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Old dogs that learn new tricks: Modified antimicrobial agents that escape pre-existing resistance mechanisms
ARTICLE IN PRESS
International Journal of Medical Microbiology 296 (2006) S2, 45–49 www.elsevier.de/ijmm
Old dogs that learn new tricks: Modified antimicrobial agents that escape pre-existing resistance mechanisms Stefan Schwarz, Corinna Kehrenberg Institute for Animal Breeding, Federal Agricultural Research Centre (FAL), Ho¨ltystr. 10, D-31535 Neustadt-Mariensee, Germany
Abstract Increasing numbers of resistant bacteria and decreasing numbers of efficient antimicrobial agents demand for the development of new highly potent antimicrobial agents. The two main directions for the development of new antimicrobial agents are either the development of completely novel antimicrobial agents, or the modification of already existing antimicrobial molecules. The second direction proved to be a more successful and cost-efficient way of generating new antimicrobial agents. For this, chemical modifications have been included which render the antimicrobial agents insensitive to prevalent bacterial resistance mechanisms. However, detailed knowledge of the resistance mechanisms is indispensable. Molecular analysis of resistance mechanisms provides the required data to identify critical target structures. Furthermore, modification of antimicrobial agents makes it possible to escape known resistance mechanisms. In this report, two examples for such successful developments, one from human medicine (telithromycin) and the other from veterinary medicine (florfenicol), are presented. r 2006 Elsevier GmbH. All rights reserved. Keywords: New antibiotics; Resistance mechanisms; Target structures
Resistance development and the need for new antimicrobial agents For most antimicrobial agents currently used in human and veterinary medicine, there is a tight time coincidence between the introduction of a certain antimicrobial agent into clinical use and the occurrence of first resistant target bacteria (Table 1). This short time span of usually not more than 3 years underlines the enormous flexibility of bacteria to adapt efficiently to new living conditions, such as survival in the presence of antimicrobial agents. In this regard, bacteria have developed resistance not only against naturally occurring antimicrobial agents produced by soil bacteria or Corresponding author. Tel.: +49 5034 871 241; fax: +49 5034 871 246. E-mail address: [email protected] (S. Schwarz).
1438-4221/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2006.01.061
fungi (e.g. penicillins, tetracyclines or aminoglycosides), but also against completely synthetic substances, such as fluoroquinolones, trimethoprim or sulfonamides. The location of many resistance genes on mobile genetic elements, such as plasmids, transposons and gene cassettes, facilitated their dissemination by horizontal gene transfer across species and genus borders. Although initially only present in the bacteria in which they were developed, many of the resistance genes known to date are found in a wide variety of bacteria. The selection of resistant bacteria is ‘‘an inevitable, Darwinian consequence of antibiotic usage’’ (Livermore, 2004). It has become obvious that virtually every antimicrobial agent can select for resistant bacteria. However, the frequency of selecting resistant strains varies depending on factors, such as the ‘‘regimens and extent of use of the respective antimicrobial agent’’ and the ‘‘effectiveness of infection control’’ (Livermore,
ARTICLE IN PRESS 46
S. Schwarz, C. Kehrenberg / International Journal of Medical Microbiology 296 (2006) S2, 45–49
Table 1. Time coincidence between the discovery/production of selected antimicrobial agents, their introduction into clinical use and the occurrence of resistant bacteria (modified from Schwarz and Chaslus-Dancla, 2001; Davies, 1997) Antimicrobial agent
Discovery/production
Introduction into clinical use
Occurrence of resistant bacteria
Penicillin Streptomycin Tetracycline Erythromycin Vancomycin Gentamicin Fluoroquinolones
1940 1944 1948 1952 1956 1963 1978
1943 1947 1952 1955 1972 1967 1982
1940 1947, 1956 1956 1956 1987 1970 1985
2004). Moreover, microbial factors such as the availability and transferability of resistance genes and their functional activity in bacteria other than their original hosts play a relevant role in this process. As a consequence, the use of every new antimicrobial agent will be followed sooner or later by the development or acquisition of resistance in bacteria. This, however, renders any antimicrobial agent newly introduced to the market less profitable over time as compared to drugs for the treatment of heart disease, pulmonary disease, mental disorders, cancer or hypertension (Clarke, 2003; Powers, 2004). Although there is a large need for new antimicrobial drugs due to increasing numbers of resistant and multiresistant bacterial pathogens in both, human and veterinary medicine, many pharmaceutical companies have either diminished or even stopped their research efforts in antimicrobial drug discovery whereas a smaller number of companies decided to persist in this field (Bush, 2004; Projan and Shlaes, 2004). As a result, only a comparatively small number of new antimicrobial agents have been approved during the last decade with most of these new agents, such as the oxazolidinone linezolid (2000), the cyclic lipopeptide daptomycin (2003) or the ketolide telithromycin (2001), being licensed exclusively for use in human medicine. In addition, few drugs have also been licensed for veterinary use only; these include florfenicol (1995), the cephalosporins ceftiofur and cefquinome (1995), the fluoroquinolones dano- and marbofloxacin (1996), difloxacin (1997), iba- and orbifloxacin (2000), the lincosamide pirlimycin (2001) and the triamilide tulathromycin (2003).
