will affect the fami ly, the commUluty and all of society (14). In this way,
The chemical 1110St often used in th ese products is tJ;closan, known for more than 30 years as a 'bio cide' and gencrally
antibiotics an: unique among pharmaceuticals. For example, if you treat a person for acne with tetracycline, eryth.romycin or cotrimoxazole, the carriage of resistant staphylOCOCCI on the skin of the patient's domestic cohabll::mts \V111 also ch.ange [151 . Studies have shown that household contacts of patients bemg treated cany significantly higher numbers of l11ultidrugresistant stap hylococci than do control groups hvmg III antibioti c- fi'ee homes [151 . The potentia l ecological effects of antibiOtiCS prompted us
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to stud y the G1Ctors leading to emergence of resistance in Nepal. We examined the frequency of resistant intestinal flora IS
weeks on tetracycline Figure 3 The effect of length of time on a Sing le antibiotic on emer · gence of multidrug resistance . Chickens fed norma l or oxytetracycline · laced feed were examined for drug resistant E. coli in their feces. With time. an imals fed tetracycline · laced feed began 10 excrete organisms resistant to mUltiple antibiotiCS. including ampicil lin. streplOmycin and sulfonamides. in addition to tetracycline.
I
of people living in villages at dtfferem dmances from Kathmandu, where antibiotics are readily available. Our snldy demonsrr;ltcd that resistance frequency correlated not so much with what was being taken by the indiVIdual, but what was be.in g consum ed by the commumty. The effect of coml11Ul11ty usc appeared to surpass that of the mdiVldual's own use [16]. Vaccines constinlte a potential approach to tl,e resIstance prob lem , j.e. to elimmate the bactenal cause, as has been
2000 Copynght by the European Society of Clinical Microbiology and Infectious Diseases. eMf. 6 (Suppl 3), 101 106
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Clinical Microbiology and Infection. Volume 6. Supplement 3. 2000
accomplished with HaemoplJillls ilif/llet1zae type b. However, a consequence of vaccines is that, while removll1g an organismsuch as a commensal organism - other bacteria will take over the ecological niche [17]. We need to be sure that the replacements are not worse than the organisms being eliminated by the vaccines. Other approaches rely on how we use antibiotics. We can try shorter courses or cycuc u age but, in the latter case, we need enough effective antibIOtics to cycle. We certainly advocate prudent use. To achieve this goal fully, it would help to know that there i a bacterial infection involved . Therefore, rapid dJagnosncs would assist greatly. We urge proper use, e.g. that the annblOnc be taken for the designated period of time. It tS difficult to implement prudent usage when the public feels that bacteria are 'out to get us' and should be exterminated. Therefore, we need to educate the public. This latter goal is essennal. Prescribers need to think more broadly - not just about what is being purposely treated, but also about the lI1advertent subjection of entire microbial conununities to the effects of the antlbionc treatment. It is th e co mbined action of the antibionc, together with the reStst.1nce gene, that result 111 a resista nce problem . If eitller one IS controlled, the problem can be limited . One example comes frol1l Australia . While tlte eastern portion of the island was, and soli is, having a problem with methicillin-resistant SlapIJylo(o((//s ell/reus (MRSA), groups in Perth have kept control of it by triaging patients. Each new II1coming patient is te ted for the presence of MRSA, and camers are treated in different lImts of the hosplt.ll, separated from the other patients. The results have been quite dramatic. While the rest of Australia has MRSA rates of 11%-25%, western Australia has recorded rates less than 1% [18] . Similarly, 111 the U A and many parts of the world, patients with MR A or VRE are placed in private hospital rooms, away from other patients. Keeping resLstance out of the rest of the hospital population is one effective local solution to curtailing the global problem. Allother approach to the resistance problem is to develop new drugs. In thiS regard, compounds can be designed to attack the resistallce mechanism ItSelf. Pioneering work in this area used a drug, sodium clavulanate, that blocked ~-lactal11ases . Against ~- lactall1 resistant Klebsiella, Protells and E. coli, neitlter clavulanate nor ampicillin alone was very effecnve [19] . However, co mbining ampici llin with eithe r I ~lg or 5 ~lg of clavulanate, reduced the amount of ampicillin required to IIlhibit growth dramatically: from 250 to 500 Ilg/mL to 0.4-0.8 Ilg/mL. TIllS is an excellent example of how one call take a remtance trait, develop a method to reverse the reSIStance mechalUslTI and so restort! the action of the antibiotic. We are taking a sumlar approach against tetracycline reSistance, which is mediated by cwo different mechani~ms , an active efflux system and nbosomal protection . In a normal
f
Sensitive
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Figure 4 Tetracycline accumulation by susceptible and resistant ce lls. 3H -Tc uptake was examined in isogenic E. coli cells with or without a resistance gene (Class B on Tn 10) . With energy. the susceptible strain actively accumulated the drug. whereas lhe resistant strain kept it out. When de-energized (e.g. with DNP) . bOlh cells took up the same amount of tetracycline by diHusion (from [20)) .
