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Epidemiology of tetracycline-resistance determinants
EVOLUTION
aminoglycoside-resistant isolates. The most common single and combination resistance mechanisms occurred in all of th e geographical regions surveyed, while the less common mechanisms tended to occur in individual regions or only in a few regions. Rates of resistance observed using NCCLS disk-diffusion criteria tended to agree well with those calculated from the phenotype of the resistance mechanisms observed, especially allowing for the occurrence of low-level permeability changes.
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References 1 Davies, J. and Smith, D.1. (1978) A~zrzrc. Rev. M~crohiol. 32, 469-,518 2 Price, K.E. eta/. (1981) /. Antimicroh. Chemother. 8 (Suppl. A), 89-105 3 Shim& K. et al. (198.5) Antimicro/]. Agents Chemother. 28, 282-288 4 Shaw, K.J. et al. (1993) Microbial. Rev. 57, 138-163
5 European Studv Group on Antibiotic Resistance (ESGAR) (1987) Eur. 1. C/in.M&h/. 6, 378-385 6 Dornbusch, K. etal. (1990) _/.Antimicrob. Chemother. 26, 131-144
Epidemiology of tetracycline-resistance determinants Marilyn C. Roberts
T
Resistance to tetracycline is generally due etracyclines are broadare readily transferred and have tither to energy-dependent efflux of few physiological barriers, is spectrum antimicrobials tetracycline or to protection of the that are active against a widely distributed’,‘,“. bacterial ribosomcs from the action of wide range of Gram-positive and Gram-negative bacteria, tetracycline. The genes that encode this Classification of Tet resistance arc normally acquired via cell-wall-free mycoplasmas, determinants transferable plasmids and/or transposons. chlamydiae, rickcttsiae and Mendcz et al.’ first examined protozoan parasites (see p. 421 Tct determinants have been found in a the genetic heterogeneity of wide range of Gram-positive and for a glossary of antibiotics)‘. tetracycline-resistance dctermiGram-negative bacteria and have Tetracyclines have been used for nants from Enterobactcriaceac reduced the effectiveness of therapy therapy in man, animals and and Pseudomonadaceae plascertain crops, as well as for with tetracycline. mids using restriction-enzyme food additives for growth proanalysis, DNA-DNA hybridizM.C. Roberts is in the Dept of Pathbiology SC-38, motion and therapy in animal ation and expression of resistSchool of Public Health and Cowzmrrnrty Medicine, husbandry’. Because of their ance to tetracyclines. DNAUniversity of Washington, Seattle, WA 982 95, USA. broad spectrum of activity, DNA hybridization using the relative safety and low cost,.tetracyclines have been structural genes as probes is now the standard method widely used throughout the world and are the second to distinguish different genes’,13*‘x. IJsing this method, most commonly used antibiotic after penicillins’. some of the resistance determinants have up to 76% Over the past 20 years, bacterial resistance to tetraDNA sequence identity but, under stringent hybridcyclines has limited their use’-4. ‘The diversity of resistization conditions, they do not crosshybridize and are ance to tetracycline was first examined in Gram-negative thus considered to be separate gencs2. enteric bacterias. A decade later, 16 tetracycline-resistance (Tet) determinants and three oxytetracyclineMechanisms of resistance to tetracycline resistance (Otr) determinants, first found in oxytetraResistance to tetracyclines is primarily due to acquisition cycline-producing Streptomyces, have been described of Tet determinants rather than to mutation of existand characterized’-4,h-1’ , with new Tet determinants ing chromosomal gcnes1-5. An exception is Neisseria being identified continually’3. Of these determinants, gonorrhoeae, where the additive effects of mutations 1.3 are frequently associated with plasmids, while others to chromosomal genes tet, mtr and penB can result are on the chromosome (Table 3 ). The Tet M and Tet in clinical resistance to tetracycline therapy”. FurQ determinants are associated with conjugative thermore, Escherichia coli has a chromosomal tetraelements’4,‘5. Wide variation in the distribution of Tct cyclinc-resistance efflux system that is associated with determinants may be related, in part, to the ease with the mar IOCUS’~.However, N. gonorrhoeae has also which particular Tet determinants are transferred acquired the Tct IM determinant”, and I:. coli has a between various isolates and genera. For example, variety of Tet determinants (Table 2). the Tet E determinant is on a large nonconjugative There are three different mechanisms of resistance plasmidlh, while the Tct M dctcrminant, which is to tetracycline: (I) energy-dependent efflux of tetracycline by proteins inserted into the cytoplasmic usually associated with conjugative transposons that
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inactivation
of tctracyclincZ2 mechanisms of drug resistance have been found in oxytetracyclineproducing St~eptonzyces~‘~‘~, which is consistent with the hypothesis that antibiotic-producing soil microorganisms could be the ancestral source of some of the Tet determinants?‘. The line between antibiotic producers and other bacteria has been blurred with the identification of Tet K and ‘I’et 1, determinants in Stveptomyces, and the Strcptomyces Otr determinants in Mycobacteriwn spccies2” (Table 4). (Table
Tet A-E Tet X TetG,H Tet K Tet L Tet M (rare) Tet 0 Tet P _
Tet Tet Tet Tet
Tet S --
Otr A-C
Efflux of tetracycline Different Gram-positive and Gram-negative genera have Tct determinants that confer resistance to tctracyclinc by removing tetracyclines from the cell using an energy-dependent efflux mechanism (Table 3). The approximately 46 kDa membrane-bound efflux pro-
membrane’U, (2) protection of the bacterial ribosomc from the action of tetracycline”, and and (3) enzymatic alteration
Table 1. Location of the tetracycline-resistance (Tet) determinants” --. .Plasmid
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Tet B (rare)
Kb L (rare) M 0
Tet Q -
“Based on information from Refs l-13,16,17,22,32,34-36. %an be associated with an integrated plasmid.
3). The same
teins consist of 12 hydrophobic
Enzymatic alteration of tetr.acycline The only example of enzymatic-mediated resistance to tetracycline is the Tct X determinant that has been cloned from anaerobic Bacteroidesz2. However, the enzyme requires oxygen, does not function in the natural host and has not been found outside Racteroides”. Because of its questionable role in conferring rcsistancc to tetracycline in Nature, it is not considered further here.
Table 2. Distribution of tetracycline-resistance (Tet) determinants among Gram-negative bacteria” .~. _-. Ribosomal protection and/or efflux _~...