New antimicrobial agents in human and veterinary medicine The two main directions for the development of new antimicrobial agents are: (1) the development of completely novel antimicrobial agents, or (2) the
modification of already existing antimicrobial molecules.
Novel antimicrobial agents or classes of antimicrobial agents The analysis of an increasing number of whole genome sequences of bacterial pathogens has led to the discovery of a number of new potential targets (Holzgrabe, 2004). The current approaches for the development of novel antimicrobial agents that act on these targets have recently been summarized by Mascaretti (2003) and Walsh (2003). Many of the novel targets are enzymes which catalyze essential steps in bacterial cell metabolism. Thus, new antimicrobial agents might interfere with either protein biosynthesis by inhibiting the enzymes peptide deformylase or methionyl and tyrosyl tRNA synthase, or with peptidoglycan synthesis by inhibiting the enzymes diaminopimelate transferase or UDP-N-acetyl-glucosamine reductase. Other approaches include the development of inhibitors of fatty acid biosynthesis, lipid A biosynthesis, two-component regulatory systems or pilus assembly (Mascaretti, 2003; Walsh, 2003). These candidates for novel antimicrobial agents are currently at the developmental stage and represent members of new rather than existing classes of antimicrobial agents (Mascaretti, 2003). One novel agent, the oxazolidinone linezolid, has been approved in 2000. Although it acts on an old target, namely the ribosome, its mode of action is new. Linezolid prevents the formation of the initiation complex composed of the 30S ribosomal subunit, the mRNA and the N-formylmethionine-tRNA and thus inhibits protein biosynthesis at a very early stage (Mascaretti, 2003; Walsh, 2003; Holzgrabe, 2004). However, the development of such novel antimicrobial agents is a cost-intensive and timeconsuming process with most of the drug candidates not surviving the clinical trials. If such a substance is finally approved for clinical use, there is also only a relatively short period of time (usually not more than 20 years
ARTICLE IN PRESS S. Schwarz, C. Kehrenberg / International Journal of Medical Microbiology 296 (2006) S2, 45–49
starting from approval of the chemical compound by the patent office) during which it is protected by patent.
New members of already existing classes of antimicrobial agents The chemical modification of members of already existing classes of antimicrobial agents proved to be a more cost-efficient and faster way of generating new antimicrobial agents. It has successfully been applied to virtually all classes of older antimicrobial agents, such as tetracyclines, macrolides or b-lactams, but also to some newer classes, such as the fluoroquinolones. Some modifications have been included in the parental substance to alter its pharmacological and pharmacokinetic properties, such as better tissue penetration, the extension of half-life time, or the improvement of the efficacy against specific groups of bacteria. However, any new molecule from an already existing class of antimicrobial agents might be a target for the preexisting class-specific resistance mechanisms. Many of the currently known resistance mechanisms are effective not only against a specific member of a certain class of antimicrobial agents, but also against many different members of the respective class. Hence, certain tetracycline exporters mediate the efflux of tetracycline, chlortetracycline, oxytetracycline, doxycycline and to a certain extent even minocycline whereas extended spectrum b-lactamases are able to hydrolyze a wide range of b-lactam antibiotics. As a consequence, indepth knowledge of the respective resistance mechanisms is an absolute pre-requisite to design new molecules which escape the known resistance mechanisms and thus also show activity against bacteria that carry the corresponding resistance genes.
Examples of new antimicrobial agents that escape class-specific resistance mechanisms Molecular analysis of resistance mechanisms provides the required data to identify critical target structures. Furthermore, modification of antimicrobial agents makes it possible to escape known resistance mechanisms. The two examples presented below – one from veterinary medicine and one from human medicine – describe how knowledge of specific resistance mechanisms has successfully been used to construct derivatives that are resistant to the respective bacterial resistance mechanisms.
Florfenicol Florfenicol is a fluorinated chloramphenicol derivative which has been licensed in Germany in 1995 and
47
2000, respectively, for the control of bovine and porcine respiratory tract infections due to Pasteurella multocida, Mannheimia haemolytica, Histophilus somni or Actinobacillus pleuropneumoniae. In 1994, chloramphenicol was banned from use in food-producing animals to protect the consumer from potential undesired sideeffects due to chloramphenicol residues in food animal carcasses. The florfenicol molecule has been modified at two positions: (1) the nitro group, which was considered to be responsible for the dose-unrelated aplastic anemia, was replaced by a sulfomethyl group at the 1-phenyl moiety, and (2) the C3 primary hydroxyl group was substituted for fluorine (Fig. 1) (Schwarz et al., 2004). The most prevalent chloramphenicol resistance mechanism in Gram-positive and Gram-negative bacteria is the enzymatic inactivation of chloramphenicol by chloramphenicol acetyltransferases (CATs). These enzymes catalyze the transfer of acetyl groups from a donor molecule (e.g. acetyl-CoA) to the hydroxyl group at C3. The replacement of this acceptor site by a fluor residue rendered florfenicol resistant to inactivation by CAT enzymes. As a consequence, chloramphenicol-resistant strains, in which resistance is exclusively based on the activity of a CAT, are susceptible to florfenicol (Schwarz et al., 2004). Recent data from specific monitoring programs to assess the susceptibility of target bacteria to florfenicol indicated that since the introduction of florfenicol into clinical veterinary use, the minimum inhibitory concentrations (MIC50, MIC90) to florfenicol remained stable for bovine as well as porcine target bacteria (Priebe and Schwarz, 2003; Kehrenberg et al., 2004; Wallmann et al., 2003, 2004). Moreover, virtually all target bacteria tested so far proved to be susceptible to florfenicol. However, it needs to be noted that florfenicol resistance due to specific exporters and other not further specified mechanisms has been identified in enteric bacteria and staphylococci (Schwarz et al., 2004). The future will show whether and – if so – when such florfenicol resistance genes will be transferred into target bacteria.
