Department of Medical Microbiology, Royal Free and University College, Royal Free Campus, London NW3 2PF, UK
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Abstract |
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Introduction |
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The purpose of the present study was to determine the incidence of various types of erythromycin resistance among a large number of unselected staphylococci isolated from patients in a university hospital, and to investigate how these resistance mechanisms affected susceptibility to antibiotics related to erythromycin A, namely another 14-membered macrolide (oleandomycin), a 16-membered macrolide (rokitamycin), a lincosamide (clindamycin), a ketolide (telithromycin) and representatives of the A and B components of the streptogramins (quinupristin and dalfopristin), alone and in combination.
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Materials and methods |
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A total of 540 strains of individual staphylococci, comprising 210 Staphylococcus aureus and 330 coagulase-negative staphylococci (CNS), isolated in the Diagnostic Microbiology Laboratory of The Royal Free Hospital, London, UK, during June 1998, were identified by their colonial appearance, Gram's staining and production of catalase. S. aureus and CNS were differentiated using DNase and Staphaurex. CNS were identified where appropriate using API Staph kits.
The origin of each strain was checked by patient's name and hospital number, and possible duplicate strains excluded. For the methicillin-resistant strains, only one of each clonal type was included in the test population.
Antibiotics
Telithromycin (HMR 3647) was given by Hoechst Marion Roussel (Romainville, France); Synercid (RP 59500, seven parts quinupristin, three parts dalfopristin), dalfopristin (RP 54476) and quinupristin (RP 57669), each as the methane sulphonate, were given by Rhone-Poulenc Rorer (Collegeville, PA, USA); erythromycin BP free base was given by Lilly Industries (Basingstoke, UK); rokitamycin was given by ISF SpA (Milan, Italy); lincomycin hydrochloride and oleandomycin phosphate were purchased from Sigma (Poole, UK). Erythromycin was dissolved in ethanol, the other compounds in water.
Discs containing oleandomycin 15 µg were purchased from Mast Laboratories (Bootle, UK), and erythromycin 15 µg and clindamycin 2 µg from Unipath Laboratories (Basingstoke, UK). Discs containing Synercid 15 µg and telithromycin 15 µg were given by Rhone-Poulenc Rorer R-D (Antony, France) and Hoechst Marion Roussel, respectively. Discs containing 15 µg of either rokitamycin, dalfopristin or quinupristin were made as required by treating Whatman AA discs with the appropriate antibiotic solution.
Chemicals
These were obtained from Sigma (Poole, UK).
Media
Nutrient agar (NA), MuellerHinton agar (MHA) and broth (MHB) were from Unipath. Blood agar was Columbia agar (Mast) + 5% whole horse blood.
Susceptibility testing
All 540 strains were tested by the breakpoint method against erythromycin (0.5 and 4 mg/L), lincomycin (1 and 2 mg/L) and Synercid (1 mg/L), on MHA inoculated with 104 cfu, incubated for 24 h in air.
The 215 strains resistant to erythromycin and the five sensitive to erythromycin but resistant to lincomycin were then screened by a disc diffusion method. An aqueous suspension of bacterial growth from blood agar was adjusted to McFarland 0.5, and inoculated by swab on MHA (60 µL in a plate of diameter 140 mm). Each plate was set with 13 discs (centres 2 cm apart) as shown in Figure 1. This arrangement allowed both the sensitivity pattern to individual compounds and interactions between the various antibiotics to be observed with a minimum amount of repetition.
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Zones were interpreted according to their size and shape, as indicating sensitive or resistant, the latter being inducible if a D-shaped zone was observed (the compound on the left being the inducer). Synergy was recorded if there was an extension of inhibition zone between two discs. The presence of satellite colonies within inhibition zones, and other evidence for the existence of sub-populations (e.g. some degree of target zone formation) as well as unusual shapes of zones were noted.
Phenotypes were denoted by abbreviations of antibiotic class to which a strain was resistant: M, erythromycin; A, oleandomycin; L, clindamycin; SA, dalfopristin; SB, quinupristin; K, telithromycin; Mac, rokitamycin.