Effluxb .~. Bacterium
Tet determinant
Bacterium
Tet determinant
Actinobacillus Aeromonas Citrobacter fdwardsiella Enterobacter Escherichia Klebsiella Moraxella Pasteurella Plesiomonas Proteus Pseudomonas Salmonella Serratia Shigella Yersinia Vibrio
Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet
Bacteroides” Campylobacter fikenella Fusobacterium” Haemophilus Kingella Neisseria Prevotella” Veil/one//a’
Tet Tet Tet Tet Tet Tet Tet Tet Tet
-
B A, A, A, B, A, A, B B, A, A, A, A, A, A, B A,
B, B, D C, B, D
D, E C, D D C. D, E
D, H B, D B, C C B, C, D, E B, C B, C, D
mcmbranc-spanning
regions separated by shorter hydrophilic Sequences-J”. ‘l’he efflux proteins exchange a proton for a tetracycline-cation complex, and are antiportcr systcmP. The Tet A-E and Tet G-I I determinants all contain a structural and a repressor gene that are expressed in oppositc directions from overlapping operator regions4,26. Both repressor and structural genes have significant scquencc similarity with each other, suggesting that the genes may derive from a common ancestor4J”,?6. The cfflux proteins have amino acid and protein structure similarities with other cfflux proteins involved in multiple-drug rcsistancc and resistance to quatcrnary ammonium, chloramphcnicol and quinolone”‘,26. The Gram-positive tetK and telI, genes encoding tetracycline-efflux proteins are regulated by mRNA attenuation in a similar way to that described for Grampositive eym genes encoding rRSA methylases, and cat genes encoding chloramphenicol acetyltransferases’:‘. The Clostridium ‘l’ct P determinant consists of two overlapping resistance-encoding gent?: tetA(P) encodes an efflux protein, while tetB(P) encodes a ribosomal-protection proteinX. The S~reptomyccs Otr B dctcrminant encodes an cfflux protcin12 that is more closely related to TetA(P) than to either the Tct(K) or the Tct( L) protein?.
_
.
M, Q, X 0 M M B, M M M Q M
B, C, D, E, G
_..__
JBased on information from Refs l-7,9,13,15-17,22,32-34,40. “Ribosomal-protection-encoding genes have not yet been found in enteric genera and, when these genes are cloned into fscherichia co/i, the level of resistance to tetracycline conferred is relatively Iowl’. “Anaerobic species.
Proteins that protect ribosomes from the action of tetracycline both in vim and in vitro form another mcchanism of resistance to tetracycline”. Tct M, 0, S, B(P) and Q, and Otr A detcrminants encode 72.5 kDa cytoplasmic proteins that have amino acid sequencc similarity to the elongation factor Tu (EF-Tu) and EF-G (Ref. 28). The Tet(,M), Tet(O) and Tct(S) proteins have more than 70% sequence identity to each othcr2; the Otr(A) and TctB(1’) proteins also have a high dcgrcc of sequence identity with each otherx, and the ret(Q) protein, with 45% amino acid sequence identity to the other groups, forms a third grouph. The Tct M determinant is thought to be regulated by mRNA attenuation”, while the Tet 0 determinant is not regulated”‘. Because these proteins are similar in amino acid scquencc to EF-Tu and EF-G (Ref. 28), we have suggested that they might be tetracycline-resistant EFs or might block the binding of tetracycline to the ril~osomc’x. Burdctt has shown that the Tet(M) protein
EVOLUTION
has ribosome-dependent G’I’I’ase activity”, while Manavathu et ~1.~” have suggested that these proteins might ‘modify ribosomal proteins or rRNA’, perhaps by phosphorylation or acetylation. However, their resuits and those of Burdett” show that ribosomes from cells carrying the Tet(M) or Tct(0) proteins are fully sensitive to tetracycline, making it unlikely that there is a transient unstable modification of the sort suggested by Manavathu. More rcccntly, Burdctt3’ has isolated an E. co/i mutant in which the ‘I’et M determinant does not confer resistance to tctracyclinc, and suggested that tRNA-modification activity is ncccssary for Tet(iM)mediated resistance to tetracycline. Location and dissemination of let determinants ‘The majority of ‘I‘et determinants are found in a variety of bacteria isolated from man, animals and the cnvironmcnt’. These data support the hypothesis that gene exchange occurs widely throughout bacteria and between ecosystems. ‘l‘hc facultative Gramnegative Tct determinants are associated primarily with transposons inserted into a diverse group of conThe Tet B determinant has jugative plasmids 1.5,7,13. the widest host range among the Gram-negative species22x23, while the genus Vihrio hosts the largest number of different Tet determinants (Table 2). The genes encoding ribosomal-protection proteins have not been found in the majority of facultative Gramnegative species. A few isolates of Haemophilus species and all highly tetracycline-resistant Moruxella (Brunhamellu) catarrhalis have a chromosomal Tet B The Tet B determinant is not determinant3,32J3. conjugative in these isolates, but can be moved by transformation.‘*J1.33. The Tct E determinant differs from the Tet A-D determinants by its association with large plasmids that are neither mobile or conjugativelh, and has rcccntly been associated with the chromosomei4, which may explain its limited distribution and its predominance in aquatic environments’Jh (Table 2). Tet G (Ref. 9) and Tet H determinants’ have only been described recently, and their distribution has not been explored fully. The Gram-positive Tet K and Tet L determinants arc found on small transmissible plasmids that can become integrated into the chromosome of staphylococci 35 or are foulid independently on the chromosome of Bacillus subtilisJh. These determinants are widely distributed among Gram-positive species associated with man, animals and the soi13.2- (Table 4), and have been found recently in rapidly growing Mycobacterium and Streptomyces species24 (Table 4), as well as in Gramnegative Haemophilus aphrophihs and Veillonella Paruth strains, all of which were isolated from Patients3’ (Table 2). This is the first documented acquisition of an antibiotic-resistance determinant in Mvcobacterium, and it suggests that gene exchange’between tetracycline-resistant Gram-positive bacteria and both Mycohacterium and Streptomyces has occurred. Recently, the Otr determinants from industrial Streptomyces have been found in clinical Mycobacterium and StreptomycesL4, and surveys are required to determine their distribution in other genera.
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Table 3. Classification of tetracycline-resistance (Tet) determinants according to their mechanism of resistance= -_. _ Ribosomal Enzymatic Unknown Efflux Tet Tet Tet Tet Tet Otr
A-E G-H K L A(P) B
Tet Tet Tet Tet Tet Otr
M 0 S Q B(P) A
Tet X
“Based on information from Refs 2-13,22. anisms involved, see the text.
