JOURNAL OF BACTERIOLOGY, May 1984. p. 513-516 Vol. 158, No. 20021-9193/84/050513-04$02.00/0Copyright t 1984, American Society for Microbiology

Low-Affinity Penicillin-Binding Protein Associated with r-LactamResistance in Staphylococcus aureus

BARRY J. HARTMAN* AND ALEXANDER TOMASZ

Division of Infectious Diseases, Cornell University Medical Center, and the Rockefeller University, New York, New York

Received 31 October 1983/Accepted 23 February 1984

Methicillin resistance in Staphylococcus aureus has been associated with alterations in the penicillin-binding proteins (PBPs). An intriguing property of all methicillin-resistant staphylococci is the dependenceof resistance on the pH value of the growth medium. Growth of such bacteria at pH 5.2 completelysuppressed the expression of methicillin resistance. We have examined the PBP patterns of methicillin-resistant staphylococci grown at pH 7.0. We detected a high-molecular-weight PBP (PBP-2a; approximatesize, 78,000 daltons) that was only present in the resistant bacteria but not in the isogenic sensitive strain. Incultures grown at pH 5.2, the extra PBP was not detectable.

Intrinsic beta-lactam resistance associated with penicillin-binding protein (PBP) alterations appears to be a newlyrecognized mechanism present in both laboratory and clini-cal isolates of resistant strains in a variety of differentbacteria (3, 5, 7, 12, 19, 21). The mechanism of resistanceappears to be due to an alteration in the target proteins(PBPs) either in their electrophoretic patterns (8) or in theiraffinities for the beta-lactam antibiotics (2, 8-10). In at leastsome cases, it was possible to demonstrate by genetictechniques that the observed PBP alterations were causallyrelated to the decreased susceptibility of the bacteria to theantibiotic (3, 5, 21). This is particularly important for methi-cillin-resistant Staphylococcus aureus strains, since theyhave become important nosocomial and community-ac-quired infectious agents (4, 17, 20).A peculiar property of methicillin-resistant staphylococci

is the rapid loss of phenotypic resistance by a shift of the pHvalue of the culture medium from the physiological pH valueof 7.0 to pH 5.2 (15, 16). This pH-dependent drop in the MICappears to be shared by all methicillin-resistant strains (seeTable 1). The mechanism of this phenomenon is unknown.In a previous study (9), we demonstrated that resistantstrains grown at both pH 7.0 and 5.2 had the same bindingpattern of the high-molecular-weight PBPs 1, 2, and 3.

In this communication, we examined the PBPs of a pair ofisogenic methicillin-resistant and methicillin-sensitive S.aureus strains as well as a resistant clinical isolate. Withimprovements in our PBP binding and electrophoretic tech-nique, we could demonstrate the presence of an additionalPBP with a low affinity for beta-lactam antibiotics in culturesgrown at pH 7.0. No such extra band was observable in theisogenic sensitive strain. Labeling of the PBPs in live grow-

ing bacteria indicated that saturation of the extra band (PBP-2a) required penicillin and methicillin concentrations in thevicinity of the MIC for these antibiotics, whereas all otherPBPs could be saturated below the MIC.We also applied our PBP binding and gel system to the

reexamination of the PBPs of the same isogenic pair ofmethicillin-resistant and methicillin-sensitive S. aureus

strains grown at either pH 7.0 (allowing expression ofresistance) or pH 5.2 (at which the bacteria are phenotypical-ly sensitive to beta-lactams). It was found that the low

* Corresponding author.

affinity PBP-2a was no longer detectable in bacteria grown atthe low pH value. Identical observations were noted when aresistant clinical isolate was used.

MATERIALS AND METHODSBacteria. An isogenic pair of strains 27 and 27R was kindly

supplied by Richard P. Novick, New York City PublicHealth Research Institute. Methicillin resistance of a clinicalisolate of strain 592 was introduced into methicillin-sensitiverecipient strain 27 by transduction with phage 80ot. Themethicillin-resistant strain Col was supplied by Leon Sabath,University of Minnesota and originated from K. G. Dyke,Strain 209-P (ATCC 4538-P) was a second sensitive controlstrain. Relevant characteristics of these strains are describedin Table 1. Culture growth was monitored by measuringoptical density or by light scattering by a Coleman nephoco-lorimeter (Coleman Instruments, Oak Brook, Ill.) (9).MICs. MICs were determined by the tube dilution method

in tryptic soy broth (Difco Laboratories, Detroit, Mich.),starting with 105 CFU/ml. Turbidity was read after 48 h ofincubation at 37°C. MICs determined by the agar dilutionmethod at 30°C on tryptic soy agar were within two- tofourfold of the values found by the tube dilution method.