When resistance was inducible by erythromycin, i was prefixed. Thus, for example, the classical inducible MLSB phenotype is denoted here as M/i(LKSBMac), and the classical constitutive MLSB phenotype is MLKSBMac.
We also used the 13 disc method to determine the phenotypes of five strains (two MRSA, three Staphylococcus haemolyticus) that had previously shown anomalous susceptibilities to telithromycin.5
MIC determinations were made following the NCCLS agar dilution method.6
Selection of resistant mutants
Cultures in MHB were spun down and resuspended at 10x original concentration. A viable count was performed, and 0.1 mL (c. 109 cfu) was spread on MHA containing 1.2 mg/L telithromycin (i.e. at least 20 x MIC) and colonies counted after 48 h incubation. The proportion of cells able to grow was calculated, and their phenotypes determined by the 13 disc method described above.
Reversal of resistance
(i) Doubling dilutions of ethidium bromide in MHA were spot-inoculated with strains under test, and incubated overnight. For each strain, sub-culture was made from the 0.5 x MIC plate on to NA, and incubated overnight. Approximately 20 colonies from these plates were spot-inoculated on to MHA ± erythromycin (8 mg/L = c. 16 x MIC for sensitive strains). Those unable to grow in the presence of erythromycin had lost their resistance; they were also tested as in second screen above.
(ii) For strains in which an efflux mechanism was suspected, the MIC of erythromycin was determined alone and in the presence of (separately) dinitrophenol (20 mg/L), reserpine (20 mg/L) and carbonyl cyanide m-chlorophenol hydrazone (0.25 mg/L). These concentrations were chosen by solubility for the first two compounds and microbiological activity for the third. A four-fold diminution in MIC was taken as indicating inhibition of efflux.
Synergy/antagonism experiments
Interactions between oleandomycin and telithromycin were investigated and analysed by chequerboard titration on MHA using doubling dilutions, as described previously.7 Results were interpreted as synergy, antagonism or indifference. The latter term was used in the sense originally defined by Jawetz & Gunnison,8 meaning that each antibiotic in combination behaves as if the other were not there.
Assays of antibiotic destruction
Destruction of lincomycin and clindamycin was tested for by a modified Gots test9 read after 48 h, and measured as described by Leclercq et al.10 For the latter test, cells from overnight cultures were concentrated 60-fold in phosphate buffer containing 20 mg/L of the antibiotic and incubated at 37°C. A similar suspension of S. aureus Oxford was tested as a negative control. Antibiotic concentrations were determined at intervals by bioassay with S. aureus Oxford as indicator.
For technical reasons it was not possible to test for dalfopristin inactivation under these conditions.
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Results |
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Sensitivity patterns are shown in Table I. CNS were significantly more often resistant to erythromycin, telithromycin, clindamycin, quinupristin or rokitamycin than were S. aureus strains (P < 0.01 by chi-squared test). Only five strains (two Staphylococcus epidermidis, two Staphylococcus sciuri and one Staphylococcus simulans) were resistant to dalfopristin (MIC 32 or 64 mg/L). All 540 strains tested were sensitive to Synercid, despite two (the S. epidermidis mentioned above, nos 152 and 538) being resistant to both components (Figure 2
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The most common type of resistance to erythromycin was the inducible variety (Table II); the constitutive and MS types were less common, especially among S. aureus strains. The incidence of all three types was higher among CNS than for S. aureus (P < 0.01).
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Results obtained by analysing patterns of resistance deduced from the 13 disc screening test defined four groups, each of which could be divided into two.
Group A: destructive mechanism. Five of the 540 staphylococcal strains tested were resistant to lincomycin although sensitive to erythromycin. This pattern suggested the possible presence of a drug inactivation mechanism.11 More detailed investigations of these strains showed this to be the case (Table III): the two S. epidermidis strains, phenotype L (group A1), inactivated clindamycin rapidly and lincomycin more slowly, while the other three strains, phenotype LSA, did not inactivate lincosamides.
