a Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK; b National Reference Centre for Antibiotic Resistance in Bacteria, Sera and Vaccines Central Research Laboratory, Chelmska 30/34, 00-725 Warsaw, Poland
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Abstract |
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Introduction |
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A number of susceptibility testing guidelines recommend that S. aureus strains are routinely tested for resistance to tetracycline, but it is not clear how to interpret the results of such tests as breakpoints for susceptibility and resistance have not been agreed.711,13
Two mechanisms of resistance to tetracyclines have been identified in Staphylococcus spp.: (i) active efflux resulting from acquisition of the plasmid-located genes, tetK1416 and tetL, and (ii) ribosomal protection mediated by transposon-located or chromosomal tetM or tetO determinants.17,18 S. aureus strains carrying tetK only have been described as resistant to tetracycline, but susceptible to minocycline.19,20 The tetM gene is believed to confer resistance to all available drugs of the group, including tetracycline and minocycline.19 Most tetM-positive isolates also carry the tetK gene and MRSA isolates are typically of tetM or tetKM genotype.19 The tetL gene has been found only in S. aureus isolates already carrying the tetM gene.19 There are no reports of tetO-positive S. aureus strains. Both drug efflux and ribosomal protection are inducible in S. aureus in vitro.15,17
In this study, 66 randomly selected tetracycline-resistant MRSA isolates were examined with regard to genotype and expression of resistance. The aim was to establish a method for the phenotypic identification of resistance to tetracyclines in S. aureus, particularly MRSA isolates.
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Materials and methods |
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Sixty-six clinical isolates were analysed, 52 of which were collected by the Sera and Vaccines Central Research Laboratory (SVCRL) between 1992 and 1997 from 16 hospitals in nine Polish towns. These isolates had already been typed by various methods, including macrorestriction analysis of SmaI-digested chromosomal DNA.2127 Of the remaining 14 isolates, three were from Turkey, three from the UK, two each from Bulgaria, Latvia and Slovenia, and one each from Hungary and Russia; these were collected between 1995 and 1997. Species identification was based on colony morphology, Gram's stain, cell morphology, presence of catalase, and the tube coagulase test with rabbit plasma (Biomed, Krakow, Poland). S. aureus 25923, S. aureus 29213, Enterococcus faecalis 29212 and Escherichia coli 25922, obtained from the American Type Culture Collection (ATCC), were used for quality control of susceptibility testing. All isolates were stored in tryptic-soy broth (Lab M, Bury, UK) frozen at 70°C with glycerol at concentrations of 1030%.
Susceptibility testing
Resistance to methicillin was detected by screening on tryptic-soy agar (TSA) (Oxoid, Basingstoke, UK) supplemented with methicillin 25 mg/L as previously described,28 and confirmed by PCR-based detection of the mecA gene.29
Resistance to tetracycline was detected by screening on MuellerHinton agar (Oxoid) supplemented with tetracycline 5 mg/L. A quarter of the screening plate was inoculated with cells from a single colony harvested from the TSA after overnight incubation. Strains were classified as resistant when any growth was observed after 20 h incubation.
For all isolates, MICs of tetracycline, doxycycline and minocycline were evaluated both with and without induction of resistance. For each strain, a single colony from overnight growth on TSA was used to inoculate three different MuellerHinton agar plates: (i) unsupplemented medium (lack of induction); (ii) MuellerHinton agar with tetracycline 5 mg/L (induction of resistance by tetracycline); and (iii) MuellerHinton agar with minocycline 0.5 mg/L (induction of resistance by minocycline). After overnight incubation, harvested cells were used to determine MICs using NCCLS guidelines.9
Resistance to tetracyclines was also determined by the disc diffusion method with tetracycline, doxycycline and minocycline discs, using NCCLS-recommended procedures.13 The double disc test technique was developed for identification of inducible resistance to minocycline. This involved placing a 30 µg tetracycline disc, a 30 µg minocycline disc and a 5 µg tetracycline disc in a straight line, 89 mm apart, with the minocycline disc in the centre (Figure). The zone diameter around the minocycline disc was measured horizontally (in the plane of the surface of the agar) and vertically, perpendicular to the first measurement.