susceptible cell, tetracycline accumulates internally (above the level of Simple diffilsion), ultimately crippling it~ reproduction . A resistant cell uses energy to keep the drug out (below even the diffusible amollnt). When you remove energy, the drug accumli lates to the same level in both the susceptible and the resistant cell (Figure 4). An efflux mechanism for tetracycline resistance exists via different resistance detenninant~ in Gramnegative and Gram-positive bacteria . It is best demonstrated in everted inner membrane vesicles of E. (o li. The vesicles are made by putting cells through a French pressure cell that tllrns the inner membrane inside out [20] . What goes into the vesicle is what the whole cell nornlally pumps out [21]. We u~ed this everted vesicle system to identify blockers of the pump . Energy-dependent uptake of tetracycline into the vesicles frol11 resistant cells was the control. We attempted to block this uptake with different dntg derivatives. With isotetracycline , which has a break in the e nng, no blocking occurred witl1 up to 200 Ilg/ mL. However, another derivative, 13-cyclopentyl tetracycline (at 0.5 or 0.2 Ilg/ mL), essentiall y stopped tetracyclllle uptake [22]: this derivative is an effective effiux blocker that acts by competitive inhibition [22] . Looking at this blocking agent against a class A efflux system in E. coli, we observed synergy, i.e. growth that occllrred at cwo dilutions from the M Ie of both 13-cyclopentyl and doxycycline (22). Synergism is based on the fact that the analog is inhibiting the efflux system. Against the class K and L efflux systems in GralTIpositive bacteria, 13-cyclopentyl was active by itself Against the very different ribosomal protecnon mechanism (specified by tll e class M detenninant), synergy was also observed [22] . Like the clavulanate example, the principle is the same: If one knows the resista nce mechamsm and can block It, tlte ' reSisted' antibioti
2000 CopYrlghl by Ihe European Society of Clinical Microbiology and Infectious Diseases. eMI. 6 (Suppl. 3) . 101-106
Levy Future of antibiotics
can work again . There are a number of new tetracycline deriva tives we are developing along the sa me lines, some of w hi ch appea r to wo rk, even as single age nt~. Our goal is to rej uvenate an antibioti c that has lost its potency to resistance. Identi fyi ng new targets may also lead to new dru gs. This app roach is being pu rsued by a number of phanllaceuti ca l companies. R.ega rdl ess of the new antib ioti cs developed, prudent use and app ropriate use must be sustained if th e dru gs will co ntinue to remain effec ti ve. O veruse and lTlisuse will , as we have seen over th e past 50 years, lead to resi tance to these new dru gs as well .
EDUCATING PROVIDERS AND CONSUMERS The maj or contributo rs to th e drug resistance problem are the consumer and the presc riber. The Alliance for the Prudent Use of Antibioti cs (HTTP: //www.apua.o rg) is working around th e globe to support local groups in dealin g with th e problem . It also produ ces info nll ati on and litera ture, such as brochures fo r co nsum ers, to involve th em in the soluti on. An oth er ac tiviry of the Alliance is to coo rdin ate wo rl dwid e, co untry-based groups to build a global surveillance system to kn ow what bac teria are out there circulatin g and wh ere are the majo r problems. APUA suppo rts local groups to deal with those prob lems. It is important to know the data: w here are th e resist-ances and w hat is the usage? T hese data are not readily available but can be obtained by local groups in their local co mmuni ties. What abou t th e prescrib er? Many health groups are now developing guidelines. For examp le, the C D C has made reco nullendatio ns for doctors for treatment of otitis media [23). When parents demand antibioti cs, the practitioner should provide edu cati onal materials and share treatment rules to expla.in why antibioti cs are not appropriate when no bac teri a are involved. D octors need to set up a dialogue with the parents, not just write a presc ripti on; one of the most impo rtant thin gs is that the physician's advice should foster coopera ti on and trust between the physician and the parent. 'Don't dismiss th e illness as only a viral infectio n' [23]. T he physician needs to explain the trea tment symptoms, give a realisti c time course and provide so mething at least to help th e child, in this case. to deal with a viral illness, wh.i ch is real. The resistance problem is a global one - any bacterium has th e potenti al to travel wo rldwide. Selection of resistance occurs wh ether it is th e cartle or chicken getting th e low-dose growth promoter. or each ac re of salmo n fa nn receiving 47 pounds of antibioti cs, or the hospi tals with their concentrated antibioti c use. If we are to succeed in reversing the resistan ce crisis, we not only need new dru gs, but we have to improve our use of th em . In this regard. we should focus not only on the orga nisms we are treating, but also the harmless
t05
'bystanders' that we do not wish to annillilate. They are our answe r - our allies in reversing resistance. O ur goal sho uld be to 'give a break to the susceptible strains.' Let us aim to make ou r antibiotic courses shorter. Let us StIive to space our antibiotic treatments so that the narural susceptible competitors of resistant strains can rerum. We must act to resto re the suscepti ble microbial £lora that preceded the antibi oti c deluge.