Otr C
For details of the mech-
The ribosomal-protection Tet M and Tct Q determinants are often associated with conjugative chromosomal elements, which code for their own transfer’4,‘S,3R. Thcsc clcments have been shown to transfer mobilizable plasmids to other bacterial isolates and species”. Recently, genes in the Tet(Q) operon have been identified that mediate excision and circularization of discrete nonadjacent scgmcnts of chromosomal DNA in Bacteroides, are important for self-transfer and the effect of which is enhanced by pre-exposure to tetracycline w. Pre-exposure to tetracycline can also enhance transfer of the Tet M determinant. The Tct M dcterminant is freely transferred between Gram-positive (donor or recipient) and Gram-negative (donor or recipient)
Table 4. Distribution of tetracycline-resistance (Tet) determinants among Gram-positive type bacteriaa Bacterium
Tet determinant
Actinomyces
Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet
Aerococcus fkJCi//US
Clos tridium b Corynebacterium Enterococcus Eubacterium Gardnerella Gemella Lactobacillus Listeria Mobiluncus b Mycobacteriumc Mycoplasmad PeptostreptococcusD Staphylococcus Streptococcus Streptomyces’ lJreaplasmaa
L M, 0 K, L K, L, M K, L, K, M M M 0 K, L, 0 K, L, M K, L, K, L, K, L, K, L, M
M, P M, 0
M, S Otr A, B M, 0 M, 0 M. 0 Otr A, B, C
“Based on information from Refs 2-4,8.10-12,24, 27,36,41. OAnaerobic species. ‘Acid-fast bacteria. bCell-wall-free bacteria with a Gram-positive metabolism. eMulticellular bacteria.
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specieG4”. It has a broad host range and is found in all groups of bacteria that have been examined, except in facultative enteric bacteria (Tables 2 and 4), and can be transferred between bacteria isolated from all different types of ecosystems and a large number of different species3. This supports the hypothesis that the Tet M determinants can cross most bacterial biochemical, genetic and morphological barriers. The Tet 0 determinant is not associated with conjugative elements and is less widely distributed than the ‘I’ct M determinant, but is also found in both Gram-positive and Gramnegative genera G” (Tables 2 and 4). My group has hypothesized that commcnsal bacteria could be reservoirs for Tet determinants’,“‘. We have examined bacteria from the urogenital tracts of females who had not taken any antibiotics for 2 weeks prcviously4’. Of the viridans streptococci, 82% hybridized with at least one Tet dctcrminant, and of the 18 that did not hybridize, only seven (7%) were susceptible to tetracycline. Of the 71 I-‘eptostreptococctis species, 79% hybridized with at least one Tet determinant, and of the 1.5 that did not hybridize, only three (4%) were susceptible to tetracycline. Similarly, only 25% of the Lactohacillus species were susceptible to tetracycline. The most common Tct determinant found was Tet M, and it could be transferred between bacteria in mating experiments4’. In contrast to the commensal species, only 46% of the opportunistic Streptococczfs agulactiae were resistant to tetracycline. ,More recently, we have examined the dental flora of normal controls and found 2-20X of the aerobically grown flora and S-56’% of the anaerobically grown flora to be resistant to tetracycline. The majority of the Tct determinants found were transferable. The discovery of the same Tet determinants in both aerobic and anaerobic commensal species and their ability to act as donors of the tetracycline-resistance-encoding genes supports the hypothesis that commensal bacteria can act as reservoirs for these genes and arc potential donors to pathogens entering the ecosystem. Conclusion In the past ten years, use of tetracycline has been rcduccd as the number of tetracycline-resistant genera and species has increasedlm4. Resistance to tetracycline is due to genes encoding either efflux or ribosomalprotection proteins, while the third possible mcchanism (enzymatic inactivation of the antibiotic) is not thought to be important in conferring resistance in Nature. Both efflux and ribosomal-protection mechanisms are found in the oxytetracycline-producing of resistance Streptomyces1’a12, while the mechanism encoded by the Streptomyces otrC gene is unknown. Most bacteria, representing nearly all of the evolutionary branches, including Mycobacterium and Streptobut myces, now carry one or more Tet determinants, the distribution of individual Tet determinants varies. However, the number of ancestral genes from which the genes encoding efflux-type resistance or ribosomalprotection-type resistance derive is still not clear4~x.2”. Examination of the DNA and protein sequences of highly related determinants, such as Tet M and Tet 0,
or the Tet A-H determinants, suggests that divergence has occurred. The Otr determinants have the same mechanisms of resistance to tetracycline as do the ‘I‘ct determinants, have sequence similarity with these genes and are associated with the oxytetracyclinc-biosynthetic pathways in Streptomyces. Based on these data, it is tempting to speculate that the Otr and Tet determinants have a common ancestor, but other possibilities cannot be ruled out2.‘. ‘ Continued widespread use of tetracyclines in human and animal medicine as well as on crops exerts sclcctivc pressure on bacteria, which will result in larger numbers of tetracycline-resistant isolates, new species acquiring tet genes and the reduced effectiveness of all applications of tetracycline therapy. Recently, modified tetracyclincs that arc not affected by efflux and ribosomal-protection mechanisms of resistance have been madc43-4’. These new derivatives or other related compounds, if approved for use, could prolong the usefulness of tetracyclincs in therapy. Acknowledgement M.C.R. was supported by grant X24136 from the National Institute of Health. References 1 Levy, S.B. (1992) ‘I’/&A~~tihiot~c Pm~dox, How Miracle Drrqs nye DestrojGzg the M~yde, Plenum Press 2 Levy,S.B.(1989) /. Anlinzicrob. Chernother. 24, l-3 3 Roberts, MC. (1989)Gerx TY~IS~CY in the Em~irowmnt (Levy, S.B. and Miller, R.V., cds), pp. 347-37.5, M&raw-Hill 4 Chopa, I., Hawkcy, P.M. and Hinton, .Ll. (1992) 1. Antinricro6. Chemother. 29, 245-277 5 Mendcz, B., Tachibana, C. and Levy, S.B. (1980) I’hstnici 3,99-10X 6 Sikolich! Ml’., Shoemaker, N.B. and Salyers, A.A. ( 1992) Anfimicrob. Agerzts Chemother. 