R1
O C CH R 3 HN CH CH CH2 R 2 OH
R1
R2
R3
- OH
= Cl2
Chloramphenicol
- NO2
Florfenicol
- SO2CH3 - F
=Cl2
Fig. 1. Structural relationships between chloramphenicol and florfenicol (modified from Schwarz et al., 2004).
ARTICLE IN PRESS 48
S. Schwarz, C. Kehrenberg / International Journal of Medical Microbiology 296 (2006) S2, 45–49
Telithromycin Telithromycin represents the first substance within the ketolides subclass of 14-membered macrolides. Ketolides are approved for the control of respiratory tract infections with Streptococcus pneumoniae being one of the main target bacteria. Telithromycin is an erythromycin derivative in which several substitutions have been introduced: (1) the L-cladinose at position C3 was replaced by a 3-keto group, and (2) a large N-substituted carbamate extension was introduced at positions C11–C12 of the macrolactone ring (Fig. 2). An excellent review on the biological and pharmacological properties of telithromycin has recently been published (Ackermann and Rodloff, 2003). Like erythromycin and other macrolides, telithromycin binds to two specific areas in the 23S rRNA, namely the domains II and V which are folded so that they form a binding pocket for these antibiotics (Ackermann and Rodloff, 2003). Dissociation kinetics showed that telithromycin binds approximately 10-fold stronger to the ribosome than erythromycin (Hansen et al., 1999) due to the carbamate extension at C11–C12 that mediates a direct contact between the nucleotides involved in drug binding, e.g. A752 in domain II as well as A2058, A2059 and G2505 in domain V. Previous work on macrolide resistance has shown that methylation of A2058 and/or A2059 by rRNA methylases is the most common resistance mechanism in staphylococci and streptococci (Roberts et al., 1999). The degree of methylation of these critical residues – mono- or dimethylation – has a large impact on the MIC for the different MLSB antibiotics. While 14membered macrolides (e.g. erythromycin, clarithromycin), but also lincosamides (e.g. lincomycin, clindamycin), cannot bind efficiently to even monomethylated ribosomes, telithromycin binds to monomethylated, but not to dimethylated ribosomes (Liu and Douthwaite, 2002). Erm(B) is the most prevalent rRNA methylase in streptococci and acts preferentially as a monomethylase in S. pneumoniae. This explains why telithromycin is active even against S. pneumoniae strains that constitu-
O
N
OR
HO
O
OH
N
O
O
O
O
O
N
O
O
N
O
O O N
O
O O
HO
tively express the Erm(B) methylase. Such strains were susceptible to telithromycin with low MIC50 and MIC90 values of 0.06 and 0.5 mg/ml, respectively, but highly resistant to erythromycin, clarithromycin and azithromycin with MIC50 and MIC90 values of 4128 mg/ml (Schmitz et al., 2002d). In staphylococci, the rRNA methylases Erm(A) and Erm(C) are the dominant ones and act in general as dimethylases. This explains why high level telithromycin resistance with MIC50 and MIC90 values of 4128 mg/ml is seen in staphylococci expressing the genes erm(A) and/or erm(C) constitutively (Schmitz et al., 2002c). Since telithromycin does not act as an inducer of erm(A) or erm(C) gene expression, staphylococcal strains that carry inducibly expressed erm(A) or erm(C) genes show low MICs which suggest that telithromycin is highly active against such strains (Schmitz et al., 2002c). However, in vitro selection studies proved that telithromycin can rapidly select for constitutive mutants that are resistant to all MLSB antibiotics including the ketolide telithromycin (Schmitz et al., 2002a, b).
Concluding remarks These examples show that detailed knowledge of resistance mechanisms can effectively be used to develop new derivatives of old drugs that overcome prevalent resistance mechanisms. However, any new selective pressure as imposed by the use of new antimicrobial agents represents a challenge for bacteria to develop/acquire new genes or mutations, which allow them to circumvent the inhibitory effects of these agents. This again results in a reduced efficacy of the new antimicrobial agents and a demand for the next generation of even more efficacious agents. There is no question that new antimicrobial agents are needed for both, human and veterinary medicine, and joint approaches of scientific research institutions and industry are necessary to identify new stable target structures in bacteria and to use them for the development of novel efficient antimicrobial agents. However, it is also necessary to bear in mind that the development of resistance in bacteria is an inevitable process. It is not possible to stop this process, but it is possible to slow it down by a judicious application of antimicrobial agents. In this regard, prudent use of antimicrobial agents in both, human and veterinary medicine, is an important requirement to slow down the speed of resistance development in bacteria and to retain the efficacy of the currently available agents as long as possible.
O
O
References O
Erythromycin
Telithromycin
N
O
Fig. 2. Structural relationships between erythromycin and telithromycin.
