1 Department of Bacteriology, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan; 2 Central Public Health Laboratory, London, UK
Received 24 January 2002; returned 22 July 2002; revised 5 September 2002; accepted 14 October 2002
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Abstract |
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Keywords: PBP2, PBP2', mecA, MRSA, autolysis
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Introduction |
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Materials and methods |
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The S. aureus strain 61/5896 is a penicillinase (PCase)-producing, methicillin-resistant (MIC 64 mg/L) clinical strain isolated in 1961 in England. Identification of the strain was confirmed by Staphylogram (Wako, Tokyo, Japan), and it was a producer of type-4 coagulase. H44 and N315 are Japanese mecA-positive S. aureus strains,57,20 and Mu3 is a heterogeneous vancomycin-resistant MRSA strain described previously.21,22 The type strain FAD209P was obtained from the National Institute of Health in Japan. The strains were stored in a 80°C freezer or freeze-dried without added drugs in our laboratory.
Stability test of methicillin resistance
Stability of methicillin resistance expressed by strain 61/5896 was tested by serial daily passage in drug-free heart-infusion (HI) broth at 37°C for up to 2 weeks. A 1/106 portion of a drug-free culture of 61/5896 in 10 mL of HI broth was transferred daily to another tube containing pre-warmed fresh HI broth and incubated with shaking at 37°C for a total period of 12 days. A portion of the culture was appropriately diluted and spread on to HI agar plates with and without 8 mg/L of methicillin on the 3rd, 7th and 12th day of passage. The temporal change in the proportion of methicillin-susceptible cells in the culture was monitored.
Obtaining derivatives from 61/5896
Thirty-six derivatives of 61/5896 were obtained by serial drug-free passage as described above. They were arbitrarily picked from the colonies that grew on drug-free HI agar plates of the 3rd, 7th and 12th overnight cultures (12 colonies from each date). Derivatives 3A, 3B and 3C were the representatives of those derived from the 3rd overnight culture, and 12A and 12B from the 12th overnight culture. Derivative 12B was intentionally sought as a non-producer of penicillinase, to test the contribution of the penicillinase plasmid to the methicillin resistance. It was obtained by screening ~1000 colonies grown from the 12th overnight culture with nitrocefin (see below) and was the sole penicillinase-non-producing colony; the penicillinase plasmid was spontaneously eliminated during the passage. Methicillin-resistant revertants, 12AR and 12BR, were obtained from methicillin-susceptible derivatives, 12A and 12B, respectively, by plating aliquots of their overnight cultures on to HI agar plates containing 25 mg/L of methicillin. The plates were then incubated at 37°C, and grown colonies were picked, purified and confirmed to be revertants.
MIC determination
MICs were determined either by an agar dilution method as described previously,20 or by Etest (AB Biodisk, Solna, Sweden). The Etest was carried out on MuellerHinton agar plates swabbed with bacterial cell suspension adjusted to McFarland Standard No. 1. After the plates had been dried briefly, Etest strips were applied, and then the plates were incubated at 37°C for 24 h. The MIC was read as the point at which the zone of complete inhibition intersected the MIC scale. To test the possible contribution of penicillinase production to methicillin resistance, MICs were compared in the presence and absence of 4 mg/L of clavulanic acid, a ß-lactamase inhibitor12 incorporated in the agar plates of the plate-dilution method.
Detection of mecA by PCR and Southern hybridization
PCR detection of mecA was carried out with DNA extracted from each strain as a template,23 and using mecA primers 5'-TGCTATCCACCCTCAAACAGG-3' and 5'-AACGTTGTAACCACCCCAAGA-3',23 and attB primers 5'-CTCGCATAATCTTAAATGCTCT-3' and 5'-AAACGACATGAAAATCACCAT-3' as previously described.6,24 Amplified DNA was detected by agarose gel electrophoresis.
Southern hybridization for the detection of mecA has been described previously.20 Extracted cellular DNA was digested with HindIII, and then hybridized with a mecA-specific probe that was prepared from pMR111 containing a 4.0 kb insert of mecA.23
Population analysis
Analysis of methicillin-resistant subpopulations of strain 61/5896 and its derivative strains was carried out as described previously.20
Detection and quantification of ß-lactamase activity
ß-Lactamase activity of strain 61/5896 was detected by a colour change of nitrocefin (BBL, Becton Dickinson, MD, USA). Quantitative analysis of the ß-lactamase activity was carried out with a macro-iodometric method25 with ampicillin and methicillin as substrates. The crude ß-lactamase enzyme was prepared as follows. Strain 61/5896 was grown in L-broth with or without 1 mg/L methicillin at 37°C for 4 h. Cells were precipitated by centrifugation at 10 000g for 20 min, washed with 0.01 M sodium phosphate buffer pH 7.0 and resuspended in the same buffer. The cells were then disrupted with a Branson Sonifier (Branson Ultrasonics Co., CT, USA) with a sonication cycle of 10 times (1 min each), with a setting of 20 KHz, 50 W and 20% efficiency. The cell debris was removed by centrifugation at 100 000g for 30 min. The supernatant was used as the crude ß-lactamase.