ACKNOWLEDGEMENTS Studi es of antibiotic resistance 10 this laboratory have been supported by grants fro m the National Institutes of H ealth.
REFERENCES Lt.'\1' SB. DJI.lIlclIlg chl' dmg .\41 2.
rt'~i\t.ln ct"
eqllJoon. Trl'uds .\lImlt,,,,/ 19")4; 2'
.2 . Levy SUo 17,r A"tiblMi( i>cmuillx : HiliI' j\/imdr Dm/<5 Aft' Nc..'w Yo rk: Ple num. 1992.
J . Levy SB , Sramng lire re""ance-If,'e. S ..... ... Levy
~B .
r:.II,~J
re~l'it.Jn(e.
Thl! ch.lllrnge- of antibiotiC
DcJ'n'}IJ".~ ,III' .\(mull'.
.\/cd 1990; 323: .1.'5- 7. &wlI!fi( .4", l')l)K: 278.
46- 53.
5. I larrio;CHl pro Ll'llcrhcrg]. (:'d" A",i/ll,t mbidl
RC." Hdllrr . IJS"r~
,I/Id Ol'/h'/1$
Wo rk'hop Report, Wal hmgton, DC: National Academy Press, 1998.
o. Levy SB. The 'eIl'nce or r.".tance In: Ilenderson OK. levy 5B cd, ResisftJIu O~dll;"''''S: C/('('dl ["'Pltrl
C,""UI"W"
,)II
l!f elm', London: Royal
Society or Mcdlcme p"'''' 1997, PI' 7- 14 7. I. . ..'\'y SB. AntibIOtic rC\l'~[.tnc(' ; Or(~ill.<, £'.."(111111011,
Resi",,",r:
JI1
rcolob'1Cll Imbalance. In ' Allub"",(
Sflt'CIi,," ",,,I SI',..ad.
Chi(he"~r: Wiley (C!ba
FOllnda tion Sympo"lHn 2(7), 1997. PI' 1 - 1~. H. Levy SB, FitzGerald ,B, Maconc AB. Ch,n!:c, "' mt<
•" '1'11'
after introduction of tecracychn~-sl1pplernemed feed on a Colnn .
E'td .11ed 1976; 295 : 583-8.
9. Levy S]3, FitzGerald GB, Macon. AB. Spread or ,ntlblotlc " ""'tance p l:l~ 1111 d~
from chicken to chicken and from chicken
(0
IllJn.
X,l/l1fr 1976;
260: 411-2.
D. Levy SU. Imer and II1cra'peri't( XlIII A(lrd So ([lS.'1) 1990; 87: 66()9- 13.
10. M.",h.,11 BM, III
Petro\\~kl
a f.1rm cnVlrOnl1lCOC
In
II . McMurry LM. Ocrhmger M. LeV)' SB. Tnclos.111 [argt.'t" lipid '),tlrhcw:..
Natllr,. 1998; 394: 53 1-2. 12. Mf: Murry LM. Ocdllngt'r M , Levy SB. O\'crcx'Pre"lon of IIh".-i , ""A B prodllces res"ranCe
10
tnrlman
111
Escllfrirhlll
,011.
(IIX")
or
rE.IlS .\lirroN.1 Leu
19'18; 166: 305-9 , 13. Sch weitzer H I'. rSl'/IIJ(lIII(l//(fS
I ntnlhlC
llt'nI,ltillM(J
141
rewitance to inhibitors offutty and
blO~ymht")ls III
dut' ro effillx: :1pphcatlon of a novd (echmqu(' ror
gCIll'f<1f10n of unmilrkcd chromosomal 1ll1lGiOOn" for [he ltrudy of dllux
'Y'tems. Alltimi",'/' A.~t.' ell,mOfllrr 199R; 42: 394- 11 14 . Levy SB. Mulndmg rc,i,t.lOcc: a "go or the nm"" 338:
137~
"',w E,~~.1 .lfc,j 199t\;
K.
15, M ll k r YW, E.dy EA, I...,cc), R W
ct
al Sequentl,1 anoblOnc therapy for
;:ICl1 e promorC4\ the c.l rn;lgc of rC\lst.1nt 'it.lphyloc(}cn on the kill of comaca.
J AII/i/",,/
CII"II",/llI'r 1996; 38: 829-37
16. Wal
In
Nepal reflect4i proXlOlI[)' to Kalhn1.lndu ( ubnutted) .
(" 2000 Copyri ght by the European Society of Clinica l Microbiology and Infectious Diseases, eMI, 6 (Suppl. 3), 101-106
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Clinical Microbiology and Infection, Volume 6, Supplement 3, 2000
17 . LlpsICch M. Bacccrial vaccin!:s and I\crotype repiaccll1t'm: IC'isom from HtJfl1l0ph illlJ "iflllrllzoe and pro.;;pcc{S for S'rl'pftlco(CIIS 1111('/lIIIOII;f1(', Elller:o!i",~
[lIf"" Df.' 1999: 5: 336-45. 1R. PC:1nnan JW, Grubb WH . Preventing dlC importation and esrabll'ih mcnt of methlcilhn-resiscant Stapilylo(o{(IIs
Of/rl'llS
(MRSA)
In
hospitalo; in Western
Auscralla. APUA ""vs!mer 1993; 11: Iff.