Ackermann, G., Rodloff, A.C., 2003. Drugs of the 21st century: telithromycin (HMR3647) – the first ketolide. J. Antimicrob. Chemother. 51, 497–511.
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Bush, K., 2004. Antibacterial drug discovery in the 21st century. Clin. Microbiol. Infect. 10 (Suppl. 4), 10–17. Clarke, T., 2003. Drug companies snub antibiotics as pipeline threatens to dry. Nature 442, 225. Davies, J.E., 1997. Origins, acquisition and dissemination of antibiotic resistance determinants. In: Chadwick, I., Goode, J. (Eds.), Antibiotic Resistance: Origins, Evolution, Selection, and Spread. Ciba Foundation Symposium, vol. 207. Wiley, Chichester, pp. 15–27. Hansen, L.H., Mauvais, P., Douthwaite, S., 1999. The macrolide–ketolide antibiotic binding site is formed by structures in domains II and V of 23S ribosomal RNA. Mol. Microbiol. 31, 623–631. Holzgrabe, U., 2004. Antibiotika-Entwicklung gestern und heute. Chemother. J. 13, 142–147. Kehrenberg, C., Mumme, J., Wallmann, J., Verspohl, J., Tegeler, R., Ku¨hn, T., Schwarz, S., 2004. Monitoring of florfenicol susceptibility among bovine and porcine respiratory tract pathogens collected in Germany during the years 2002 and 2003. J. Antimicrob. Chemother. 54, 572–574. Liu, M., Douthwaite, S., 2002. Activity of the ketolide telithromycin is refractory to Erm monomethylation of bacterial rRNA. Antimicrob. Agents Chemother. 46, 1629–1633. Livermore, D.M., 2004. The need for new antibiotics. Clin. Microbiol. Infect. 10 (Suppl. 4), 1–9. Mascaretti, O., 2003. Bacteria Versus Antibacterial Agents: an Integrated Approach. ASM Press, Washington, DC. Powers, J.H., 2004. Antimicrobial drug development – the past, the present and the future. Clin. Microbiol. Infect. 10 (Suppl. 4), 23–31. Priebe, S., Schwarz, S., 2003. In vitro activities of florfenicol against bovine and porcine respiratory tract pathogens. Antimicrob. Agents Chemother. 47, 2703–2705. Projan, S.J., Shlaes, D.M., 2004. Antibacterial drug discovery: is it all downhill from here? Clin. Microbiol. Infect. 10 (Suppl. 4), 18–22. Roberts, M.C., Sutcliffe, J., Courvalin, P., Jensen, L.B., Rood, J.I., Seppala, H., 1999. Nomenclature for macrolide and macrolide-lincosamide streptogramin B antibiotic resistance determinants. Antimicrob. Agents Chemother. 43, 2823–2830.
49
Schmitz, F.J., Petridou, J., Astfalk, N., Scheuring, S., Ko¨hrer, K., Schwarz, S., 2002a. Molecular analysis of constitutively expressed erm(C) genes selected in-vitro by incubation in the presence of the non-inducers quinupristin, telithromycin or ABT-773. Microb. Drug Resist. 8, 171–177. Schmitz, F.J., Petridou, J., Jagusch, H., Astfalk, N., Scheuring, S., Schwarz, S., 2002b. Molecular characterization of ketolide-resistant erm(A)-carrying Staphylococcus aureus isolates selected in vitro by telithromycin, ABT-773, quinupristin and clindamycin. J. Antimicrob. Chemother. 49, 611–617. Schmitz, F.J., Petridou, J., Milatovic, D., Verhoef, J., Fluit, A.C., Schwarz, S., 2002c. In vitro activity of new ketolides against macrolide-susceptible and macrolide-resistant Staphylococcus aureus isolates with defined resistance gene status. J. Antimicrob. Chemother. 49, 580–582. Schmitz, F.J., Schwarz, S., Milatovic, D., Verhoef, J., Fluit, A.C., 2002d. In vitro activities of the ketolides ABT-773 and telithromycin and of three macrolides against genetically characterized isolates of Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, and Moraxella catarrhalis. J. Antimicrob. Chemother. 50, 145–148. Schwarz, S., Chaslus-Dancla, E., 2001. Use of antimicrobials in veterinary medicine and mechanisms of resistance. Vet. Res. 32, 201–225. Schwarz, S., Kehrenberg, C., Doublet, B., Cloeckaert, A., 2004. Molecular basis of bacterial resistance to chloramphenicol and florfenicol. FEMS Microbiol. Rev. 28, 519–542. Wallmann, J., Schro¨ter, K., Wieler, L.H., Kroker, R., 2003. Antibiotikaempfindlichkeit ausgewa¨hlter pathogener Bakterien von erkrankten Lebensmittel liefernden Tieren in Deutschland: Ergebnisse aus der Modellstudie 2001 des nationalen Resistenzmonitorings. Tiera¨rztl. Praxis 31 (G), 122–131. Wallmann, J., Kaspar, H., Kroker, R., 2004. Pra¨valenzdaten zur Antibiotikaempfindlichkeit von bei Rindern und Schweinen isolierten bakteriellen Infektionserregern: Nationales BVL Resistenzmonitoring 2002/2003. Berl. Mu¨nch. Tiera¨rztl. Wochenschr. 117, 480–492. Walsh, C., 2003. Antibiotics: Actions, Origins, Resistance. ASM Press, Washington, DC.