36, 1005-l 012 7 Hansen, L&l. et al. (1993) Aufimicroh. Agents Chernother. 37, 2699-2705 8 Sloan, J.. hlcMurry, L.M. and Lyras, D. (1994) Mol. :Micyohiol. 11,403-415 9 Zhao, J. and Aoki, T. (1992) Microhrol/~w~Iuw~.36, 1051-1060 10 Charperntier, E., Gerbaud, G. and Courvalin, I’. (1993) Gene 131,27-34 Agerzfs 11 Dittrich, W. and Schrempf, H. (1992) Antinmnb. Cbemotbe~. 36, 1119-l 124 12 Butler, MJ. etal. (1989) Mol. Gen. Genet. 215,231-238 13 Jones, C.S., Osborne, U.J. and Stat&y, J. (1992) M. Cell. Probes 6, 3 13-3 17 14 Clcwell, D.B. rl al. (I 988) 1. Rmteriol. 170, 3046-3052 15 Salyers, AA. and Shoemaker, K.B. (1992) Erru. 1. C/in. Micyobiol. Infect. DIS. 11, 1032-l 038 16 Sorum, H., Roberts, MC. and Cross, J.H. (1992) Antbnicrob. Agents Cbemother. 36, 61 l-615 Agents Chender. 30, 17 Morse, S.A. et ul. (1986) Admicroh. 664-670 18 Levy, S.B. et al. (19891A~~fmicroh. Agents Chrmother. 33, 1373-1374 19 George, AM and Levy, S.B. (1983) 1. Racteriol. 155, 531-540 20 Levy, S.R.(1992) Anfmicyob. Ageents Chemother. 36, 695-703 21 Burdett, V. (1991)]. Biol. Chem. 266,2872-2877 22 Spccr, B.S., Bcdqk, L. and Salyers, A.A. (1991) 1. Hocferd. 173, 176-183 23 Bcnvcnistc, R. and Davies, J. (1973) Proc. lVaf/ Ad. Sci. liSA 172,3628-3632 24 Pang, Y. et al. ( 1994) Autinzrcroh. Agents Chemothcr. 38, 1408-1412
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2.j Yamaguchi, A. et al. ( 1992) /. Bid. Cbem. 267, 7490-7498 5, 895-900 26 Sheridan, R.P. and Chopra, I. (199 1) !ziiol.Microbid. 27 Schwarz, S., Cardoso, M. and Wcgcner, H.C. (1992) Antimicroh. Agmts Chemther. 36, .580-SSY 28 Sanchez-Pcscador, R. et al. (1988) Ntrcle~cAcids Kes. 16. 1218 29 51, Y.A., He, I’. and C~cwcll, I).B. (1992) Antimwoh. Agents Chemotber. 36, 769-778 Agents Chemother. 30 Manavathu. E.K. et al. ( 1990) Antirnicrob. 34,71-77 31
hrdett, V. (1993)]. Rncterid.
175, 7209-7215 32 .Ilarshall, 1%. et a/. (1984) 1. Infect. Dis. 149, 1028-1029 33 Roberts, M.C. et a/. (1991) Antrm~croh. Agents Chernother. 3.5, 2453-2455 34 Lee, C., Langlois, B.E. and Dawson, K.I.. (I 993) Appl. ~wiron. h4icr&d. 59, 1467-1472 3.5 Gllcspie, .v.T., h4ay, J.W. and Skurray, R. (lY86)j. Gen.
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iC!~crobiol.132, 1723-1728 36 Sakaguchi, R. and Shishido, K. (1988) Biochim. Hmphys. Acta 949, 9-si 37 Pang, Y., Bosch, T. and Kobcrts, M.C. ~%lol.Cell. Probes (in press) 38 Le Bougucncc, C., dc Ccspedes, G. and Horaud, T. (1990) /. Bacterial. 172, 727-734 39 Naglich, J.G. and hndrews, R.E., Jr (1988) P/amid 20, 113-126 40 Srevens, 01. et al. (1992) 1. Racteriol. 174,2935-2942 41 Kuberts, MC. and Lansciardi, J. (1990) Antimlcrob. ilp~ts Chemother. 34, 1836-I 838 42 Rohcrts, .Ll.C. and Hillier, 51.. (1990) Autimicrob. Agents Chemotber. 34, 261-264 Ayeuts Chemother. 37, 43 Testa, R.T. et nl. (1993) Antimicrob. 2270-2277 44 Nelson, I1.L. et ~71. (1993) 1. Med. Chem. 36, 370-377 45 Tymiak, A.h. et al. (1993)_/. Org. Chem. S&535-,537
Extrachromosomal resistance in Gram-negative organisms: the evolution of P-lactamase George A. Jacoby uch of the antibiotic b-Lactamascs are the major defense used point (PI); or .by gcnctic criteria, resistance in bacteria by bacteria to overcome the effects of such as inducrbrlity and location penicillins, cephalosporins and related is encoded by plasof their genes on plasmids or mids that can package multiple b-lactam antibiotics. In the antibiotic era, on the bacterial chromosome. antibiotic-resistance or viruthe enzymes have evolved to become more Several schemes have been pro lence determinants and can prevalent, to appear in new hosts, to be posed to classify B-lactamases. facilitate their transfer beexprcsscd at higher Icvcls, to be acquired One current scheme (K. Bush, tween organisms. Plasmids and by plasmids and to change catalytic G.A. Jacoby and A.A. Medeiros, B-lactamascs have been around properties to incrcasc affinity for what submitted) allocates the enzymes for a long time. Conjugative were meant to be nonhydrolysablc into 11 groups on the basis plasmids, free from antibioticsubstrates or to reduce affinity of their functional properties resistance determinants, have for b-lactamase inhibitors. (Table 1). Another classifibeen recovered from bacteria cation, initiated by Ambler’, preserved from the pre-antidivides the enzymes into four biotic era’, and soil organisms structural classes (A-D) on making P-lactamase have been the basis of amino acid simirevived from plant specimens stored in the 17th larities. Class B enzymes are metallo-B-lactamases, century2. What the enzyme was doing then is not while enzymes of classes A, C and D are members known, but B-lactamases are now the major mcchanof the superfamily of penicilloyl serine transferases ism of resistance to penicillins, ccphalosporins and that includes penicillin-binding proteins involved in other B-lactam antibiotics (see p. 421 for a glossary of the terminal steps of bacterial-cell-wall synthesis4. antibiotics), and the clinical use of these drugs has Some pairs of enzymes, such as TEM-1 and TIN-2 provided powerful selection for the evolution of this or I’%-1 and PSE-4 (see Box 1 for an explanation enzyme. of the nomenclature), differ in a single amino acid that changes the pl, but most B-lactamascs vary at P-Lactamase classification many residues. Certain amino acids that contribute There are many B-lactamascs. They can be distinguished to the structure of the active site are strongly conby biochemical criteria, such as substrate spectrum, served, while other regions have diverged to prokinetic properties and response to inhibitors; by physiduce the functional variety that is shown in Table 1. Sites in the enzyme that are critical for differential cal properties, such as molecular size and isoelectric
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OYh6 842X/04/$07.00
aminoglycoside-resistant isolates. The most common single and combination resistance mechanisms occurred in all of th e geographical regions surveyed, while the less common mechanisms tended to occur in individual regions or only in a few regions. Rates of resistance observed using NCCLS disk-diffusion criteria tended to agree well with those calculated from the phenotype of the resistance mechanisms observed, especially allowing for the occurrence of low-level permeability changes.