19. Readmg C. Cole M. Clav.linic acid: a beca- Iactam",e inhibiting bccalacGlm (rOin SUt'J'lol1l),ces d(J"(J/~ems. A"fim;crob Agell/s CllCII/(l,lta 1977; 11: 1152-7. 20. McMurry LM. Pecrucei R . Levy SB. Amve efllux of cetraeyeline encoded
by fOllr gcnl!tically ditTt:rent tetracyclint' resistance dercfmin:mrs in E. Coli. Pro( Nat! .'lrnd Sci (liSA) 1980: 77: 3974-7. 2 1. Levy Sl3. Active efflux IllCChallhlw; for antimicrobia l resista nce. Alllilllir.ro/, Ag"II" Che",,,,her 1992; 36: 695-703. 22. Nelson M L. Levy SB. Reve,,"1 of tetracycline resi.e.nce mediated by differem bacterial tetracycline resistance determ inants by an inhibicor of rhe.! T e[ (13) anci porc protein. All till/kroh Agts C!""",,her 1999: 43 : 1523-4. 23. Dowell SF. Marcy SM. Phillips WR. Gerbe r MA. Schwartz B. Otitis media - principles of Judicious USc of antimicrobial ag~1t~. Pedia/rirs 199R; 101 (Suppl.): 165-71.
2000 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, eM!. 6 (Suppl. 3) . 101- 106
Center for Adaptation Genetics and Drug Resistance, Tufts University School of Medicine, Boston, MA, USA -IiI I Mir,.,bio/ b!fcrt 2000: 6 (Supplemcm 3): 101-106
Antibiotic resi tance originates locally, but manifests globally. R.esistance frequencie~ can vary widely in different parts of the world, and even in hospitals in the same city. A good example of this point is demonstrated by the l1lultidrug resistant pneumococcus. There are wide variations in the percentage frequency of resistance to multiple antibiotics from different parts of the world as reported for the t 9905. Large differences exist among cities in the USA, Europe and Africa (Figure I). The approach to the problem depends on empowering local groups to examine the situation and find the solution appropriate for that locality. Resistance traits are not a recent microbia l invention to confront the antibiotics we are developing and using. They
have been around for the many nullennia of bactenal existence, dUling which time they have preslImably served other purposes. However, III any parncular envIronment whether it be a home, a city or a hospital - the applicaoon of an antibiotic will select resistance genes and propagate strains beating resistance to that antlbioric [1]. Resistance as a clinical problem is not inevitable. Just because an antibiotic is lIsed does not mean that a patiem will present with a drug-resistant infection. The reason we con£i:om the problem so often today stems from the extensIVe overuse of antibiotics, which encourage, the resistant strams to sUlvive. Environments teem ,vith large numbers of resistant bacteria. This preponderance of resistant strams dramatically
50
02 resistances 03 resistances
40
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Figure 1 Frequency of mullldrug resislanl S. pneumoniae In various countries in l he 1990s. (Dala organized from published studies Marshall and Levy. In preparation)
I 2000 Copyrigh l by Ihe European Soci ty of Clinical Microbiology and InfectiOUs Diseases, eMJ, 6 (Suppl . 3) , 101106
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Clinical Microbiology and Infection, Volume 6, Supplement 3, 2000
increases the chances that a patient's infection (which, after aJJ, derives from the environment) will be drug resistant. Another feature of resistance that underm.ines our efforts, but certainly aids the microbial world, is that resistance traits can be exchanged among aJJ types of bacteria - from Gramnegative bacteria to Gram-positive and vice versa, including MycobacteriulII. Unless it is a chromosomal mutation, a resistance determinant that appears in one organism will not stay solely in that organism. R esistances are often carried on transferable units of DNA called plasmids and transposons, which can go directly or indirectly from one bacterial genus to another. They can be transferred by bacteriophages and even through cell acquisition of naked chromosomal DNA (Figure 2) [2). Antibiotics produce major changes in microbial ecology, dismpting the equilibria among species and genera, and among resistant and susceptible strains (3). Tills effect stems from the large amount of antibiotics used. Tn tlle USA, an estimated 50 million pounds of antibiotics are produced each year. About half of them are used in humans, and the other half for plants and animals [4,5) . In humans, outpatient prescriptions in the USA number about 200 million annually. In animals, ,about 80% of the antibiotics used are for growth promotion. More subtle, and less well known, is antibiotic use in agriculture. An estimated 300 000 pound~ of antibiotics, called 'pesticides', are annuaJJy sprayed onto fruit trees [5). This latter use must have a large environmental impact per geographic unit. The end re;ult of all this antibiotic use is that the surviving bacteria are dmg resistant and can continue to transfer their plasmids and transposons to other bacteria. There is another feature of resistance that is problematic, if not paradoxical. Antibiotics are quite stable, and most of the mechanisms of resistance leave them untouched. Whether applied to human or animal systems or dispersed in the environment, antibiotics remain active and capable of
continuing the resistance selection process anywhere in the environment [6). Dmg inactivation could be an answer to this problem, but only a small number of resistance determ.inants have the dmg itself as their target (e.g. the ~-lactamases for penicillins and cephalosporins, acetyltransferases for chloramphenicol, different inactivating enzymes for aminoglycosides and macrolides). AU others, e.g. rifampicin, quinolones, trimethoprim, tetracycline, involve mechanisms that either act on the target of tlle dmg or keep the dmg out of the cell. The net result is that an active dmg remains in the environment where it continues to select for any strains that are resistant to it [6.7) .
