Antimicrobial Research Centre and Division of Microbiology, University of Leeds, Leeds LS2 9JT, UK
Received 21 June 2002; returned 10 September 2002; revised 13 September 2002; accepted 14 September 2002
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Abstract |
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Keywords: cephalosporin P1, fusidic acid, Staphylococcus aureus, resistance
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Introduction |
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Several new strategies for controlling staphylococcal infections have been considered in recent years. These include the use of antibiotic combinations,57 the development of new members of existing antibiotic classes2,4,8 and the discovery of novel agents through genomic approaches.9 Another approach is the re-evaluation of older, unexploited agents, with a view to developing them for use against organisms resistant to current agents.10,11 This concept has already been applied to daptomycin, an antibiotic discovered in the early 1980s, which is now undergoing clinical trials for application as an agent to control infections caused by Gram-positive pathogens, including S. aureus.12 Recently, at the pre-clinical level, we have assessed the potential of older, unexploited, natural product RNA polymerase inhibitors as anti-staphylococcal agents.8,13
Cephalosporin P1 (The nomenclature here is confusing. Cephalosporin P1 is not related to the cephalosporins that inhibit peptidoglycan synthesis.) is a triterpenoid antibiotic (Figure 1) discovered in 1951 from the culture fluid of Cephalosporium acremonium.14 Early work demonstrated that cephalosporin P1 has an antibacterial spectrum that predominantly encompasses Gram-positive organisms, with particularly potent activity against S. aureus.14,15 However, no recent work on cephalosporin P1 has been conducted. In view of its excellent activity against S. aureus, including in vivo activity,15 we decided to re-evaluate this antibiotic as a potential anti-staphylococcal agent.
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Results reported in this paper confirm the excellent anti-staphylococcal activity of cephalosporin P1 and address in detail the issue of cross-resistance between fusidic acid and cephalosporin P1 in S. aureus. For this purpose, we have used laboratory mutants with defined mutations in fusA,22 a strain harbouring pUB101, which encodes the fusB plasmid-mediated mechanism,21 and a set of fusidic acid-resistant clinical isolates.18 In all cases we observed cross-resistance between fusidic acid and cephalosporin P1, and novel mutations in fusA conferring resistance to both antibiotics were identified. In addition, using a mutant deleted for the AcrAB efflux system in Escherichia coli, we demonstrated that cross-resistance between the two antibiotics extends to a Gram-negative efflux pump. Some of the results reported here were presented in a preliminary communication.22
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Materials and methods |
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S. aureus and E. coli laboratory strains are listed in Table 1. Clinical bacteria used for comparative susceptibility testing were isolates maintained in a culture collection belonging to the University of Leeds. S. aureus clinical isolates resistant to fusidic acid (prefix H) were obtained from the Departments of Dermatology and Microbiology, Harrogate District Hospital, UK.18
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MuellerHinton broth (MHB) and agar (MHA) were from Fisher, Loughborough, UK. Iso-Sensitest broth (ISB) and Iso-Sensitest agar (ISA) were purchased from Oxoid, Basingstoke, UK.
Ciprofloxacin, mupirocin, linezolid and cephalosporin P1 were gifts, respectively, from Bayer AG (Leverkusen, Germany), SmithKline Beecham Pharmaceuticals (Harlow, UK), Pharmacia and Upjohn Inc. (Kalamazoo, MI, USA) and Biochemie GmbH (Kundl, Austria). Other antibiotics were purchased from Sigma-Aldrich, Poole, UK.
Determination of susceptibility to antimicrobial agents
MICs were determined by agar dilution on MHA or ISA using an inoculum in MHB or ISB of 106 cfu/spot for S. aureus and 104 cfu/spot for E. coli.23 The MIC was defined as the lowest concentration of antibiotic completely inhibiting visible growth after 1824 h incubation at 37°C.
Determination of mutation frequencies for resistance to antimicrobial agents
This was carried out as described by ONeill et al.5 using ISA. Both standard and concentrated cell techniques were used, whereby mutation frequencies as low as 1 in 1011 can be detected. Mutant colonies were normally counted after incubation of plates for 24 h at 37°C. However, mutants resistant to ciprofloxacin were slow growing and in this case colonies were quantified after 48 h incubation.