International Journal of Medical Microbiology 296 (2006) S2, 45–49 www.elsevier.de/ijmm
Old dogs that learn new tricks: Modified antimicrobial agents that escape pre-existing resistance mechanisms Stefan Schwarz, Corinna Kehrenberg Institute for Animal Breeding, Federal Agricultural Research Centre (FAL), Ho¨ltystr. 10, D-31535 Neustadt-Mariensee, Germany
Abstract Increasing numbers of resistant bacteria and decreasing numbers of efficient antimicrobial agents demand for the development of new highly potent antimicrobial agents. The two main directions for the development of new antimicrobial agents are either the development of completely novel antimicrobial agents, or the modification of already existing antimicrobial molecules. The second direction proved to be a more successful and cost-efficient way of generating new antimicrobial agents. For this, chemical modifications have been included which render the antimicrobial agents insensitive to prevalent bacterial resistance mechanisms. However, detailed knowledge of the resistance mechanisms is indispensable. Molecular analysis of resistance mechanisms provides the required data to identify critical target structures. Furthermore, modification of antimicrobial agents makes it possible to escape known resistance mechanisms. In this report, two examples for such successful developments, one from human medicine (telithromycin) and the other from veterinary medicine (florfenicol), are presented. r 2006 Elsevier GmbH. All rights reserved. Keywords: New antibiotics; Resistance mechanisms; Target structures
Resistance development and the need for new antimicrobial agents For most antimicrobial agents currently used in human and veterinary medicine, there is a tight time coincidence between the introduction of a certain antimicrobial agent into clinical use and the occurrence of first resistant target bacteria (Table 1). This short time span of usually not more than 3 years underlines the enormous flexibility of bacteria to adapt efficiently to new living conditions, such as survival in the presence of antimicrobial agents. In this regard, bacteria have developed resistance not only against naturally occurring antimicrobial agents produced by soil bacteria or Corresponding author. Tel.: +49 5034 871 241; fax: +49 5034 871 246. E-mail address: [email protected] (S. Schwarz).
1438-4221/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijmm.2006.01.061
fungi (e.g. penicillins, tetracyclines or aminoglycosides), but also against completely synthetic substances, such as fluoroquinolones, trimethoprim or sulfonamides. The location of many resistance genes on mobile genetic elements, such as plasmids, transposons and gene cassettes, facilitated their dissemination by horizontal gene transfer across species and genus borders. Although initially only present in the bacteria in which they were developed, many of the resistance genes known to date are found in a wide variety of bacteria. The selection of resistant bacteria is ‘‘an inevitable, Darwinian consequence of antibiotic usage’’ (Livermore, 2004). It has become obvious that virtually every antimicrobial agent can select for resistant bacteria. However, the frequency of selecting resistant strains varies depending on factors, such as the ‘‘regimens and extent of use of the respective antimicrobial agent’’ and the ‘‘effectiveness of infection control’’ (Livermore,
ARTICLE IN PRESS 46
S. Schwarz, C. Kehrenberg / International Journal of Medical Microbiology 296 (2006) S2, 45–49
Table 1. Time coincidence between the discovery/production of selected antimicrobial agents, their introduction into clinical use and the occurrence of resistant bacteria (modified from Schwarz and Chaslus-Dancla, 2001; Davies, 1997) Antimicrobial agent
Discovery/production
Introduction into clinical use
Occurrence of resistant bacteria
Penicillin Streptomycin Tetracycline Erythromycin Vancomycin Gentamicin Fluoroquinolones
1940 1944 1948 1952 1956 1963 1978
1943 1947 1952 1955 1972 1967 1982
1940 1947, 1956 1956 1956 1987 1970 1985
2004). Moreover, microbial factors such as the availability and transferability of resistance genes and their functional activity in bacteria other than their original hosts play a relevant role in this process. As a consequence, the use of every new antimicrobial agent will be followed sooner or later by the development or acquisition of resistance in bacteria. This, however, renders any antimicrobial agent newly introduced to the market less profitable over time as compared to drugs for the treatment of heart disease, pulmonary disease, mental disorders, cancer or hypertension (Clarke, 2003; Powers, 2004). Although there is a large need for new antimicrobial drugs due to increasing numbers of resistant and multiresistant bacterial pathogens in both, human and veterinary medicine, many pharmaceutical companies have either diminished or even stopped their research efforts in antimicrobial drug discovery whereas a smaller number of companies decided to persist in this field (Bush, 2004; Projan and Shlaes, 2004). As a result, only a comparatively small number of new antimicrobial agents have been approved during the last decade with most of these new agents, such as the oxazolidinone linezolid (2000), the cyclic lipopeptide daptomycin (2003) or the ketolide telithromycin (2001), being licensed exclusively for use in human medicine. In addition, few drugs have also been licensed for veterinary use only; these include florfenicol (1995), the cephalosporins ceftiofur and cefquinome (1995), the fluoroquinolones dano- and marbofloxacin (1996), difloxacin (1997), iba- and orbifloxacin (2000), the lincosamide pirlimycin (2001) and the triamilide tulathromycin (2003).