Detection of PBP2 with western blotting
The bacterial cells (1 x 109 cfu) were harvested in exponential growth phase, washed with and resuspended in 0.05 M TrisHCl, pH 7.4. The membrane fraction was obtained by centrifugation at 100 000g for 30 min following disruption of bacteria by sonication. The membrane preparation (20 µg of protein) was subjected to SDSPAGE, and the separated proteins were transferred to PVDF membrane (Immobilion-P; Millipore Corp., Bedford, MA, USA). The membrane was incubated in PBST buffer (PBS pH 7.2, 0.05% Tween 20) supplemented with 3% skimmed milk (SM) to block non-specific reaction and then incubated with PBP2-specific rat antibody26 in PBST with 1% SM for 1 h at room temperature. The membrane was washed with PBST, and incubated with peroxidase-conjugated goat anti-rat IgG (Chemicon International Inc., Poway, CA, USA) in PBST containing 1% SM for 1 h at room temperature. After extensive washing, the PBP2 band was visualized with 3,3'-diaminobenzidine tetrahydrochloride, cobalt chloride and 0.2% hydrogen peroxide. Comparison of the amounts of PBP2 produced by the test strains was made by densitometry using Scanalytics (CSPI, Billerica, MA, USA).
Transmission electron microscopy
The bacterial cells, in exponential growth phase (OD600 = 0.5) were fixed in 2% glutaraldehyde in 0.1 M sodium phosphate buffer pH 7.4 for 2 h, and treated with 1% osmium tetroxide for 2 h at 4°C. Cells were dehydrated with graded (70100%) concentrations of ethanol, and embedded in EPOK812 (Ohken, Tokyo, Japan). Ultra-thin sections were stained with uranyl acetate and lead citrate, and then examined with a transmission electron microscope, JEM-1230EX (JEOL, Tokyo, Japan).
Autolysis assay
Autolytic activities of 61/5896 and its derivative strains were tested as follows. Cells in the exponentially growing culture (OD600 = 0.7) were harvested by centrifugation at 3700g for 5 min, washed once with saline and resuspended in 0.01 M sodium phosphate buffer pH 7.0. Autolysis was monitored as a decrease in OD600 with Bio-Photo Recorder TN-2612 (Advantec, Osaka, Japan) during aerated incubation at 37°C.
Detection of bacteriolytic enzyme activity
A zymogram detecting autolysin activity was carried out as described previously21 with a slight modification. Briefly, S. aureus strains were grown to OD600 = 1.0 in 250 mL of HI broth at 37°C. After harvesting cells by centrifugation and washing once in 50 mM TrisHCl (pH 6.8), the cell-wall-associated autolysin molecules were extracted with 1 mL of 4% SDS in 100 mM TrisHCl pH 6.8 at 37°C for 30 min.
Incorporation of [14C]N-acetylglucosamine into cells in the resting medium
The bacterial strains were cultivated at 37°C for 18 h in MuellerHinton broth. The culture was diluted 20-fold with fresh pre-warmed MuellerHinton broth, and further cultivated until OD578 = 0.7 was reached. The cells were harvested by centrifugation at 12 000g for 10 min. The pellet was washed once with cold resting medium (RM; 28.5 mM glucose, 1 mM glycine, 1 mM D-glutamic acid, 0.5 mM DL-Ala-DL-Ala, 0.2 mM L-lysine, 1 mM MgCl2, 0.1 mM MnCl2, 0.17 mM uracil, 80 mM K2PO4, 8.2 µM nicotinamide, 3 µM thiamine, pH 7.4) and re-suspended in 4 mL of RM.27 The cell suspension was added to 8 µL of [14C]N-acetylglucosamine (1.85 MBq/mL; Amersham, Arlington Heights, IL, USA) to a final concentration of 0.44 µg/mL and to 12 µL of cold N-acetylglucosamine (Sigma Chemical Co., St Louis, MO, USA) to a final concentration of 12 µg/mL, followed by incubation at 37°C with gentle shaking. After 15, 30, 60 and 90 min incubation, a 0.5 mL portion of the cell suspension was transferred to a microfuge tube containing a mixture of 25 µL of 10 mg/mL N-acetylglucosamine and 0.5 mL of 0.1% SDS. After centrifugation at 12 000g for 3 min, the supernatant was discarded. The cell pellet was washed once with distilled water, and resuspended in 0.5 mL of distilled water. The cell suspension was then mixed with 5 mL of AQUA SOL2 (Packard Inc., Palo Alto, CA, USA) for radioactivity measurement using a liquid scintillation counter LS3801 (Beckman Instruments Inc., Palo Alto, CA, USA).