ANTIBIOTIC AVAILABILITY Several years ago, on a visit to Santiago, Chile, to give a lecture, I was placed in a hotel across the street from the local pharmacy. ) decided to go over to see what drugs were available. I did not even have to enter the store. There was a 'desquento', a sale, on antibiotics. In particular, there was a 30% savings on the 10caJJy made chloramphenicol sold over the counter. In tlle window display I could see ampicillin and tetracycline [21 . It is notable that over-the-counter availability of antibiotics in Chile is no longer permitted. However, the same easy access to dmgs occ urs in Mexico and other Latin and South American countries. In many developing cou ntries, antibiotics are sold over tlle counter like other commoditi es. Tn fact, even in the USA one can get antibiotics quite readily frOI11 physicians and other providers; while one nuy not necessarily be able to buy them over the co unter, one ca n demand them from physicians. Once obtained, they may be improperly used andlor stored for later use (2). Some time ago, we underwent the pleasures and ttials of raising chickens to evaluate the effect of antibiotic use as
bacterlopl1age
Figure 2 The genetics of antibiotic resis-
-- .....
"fre." DNA
tance transfer. Resistance genes can enter new hosts via plasm ids, phages or naked DNA. Once inside the cell, many resistance genes reside on transposons that can move from one DNA molecule to anoth er. (Adapted from Figure 4.3 in [2].)
<" 2000 Copyright by the European Society of Clinical Microbiology and InfectiOUs Diseases, eMI. 6 (Suppl. 3), 101 - 106
Levy Future of antibiotics
growth promoters. Starting with eggs, we raised 3()O chi ckens. H alf of these were treated with low dose oxytetracyclin e in their feed. Whi le studyi ng the mi crob io logy of the treated and control groups, it became rapidly clear that th e low, subth erapeuri c dose of tetracycline led to a dramatic change: resistant orgalllsms appeared in the chi ckens within 24 h of eating the antibiotic-containi ng feed [8] . What was perhaps more interesting was the finding that as we contin ucd th e chi ckens on the tetracycline-laced feed, Esrherirhia coli em erged
103
co nsidered as lacking a specific cellular target. Its use was previously reserved fo r hospitals. We examined this chemical fo r its abi lity to selec mutants of E. roli and succeeded in obtaining them. Resistance emerged by a mutation in the Jab! gene involved in fatty acid biosynthesis [J 1]. Tlus findmg led to a confirmation that the protein product of the gene, enoyl reductase, was the target of triclosan. Perhaps more important is the emcrgence of triclosan resIstance through activation of the /liar locus, whic h leads to multiple resistance to antibiotics
with resistance, not just to tetracycl in e, but to multipl e o th er
as well [1 2]. Overexpression of the
antibiotics including ampi ci llin, streptomycin and sulfonamides (Figure 3). Within 4-6 weeks, multidrug resistant E. roli
expression of the AcrAB dmg efflux protein, which can pump out pine oi l, triclosan, quaternary ammonium compounds,
were recovered from worke rs and their family members, but not trom their neighbors (8). H ere, then, IS an illustration of an
chlorhexidine and organic solvents in addition to antibiotics.
ecological effect. In the animals, we see a direct effect of the antibiotic on the kinds of bacteria associated with them. At the same time we also see that the immediate enviro nm ent is
IIlar
locus causes increased
Consequently, these antibacterials are affecting the bacterial world in a fashion similar to antibiotics. SIIlUlarly, multidrug efflux systems can and do provide triclosan resistance m
Pseudomollas [13] . The intrinsIC pumps are presumably the reason o;closan is not particularly active against tim organism.
being changed by the presence of an antibiotic - even de livered at subtherapeutic amounts. [n a couple of the fa1111 workers, we traced a biochemically marked 111ultidrugresistant E. coli in their feces to chickens on the fa1111 [9). Thus. bacteria emerging in one animal group can m ove to another. This phenomenon was also clearly demonstrated in a
We have to recognize that we have entered the IlUCrobla.l world - tlley are not visitors in ours. We cannot continue to
later study performed on the same £ann ri O]. As we look into the futurc, we see anoth er spectre arisi ng
'a ntimi crobe' battle. Bacteria will always be victorious - they
fr0111 the past decade: another form of antimic robia l activity
are mo re numerous and genetically more flexible - and they
has made its way through the USA, Europe and Asia. Under the guise of hygiene, the re is increased substitu tion of
perform important fennentation and degradation fi.mctions that are essential to life processes .