Labeling of PBPs in growing bacteria. In our experimentswe used whole living cells. Bacterial strains were stored at-70°C. Cultures were grown to late logarithmic phase intryptic soy broth at pH 7.0 or 5.2 at 37°C with vigorousshaking. These were then rediluted by placing 1.0 ml ofbacteria into 100 ml of fresh warm tryptic soy broth at theappropriate pH value and grown again to the midlogarithmicphase of growth. The bacteria were centrifuged at 10,000rpm at 0°C for 15 min, and the pellets were resuspended in1.0 ml of clean tryptic soy broth (1OOX concentration) at theappropriate pH.Samples of cells (25 RI) were incubated with various

concentrations of [3H]benzylpenicillin (ethylpiperidiniumsalt) with a specific activity of 25 Ci/mmol (14). Immediatelybefore the [3H]benzylpenicillin was used, the acetone sol-vent was evaporated and replaced by an equal volume ofdistilled water. The samples were incubated with [3H]ben-zylpenicillin at 37°C for 10 min and then were boiled for 2min. This heat inactivation step considerably improved theresolution of the gels. Next, 25 [LI of 0.1 M potassiumphosphate buffer (pH 7.6) was added to all tubes to allowlysis by lysostaphin (100 pLg/ml), and the samples were

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514 HARTMAN AND TOMASZ

TABLE 1. S. aureus strainsMIC (,ug/ml) of:

Straina Methicillin BenzylpenicillinpH 7.0 pH 5.2 pH 7.0 pH 5.2

209-P 0.8 0.2 0.2 0.227 1.6 0.4 0.025 0.02527R 625 3.1 10 0.025Col 1250 1.6 31 0.025

a All strains were beta-lactamase negative by the Nitrocefin assay(13).

incubated at 37°C for another 30 min. After the addition of 25,u of sample dilution buffer (3), the entire sample was boiledagain for 2 min.

In one type of experiment, the methicillin and [3H]benzyl-penicillin (at a constant concentration of 10 ,ug/ml) wereadded simultaneously to the bacteria, and incubation was for10 min. In a second type of experiment (sequential competi-tion) the bacterial sample was preincubated with the methi-cillin for 10 min, followed by the addition of [3H]benzyl-penicillin (10 jig/ml) and an additional 10-min incubation. Inboth methods the samples were boiled, and the next proce-dure was followed as above.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(11) was done with a 10% acrylamide-0.13% bis-acrylamideconcentration in the running gel measuring 13 by 18 cm. Thestacking gel was composed of 5% acrylamide and 0.07% bis-acrylamide. Samples were added to the gel lanes and runthrough the stacking gel at 75 mV and through the runninggel at 120 mV. The samples were run for 45 to 60 min afterthe leading edge had passed the bottom of the running gel(total time through the running gel, ca. 4 h).The gels were stained with Coomassie brilliant blue (6) and

destained overnight with methanol-acetic acid-water(30:5:65%). The gels were processed by first removing thewater by two 30-min soaks in dimethyl sulfoxide, followedby a 2-h soak in a 20% (wt/vol) solution of the scintillatorPPO (2,5-diphenyloxazole) in dimethyl sulfoxide. The gelswere then rehydrated in 1% glycerol solution in water for 45to 60 min, dried thoroughly, and exposed to a Kodak X-OMAT XAR-2 film for 3 to 14 days at -70°C (1).

Assay for penicillinase. The presence of penicillinase wasassayed with Nitrocefin (Glaxo Research Ltd., Greenford,Middlesex, England) (13).