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Results |
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Strain 61/5896 expressed a significant level of methicillin resistance that was comparable to that expressed by MRSA strains carrying mecA integrated in the chromosome. The MICs of representative antibiotics, as determined with a plate-dilution method, were 64 mg/L for methicillin, 32 mg/L for oxacillin, 64 mg/L for ampicillin, 1024 mg/L for ceftizoxime, 1.5 mg/L for vancomycin and 1.5 mg/L for teicoplanin. To further characterize the phenotype of ß-lactam resistance expressed by the strain, we analysed the population profile of the strain against oxacillin and methicillin. Figure 1 illustrates the shape of the population curve for oxacillin, which was essentially the same as that for methicillin (data not shown). Strain 61/5896 yielded a population curve typical of heterogeneous methicillin resistance (hetero-resistance) expressed by MRSA strains.
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PCR was carried out with DNA extracted from strain 61/5896 and control mecA-positive strains H44 and N315 as templates and using the set of mecA-specific primers. Although the expected band of 298 bp was amplified from H44 and N315, no band was amplified from 61/5896. Southern blot hybridization with a mecA-gene probe also failed to detect the gene in 61/5896 (data not shown). Furthermore, mec right extremity polymorphism (MREP) typing, used to detect the right end of the mobile genetic element SCCmec carrying mecA,6,24 did not detect any SCCmec;28 and the primers flanking the integration site (attBscc) for SCCmec amplified the chromosomal DNA fragment without an integrated copy of SCCmec24 (data not shown). From these results, it was concluded that mecA was absent from the chromosome of strain 61/5896.
Unstable nature of methicillin resistance of 61/5896
To investigate the stability of the phenotypic expression of methicillin resistance of strain 61/5896, the strain was subjected to serial daily passage in drug-free HI medium for 12 days. On days 3, 7 and 12, part of the culture was screened for the emergence of methicillin-susceptible derivatives by replicating colonies grown on the drug-free HI agar plates on to agar plates containing 8 mg/L methicillin. The proportion of colonies susceptible to 8 mg/L methicillin increased with time: 0.15% (six of 3982 colonies) on day 3, 79.7% (306 of 384) on day 7 and 100% (496 of 496), on day 12. Thus, the methicillin-resistance phenotype of 61/5896 was unstable in the drug-free HI medium and was lost completely by day 12.
ß-Lactam and glycopeptide susceptibilities of the derivatives of 61/5896
To investigate how the phenotype of 61/5896 changed during drug-free passage, 36 derivative strains were obtained on the 3rd, 7th and 12th day of passage. MICs of methicillin, oxacillin, vancomycin and teicoplanin were determined by Etest. Glycopeptide MICs were measured because they are correlated with ß-lactam resistance in some MRSA strains.22 Table 1 compares MICs for 61/5896 and its representative derivatives studied in detail. For all the derivatives, susceptibilities to methicillin and oxacillin correlated well with each other [correlation coefficient = 0.939 (P < 0.0001), data not shown]. Some derivatives obtained on the 3rd day of passage had greater oxacillin and methicillin MICs than the parent strain (Table 1). The derivatives obtained on the 12th day of passage, on the other hand, were below the breakpoint for methicillin resistance (4 mg/L).
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Seven derivatives of 61/5896 were subjected to population analysis (Figure 1). Two of the three derivatives in the 3rd overnight culture had a population curve comparable with that of the parent strain, whereas derivative 3B lost oxacillin resistance.
Most cells of derivatives 12A and 12B, obtained from the 12th serial culture, were susceptible to oxacillin; but 12A still retained small subpopulations of cells resistant to 832 mg/L of oxacillin, and was thus heterogeneously resistant to oxacillin (Figure 1). Derivative 12B, a non-producer of ß-lactamase, did not contain any subpopulation resistant to 2 mg/L oxacillin (Figure 1). The methicillin-resistant revertant, 12AR, had increased resistant subpopulations against oxacillin concentrations from 0.5 to 32 mg/L compared with its parent, 12A. Revertant 12BR had a population pattern of heterogeneous oxacillin resistance (Figure 1).