New antimicrobial agents in human and veterinary medicine The two main directions for the development of new antimicrobial agents are: (1) the development of completely novel antimicrobial agents, or (2) the
modification of already existing antimicrobial molecules.
Novel antimicrobial agents or classes of antimicrobial agents The analysis of an increasing number of whole genome sequences of bacterial pathogens has led to the discovery of a number of new potential targets (Holzgrabe, 2004). The current approaches for the development of novel antimicrobial agents that act on these targets have recently been summarized by Mascaretti (2003) and Walsh (2003). Many of the novel targets are enzymes which catalyze essential steps in bacterial cell metabolism. Thus, new antimicrobial agents might interfere with either protein biosynthesis by inhibiting the enzymes peptide deformylase or methionyl and tyrosyl tRNA synthase, or with peptidoglycan synthesis by inhibiting the enzymes diaminopimelate transferase or UDP-N-acetyl-glucosamine reductase. Other approaches include the development of inhibitors of fatty acid biosynthesis, lipid A biosynthesis, two-component regulatory systems or pilus assembly (Mascaretti, 2003; Walsh, 2003). These candidates for novel antimicrobial agents are currently at the developmental stage and represent members of new rather than existing classes of antimicrobial agents (Mascaretti, 2003). One novel agent, the oxazolidinone linezolid, has been approved in 2000. Although it acts on an old target, namely the ribosome, its mode of action is new. Linezolid prevents the formation of the initiation complex composed of the 30S ribosomal subunit, the mRNA and the N-formylmethionine-tRNA and thus inhibits protein biosynthesis at a very early stage (Mascaretti, 2003; Walsh, 2003; Holzgrabe, 2004). However, the development of such novel antimicrobial agents is a cost-intensive and timeconsuming process with most of the drug candidates not surviving the clinical trials. If such a substance is finally approved for clinical use, there is also only a relatively short period of time (usually not more than 20 years
ARTICLE IN PRESS S. Schwarz, C. Kehrenberg / International Journal of Medical Microbiology 296 (2006) S2, 45–49
starting from approval of the chemical compound by the patent office) during which it is protected by patent.
New members of already existing classes of antimicrobial agents The chemical modification of members of already existing classes of antimicrobial agents proved to be a more cost-efficient and faster way of generating new antimicrobial agents. It has successfully been applied to virtually all classes of older antimicrobial agents, such as tetracyclines, macrolides or b-lactams, but also to some newer classes, such as the fluoroquinolones. Some modifications have been included in the parental substance to alter its pharmacological and pharmacokinetic properties, such as better tissue penetration, the extension of half-life time, or the improvement of the efficacy against specific groups of bacteria. However, any new molecule from an already existing class of antimicrobial agents might be a target for the preexisting class-specific resistance mechanisms. Many of the currently known resistance mechanisms are effective not only against a specific member of a certain class of antimicrobial agents, but also against many different members of the respective class. Hence, certain tetracycline exporters mediate the efflux of tetracycline, chlortetracycline, oxytetracycline, doxycycline and to a certain extent even minocycline whereas extended spectrum b-lactamases are able to hydrolyze a wide range of b-lactam antibiotics. As a consequence, indepth knowledge of the respective resistance mechanisms is an absolute pre-requisite to design new molecules which escape the known resistance mechanisms and thus also show activity against bacteria that carry the corresponding resistance genes.
Examples of new antimicrobial agents that escape class-specific resistance mechanisms Molecular analysis of resistance mechanisms provides the required data to identify critical target structures. Furthermore, modification of antimicrobial agents makes it possible to escape known resistance mechanisms. The two examples presented below – one from veterinary medicine and one from human medicine – describe how knowledge of specific resistance mechanisms has successfully been used to construct derivatives that are resistant to the respective bacterial resistance mechanisms.
Florfenicol Florfenicol is a fluorinated chloramphenicol derivative which has been licensed in Germany in 1995 and
47
2000, respectively, for the control of bovine and porcine respiratory tract infections due to Pasteurella multocida, Mannheimia haemolytica, Histophilus somni or Actinobacillus pleuropneumoniae. In 1994, chloramphenicol was banned from use in food-producing animals to protect the consumer from potential undesired sideeffects due to chloramphenicol residues in food animal carcasses. The florfenicol molecule has been modified at two positions: (1) the nitro group, which was considered to be responsible for the dose-unrelated aplastic anemia, was replaced by a sulfomethyl group at the 1-phenyl moiety, and (2) the C3 primary hydroxyl group was substituted for fluorine (Fig. 1) (Schwarz et al., 2004). The most prevalent chloramphenicol resistance mechanism in Gram-positive and Gram-negative bacteria is the enzymatic inactivation of chloramphenicol by chloramphenicol acetyltransferases (CATs). These enzymes catalyze the transfer of acetyl groups from a donor molecule (e.g. acetyl-CoA) to the hydroxyl group at C3. The replacement of this acceptor site by a fluor residue rendered florfenicol resistant to inactivation by CAT enzymes. As a consequence, chloramphenicol-resistant strains, in which resistance is exclusively based on the activity of a CAT, are susceptible to florfenicol (Schwarz et al., 2004). Recent data from specific monitoring programs to assess the susceptibility of target bacteria to florfenicol indicated that since the introduction of florfenicol into clinical veterinary use, the minimum inhibitory concentrations (MIC50, MIC90) to florfenicol remained stable for bovine as well as porcine target bacteria (Priebe and Schwarz, 2003; Kehrenberg et al., 2004; Wallmann et al., 2003, 2004). Moreover, virtually all target bacteria tested so far proved to be susceptible to florfenicol. However, it needs to be noted that florfenicol resistance due to specific exporters and other not further specified mechanisms has been identified in enteric bacteria and staphylococci (Schwarz et al., 2004). The future will show whether and – if so – when such florfenicol resistance genes will be transferred into target bacteria.