RESULTS

27R, both grown at pH 7.0. Exposure of the preparations to arange of [3H]benzylpenicillin concentrations (0.01 to 1.0 P,g/ml) revealed a rather similar band pattern. However, expo-sure to higher concentrations of the antibiotic (2 to 20 ,ug/ml)resulted in the appearance of an extra radioactive band(PBP-2a) in the methicillin-resistant strain. This PBP-2a ranslightly ahead of PBP-2 with an approximate molecularweight of 78,000. No PBP with similar mobility was ob-served in either strain 27 or in a second penicillin-sensitivecontrol strain 209-P (data not shown). The detection of PBP-2a required exposure to a minimum concentration of 2 to 5p.g of penicillin per ml. This is close to the MIC for theresistant strain 27R (Table 1).An extra PBP, indistinguishable from PBP-2a, was also

detectable in strain Col, a naturally occurring clinical isolate(Fig. 2).The low affinity of PBP-2a to methicillin could be demon-

strated by competition experiments at pH 7.0 (Fig. 2).Exposure of the methicillin-resistant Col strain to variousconcentrations of methicillin 10 min before the addition of 10,pg of [3H]benzylpenicillin per ml resulted in a titrationprofile in which the familiar PBPs (1 and 3) were completelysaturated by 50 ,ug of methicillin per ml; PBP-2 required 100to 600 ,ug of methicillin per ml to achieve similar saturation.However, binding of PBP-2a required still higher concentra-tions, in the range of 1,000 ,ug of methicillin per ml. Virtuallyidentical results were obtained with the methicillin-resistanttransductant strain 27R (data not shown).The low affinity of PBP-2a for methicillin could also be

demonstrated in simultaneous competition experiments (Fig.3) in which resistant strain 27R was exposed simultaneouslyto methicillin (at various concentrations) plus [3H]benzyl-penicillin. In this case, saturation of PBP-2a occurred at orabove the MIC (625 ,ug/ml) of the organism for the antibiotic.A comparison of Fig. 3 with Fig. 2 indicates that in thesimultaneous competition experiment, substantially higherconcentrations of methicillin (up to 600 ,ug/ml) were alsoneeded for the saturation of PBPs 1, 2, and 3 than were

pH 7

0 1 5 1500.10.61 5

Figure 1 shows the PBPs of the methicillin-sensitive strain27 and the isogenic methicillin-resistant transductant strain V

0.1 0.2 0.4 0.8 1 2 5 10 20r sr S r s r s r s r s r s rss .s

2a-.s - - -- - _ * i # *

4-

FIG. 1. PBP patterns of methicillin-sensitive strain 27 (s) andmethicillin-resistant strain 27R (r) of S. aureus at pH 7.0. Concentra-tions of [3H]benzylpenicillin range from 0.1 to 20 p.g/ml. PBPs 1, 2,2a, 3, and 4 are labeled.

A AFIG. 2. PBP pattern of methicillin-resistant strain Col in a com-

petition experiment with various concentrations of methicillin (0 to 5mg/ml). [3H]benzylpenicillin (10 ,ug/ml) was added after exposure tomethicillin for 10 min (sequential competition). Lane 0 is a controllane in which only [3H]benzylpenicillin at 10 p.g/ml is added.Symbols: A, Concentration of methicillin at which PBP-2 disap-peared; A, concentration at which PBP-2a disappeared.

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METHICILLIN-RESISTANT S. AUREUS: PBPs AT pH 7 AND 5.2 515

needed in the sequential competition experiments (Fig. 2 anddata not shown). Similar results have already been described(9).The visualization of PBP-2a made it necessary to use high

concentrations of radioactive penicillin which, in turn, hasled to the appearance of increased background radioactivity.Similar situations have also been noted with other bacterialspecies (18). The extra bands detectable at high penicillinconcentrations represent, presumably, nonspecific (nonen-zymatic) binding of the antibiotic. This is supported by theobservation that in the methicillin competition experiments(see, e.g., Fig. 2) the intensity of background bands did notdecrease in contrast to the behavior of the true PBPs.

Figure 4 shows the PBP profiles of methicillin-sensitivestrain 27 and its isogenic transductant 27R grown at pH 5.2.Examination of the fluorogram indicates the similarity ofPBP patterns and the absence of PBP-2a from among thePBPs of strain 27R. By comparing Fig. 1 with Fig. 4, one cansee the extra binding protein (PBP-2a) in the methicillin-resistant transductant grown at pH 7.0.A culture of the resistant clinical isolate (Col) grown at

two pH values also yielded a similar PBP pattern: PBP-2awas only demonstrable in the cultures grown at pH 7.0 (Fig.5, lane 0). The absence of PBP-2a was not due to slowerpenicillin binding since this protein was not detectable evenupon prolonged (1 h) exposure to penicillin (data not shown).The methicillin competition experiments were run with thesame clinical isolate (Col) grown at two pH values. PBP-2a(demonstrable in the pH 7 cultures) had a low affinity formethicillin, since saturation of this binding protein requiredmore than 1 mg of methicillin per ml (Fig. 5). At pH 7, all theother high-molecular-weight PBPs (1, 2, and 3) were saturat-ed below 100 xg of methicillin per ml. In the cultures grownat pH 5.2, PBP-2a was not detectable, and the other threehigh-molecular-weight PBPs were saturated below a concen-tration of 100 .g of methicillin per ml.