The role of penicillinase and penicillinase plasmid in the methicillin resistance of 61/5896
Penicillinase overproduction has been reported to cause borderline methicillin resistance in some S. aureus clinical strains.12 To evaluate the possible contribution of penicillinase to the methicillin resistance of 61/5896, its methicillin MIC was determined in the presence of 2 and 4 mg/L of clavulanic acid. There was no change in the methicillin MIC for 61/5896 in the presence of clavulanic acid. To test directly whether the penicillinase produced by the strain could hydrolyse methicillin, the crude penicillinase preparation was added to solutions of methicillin and ampicillin, and residual ß-lactamase activity was assayed both with macro-iodometric and bioassay methods. The hydrolysis rate of ampicillin and methicillin was 11.88 and 0.03 µmol/min/mg, respectively. This hydrolysis rate of methicillin was interpreted as the background level since the same value was recorded with the cell extract from strain 12B, the penicillinase-negative derivative of 61/5896. Table 2 compares the ampicillin hydrolysis activity of 61/5896 and its derivative strains. The penicillinase activity produced by methicillin-susceptible strain 12A was similar to that of the resistant strains; thus, there was no change in the production of penicillinase between 12A and 61/5896.
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Figure 2 shows western blot detection of PBP2 in 61/5896 and its derivatives. The amount of PBP2 produced by methicillin-susceptible derivative strains 12A and 12B was less than one-third of that produced by the parent strain 61/5896. PBP2 was again produced in significantly higher amount by methicillin-resistant revertants 12AR and 12BR; the amount was comparable to that of 3A and 3B (Figure 2). However, the correlation between PBP2 production and methicillin resistance was incomplete since PBP2 was produced in comparable amounts by 61/5896 and by its methicillin-susceptible derivative, 3B (Figure 2).
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Figure 3 shows transmission electron micrographs and the cell-wall thickness (mean ± S.D. in nm) of strain 61/5896 and its derivatives cultivated in drug-free L-broth. Inspection of cut cell surfaces in the micrographs revealed a thick cell wall characteristic of 61/5896 (represented by the cell in Figure 3a), which was not seen in methicillin-susceptible strains 3B, 12A and 12B (in Figure 3c, d and e, respectively). Also the rough outer surface of the cell wall, which was a morphological characteristic of 61/5896, was not observed in its derivatives. Abnormal septum formation was observed in 210% of the 61/5896 cells in the absence of methicillin (data not shown).
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The autolytic activity of strain 61/5896 and its derivatives was compared by monitoring the rate of decrease in the optical density of the cell suspensions in phosphate buffer (Figure 4). Strain 61/5896 had remarkably little autolytic activity in comparison with its derivatives. Autolytic enzyme production by these strains was analysed with zymograms. Strain 61/5896 did not produce autolytic bands, whereas its derivatives showed evident autolytic bands similar to those of the control strain, FDA209P (data not shown). The absence or presence of autolytic bands and the levels of autolytic activity demonstrated in Figure 4 correlated well. Derivatives 12A and 12B produced a large amount of the lytic enzyme.
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Figure 5 shows the rate of incorporation of [14C]N-acetylglucosamine into a fixed number (108 cfu) of cells. The incorporation of 14C-labelled N-acetylglucosamine into the cell wall increased linearly in the initial 90 min for all the strains tested. The total radioactivities of cell walls measured at 90 min with cells of 61/5896, 3A, 12B and 12BR were 5.4, 4.6, 4.0 and 8.7 times higher, respectively, than that of FAD209P. However, the rate of incorporation of N-acetylglucosamine of 61/5896 was only 25% of that observed with Mu3, the heterogeneously vancomycin-resistant MRSA strain (hetero-VRSA).22
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Discussion |
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Strain 61/5896 possessed an unusually reduced autolytic activity, which correlated with decreased autolysin production since no autolytic band was detected by zymography. Derivative strains showed evident bands similar to those produced by control strain FDA209P.30 Although a reduction of autolytic activity has been associated with high or homogeneous methicillin resistance in MRSA,5,3133 the degree of reduction in 61/5896 was only comparable to that observed in autolysin-defective mutant strains.34 The characteristic rough outer cell surface of 61/5896, which disappeared with a concomitant increase in autolysis, is also a characteristic of autolysin-defective S. aureus strains.34 It was curious that the increase in autolytic activity was accompanied by an increase in methicillin resistance (Table 1). On the other hand, derivative strains with methicillin susceptibility and increased autolysin production were also obtained, such as 12A and 12B. Therefore, it seems that suppression of autolysis in 61/5896 is not the direct cause of methicillin resistance in the strain. In contrast, a significant decrease in teicoplanin MIC was observed with increased autolytic activity: the teicoplanin MIC was 1.5 mg/L for 61/5896, whereas values for the derivatives were <0.4 mg/L. Suppression of autolysis may therefore be more closely associated with the change in sensitivity of the S. aureus cell wall to teicoplanin.