R1
O C CH R 3 HN CH CH CH2 R 2 OH
R1
R2
R3
- OH
= Cl2
Chloramphenicol
- NO2
Florfenicol
- SO2CH3 - F
=Cl2
Fig. 1. Structural relationships between chloramphenicol and florfenicol (modified from Schwarz et al., 2004).
ARTICLE IN PRESS 48
S. Schwarz, C. Kehrenberg / International Journal of Medical Microbiology 296 (2006) S2, 45–49
Telithromycin Telithromycin represents the first substance within the ketolides subclass of 14-membered macrolides. Ketolides are approved for the control of respiratory tract infections with Streptococcus pneumoniae being one of the main target bacteria. Telithromycin is an erythromycin derivative in which several substitutions have been introduced: (1) the L-cladinose at position C3 was replaced by a 3-keto group, and (2) a large N-substituted carbamate extension was introduced at positions C11–C12 of the macrolactone ring (Fig. 2). An excellent review on the biological and pharmacological properties of telithromycin has recently been published (Ackermann and Rodloff, 2003). Like erythromycin and other macrolides, telithromycin binds to two specific areas in the 23S rRNA, namely the domains II and V which are folded so that they form a binding pocket for these antibiotics (Ackermann and Rodloff, 2003). Dissociation kinetics showed that telithromycin binds approximately 10-fold stronger to the ribosome than erythromycin (Hansen et al., 1999) due to the carbamate extension at C11–C12 that mediates a direct contact between the nucleotides involved in drug binding, e.g. A752 in domain II as well as A2058, A2059 and G2505 in domain V. Previous work on macrolide resistance has shown that methylation of A2058 and/or A2059 by rRNA methylases is the most common resistance mechanism in staphylococci and streptococci (Roberts et al., 1999). The degree of methylation of these critical residues – mono- or dimethylation – has a large impact on the MIC for the different MLSB antibiotics. While 14membered macrolides (e.g. erythromycin, clarithromycin), but also lincosamides (e.g. lincomycin, clindamycin), cannot bind efficiently to even monomethylated ribosomes, telithromycin binds to monomethylated, but not to dimethylated ribosomes (Liu and Douthwaite, 2002). Erm(B) is the most prevalent rRNA methylase in streptococci and acts preferentially as a monomethylase in S. pneumoniae. This explains why telithromycin is active even against S. pneumoniae strains that constitu-
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tively express the Erm(B) methylase. Such strains were susceptible to telithromycin with low MIC50 and MIC90 values of 0.06 and 0.5 mg/ml, respectively, but highly resistant to erythromycin, clarithromycin and azithromycin with MIC50 and MIC90 values of 4128 mg/ml (Schmitz et al., 2002d). In staphylococci, the rRNA methylases Erm(A) and Erm(C) are the dominant ones and act in general as dimethylases. This explains why high level telithromycin resistance with MIC50 and MIC90 values of 4128 mg/ml is seen in staphylococci expressing the genes erm(A) and/or erm(C) constitutively (Schmitz et al., 2002c). Since telithromycin does not act as an inducer of erm(A) or erm(C) gene expression, staphylococcal strains that carry inducibly expressed erm(A) or erm(C) genes show low MICs which suggest that telithromycin is highly active against such strains (Schmitz et al., 2002c). However, in vitro selection studies proved that telithromycin can rapidly select for constitutive mutants that are resistant to all MLSB antibiotics including the ketolide telithromycin (Schmitz et al., 2002a, b).
Concluding remarks These examples show that detailed knowledge of resistance mechanisms can effectively be used to develop new derivatives of old drugs that overcome prevalent resistance mechanisms. However, any new selective pressure as imposed by the use of new antimicrobial agents represents a challenge for bacteria to develop/acquire new genes or mutations, which allow them to circumvent the inhibitory effects of these agents. This again results in a reduced efficacy of the new antimicrobial agents and a demand for the next generation of even more efficacious agents. There is no question that new antimicrobial agents are needed for both, human and veterinary medicine, and joint approaches of scientific research institutions and industry are necessary to identify new stable target structures in bacteria and to use them for the development of novel efficient antimicrobial agents. However, it is also necessary to bear in mind that the development of resistance in bacteria is an inevitable process. It is not possible to stop this process, but it is possible to slow it down by a judicious application of antimicrobial agents. In this regard, prudent use of antimicrobial agents in both, human and veterinary medicine, is an important requirement to slow down the speed of resistance development in bacteria and to retain the efficacy of the currently available agents as long as possible.
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Fig. 2. Structural relationships between erythromycin and telithromycin.
Ackermann, G., Rodloff, A.C., 2003. Drugs of the 21st century: telithromycin (HMR3647) – the first ketolide. J. Antimicrob. Chemother. 51, 497–511.