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Cefotaxime, doxycycline, minocycline and oxacillin powders were obtained from SigmaAldrich (Poole, UK) tetracycline from NBL Gene Sciences (Cramlington, UK) and methicillin from SmithKline Beecham Pharmaceuticals (Betchworth, UK). Tetracycline (30 and 5 µg), doxycycline (30 µg) and minocycline (30 µg) discs were from Oxoid.
Statistical methods
Friedman's test30 was used to determine the significance of changes in MICs after preincubation of cells of the same genotype with tetracycline and minocycline. The Kruskal Wallis test30 was used to compare differences between MICs for strains of different genotypes subjected to the same treatments. When these differences proved significant, Dunn's procedure30 was used to make pairwise comparisons between treatments in the first case, and between genotypes in the second. A P value of <0.05 was considered significant.
Detection of tetK, tetL, tetM and tetO by PCR
DNA templates were prepared as follows. Tryptic soy broth supplemented with oxacillin and cefotaxime at 6 mg/L was inoculated with a single colony of an MRSA strain. After incubation with vigorous shaking for 24 days at 30°C, 500 µL of culture was centrifuged at 15000g for 3 min. Pellets were resuspended in 400 µL of PBS, harvested by centrifugation, resuspended in 100 µL of 20 mM TrisHCl (pH 8.4), 50 mM KCl and 3 mM MgCl2, incubated for 10 min at 99°C in a Touchdown thermocycler (Hybaid, Teddington, UK), and then immediately centrifuged for 2 min at 15000g at 4°C. Supernatants were used as DNA template for PCR. Each reaction was carried out in 50 µL of mix containing 20 mM TrisHCl (pH 8.4) and 50 mM KCl, 3 mM MgCl2, 0.2 mM each of dATP, dCTP, dGTP and dTTP, 0.5 mM of each primer, 2.5 U of Taq DNA polymerase (Gibco BRL, Paisley, UK) and 2 µL of DNA template. The following primers were used: for tetK detection, tetK-up (5'-TATTTTGGCTTTGTATTCTTTCAT-3') and tetK-rev (5'-GCTATACCTGTTCCCTCTGATAA-3'); for tetL detection, tetL-up (5'-ATAAATTGTTTCGGGTCGGTAAT-3') and tetL-rev (5'-AACCAGCCAACTAATGACAATGAT-3'); for tetM detection, tetM-up (5'-AGTTTTAGCTCATGTTGATG-3') and tetM-rev (5'-TCCGACTATTTAGACGACGG-3') and for tetO detection, tetO-up (5'-AGCGTCAAAGGGGAATCACTATCC-3') and tetO-rev (5'-CGGCGGGGTTGGCAAATA-3'). The PCR consisted of 35 cycles of 1 min at 95°C, 1 min at 50°C, 1 min 30 s at 72°C, followed by a final 5 min at 72°C, except for tetO, for which the annealing temperature was 55°C. Amplified products were run in 1.5% ultraPure agarose (Gibco BRL) with ethidium bromide and photographed under UV light. Detection of the 1862 bp fragment from positions 211882 of the published sequence of the S. aureus tetM gene17 and a 1159 bp amplicon from positions 471205 of the published sequence of the S. aureus tetK gene16 was taken as indicative of the presence of the tetM and tetK gene, respectively. The expected product for the tetO gene was 1723 bp (base pairs 1461868 of the published sequence of the Streptococcus mutans tetO gene31) and that for the tetL gene was an amplicon of 1077 bp (base pairs 2621338 of the published sequence of the Enterococcus faecalis tetL gene32). A 1 kb DNA ladder (Gibco BRL) was used as a molecular weight marker.
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Results |
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MIC results for all 66 MRSA isolates are presented in Table I. In the absence of induction, the MICs of all three antibiotics varied among genotypes. However, pairwise comparisons between tetracycline MICs for isolates of tetK and tetM genotypes, and minocycline MICs for isolates of tetM and tetKM genotype, showed no significant differences (P > 0.2). In contrast, levels of tetracycline resistance in tetKM genotype isolates were significantly higher than those for both single-gene clusters (P < 0.0001), and the MIC geometric mean was over four-fold greater. MICs of doxycycline for tetM isolates were significantly higher than those for tetK isolates (P < 0.05). Isolates of the tetKM genotype again had significantly higher MICs than both of the single-gene isolates (P < 0.0001), and the geometric mean was also over four-fold greater. All MRSA of the tetK genotype were susceptible to minocycline
0.25 mg/L; they were significantly more susceptible than the other genotypes (P < 0.0001). The highest MICs of tetracyclines were observed in isolates of the tetKM genotype.