Selection of fusidic acid- and cephalosporin P1-resistant mutants
Fusidic acid-resistant mutants were generated by growing S. aureus 8325-4 in ISB at 37°C with shaking to a culture density at 600 nm of 1, and plating 100 µL aliquots onto ISA at either 0.25 or 10 mg/L. For cephalosporin P1, resistant mutants were selected at 0.25 mg/L. After 24 h incubation at 37°C, colonies were picked at random from the selective plates and their susceptibilities (MICs) to fusidic acid and cephalosporin P1 established on ISA.
Plasmid curing
Loss of resistance to fusidic acid (plasmid curing) in clinical isolates of S. aureus following incubation at 43°C was carried out essentially as described by Lacey & Grinsted.24 However, loss of resistance was monitored by replica plating onto ISA containing fusidic acid at 0.516 mg/L to identify colonies that had become more susceptible to fusidic acid.
PCR and DNA sequencing
Oligonucleotide primers for PCR and DNA sequencing were designed using Oligo 5.0 (MBI, Cascade, ID, USA) and purchased from MWG Biotech (Milton Keynes, UK). DNA templates were prepared using the QIAamp DNA mini kit (Qiagen) according to the manufacturers instructions. Extensor Hi-fidelity Master mix (ABgene, Epsom, UK) was used for all amplifications under standard PCR cycling conditions.25 Primers for amplification of the entire 2.1 kb fusA gene were fusF (5'-CGCGGATCCTATCGTATTTATTCAGTAAT) and fusR (5'-AAGGATCCCTTGTATTTTAACCTAGGCTA) and were based on sequence data from the S. aureus NCTC 8325 genome (http:// www.genome.ou.edu/staph.html). Successful amplification was confirmed by agarose gel electrophoresis.25 DNA was eluted from gels by solubilizing the agarose in QG buffer (Qiagen), and purified using the QIAquick PCR Purification Kit (Qiagen).
DNA sequencing of fusA was carried out using the amplification primers and three additional sequencing primers (5'-GCGTCAGGCTACAACTTATGG, 5'-CAATAGTTACTTTCATCATTGG and 5'-TTATTGGTCACCGTGCTAGCAACC) by Lark Technologies Incorporated (Saffron Walden, UK) on an Applied Biosystems 377 DNA sequencer.
Deletion of acrAB in E. coli 1411
Although strain JZM120 (JC7623 acrAB::Tn903kanr)26 (Table 1) was donated to us, its parent (JC7623) was not available. Therefore, to determine the effects of deleting acrAB on antibiotic susceptibility, we constructed an acrAB knock-out in one of our laboratory strains (E. coli 1411; Table 1) to provide an isogenic pair. A
acrAB 1411 strain was constructed by P1 transduction27 of
acrAB::Tn903kanr from JZM120, resulting in strain SM1411. Deletion of acrAB was confirmed by generation of a PCR amplicon from a primer specific for the Tn903 kanamycin kinase gene (5'-TCCGACCATCAAGCATTTTA) and one specific for the acrB gene (5'-CAGCAGCACGAACATACCA), and by increased susceptibility to puromycin, erythromycin, novobiocin and linezolid, as reported previously28,29 (Table 6).
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Results |
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The in vitro anti-staphylococcal activity of cephalosporin P1 has been reported for only a limited number of strains and the data are not expressed in conventional MIC format.14,15 Furthermore, comparative data with other anti-staphylococcal agents are not available. Therefore the activity of cephalosporin P1 against a number of clinical isolates of S. aureus (MSSA, MRSA and VISA) was determined by dilution in MHA and compared with that of cefotaxime, ceftriaxone, cefepime, fusidic acid, linezolid, oxacillin, quinupristin/dalfopristin and vancomycin (Table 2). Cephalosporin P1 was slightly less active than fusidic acid against the panel of 67 strains tested. Nevertheless, it displayed excellent anti-staphylococcal activity, including those strains resistant to established agents.