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Group B: classical inducible MLSB. The 129 strains in this category (26 S. aureus, 103 CNS) were resistant to erythromycin and oleandomycin, and sensitive to clindamycin, telithromycin, quinupristin, dalfopristin and rokitamycin. For several strains, satellite colonies were observed in the truncated part of the zone between the erythromycin and clindamycin discs (e.g. as in Figure 1).
Strains could be divided into two groups on the basis of the inducing behaviour of erythromycin and oleandomycin. In organisms in group B1 (15 S. aureus, 99 CNS), resistance to clindamycin, telithromycin, quinupristin and rokitamycin was induced by erythromycin and by oleandomycin.
The 15 strains in group B2 (11 S. aureus, two S. haemolyticus, two S. simulans) were induced by erythromycin, but oleandomycin either did not induce (Figure 3) or had a variable effect, inducing resistance to some but not all of the agents depending on the strain. There was a disproportionate number of S. aureus strains in group B273% compared with 20.1% in group B overall. All the group B strains had the phenotype M/i(LKSBMac).
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A zone of inhibition shaped like a shield (Figure 4) was seen when dalfopristin and quinupristin were tested side by side against group C1 strains. The enhanced area of inhibition between the discs represents synergy between dalfopristin and quinupristin, despite the strains being resistant to the latter.
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Group D: efflux mechanisms. Forty-six strains (two S. aureus, 44 CNS) were resistant to erythromycin and there was no induction of resistance to clindamycin. This pattern suggests active efflux.11 In the CNS strains, resistance to telithromycin and to quinupristin was induced by either erythromycin or oleandomycin, giving a phenotype of M/i(KSB); these were designated group D1. In the S. aureus strains, however, no such induction occurred; these therefore had phenotype M, and were assigned to group D2.
Further investigations on strains of different phenotypes
Inducibly resistant strains (group B). Twenty-one strains (13 S. aureus, eight CNS), made up of six B1 and all 15 B2 strains, were tested for the presence of mutants constitutively resistant to telithromycin. Ketolide-resistant colonies were isolated from 17 strains (81%), in greater numbers from B1 strains (range 1 per 6 x 1051 per 107, median 1 per 4 x 106) than in B2 strains (range 1 per 1061 per 109, median 1 per 108). The colonies isolated from 15 of these strains were C phenotype (MLKSBMac) i.e. constitutively resistant. Other novel phenotypes were found from colonies isolated during these experiments, including MLKMac/iSB (called C5), from three S. haemolyticus strains.
Seventeen strains (nine S. aureus, eight CNS) were grown in the presence of ethidium bromide: clones sensitive to all MLKSMac antibiotics were isolated from one S. aureus and one S. haemolyticus.
The interactions between oleandomycin and telithromycin against B2 strains were further investigated by the chequerboard method. For three strains (S. simulans nos 190 and 416, and S. haemolyticus no. 29), antagonism was found; for four (S. aureus nos 212, 342, 482 and 545) there was synergy; against the remaining eight strains each antibiotic behaved as if the other was not there (indifference). These results can be correlated with MICs of oleandomycin (Table IV): antagonism occurred for the highly resistant strains (MIC of oleandomycin
128 mg/L), synergy for the least resistant strains (MICs 48 mg/L) and indifference in those strains for which MICs were intermediate (usually 8 or 16 mg/L).
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Constitutively resistant strains (group C). Nine strains with constitutive resistance grown in the presence of ethidium bromide retained their phenotype.
Strains with efflux mechanisms. MICs were determined for 18 D1 strains and the two D2 strains (Table V). Six strains of the former sub-group and one of the latter (S. aureus no. 514) were plated on to agar containing 20 x MIC of telithromycin. Telithromycin-resistant colonies were found only from S. aureus no. 514; these were also resistant to quinupristin. Thus, a change of phenotype from M to MSBK had occurred.