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Comparison of MIC geometric means for isolates of the tetM genotype before and after induction showed that the greatest increase in MICs of minocycline occurred when cells were preincubated with tetracycline (ratio 4.4) and after induction by minocycline (ratio 3.9). The lowest increase was observed for MICs of tetracycline when induced by tetracycline (ratio 1.2) or minocycline (ratio 1.5). For doxycycline MICs, a 1.7-fold increase in the geometric mean after preincubation with tetracycline and a 2.5-fold increase after minocycline treatment were observed. All six of these increases were statistically significant (P < 0.05).
Induction of tetracycline resistance was weak in tetKM isolates; although there was significant variation between the three treatment groups (P = 0.025), neither of the pairwise comparisons between induced and uninduced cells showed significant differences on their own (P > 0.2 for tetracycline preincubation; 0.05 < P < 0.1 for minocycline preincubation). Corresponding increases in the MIC geometric means were 1.1-fold and 1.2-fold, respectively. In MRSA of the same genotype, MICs of doxycycline increased 1.4- and 2.4-fold, and MICs of minocycline 1.6- and 2.8-fold after tetracycline and minocycline pretreatment, respectively. All but the first of these were significant at the 5% level.
No significant differences were found in MICs of tetracycline and minocycline between isolates induced with tetracycline and those induced with minocycline. However, isolates induced with minocycline showed significantly higher doxycycline MICs than those induced with tetracycline for both tetKM and tetM isolates (P < 0.01 and P < 0.05, respectively).
In 12 out of 21 tetKM isolates, MICs of minocycline were 8 mg/L, whereas only two tetM isolates had MICs as high as 8 mg/L. Both the MIC50 and MIC90 were 8 mg/L for tetKM isolates; for tetM isolates the corresponding values were 2 and 4 mg/L, respectively. For four of the tetKM isolates, MICs of minocycline were as low as
1 mg/L for uninduced cells, and no more than one dilution higher when tetracycline was used as an inducer. In two of these, preincubation with minocycline clearly induced resistance, giving an eight-fold increase from 1 to 8 mg/L for the first, and a 64-fold increase from 0.5 to 32 mg/L for the second. For the other two isolates, induction by minocycline was weaker (leading to a two-fold increase in MIC).
Results of tetracycline susceptibility testing by the disc diffusion method are presented in Table II. Zone diameters around the 30 µg minocycline disc were
27 mm for tetK isolates and
25 mm for tetM isolates. Lack of inhibition around the 30 µg tetracycline disc was indicative of the tetKM genotype as a zone of inhibition was observed with all other genotypes. In the double disc test, an oval zone of inhibition around the minocycline disc was observed for all tetM isolates (Figure
): horizontal zone diameters were 15 mm (mean 3.3 mm) smaller than vertical diameters. For isolates of the tetK and tetKM genotype, zones around the minocycline disc appeared circular. Stronger induction of resistance to minocycline was achieved using a 5 µg tetracycline disc than with a 30 µg disc (data not shown). Analysis of the results for all three tetracyclines generally showed good correlation between the agar dilution method using uninduced cells and the disc diffusion method. An exception was doxycyline testing of isolates of the tetK genotype, for which zone diameters around the 30 µg doxycycline disc were smaller than would be expected from the doxycycline MICs.