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ECOLOGY
References 1 Davies, J. and Smith, D.1. (1978) A~zrzrc. Rev. M~crohiol. 32, 469-,518 2 Price, K.E. eta/. (1981) /. Antimicroh. Chemother. 8 (Suppl. A), 89-105 3 Shim& K. et al. (198.5) Antimicro/]. Agents Chemother. 28, 282-288 4 Shaw, K.J. et al. (1993) Microbial. Rev. 57, 138-163
5 European Studv Group on Antibiotic Resistance (ESGAR) (1987) Eur. 1. C/in.M&h/. 6, 378-385 6 Dornbusch, K. etal. (1990) _/.Antimicrob. Chemother. 26, 131-144
Epidemiology of tetracycline-resistance determinants Marilyn C. Roberts
T
Resistance to tetracycline is generally due etracyclines are broadare readily transferred and have tither to energy-dependent efflux of few physiological barriers, is spectrum antimicrobials tetracycline or to protection of the that are active against a widely distributed’,‘,“. bacterial ribosomcs from the action of wide range of Gram-positive and Gram-negative bacteria, tetracycline. The genes that encode this Classification of Tet resistance arc normally acquired via cell-wall-free mycoplasmas, determinants transferable plasmids and/or transposons. chlamydiae, rickcttsiae and Mendcz et al.’ first examined protozoan parasites (see p. 421 Tct determinants have been found in a the genetic heterogeneity of wide range of Gram-positive and for a glossary of antibiotics)‘. tetracycline-resistance dctermiGram-negative bacteria and have Tetracyclines have been used for nants from Enterobactcriaceac reduced the effectiveness of therapy therapy in man, animals and and Pseudomonadaceae plascertain crops, as well as for with tetracycline. mids using restriction-enzyme food additives for growth proanalysis, DNA-DNA hybridizM.C. Roberts is in the Dept of Pathbiology SC-38, motion and therapy in animal ation and expression of resistSchool of Public Health and Cowzmrrnrty Medicine, husbandry’. Because of their ance to tetracyclines. DNAUniversity of Washington, Seattle, WA 982 95, USA. broad spectrum of activity, DNA hybridization using the relative safety and low cost,.tetracyclines have been structural genes as probes is now the standard method widely used throughout the world and are the second to distinguish different genes’,13*‘x. IJsing this method, most commonly used antibiotic after penicillins’. some of the resistance determinants have up to 76% Over the past 20 years, bacterial resistance to tetraDNA sequence identity but, under stringent hybridcyclines has limited their use’-4. ‘The diversity of resistization conditions, they do not crosshybridize and are ance to tetracycline was first examined in Gram-negative thus considered to be separate gencs2. enteric bacterias. A decade later, 16 tetracycline-resistance (Tet) determinants and three oxytetracyclineMechanisms of resistance to tetracycline resistance (Otr) determinants, first found in oxytetraResistance to tetracyclines is primarily due to acquisition cycline-producing Streptomyces, have been described of Tet determinants rather than to mutation of existand characterized’-4,h-1’ , with new Tet determinants ing chromosomal gcnes1-5. An exception is Neisseria being identified continually’3. Of these determinants, gonorrhoeae, where the additive effects of mutations 1.3 are frequently associated with plasmids, while others to chromosomal genes tet, mtr and penB can result are on the chromosome (Table 3 ). The Tet M and Tet in clinical resistance to tetracycline therapy”. FurQ determinants are associated with conjugative thermore, Escherichia coli has a chromosomal tetraelements’4,‘5. Wide variation in the distribution of Tct cyclinc-resistance efflux system that is associated with determinants may be related, in part, to the ease with the mar IOCUS’~.However, N. gonorrhoeae has also which particular Tet determinants are transferred acquired the Tct IM determinant”, and I:. coli has a between various isolates and genera. For example, variety of Tet determinants (Table 2). the Tet E determinant is on a large nonconjugative There are three different mechanisms of resistance plasmidlh, while the Tct M dctcrminant, which is to tetracycline: (I) energy-dependent efflux of tetracycline by proteins inserted into the cytoplasmic usually associated with conjugative transposons that
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Chromosome
inactivation
of tctracyclincZ2 mechanisms of drug resistance have been found in oxytetracyclineproducing St~eptonzyces~‘~‘~, which is consistent with the hypothesis that antibiotic-producing soil microorganisms could be the ancestral source of some of the Tet determinants?‘. The line between antibiotic producers and other bacteria has been blurred with the identification of Tet K and ‘I’et 1, determinants in Stveptomyces, and the Strcptomyces Otr determinants in Mycobacteriwn spccies2” (Table 4). (Table
Tet A-E Tet X TetG,H Tet K Tet L Tet M (rare) Tet 0 Tet P _
Tet Tet Tet Tet
Tet S --
Otr A-C
Efflux of tetracycline Different Gram-positive and Gram-negative genera have Tct determinants that confer resistance to tctracyclinc by removing tetracyclines from the cell using an energy-dependent efflux mechanism (Table 3). The approximately 46 kDa membrane-bound efflux pro-
membrane’U, (2) protection of the bacterial ribosomc from the action of tetracycline”, and and (3) enzymatic alteration
Table 1. Location of the tetracycline-resistance (Tet) determinants” --. .Plasmid
ECOLOGY
Tet B (rare)
Kb L (rare) M 0
Tet Q -
“Based on information from Refs l-13,16,17,22,32,34-36. %an be associated with an integrated plasmid.
3). The same
teins consist of 12 hydrophobic
Enzymatic alteration of tetr.acycline The only example of enzymatic-mediated resistance to tetracycline is the Tct X determinant that has been cloned from anaerobic Bacteroidesz2. However, the enzyme requires oxygen, does not function in the natural host and has not been found outside Racteroides”. Because of its questionable role in conferring rcsistancc to tetracycline in Nature, it is not considered further here.
Table 2. Distribution of tetracycline-resistance (Tet) determinants among Gram-negative bacteria” .~. _-. Ribosomal protection and/or efflux _~...