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Discussion |
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As expected, there was complete cross-resistance between the two 14-membered macrolides erythromycin and oleandomycin, while telithromycin, the 16-membered rokitamycin, clindamycin and quinupristin remained active against all but the strains with a constitutive MLSB phenotype. This is because the latter four compounds do not induce the staphylococcal enzyme that confers resistance by ribosomal methylation.19
The experimental plan adopted in this investigationtesting the activities of and interactions between seven MLSK antibiotics including oleandomycinhas revealed novel phenotypes among clinical isolates and their laboratory derivatives. This further illustrates the considerable and apparently increasing complexity of resistance manifestations to this group of antibiotics,11,1923 as well as the shortcomings of conventional phenotyping in terms of susceptibility to erythromycin and clindamycin only (Table VI).
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Staphylococci of phenotype LSA are unusual, and have previously been found mainly in S. aureus2426 and only where pristinamycin has been used clinically.27 The three CNS strains found in the present study (two S. sciuri and one S. simulans) add to the six (four S. sciuri, two S. haemolyticus) reported previously.27,28 Recent work29 on S. sciuri suggests that this species is usually intrinsically resistant to dalfopristin, showing the LSA phenotype.
Phenotype B
The behaviour of oleandomycin as an inducing agent enabled strains traditionally allotted to the inducible MLSB phenotype to be split into two groups. Despite having the same phenotypeM/i(LKSBMac)strains in group B2 were less highly resistant to erythromycin than were group B1 strains: none grew at 128 mg/L, and trailing end-points suggested heterogeneity. In contrast both to erythromycin-sensitive strains and B1 strains, B2 strains were more susceptible to oleandomycin than to erythromycin, and often showed a small zone of inhibition around the oleandomycin disc. Whereas erythromycin induced resistance to clindamycin, telithromycin, quinupristin and rokitamycin in all group B strains, oleandomycin did so only in group B1 strains: there was great variability in the interaction between oleandomycin and the other antibiotics for the group B2 strains, and synergy was observed for some with telithromycin. Japanese workers30,31 showed more than 30 years ago that oleandomycin and erythromycin may have different inducing abilities for certain staphylococci, but since then this phenomenon has been largely ignored.
Phenotype C
All the strains in this grouping would be classified as constitutively resistant by conventional testing using only erythromycin and clindamycin. C1 and C2 strains had very similar phenotypesMLKSBMac and MLKSABMac, respectively. Another phenotype found during this investigation that would be classified loosely as constitutively resistant, but is in fact novel is that of the ketolide-resistant mutants selected from three S. haemolyticus strains from group B, whose phenotype was MLKMac/iSB. It should be noted, however, that the phenotype found in the majority of phenotype B strains selected with telithromycin was the classical C1 pattern.
Phenotype D
Strains that are resistant to erythromycin and sensitive to clindamycin (no induction) have been generally called MS or PMS in staphylococci,32 and M phenotype or novel resistance (NR) in streptococci.33,34 The two S. aureus strains in this group (D2) were resistant only to erythromycin and oleandomycin, in contrast to the CNS strains (D1) in which resistance could be induced to quinupristin and telithromycin. Another difference was that the uncoupling agent dinitrophenol reduced the MICs of erythromycin for D2 but not for D1 strains.
Two additional novel phenotypes, MSB/iK in S. haemolyticus and Staphylococcus saprophyticus (both D1) and MSBK in S. aureus, were produced by selection of these strains with telithromycin (Table VI).
Ketolides, the latest members of the macrolide group, show good activity against a wide range of Gram-positive species, including important respiratory pathogens. The survey reported above shows that telithromycin, which has the advantage over the quinupristindalfopristin combination of being orally bioavailable, is active against the great majority of staphylococci. Adding telithromycin to the battery of tests for resistance to the MLS antibiotic group has revealed some novel phenotypes, which may be of epidemiological interest.
From a clinical viewpoint, careful monitoring must be continued to determine the incidence of constitutive resistance, as such strains are insensitive to the MLKSB antibiotics. The relative ease of selection of constitutive mutants from inducible strains (group BC conversion) also suggests that vigilance be exercised when novel members of this group are used to treat infections caused by inducible strains.
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Acknowledgments |
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Notes |
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References |
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Received 18 April 2000; returned 30 July 2000; revised 9 August 2000; accepted 19 August 2000