Teicoplanin resistance (and vancomycin resistance to a smaller degree) of S. aureus is caused by increased production of PBP2.35,36 We have shown that experimental over-production of PBP2 in S. aureus cells increases the MIC from 2 to 8 mg/L.21 In this study, a relatively increased amount of PBP2 was demonstrated in 61/5896 compared with its derivatives susceptible to methicillin and glycopeptides (12A and 12B; Figure 2). Moreover, the amount of PBP2 was significantly increased in revertants 12AR and 12BR, which had increased teicoplanin MICs as well as increased methicillin resistance. A significant reduction in PBP2 production in strains 12A and 12B is consistent with their hyper-susceptibility to teicoplanin. Thus, the amount of PBP2 and teicoplanin susceptibility correlated well.
On the other hand, the relationship between the amount of PBP2 and methicillin resistance was more complex. Derivative 3B was susceptible to methicillin despite producing a comparable amount of PBP2 to 61/5896 (Figure 2). This indicates that an increase in PBP2 production per se does not raise methicillin resistance. This is supported by an experiment in which we introduced a multi-copy recombinant plasmid carrying pbp2 into an S. aureus strain N315.20 The transformant had increased teicoplanin resistance (the MIC was raised from 2 to 8 mg/L), but its methicillin susceptibility was not changed (K. Kuwahara-Arai, unpublished data).
Tonin & Tomasz17 reported increased penicillin-binding capacities of PBP2 and PBP4 in an in vitro-trained methicillin-resistant mecA-negative mutant strain (MIC, 100 mg/L). Berger-Bächi et al.16 also reported increased penicillin-binding capacities of PBP2 and PBP4 in the methicillin-resistant mutants obtained by step-wise selection of S. aureus strain NCTC 8325 with increasing concentrations of methicillin. By measuring the penicillin G-binding kinetics, Chambers et al.15 observed increased production of PBP4 and slower acylation and more rapid deacylation rather than increased production of PBP1 and PBP2 in such a laboratory mutant strain compared with its methicillin-susceptible parent strain. Therefore, besides PBP2, mecA-independent methicillin resistance seems to be associated with the alteration of other PBPs as well. Pure strain 61/5896 was distinct from the strains produced in vitro with ß-lactam selective pressure. Our current hypothesis is that the PBP2 overproduction observed after one-step methicillin selection of derivatives 12A and 12B to obtain 12AR and 12BR, is one of multiple phenotypic changes caused by a mutation that confers methicillin resistance to the cell. We consider that the PBP2 overproduction is effective in raising methicillin resistance only when combined with other concomitant phenotypic changes, whereas it per se is sufficient to confer teicoplanin resistance. Significant thickening of the cell wall as observed with electron microscopy, and increased cell-wall synthesis,36 as reflected in the increased incorporation of N-acetylglucosamine, may be some of those concomitant phenotypic changes.
In this report, we describe a novel class of methicillin resistance in S. aureus, which is distinct from either mecA-mediated resistance or penicillinase-mediated resistance. It is also discriminated from any of the in vitro selected mutant strains based on its characteristic features. The most striking feature of the resistance was its instability. By cultivation of strain 61/5896 in drug-free medium, we generated a series of derivatives whose phenotypes varied from that of the parent strain in terms of autolysis, cell-wall thickness, PBP2 production and degrees of methicillin (oxacillin) and glycopeptide susceptibility. However, none of these phenotypes correlated simply with the methicillin resistance of the strain. This indicates that resistance expression is caused by a certain physiological status of the strain, which the cell is forced to take by external selective pressure and which is easily replaced when that selective pressure is removed. Elucidation of the precise mechanism of resistance of this strain requires another approach to those described here. Taking advantage of the whole genome sequence of S. aureus,37 we are now analysing the transcriptome of 61/5896 in comparison with those of its derivatives and other S. aureus strains. Pursuit of the resistance mechanism of 61/5896 will not only reveal the physiology of this particular strain, but may reveal much about the as-yet unclear mechanism of hetero-methicillin resistance expressed by many mecA-carrying MRSA strains.
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Acknowledgements |
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Footnotes |
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References |
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