Effluxb .~. Bacterium
Tet determinant
Bacterium
Tet determinant
Actinobacillus Aeromonas Citrobacter fdwardsiella Enterobacter Escherichia Klebsiella Moraxella Pasteurella Plesiomonas Proteus Pseudomonas Salmonella Serratia Shigella Yersinia Vibrio
Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet
Bacteroides” Campylobacter fikenella Fusobacterium” Haemophilus Kingella Neisseria Prevotella” Veil/one//a’
Tet Tet Tet Tet Tet Tet Tet Tet Tet
-
B A, A, A, B, A, A, B B, A, A, A, A, A, A, B A,
B, B, D C, B, D
D, E C, D D C. D, E
D, H B, D B, C C B, C, D, E B, C B, C, D
mcmbranc-spanning
regions separated by shorter hydrophilic Sequences-J”. ‘l’he efflux proteins exchange a proton for a tetracycline-cation complex, and are antiportcr systcmP. The Tet A-E and Tet G-I I determinants all contain a structural and a repressor gene that are expressed in oppositc directions from overlapping operator regions4,26. Both repressor and structural genes have significant scquencc similarity with each other, suggesting that the genes may derive from a common ancestor4J”,?6. The cfflux proteins have amino acid and protein structure similarities with other cfflux proteins involved in multiple-drug rcsistancc and resistance to quatcrnary ammonium, chloramphcnicol and quinolone”‘,26. The Gram-positive tetK and telI, genes encoding tetracycline-efflux proteins are regulated by mRNA attenuation in a similar way to that described for Grampositive eym genes encoding rRSA methylases, and cat genes encoding chloramphenicol acetyltransferases’:‘. The Clostridium ‘l’ct P determinant consists of two overlapping resistance-encoding gent?: tetA(P) encodes an efflux protein, while tetB(P) encodes a ribosomal-protection proteinX. The S~reptomyccs Otr B dctcrminant encodes an cfflux protcin12 that is more closely related to TetA(P) than to either the Tct(K) or the Tct( L) protein?.
_
.
M, Q, X 0 M M B, M M M Q M
B, C, D, E, G
_..__
JBased on information from Refs l-7,9,13,15-17,22,32-34,40. “Ribosomal-protection-encoding genes have not yet been found in enteric genera and, when these genes are cloned into fscherichia co/i, the level of resistance to tetracycline conferred is relatively Iowl’. “Anaerobic species.
Proteins that protect ribosomes from the action of tetracycline both in vim and in vitro form another mcchanism of resistance to tetracycline”. Tct M, 0, S, B(P) and Q, and Otr A detcrminants encode 72.5 kDa cytoplasmic proteins that have amino acid sequencc similarity to the elongation factor Tu (EF-Tu) and EF-G (Ref. 28). The Tet(,M), Tet(O) and Tct(S) proteins have more than 70% sequence identity to each othcr2; the Otr(A) and TctB(1’) proteins also have a high dcgrcc of sequence identity with each otherx, and the ret(Q) protein, with 45% amino acid sequence identity to the other groups, forms a third grouph. The Tct M determinant is thought to be regulated by mRNA attenuation”, while the Tet 0 determinant is not regulated”‘. Because these proteins are similar in amino acid scquencc to EF-Tu and EF-G (Ref. 28), we have suggested that they might be tetracycline-resistant EFs or might block the binding of tetracycline to the ril~osomc’x. Burdctt has shown that the Tet(M) protein
EVOLUTION
has ribosome-dependent G’I’I’ase activity”, while Manavathu et ~1.~” have suggested that these proteins might ‘modify ribosomal proteins or rRNA’, perhaps by phosphorylation or acetylation. However, their resuits and those of Burdett” show that ribosomes from cells carrying the Tet(M) or Tct(0) proteins are fully sensitive to tetracycline, making it unlikely that there is a transient unstable modification of the sort suggested by Manavathu. More rcccntly, Burdctt3’ has isolated an E. co/i mutant in which the ‘I’et M determinant does not confer resistance to tctracyclinc, and suggested that tRNA-modification activity is ncccssary for Tet(iM)mediated resistance to tetracycline. Location and dissemination of let determinants ‘The majority of ‘I‘et determinants are found in a variety of bacteria isolated from man, animals and the cnvironmcnt’. These data support the hypothesis that gene exchange occurs widely throughout bacteria and between ecosystems. ‘l‘hc facultative Gramnegative Tct determinants are associated primarily with transposons inserted into a diverse group of conThe Tet B determinant has jugative plasmids 1.5,7,13. the widest host range among the Gram-negative species22x23, while the genus Vihrio hosts the largest number of different Tet determinants (Table 2). The genes encoding ribosomal-protection proteins have not been found in the majority of facultative Gramnegative species. A few isolates of Haemophilus species and all highly tetracycline-resistant Moruxella (Brunhamellu) catarrhalis have a chromosomal Tet B The Tet B determinant is not determinant3,32J3. conjugative in these isolates, but can be moved by transformation.‘*J1.33. The Tct E determinant differs from the Tet A-D determinants by its association with large plasmids that are neither mobile or conjugativelh, and has rcccntly been associated with the chromosomei4, which may explain its limited distribution and its predominance in aquatic environments’Jh (Table 2). Tet G (Ref. 9) and Tet H determinants’ have only been described recently, and their distribution has not been explored fully. The Gram-positive Tet K and Tet L determinants arc found on small transmissible plasmids that can become integrated into the chromosome of staphylococci 35 or are foulid independently on the chromosome of Bacillus subtilisJh. These determinants are widely distributed among Gram-positive species associated with man, animals and the soi13.2- (Table 4), and have been found recently in rapidly growing Mycobacterium and Streptomyces species24 (Table 4), as well as in Gramnegative Haemophilus aphrophihs and Veillonella Paruth strains, all of which were isolated from Patients3’ (Table 2). This is the first documented acquisition of an antibiotic-resistance determinant in Mvcobacterium, and it suggests that gene exchange’between tetracycline-resistant Gram-positive bacteria and both Mycohacterium and Streptomyces has occurred. Recently, the Otr determinants from industrial Streptomyces have been found in clinical Mycobacterium and StreptomycesL4, and surveys are required to determine their distribution in other genera.
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Table 3. Classification of tetracycline-resistance (Tet) determinants according to their mechanism of resistance= -_. _ Ribosomal Enzymatic Unknown Efflux Tet Tet Tet Tet Tet Otr
A-E G-H K L A(P) B
Tet Tet Tet Tet Tet Otr
M 0 S Q B(P) A
Tet X
“Based on information from Refs 2-13,22. anisms involved, see the text.