WHAT CAN WE DO?
try to steri li ze o ur world in an attempt to win some kind of
'antibacterial' chemicals for soap and water, added to all
We have to rea.lize that antibiotics are socIetal and
manner of cleansers. Moreover, mattresses, cutting boards,
ecological dmgs as well as therapeutics, and choo e our
utensIls and toys 3re among the many items impregnated with an antibacteria l to offer 'b uilt-in anti bacte ri al protection'.
actions accordingly. Treatment of the indIvidual will affect the fami ly, the commUluty and all of society (14). In this way,
The chemical 1110St often used in th ese products is tJ;closan, known for more than 30 years as a 'bio cide' and gencrally
antibiotics an: unique among pharmaceuticals. For example, if you treat a person for acne with tetracycline, eryth.romycin or cotrimoxazole, the carriage of resistant staphylOCOCCI on the skin of the patient's domestic cohabll::mts \V111 also ch.ange [151 . Studies have shown that household contacts of patients bemg treated cany significantly higher numbers of l11ultidrugresistant stap hylococci than do control groups hvmg III antibioti c- fi'ee homes [151 . The potentia l ecological effects of antibiOtiCS prompted us
--.
60
~ 0
+tet
'-'
'"
40
~
'"
.
'i;j ~
20
-tet
c;,\
to stud y the G1Ctors leading to emergence of resistance in Nepal. We examined the frequency of resistant intestinal flora IS
weeks on tetracycline Figure 3 The effect of length of time on a Sing le antibiotic on emer · gence of multidrug resistance . Chickens fed norma l or oxytetracycline · laced feed were examined for drug resistant E. coli in their feces. With time. an imals fed tetracycline · laced feed began 10 excrete organisms resistant to mUltiple antibiotiCS. including ampicil lin. streplOmycin and sulfonamides. in addition to tetracycline.
I
of people living in villages at dtfferem dmances from Kathmandu, where antibiotics are readily available. Our snldy demonsrr;ltcd that resistance frequency correlated not so much with what was being taken by the indiVIdual, but what was be.in g consum ed by the commumty. The effect of coml11Ul11ty usc appeared to surpass that of the mdiVldual's own use [16]. Vaccines constinlte a potential approach to tl,e resIstance prob lem , j.e. to elimmate the bactenal cause, as has been
2000 Copynght by the European Society of Clinical Microbiology and Infectious Diseases. eMf. 6 (Suppl 3), 101 106
104
Clinical Microbiology and Infection. Volume 6. Supplement 3. 2000
accomplished with HaemoplJillls ilif/llet1zae type b. However, a consequence of vaccines is that, while removll1g an organismsuch as a commensal organism - other bacteria will take over the ecological niche [17]. We need to be sure that the replacements are not worse than the organisms being eliminated by the vaccines. Other approaches rely on how we use antibiotics. We can try shorter courses or cycuc u age but, in the latter case, we need enough effective antibIOtics to cycle. We certainly advocate prudent use. To achieve this goal fully, it would help to know that there i a bacterial infection involved . Therefore, rapid dJagnosncs would assist greatly. We urge proper use, e.g. that the annblOnc be taken for the designated period of time. It tS difficult to implement prudent usage when the public feels that bacteria are 'out to get us' and should be exterminated. Therefore, we need to educate the public. This latter goal is essennal. Prescribers need to think more broadly - not just about what is being purposely treated, but also about the lI1advertent subjection of entire microbial conununities to the effects of the antlbionc treatment. It is th e co mbined action of the antibionc, together with the reStst.1nce gene, that result 111 a resista nce problem . If eitller one IS controlled, the problem can be limited . One example comes frol1l Australia . While tlte eastern portion of the island was, and soli is, having a problem with methicillin-resistant SlapIJylo(o((//s ell/reus (MRSA), groups in Perth have kept control of it by triaging patients. Each new II1coming patient is te ted for the presence of MRSA, and camers are treated in different lImts of the hosplt.ll, separated from the other patients. The results have been quite dramatic. While the rest of Australia has MRSA rates of 11%-25%, western Australia has recorded rates less than 1% [18] . Similarly, 111 the U A and many parts of the world, patients with MR A or VRE are placed in private hospital rooms, away from other patients. Keeping resLstance out of the rest of the hospital population is one effective local solution to curtailing the global problem. Allother approach to the resistance problem is to develop new drugs. In thiS regard, compounds can be designed to attack the resistallce mechanism ItSelf. Pioneering work in this area used a drug, sodium clavulanate, that blocked ~-lactal11ases . Against ~- lactall1 resistant Klebsiella, Protells and E. coli, neitlter clavulanate nor ampicillin alone was very effecnve [19] . However, co mbining ampici llin with eithe r I ~lg or 5 ~lg of clavulanate, reduced the amount of ampicillin required to IIlhibit growth dramatically: from 250 to 500 Ilg/mL to 0.4-0.8 Ilg/mL. TIllS is an excellent example of how one call take a remtance trait, develop a method to reverse the reSIStance mechalUslTI and so restort! the action of the antibiotic. We are taking a sumlar approach against tetracycline reSistance, which is mediated by cwo different mechani~ms , an active efflux system and nbosomal protection . In a normal
f
Sensitive
2
~
_ __
Resistant (R222)
30 +DNP~,.() " "
+DNP. o
//,.JY"-
------
/($',""0
E ~
I
I
10 "
I
/
I
I
I
oS
I I
10
20 Time (min)
10
20
Figure 4 Tetracycline accumulation by susceptible and resistant ce lls. 3H -Tc uptake was examined in isogenic E. coli cells with or without a resistance gene (Class B on Tn 10) . With energy. the susceptible strain actively accumulated the drug. whereas lhe resistant strain kept it out. When de-energized (e.g. with DNP) . bOlh cells took up the same amount of tetracycline by diHusion (from [20)) .