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Discussion |
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PCR characterization of the tet determinants in the isolates selected for this study showed that they comprised three different genotypes. The tetK genotype was identified only in Polish isolates heterogeneously resistant to methicillin. This is the first report of a pure tetK genotype in MRSA. The other two genotypes identified, namely tetM alone and tetKM, were observed in Polish S. aureus isolates homogeneously resistant to methicillin and in isolates from other European countries.19
As reported previously,19,20 S. aureus isolates of the tetK or tetM genotype were resistant to tetracycline both by breakpoint and agar dilution methods, and all isolates of the tetK genotype were susceptible to minocycline, irrespective of the breakpoints used. However, when French breakpoints were applied, the agar dilution method led us to identify tetK- and tetM-positive isolates as susceptible to doxycycline, and both agar dilution and diffusion methods indicated that some tetM and tetKM isolates were susceptible to minocycline.11 Using NCCLS recommendations, some tetM isolates would be identified as intermediately susceptible to tetracycline by the disc diffusion method, and some tetK and tetM as intermediately susceptible to doxycycline by both methods.9,13 In contrast, all 66 isolates would be identified as resistant to tetracycline and resistant or intermediately susceptible to doxycycline according to the Scandinavian breakpoints for both MICs and zone diameters.9 There are no NCCLS or Scandinavian recommendations for minocycline breakpoints. Current BSAC guidelines do not mention testing staphylococci against tetracyclines.8 However, according to MIC breakpoints previously recommended by the BSAC, all strains in this study would be identified as resistant to tetracycline.36 All the breakpoints applied, for both agar dilution and disc diffusion, unanimously indicated full resistance to tetracycline and doxycycline only in the tetKM genotype.
It was confirmed that higher levels of resistance can be induced by subinhibitory concentrations of tetracyclines for both the resistance mechanisms described. After induction, all isolates were resistant to tetracycline and doxycycline according to all the guidelines applied, and an increase in resistance to minocycline was observed in tetM isolates. This is the first time that minocycline and tetracycline have been documented to cross-induce resistance to each other in tetM-positive S. aureus isolates in vitro. An elevated level of tetracycline resistance in strains harbouring both genes has been described previously.19,20 Here, higher levels of resistance for tetKM isolates were also observed for doxycycline and minocycline.
For the identification of tetracycline-resistant genotypes, the double disc test appears to be an alternative to molecular methods. The data suggest that lack of any zone of inhibition around a 30 µg tetracycline disc or a high level of resistance to tetracycline (MIC 128 mg/L) and doxycycline (
16 mg/L) may predict the presence of tetKM. The presence of any zone of inhibition around a 30 µg tetracycline disc or low-level resistance to tetracycline (MICs
64 mg/L) or doxycycline (MICs
8 mg/L), together with a positive result in the double disc test (i.e. identification of inducible resistance to minocycline), predicts the tetM genotype. This result would also indicate resistance to all tetracyclines. In the case of low-level resistance to tetracycline and negative results in the double disc test (i.e. lack of induction), the tetK genotype and minocycline susceptibility might be predicted. MRSA strains in which resistance to tetracycline was identified were always resistant to doxycycline.
Double disc tests are already used for detecting other mechanisms of resistance (e.g. inducible-MLSB in Gram-positive cocci,37 chromosomally mediated ß-lactamases38 and extended-spectrum ß-lactamases in Gram-negative bacilli39). The double disc test seems to be useful in the proper identification of the inducible tetM genotype, at least in MRSA. There is no need for the precise measurement of a zone of inhibition around a 5 µg tetracycline disc; using a 5 µg disc instead of a 30 µg one improves the expression of inducible resistance (Figure). If a 5 µg disc is not available, it could be replaced by a quarter of a 30 µg tetracycline one, though standardization of such a test might be difficult. The zones of inhibition around the doxycycline discs were relatively small considering the low doxycycline MICs for tetK isolates. This can be explained by induction of resistance by the drug as it diffuses into the agar.
Because MICs were determined using two-fold concentration increments, the statistical significance of the results was assessed using non-parametric tests. In eight of the nine genotypeantibiotic combinations, significant differences in the MICs between the three different treatments were found. In such cases pairwise comparisons between treatments can also be made. There is a strong case for using a higher overall level of significance than the traditional 5% when making such comparisons, since significant variation has already been found.30 For this reason we report P values of <0.2 in Table I.
The data presented here indicate the importance of the proper recognition of tetracycline resistance mechanisms in S. aureus strains, and the necessity of revising existing breakpoints and interpretation guidelines. The consequences of failing to identify these mechanisms correctly, and of misclassifying strains likely to have inducible resistance as susceptible (as observed for minocycline in tetM-positive isolates19), need to be considered. It is possible that the prevalence of resistance to tetracyclines in S. aureus has been underestimated owing to the false identification of susceptibility. The importance of the induction of resistance for the outcome of infections when treated by drugs of this group should not be underestimated.
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Acknowledgments |
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Notes |
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References |
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Received 30 September 1999; returned 26 November 1999; revised 23 December 1999; accepted 20 January 2000