Otr C
For details of the mech-
The ribosomal-protection Tet M and Tct Q determinants are often associated with conjugative chromosomal elements, which code for their own transfer’4,‘S,3R. Thcsc clcments have been shown to transfer mobilizable plasmids to other bacterial isolates and species”. Recently, genes in the Tet(Q) operon have been identified that mediate excision and circularization of discrete nonadjacent scgmcnts of chromosomal DNA in Bacteroides, are important for self-transfer and the effect of which is enhanced by pre-exposure to tetracycline w. Pre-exposure to tetracycline can also enhance transfer of the Tet M determinant. The Tct M dcterminant is freely transferred between Gram-positive (donor or recipient) and Gram-negative (donor or recipient)
Table 4. Distribution of tetracycline-resistance (Tet) determinants among Gram-positive type bacteriaa Bacterium
Tet determinant
Actinomyces
Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet Tet
Aerococcus fkJCi//US
Clos tridium b Corynebacterium Enterococcus Eubacterium Gardnerella Gemella Lactobacillus Listeria Mobiluncus b Mycobacteriumc Mycoplasmad PeptostreptococcusD Staphylococcus Streptococcus Streptomyces’ lJreaplasmaa
L M, 0 K, L K, L, M K, L, K, M M M 0 K, L, 0 K, L, M K, L, K, L, K, L, K, L, M
M, P M, 0
M, S Otr A, B M, 0 M, 0 M. 0 Otr A, B, C
“Based on information from Refs 2-4,8.10-12,24, 27,36,41. OAnaerobic species. ‘Acid-fast bacteria. bCell-wall-free bacteria with a Gram-positive metabolism. eMulticellular bacteria.
EVOLUTION
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ECOLOGY
specieG4”. It has a broad host range and is found in all groups of bacteria that have been examined, except in facultative enteric bacteria (Tables 2 and 4), and can be transferred between bacteria isolated from all different types of ecosystems and a large number of different species3. This supports the hypothesis that the Tet M determinants can cross most bacterial biochemical, genetic and morphological barriers. The Tet 0 determinant is not associated with conjugative elements and is less widely distributed than the ‘I’ct M determinant, but is also found in both Gram-positive and Gramnegative genera G” (Tables 2 and 4). My group has hypothesized that commcnsal bacteria could be reservoirs for Tet determinants’,“‘. We have examined bacteria from the urogenital tracts of females who had not taken any antibiotics for 2 weeks prcviously4’. Of the viridans streptococci, 82% hybridized with at least one Tet dctcrminant, and of the 18 that did not hybridize, only seven (7%) were susceptible to tetracycline. Of the 71 I-‘eptostreptococctis species, 79% hybridized with at least one Tet determinant, and of the 1.5 that did not hybridize, only three (4%) were susceptible to tetracycline. Similarly, only 25% of the Lactohacillus species were susceptible to tetracycline. The most common Tct determinant found was Tet M, and it could be transferred between bacteria in mating experiments4’. In contrast to the commensal species, only 46% of the opportunistic Streptococczfs agulactiae were resistant to tetracycline. ,More recently, we have examined the dental flora of normal controls and found 2-20X of the aerobically grown flora and S-56’% of the anaerobically grown flora to be resistant to tetracycline. The majority of the Tct determinants found were transferable. The discovery of the same Tet determinants in both aerobic and anaerobic commensal species and their ability to act as donors of the tetracycline-resistance-encoding genes supports the hypothesis that commensal bacteria can act as reservoirs for these genes and arc potential donors to pathogens entering the ecosystem. Conclusion In the past ten years, use of tetracycline has been rcduccd as the number of tetracycline-resistant genera and species has increasedlm4. Resistance to tetracycline is due to genes encoding either efflux or ribosomalprotection proteins, while the third possible mcchanism (enzymatic inactivation of the antibiotic) is not thought to be important in conferring resistance in Nature. Both efflux and ribosomal-protection mechanisms are found in the oxytetracycline-producing of resistance Streptomyces1’a12, while the mechanism encoded by the Streptomyces otrC gene is unknown. Most bacteria, representing nearly all of the evolutionary branches, including Mycobacterium and Streptobut myces, now carry one or more Tet determinants, the distribution of individual Tet determinants varies. However, the number of ancestral genes from which the genes encoding efflux-type resistance or ribosomalprotection-type resistance derive is still not clear4~x.2”. Examination of the DNA and protein sequences of highly related determinants, such as Tet M and Tet 0,
or the Tet A-H determinants, suggests that divergence has occurred. The Otr determinants have the same mechanisms of resistance to tetracycline as do the ‘I‘ct determinants, have sequence similarity with these genes and are associated with the oxytetracyclinc-biosynthetic pathways in Streptomyces. Based on these data, it is tempting to speculate that the Otr and Tet determinants have a common ancestor, but other possibilities cannot be ruled out2.‘. ‘ Continued widespread use of tetracyclines in human and animal medicine as well as on crops exerts sclcctivc pressure on bacteria, which will result in larger numbers of tetracycline-resistant isolates, new species acquiring tet genes and the reduced effectiveness of all applications of tetracycline therapy. Recently, modified tetracyclincs that arc not affected by efflux and ribosomal-protection mechanisms of resistance have been madc43-4’. These new derivatives or other related compounds, if approved for use, could prolong the usefulness of tetracyclincs in therapy. Acknowledgement M.C.R. was supported by grant X24136 from the National Institute of Health. References 1 Levy, S.B. (1992) ‘I’/&A~~tihiot~c Pm~dox, How Miracle Drrqs nye DestrojGzg the M~yde, Plenum Press 2 Levy,S.B.(1989) /. Anlinzicrob. Chernother. 