susceptible cell, tetracycline accumulates internally (above the level of Simple diffilsion), ultimately crippling it~ reproduction . A resistant cell uses energy to keep the drug out (below even the diffusible amollnt). When you remove energy, the drug accumli lates to the same level in both the susceptible and the resistant cell (Figure 4). An efflux mechanism for tetracycline resistance exists via different resistance detenninant~ in Gramnegative and Gram-positive bacteria . It is best demonstrated in everted inner membrane vesicles of E. (o li. The vesicles are made by putting cells through a French pressure cell that tllrns the inner membrane inside out [20] . What goes into the vesicle is what the whole cell nornlally pumps out [21]. We u~ed this everted vesicle system to identify blockers of the pump . Energy-dependent uptake of tetracycline into the vesicles frol11 resistant cells was the control. We attempted to block this uptake with different dntg derivatives. With isotetracycline , which has a break in the e nng, no blocking occurred witl1 up to 200 Ilg/ mL. However, another derivative, 13-cyclopentyl tetracycline (at 0.5 or 0.2 Ilg/ mL), essentiall y stopped tetracyclllle uptake [22]: this derivative is an effective effiux blocker that acts by competitive inhibition [22] . Looking at this blocking agent against a class A efflux system in E. coli, we observed synergy, i.e. growth that occllrred at cwo dilutions from the M Ie of both 13-cyclopentyl and doxycycline (22). Synergism is based on the fact that the analog is inhibiting the efflux system. Against the class K and L efflux systems in GralTIpositive bacteria, 13-cyclopentyl was active by itself Against the very different ribosomal protecnon mechanism (specified by tll e class M detenninant), synergy was also observed [22] . Like the clavulanate example, the principle is the same: If one knows the resista nce mechamsm and can block It, tlte ' reSisted' antibioti
2000 CopYrlghl by Ihe European Society of Clinical Microbiology and Infectious Diseases. eMI. 6 (Suppl. 3) . 101-106
Levy Future of antibiotics
can work again . There are a number of new tetracycline deriva tives we are developing along the sa me lines, some of w hi ch appea r to wo rk, even as single age nt~. Our goal is to rej uvenate an antibioti c that has lost its potency to resistance. Identi fyi ng new targets may also lead to new dru gs. This app roach is being pu rsued by a number of phanllaceuti ca l companies. R.ega rdl ess of the new antib ioti cs developed, prudent use and app ropriate use must be sustained if th e dru gs will co ntinue to remain effec ti ve. O veruse and lTlisuse will , as we have seen over th e past 50 years, lead to resi tance to these new dru gs as well .
EDUCATING PROVIDERS AND CONSUMERS The maj or contributo rs to th e drug resistance problem are the consumer and the presc riber. The Alliance for the Prudent Use of Antibioti cs (HTTP: //www.apua.o rg) is working around th e globe to support local groups in dealin g with th e problem . It also produ ces info nll ati on and litera ture, such as brochures fo r co nsum ers, to involve th em in the soluti on. An oth er ac tiviry of the Alliance is to coo rdin ate wo rl dwid e, co untry-based groups to build a global surveillance system to kn ow what bac teria are out there circulatin g and wh ere are the majo r problems. APUA suppo rts local groups to deal with those prob lems. It is important to know the data: w here are th e resist-ances and w hat is the usage? T hese data are not readily available but can be obtained by local groups in their local co mmuni ties. What abou t th e prescrib er? Many health groups are now developing guidelines. For examp le, the C D C has made reco nullendatio ns for doctors for treatment of otitis media [23). When parents demand antibioti cs, the practitioner should provide edu cati onal materials and share treatment rules to expla.in why antibioti cs are not appropriate when no bac teri a are involved. D octors need to set up a dialogue with the parents, not just write a presc ripti on; one of the most impo rtant thin gs is that the physician's advice should foster coopera ti on and trust between the physician and the parent. 'Don't dismiss th e illness as only a viral infectio n' [23]. T he physician needs to explain the trea tment symptoms, give a realisti c time course and provide so mething at least to help th e child, in this case. to deal with a viral illness, wh.i ch is real. The resistance problem is a global one - any bacterium has th e potenti al to travel wo rldwide. Selection of resistance occurs wh ether it is th e cartle or chicken getting th e low-dose growth promoter. or each ac re of salmo n fa nn receiving 47 pounds of antibioti cs, or the hospi tals with their concentrated antibioti c use. If we are to succeed in reversing the resistan ce crisis, we not only need new dru gs, but we have to improve our use of th em . In this regard. we should focus not only on the orga nisms we are treating, but also the harmless
t05
'bystanders' that we do not wish to annillilate. They are our answe r - our allies in reversing resistance. O ur goal sho uld be to 'give a break to the susceptible strains.' Let us aim to make ou r antibiotic courses shorter. Let us StIive to space our antibiotic treatments so that the narural susceptible competitors of resistant strains can rerum. We must act to resto re the suscepti ble microbial £lora that preceded the antibi oti c deluge.