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Selection of S. aureus mutants resistant to cephalosporin P1 has been reported,30 but the frequency with which mutants arise has not been determined. Spontaneous mutants of S. aureus 8325-4 resistant to cephalosporin P1 were recovered by direct plating of organisms on to ISA containing the antibiotic at 4 x MIC. Mutants arose with a frequency of 1.6 x 106 (Table 3). Frequencies of mutational resistance to a number of other anti-staphylococcal agents, including fusidic acid, were determined under identical conditions (Table 3). Mutation frequencies were cephalosporin P1 > fusidic acid > rifampicin > norfloxacin > mupirocin > ciprofloxacin. No mutants were recovered (frequencies <1011) when bacteria were selected on vancomycin or penicillin at 4 x MIC.
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To assist the evaluation of cephalosporin P1, we cross-screened the antibiotic against a collection of fusidic acid-resistant mutants of S. aureus 8325-4, generated in an isogenic background (Table 4; Figure 2). Mutations in fusA were identified in 11 of 14 mutants selected for study and were located in the three clusters (IIII) reported previously for mutations conferring resistance to fusidic acid in S. aureus and Salmonella typhimurium.19,31,32 Several of the mutations we identified; F88L (F26), P406
L (F13), T436
I (F20, F16) and H457
Y (F11, F28, F38) were identical to those previously reported for S. aureus. However, in other cases we observed novel mutations either involving variant substitutions at a known mutational site, i.e. H457
L (F32) and H457
N (F34), or new mutations not previously reported in any species, i.e. R483
L (F3) and G664
S (F7). In three mutants (F1, F4, F17), we were unable to detect changes in fusA (Table 4), similar to the observations of Laurberg et al.33 who also reported unidentified chromosomal loci that confer fusidic acid resistance in S. aureus.
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Cross-resistance between cephalosporin P1 and fusidic acid in chromosomal mutants selected with cephalosporin P1
In addition to determining cross-resistance patterns for mutants initially selected with fusidic acid, we reversed the approach by selecting mutants resistant to cephalosporin P1 followed by examination of cross-resistance to fusidic acid (Table 4). In all cases, mutants selected on the basis of resistance to cephalosporin P1 were also resistant to fusidic acid. The levels of resistance to cephalosporin P1 displayed by these mutants were greater than those for fusidic acid, as noted previously for mutants selected with fusidic acid as the primary agent. To determine whether the changes responsible for resistance in mutants selected with cephalosporin P1 arose in fusA, the complete gene was sequenced in 10 mutants covering the cephalosporin P1 MIC range (264 mg/L) observed for these mutants. Mutations were identified in each of the fusA clusters IIII at amino acid residues 118, 404, 406, 436, 452, 478, 656 (three mutants) and 666. With the exception of T436-I and G452-S, which confer fusidic acid resistance in S. aureus, the mutations did not correspond exactly to those we, or others, observed when fusidic acid was used as the primary selective agent (Table 4; Figure 2). Thus, the mutations at residues 478, 656 and 666 are novel since they have not previously been associated with resistance to fusidic acid, whilst T118-I, which has been reported to confer fusidic acid resistance in S. typhimurium, was detected for the first time in S. aureus. Mutations at amino acid residues 404, 406 and 452 have previously been associated with resistance to fusidic acid in S. aureus, as described above and in Figure 2. However, the actual substitutions identified following selection with cephalosporin P1 were novel. In contrast to selections with fusidic acid, mutants selected with cephalosporin P1 appeared to contain mutations only in fusA.
Cross-resistance between fusidic acid and cephalosporin P1 mediated by plasmid pUB101
Although the molecular basis of plasmid-mediated resistance to fusidic acid in S. aureus has not been established, it is not related to that conferred by mutant EF-G.21 It was therefore of interest to determine whether the plasmid-mediated fusidic acid resistance mechanism also conferred cross-resistance to cephalosporin P1. For this purpose, we used plasmid pUB101, which specifies resistance to fusidic acid.21 Plasmid pUB101 conferred resistance to both fusidic acid and cephalosporin P1 when present in S. aureus strain 649, raising the MICs of these drugs by factors of 128 and 256, respectively (Table 5).