24, l-3 3 Roberts, MC. (1989)Gerx TY~IS~CY in the Em~irowmnt (Levy, S.B. and Miller, R.V., cds), pp. 347-37.5, M&raw-Hill 4 Chopa, I., Hawkcy, P.M. and Hinton, .Ll. (1992) 1. Antinricro6. Chemother. 29, 245-277 5 Mendcz, B., Tachibana, C. and Levy, S.B. (1980) I’hstnici 3,99-10X 6 Sikolich! Ml’., Shoemaker, N.B. and Salyers, A.A. ( 1992) Anfimicrob. Agerzts Chemother. 36, 1005-l 012 7 Hansen, L&l. et al. (1993) Aufimicroh. Agents Chernother. 37, 2699-2705 8 Sloan, J.. hlcMurry, L.M. and Lyras, D. (1994) Mol. :Micyohiol. 11,403-415 9 Zhao, J. and Aoki, T. (1992) Microhrol/~w~Iuw~.36, 1051-1060 10 Charperntier, E., Gerbaud, G. and Courvalin, I’. (1993) Gene 131,27-34 Agerzfs 11 Dittrich, W. and Schrempf, H. (1992) Antinmnb. Cbemotbe~. 36, 1119-l 124 12 Butler, MJ. etal. (1989) Mol. Gen. Genet. 215,231-238 13 Jones, C.S., Osborne, U.J. and Stat&y, J. (1992) M. Cell. Probes 6, 3 13-3 17 14 Clcwell, D.B. rl al. (I 988) 1. Rmteriol. 170, 3046-3052 15 Salyers, AA. and Shoemaker, K.B. (1992) Erru. 1. C/in. Micyobiol. Infect. DIS. 11, 1032-l 038 16 Sorum, H., Roberts, MC. and Cross, J.H. (1992) Antbnicrob. Agents Cbemother. 36, 61 l-615 Agents Chender. 30, 17 Morse, S.A. et ul. (1986) Admicroh. 664-670 18 Levy, S.B. et al. (19891A~~fmicroh. Agents Chrmother. 33, 1373-1374 19 George, AM and Levy, S.B. (1983) 1. Racteriol. 155, 531-540 20 Levy, S.R.(1992) Anfmicyob. Ageents Chemother. 36, 695-703 21 Burdett, V. (1991)]. Biol. Chem. 266,2872-2877 22 Spccr, B.S., Bcdqk, L. and Salyers, A.A. (1991) 1. Hocferd. 173, 176-183 23 Bcnvcnistc, R. and Davies, J. (1973) Proc. lVaf/ Ad. Sci. liSA 172,3628-3632 24 Pang, Y. et al. ( 1994) Autinzrcroh. Agents Chemothcr. 38, 1408-1412
EVOLUTION
2.j Yamaguchi, A. et al. ( 1992) /. Bid. Cbem. 267, 7490-7498 5, 895-900 26 Sheridan, R.P. and Chopra, I. (199 1) !ziiol.Microbid. 27 Schwarz, S., Cardoso, M. and Wcgcner, H.C. (1992) Antimicroh. Agmts Chemther. 36, .580-SSY 28 Sanchez-Pcscador, R. et al. (1988) Ntrcle~cAcids Kes. 16. 1218 29 51, Y.A., He, I’. and C~cwcll, I).B. (1992) Antimwoh. Agents Chemotber. 36, 769-778 Agents Chemother. 30 Manavathu. E.K. et al. ( 1990) Antirnicrob. 34,71-77 31
hrdett, V. (1993)]. Rncterid.
175, 7209-7215 32 .Ilarshall, 1%. et a/. (1984) 1. Infect. Dis. 149, 1028-1029 33 Roberts, M.C. et a/. (1991) Antrm~croh. Agents Chernother. 3.5, 2453-2455 34 Lee, C., Langlois, B.E. and Dawson, K.I.. (I 993) Appl. ~wiron. h4icr&d. 59, 1467-1472 3.5 Gllcspie, .v.T., h4ay, J.W. and Skurray, R. (lY86)j. Gen.
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iC!~crobiol.132, 1723-1728 36 Sakaguchi, R. and Shishido, K. (1988) Biochim. Hmphys. Acta 949, 9-si 37 Pang, Y., Bosch, T. and Kobcrts, M.C. ~%lol.Cell. Probes (in press) 38 Le Bougucncc, C., dc Ccspedes, G. and Horaud, T. (1990) /. Bacterial. 172, 727-734 39 Naglich, J.G. and hndrews, R.E., Jr (1988) P/amid 20, 113-126 40 Srevens, 01. et al. (1992) 1. Racteriol. 174,2935-2942 41 Kuberts, MC. and Lansciardi, J. (1990) Antimlcrob. ilp~ts Chemother. 34, 1836-I 838 42 Rohcrts, .Ll.C. and Hillier, 51.. (1990) Autimicrob. Agents Chemotber. 34, 261-264 Ayeuts Chemother. 37, 43 Testa, R.T. et nl. (1993) Antimicrob. 2270-2277 44 Nelson, I1.L. et ~71. (1993) 1. Med. Chem. 36, 370-377 45 Tymiak, A.h. et al. (1993)_/. Org. Chem. S&535-,537
Extrachromosomal resistance in Gram-negative organisms: the evolution of P-lactamase George A. Jacoby uch of the antibiotic b-Lactamascs are the major defense used point (PI); or .by gcnctic criteria, resistance in bacteria by bacteria to overcome the effects of such as inducrbrlity and location penicillins, cephalosporins and related is encoded by plasof their genes on plasmids or mids that can package multiple b-lactam antibiotics. In the antibiotic era, on the bacterial chromosome. antibiotic-resistance or viruthe enzymes have evolved to become more Several schemes have been pro lence determinants and can prevalent, to appear in new hosts, to be posed to classify B-lactamases. facilitate their transfer beexprcsscd at higher Icvcls, to be acquired One current scheme (K. Bush, tween organisms. Plasmids and by plasmids and to change catalytic G.A. Jacoby and A.A. Medeiros, B-lactamascs have been around properties to incrcasc affinity for what submitted) allocates the enzymes for a long time. Conjugative were meant to be nonhydrolysablc into 11 groups on the basis plasmids, free from antibioticsubstrates or to reduce affinity of their functional properties resistance determinants, have for b-lactamase inhibitors. (Table 1). Another classifibeen recovered from bacteria cation, initiated by Ambler’, preserved from the pre-antidivides the enzymes into four biotic era’, and soil organisms structural classes (A-D) on making P-lactamase have been the basis of amino acid simirevived from plant specimens stored in the 17th larities. Class B enzymes are metallo-B-lactamases, century2. What the enzyme was doing then is not while enzymes of classes A, C and D are members known, but B-lactamases are now the major mcchanof the superfamily of penicilloyl serine transferases ism of resistance to penicillins, ccphalosporins and that includes penicillin-binding proteins involved in other B-lactam antibiotics (see p. 421 for a glossary of the terminal steps of bacterial-cell-wall synthesis4. antibiotics), and the clinical use of these drugs has Some pairs of enzymes, such as TEM-1 and TIN-2 provided powerful selection for the evolution of this or I’%-1 and PSE-4 (see Box 1 for an explanation enzyme. of the nomenclature), differ in a single amino acid that changes the pl, but most B-lactamascs vary at P-Lactamase classification many residues. Certain amino acids that contribute There are many B-lactamascs. They can be distinguished to the structure of the active site are strongly conby biochemical criteria, such as substrate spectrum, served, while other regions have diverged to prokinetic properties and response to inhibitors; by physiduce the functional variety that is shown in Table 1. Sites in the enzyme that are critical for differential cal properties, such as molecular size and isoelectric
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