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Bush, K., 2004. Antibacterial drug discovery in the 21st century. Clin. Microbiol. Infect. 10 (Suppl. 4), 10–17. Clarke, T., 2003. Drug companies snub antibiotics as pipeline threatens to dry. Nature 442, 225. Davies, J.E., 1997. Origins, acquisition and dissemination of antibiotic resistance determinants. In: Chadwick, I., Goode, J. (Eds.), Antibiotic Resistance: Origins, Evolution, Selection, and Spread. Ciba Foundation Symposium, vol. 207. Wiley, Chichester, pp. 15–27. Hansen, L.H., Mauvais, P., Douthwaite, S., 1999. The macrolide–ketolide antibiotic binding site is formed by structures in domains II and V of 23S ribosomal RNA. Mol. Microbiol. 31, 623–631. Holzgrabe, U., 2004. Antibiotika-Entwicklung gestern und heute. Chemother. J. 13, 142–147. Kehrenberg, C., Mumme, J., Wallmann, J., Verspohl, J., Tegeler, R., Ku¨hn, T., Schwarz, S., 2004. Monitoring of florfenicol susceptibility among bovine and porcine respiratory tract pathogens collected in Germany during the years 2002 and 2003. J. Antimicrob. Chemother. 54, 572–574. Liu, M., Douthwaite, S., 2002. Activity of the ketolide telithromycin is refractory to Erm monomethylation of bacterial rRNA. Antimicrob. Agents Chemother. 46, 1629–1633. Livermore, D.M., 2004. The need for new antibiotics. Clin. Microbiol. Infect. 10 (Suppl. 4), 1–9. Mascaretti, O., 2003. Bacteria Versus Antibacterial Agents: an Integrated Approach. ASM Press, Washington, DC. Powers, J.H., 2004. Antimicrobial drug development – the past, the present and the future. Clin. Microbiol. Infect. 10 (Suppl. 4), 23–31. Priebe, S., Schwarz, S., 2003. In vitro activities of florfenicol against bovine and porcine respiratory tract pathogens. Antimicrob. Agents Chemother. 47, 2703–2705. Projan, S.J., Shlaes, D.M., 2004. Antibacterial drug discovery: is it all downhill from here? Clin. Microbiol. Infect. 10 (Suppl. 4), 18–22. Roberts, M.C., Sutcliffe, J., Courvalin, P., Jensen, L.B., Rood, J.I., Seppala, H., 1999. Nomenclature for macrolide and macrolide-lincosamide streptogramin B antibiotic resistance determinants. Antimicrob. Agents Chemother. 43, 2823–2830.
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Schmitz, F.J., Petridou, J., Astfalk, N., Scheuring, S., Ko¨hrer, K., Schwarz, S., 2002a. Molecular analysis of constitutively expressed erm(C) genes selected in-vitro by incubation in the presence of the non-inducers quinupristin, telithromycin or ABT-773. Microb. Drug Resist. 8, 171–177. Schmitz, F.J., Petridou, J., Jagusch, H., Astfalk, N., Scheuring, S., Schwarz, S., 2002b. Molecular characterization of ketolide-resistant erm(A)-carrying Staphylococcus aureus isolates selected in vitro by telithromycin, ABT-773, quinupristin and clindamycin. J. Antimicrob. Chemother. 49, 611–617. Schmitz, F.J., Petridou, J., Milatovic, D., Verhoef, J., Fluit, A.C., Schwarz, S., 2002c. In vitro activity of new ketolides against macrolide-susceptible and macrolide-resistant Staphylococcus aureus isolates with defined resistance gene status. J. Antimicrob. Chemother. 49, 580–582. Schmitz, F.J., Schwarz, S., Milatovic, D., Verhoef, J., Fluit, A.C., 2002d. In vitro activities of the ketolides ABT-773 and telithromycin and of three macrolides against genetically characterized isolates of Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae, and Moraxella catarrhalis. J. Antimicrob. Chemother. 50, 145–148. Schwarz, S., Chaslus-Dancla, E., 2001. Use of antimicrobials in veterinary medicine and mechanisms of resistance. Vet. Res. 32, 201–225. Schwarz, S., Kehrenberg, C., Doublet, B., Cloeckaert, A., 2004. Molecular basis of bacterial resistance to chloramphenicol and florfenicol. FEMS Microbiol. Rev. 28, 519–542. Wallmann, J., Schro¨ter, K., Wieler, L.H., Kroker, R., 2003. Antibiotikaempfindlichkeit ausgewa¨hlter pathogener Bakterien von erkrankten Lebensmittel liefernden Tieren in Deutschland: Ergebnisse aus der Modellstudie 2001 des nationalen Resistenzmonitorings. Tiera¨rztl. Praxis 31 (G), 122–131. Wallmann, J., Kaspar, H., Kroker, R., 2004. Pra¨valenzdaten zur Antibiotikaempfindlichkeit von bei Rindern und Schweinen isolierten bakteriellen Infektionserregern: Nationales BVL Resistenzmonitoring 2002/2003. Berl. Mu¨nch. Tiera¨rztl. Wochenschr. 117, 480–492. Walsh, C., 2003. Antibiotics: Actions, Origins, Resistance. ASM Press, Washington, DC.