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Results described above suggested that clinical isolates of S. aureus resistant to fusidic acid would also exhibit cross-resistance to cephalosporin P1. This was explored by screening some of the fusidic acid-resistant S. aureus strains originally isolated from clinical material by Ravenscroft et al.18 Strains chosen for analysis exhibited a range of resistance levels to fusidic acid (MICs 2256 mg/L). High-level fusidic acid resistance (16256 mg/L) appeared to be associated with possession of plasmids, since heat treatment of strains H5, H10, H56 and H71, under conditions that promote plasmid curing,24 resulted in decreased resistance to fusidic acid (Table 5). In contrast, with the exception of strain H84 (fusidic acid MIC 8 mg/L), heat treatment of isolates exhibiting low-level resistance to fusidic acid (MIC 28 mg/L) failed to decrease the resistance levels. Thus, presumptive evidence was obtained for the occurrence of both chromosomal and plasmid-mediated mechanisms of fusidic acid resistance in the clinical isolates. In all cases cross-resistance with cephalosporin P1 was observed (Table 5). Residual resistance to both fusidic acid and cephalosporin P1 was observed in those strains that had been subjected to plasmid curing (Table 5).
The AcrAB efflux pump in E. coli mediates resistance to both fusidic acid and cephalosporin P1
In addition to the mechanisms of fusidic resistance described above, the AcrAB efflux system in E. coli mediates resistance to fusidic acid.28,34 To determine whether AcrAB also mediates resistance to cephalosporin P1, we compared its activities against a wild-type E. coli K12 strain (1411) and an isogenic derivative deleted in acrAB (SM1411). Deletion of acrAB resulted in an 10-fold increase in susceptibility to cephalosporin P1 (Table 6). The activities of fusidic acid, puromycin, novobiocin, erythromycin and linezolid were all enhanced (16- to 64-fold) against strain SM1411 (
acrAB) (Table 6). These antibiotics are all substrates of the AcrAB efflux pump.28,29,34
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Discussion |
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Bacterial resistance to one member of an antibiotic structural series does not necessarily mean that cross-resistance will be exhibited to other analogues within the family, e.g. the glycylcyclines, although sharing many structural features with earlier tetracyclines, are not subject to the efflux and ribosomal protection mechanisms that confer resistance to earlier members.35 However, the structural similarity between fusidic acid and cephalosporin P1 (Figure 1) suggests the possibility of cross-resistance between the two antibiotics. This situation has not previously been explored in detail, but data reported here demonstrate that mutations in all three clusters of EF-G that confer resistance to fusidic acid also confer cross-resistance to cephalosporin P1. This situation extends to mutants selected with cephalosporin P1, which all mapped in fusA (encoding EF-G) and displayed cross-resistance to fusidic acid. A further mechanism of resistance to fusidic acid in S. aureus involves a plasmid-mediated system21 that has been designated fusB.36 Although the molecular basis of fusB-encoded resistance has yet to be determined, early studies clearly differentiate it from EF-G-based mutational resistance.21 Nevertheless, cross-resistance with cephalosporin P1 was also exhibited at the level of fusB.
Since mutations in fusA and acquisition of fusB are both responsible for resistance to fusidic acid in clinical isolates of S. aureus,19,20 we were not surprised to observe that a collection of fusidic acid-resistant clinical strains also exhibited cross-resistance to cephalosporin P1. We have not yet determined the exact status of these clinical isolates with respect to mutations in fusA and carriage of fusB. However, resistance of some strains (H3, H36 and H69) to both antibiotics was unaffected by treatment leading to plasmid loss. This suggests that these strains may only contain fusA mutations. In contrast, derivatives of other clinical isolates (H5, H10, H56, H71 and H84) that were apparently cured of plasmid determinants nevertheless retained residual resistance to both fusidic acid and cephalosporin P1 (Table 5). These results are therefore consistent with the possibility that the original clinical isolates (H5, H10, H56, H71 and H84) contained both chromosomal fusA mutations and plasmid-located fusB determinants. The collection of clinical isolates studied here is therefore likely to contain the fusidic acid resistance genotypes commonly occurring during therapy with fusidic acid. Consequently, the increasing incidence of fusidic acid resistance in clinical isolates of S. aureus17,18 is undoubtedly an issue when considering whether cephalosporin P1 might be developed as an antibiotic candidate.