ACKNOWLEDGEMENTS Studi es of antibiotic resistance 10 this laboratory have been supported by grants fro m the National Institutes of H ealth.
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rt'~i\t.ln ct"
eqllJoon. Trl'uds .\lImlt,,,,/ 19")4; 2'
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~B .
r:.II,~J
re~l'it.Jn(e.
Thl! ch.lllrnge- of antibiotiC
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•" '1'11'
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(0
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Petro\\~kl
a f.1rm cnVlrOnl1lCOC
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II . McMurry LM. Ocrhmger M. LeV)' SB. Tnclos.111 [argt.'t" lipid '),tlrhcw:..
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10
tnrlman
111
Escllfrirhlll
,011.
(IIX")
or
rE.IlS .\lirroN.1 Leu
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I ntnlhlC
llt'nI,ltillM(J
141
rewitance to inhibitors offutty and
blO~ymht")ls III
dut' ro effillx: :1pphcatlon of a novd (echmqu(' ror
gCIll'f<1f10n of unmilrkcd chromosomal 1ll1lGiOOn" for [he ltrudy of dllux
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137~
"',w E,~~.1 .lfc,j 199t\;
K.
15, M ll k r YW, E.dy EA, I...,cc), R W
ct
al Sequentl,1 anoblOnc therapy for
;:ICl1 e promorC4\ the c.l rn;lgc of rC\lst.1nt 'it.lphyloc(}cn on the kill of comaca.
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CII"II",/llI'r 1996; 38: 829-37
16. Wal
In
Nepal reflect4i proXlOlI[)' to Kalhn1.lndu ( ubnutted) .
(" 2000 Copyri ght by the European Society of Clinica l Microbiology and Infectious Diseases, eMI, 6 (Suppl. 3), 101-106
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Clinical Microbiology and Infection, Volume 6, Supplement 3, 2000
17 . LlpsICch M. Bacccrial vaccin!:s and I\crotype repiaccll1t'm: IC'isom from HtJfl1l0ph illlJ "iflllrllzoe and pro.;;pcc{S for S'rl'pftlco(CIIS 1111('/lIIIOII;f1(', Elller:o!i",~
[lIf"" Df.' 1999: 5: 336-45. 1R. PC:1nnan JW, Grubb WH . Preventing dlC importation and esrabll'ih mcnt of methlcilhn-resiscant Stapilylo(o{(IIs
Of/rl'llS
(MRSA)
In
hospitalo; in Western
Auscralla. APUA ""vs!mer 1993; 11: Iff.
19. Readmg C. Cole M. Clav.linic acid: a beca- Iactam",e inhibiting bccalacGlm (rOin SUt'J'lol1l),ces d(J"(J/~ems. A"fim;crob Agell/s CllCII/(l,lta 1977; 11: 1152-7. 20. McMurry LM. Pecrucei R . Levy SB. Amve efllux of cetraeyeline encoded
by fOllr gcnl!tically ditTt:rent tetracyclint' resistance dercfmin:mrs in E. Coli. Pro( Nat! .'lrnd Sci (liSA) 1980: 77: 3974-7. 2 1. Levy Sl3. Active efflux IllCChallhlw; for antimicrobia l resista nce. Alllilllir.ro/, Ag"II" Che",,,,her 1992; 36: 695-703. 22. Nelson M L. Levy SB. Reve,,"1 of tetracycline resi.e.nce mediated by differem bacterial tetracycline resistance determ inants by an inhibicor of rhe.! T e[ (13) anci porc protein. All till/kroh Agts C!""",,her 1999: 43 : 1523-4. 23. Dowell SF. Marcy SM. Phillips WR. Gerbe r MA. Schwartz B. Otitis media - principles of Judicious USc of antimicrobial ag~1t~. Pedia/rirs 199R; 101 (Suppl.): 165-71.
2000 Copyright by the European Society of Clinical Microbiology and Infectious Diseases, eM!. 6 (Suppl. 3) . 101- 106