In addition to mutations in fusA, other uncharacterized chromosomal mutations confer resistance to fusidic acid in staphylococci.19,22,33 Although it is not clear whether such mutants arise in the clinic, these strains were also cross-resistant to cephalosporin P1 (Table 4). Finally, although fusidic acid has no application in the treatment of infections caused by E. coli,20 we demonstrated a further cross-resistance relationship between fusidic acid and cephalosporin P1 at the level of the AcrAB efflux pump. Furthermore, we noted that even when the AcrAB efflux pump was deleted, the MICs of fusidic acid and cephalosporin P1 for this E. coli mutant were greater than those observed for the antibiotic-sensitive staphylococcal strains 8325-4 and 649. It is known that the lipid bilayer region of the Gram-negative outer membrane is relatively impermeable to lipophilic molecules.37 Since fusidic acid and cephalosporin P1 are lipophilic molecules, the residual insusceptibility of E. coli SM1411 (acrAB) to these antibiotics probably reflects poor uptake across the Gram-negative outer membrane.
Apart from exploring the potential of cephalosporin P1 as an anti-staphylococcal agent, this study has provided further insights into structurefunction relationships in fusA. This relates to the nature and location of mutants selected with fusidic acid and cephalosporin P1 (Figure 2).
Despite the spread of resistance loci across EF-G, some sites appear to be especially important for the development of fusidic acid resistance, particularly the conserved region38 centred on residues 451464 (Figure 2). This region, and in particular H457, is thought to interact directly with fusidic acid.33 Removal of histidine per se from this site is not sufficient to generate high-level resistance, as the nature of the substitution at residue 457 influences susceptibility to fusidic acid. For example, an HY replacement results in a mutant with a fusidic acid MIC of 64 mg/L, whereas an H
N replacement generates a mutant with an MIC of 256 mg/L (Table 4). Despite the fact that H457 is highly conserved, EF-G function is nonetheless retained with any of three different residues in its place (Table 4). However, analysis of mutant F32 (Table 4) suggests that the nature of the histidine-replacement residue is not immaterial; this strain carries two nucleotide substitutions (CACCTA) in a single codon. It is likely that mutation CACCAA (H457
Q) took place first, rather than CACCTC (H457
L); if histidine was first replaced with leucine, a second, silent nucleotide substitution would need to arise subsequently in the same codon. Conceivably, mutation Q457, despite conferring a degree of fusidic acid resistance, exhibited unsatisfactory translocase activity, resulting in subsequent selection of the compensatory mutation CAACTA (Q457
L).
Although we observed cross-resistance between fusidic acid and cephalosporin P1, there appeared to be differences in the nature and location of mutations selected by the two agents in S. aureus (Figure 2). The majority of mutations selected with cephalosporin P1 either arose at novel sites within fusA, or involved different substitutions from those arising with fusidic acid selection. Furthermore, all the mutants selected with cephalosporin P1 contained mutations in fusA, in contrast to three of the 14 mutants selected with fusidic acid that did not map to fusA (Table 4). Examination of the effects of fusidic acid and cephalosporin P1 on translocation and peptide bond formation using cell-free assays suggests that the interaction of the two antibiotics with EF-G may differ.39 Possibly, the differences in EF-G binding characteristics of cephalosporin P1 favour the selection of different types of mutation in EF-G compared with selection with fusidic acid. However, whether there is a real difference in the spectrum of mutations selected by each agent will require examination of larger numbers of mutants.
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Acknowledgements |
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Footnotes |
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