DISCUSSIONPrevious work with the PBPs of methicillin-resistant

staphylococcal strains has resulted in the description of avariety of PBP alterations associated with resistance to beta-lactam antibiotics. Brown and Reynolds compared the PBPsof a resistant and a sensitive strain (2). They reported both anormal PBP pattern and a high beta-lactam affinity in thePBPs of both strains, with the exception of PBP-3 which, inthe resistant strain, appeared to have lower affinity forpenicillin and other beta-lactam antibiotics. In addition,under growth conditions in which the resistant strain couldexpress its methicillin resistance (growth at 30°C), a proteinwith an electrophoretic mobility similar to PBP-3 appeared,and a corresponding heavy band appeared on the Coomassiebrilliant blue-stained gel. These authors suggested that in

ug'" -- - - I--....... -MQ......0 Oi 1 5 10 20 50 01.0.2 0.6 1 5

FIG. 3. PBP pattern for strain 27R in a competition experimentwith various concentrations of methicillin (O to 5 mg/ml) for 10 min.[3H]benzylpenicillin (10 ,ug/ml) was added simultaneously with themethicillin (simultaneous competition).

0.1 0.2 OA 0.8 1 2 5 IO- aOr s r s r s r s r s r s r s r

1- ` ..

2- ak af O ___ _ot a

3- ~w3FIG. 4. PBP profiles of methicillin-sensitive strain 27 (s) and

methicillin-resistant strain 27R (r) in a [3H]benzylpenicillin titrationexperiment (1 to 20 p.g/ml). The titration was performed in growingbacterial cultures at pH 5.2. PBPs 1, 2, and 3 are labeled. PBP-4 isnot seen well for either strain. PBP-2a is absent for both strains.

their strains, resistance may result from either an increasedamount of PBP-3 or the presence of a new low affinity PBPwith a molecular weight identical to PBP-3.Georgopapadakou et al. (8) reported that PBP-3 may be

missing (or present with a reduced binding affinity) in a

cephradine-resistant clinical isolate of S. aureus. This isolatealso appeared to have an increased concentration of a lowaffinity PBP-2, as well as a satellite band (PBP-2') with an

approximate molecular weight of 78,000. These authorssuggested that antibiotic resistance was related to poorbinding to PBP-2. PBP-2' did not show decreased affinity forcephradine and other relevant cephalosporins. This strainhad relatively low MICs for methicillin and many other beta-lactam antibiotics and thus may not be directly comparableto methicillin-resistant strains studied by others (2, 9, 10). Inaddition, the strains compared were not isogenic.Hayes et al. (10), studying another methicillin-resistant

clinical strain, showed that PBP-3 had relatively low affini-ties for beta-lactam antibiotics which were consistent withthe respective MICs. As a comparison, nonisogenic methi-cillin-sensitive strain H was used.

Utsui et al. (Y. Utsui, M. Tajima, R. Sekiguchi, E. Suzuki,and T. Yokota. Abstr. Int. Congr. Chemother. 13th, Vienna,Austria, abstr. no. 2.11/3-2, 1983) reported that cephem-resistant clinical isolates of S. aureus possessed a new

78,000-dalton protein with low affinities to benzylpenicillinand some other beta-lactam antibiotics. Spontaneous rever-

tants were found to have lost the 78,000 PBP. However, this78,000 PBP had a slower mobility than PBP-2 in contrast tothe PBP-2' (8) observed in cephradine-resistant S. aureus,which migrated ahead of PBP-2. The relationship of thecephem-resistant organisms to the methicillin-resistantstaphylococci is not clear.

*PH 7 PH15.2

I ID W0 QS1 5 O 1 KIO 50al"5 5

77'0*~~ ~ ~ ~ - - . ~ --2

FIG. 5. PBP pattern of methicillin-resistant strain Col in a com-petition experiment with various concentrations of methicillin (0 to 5mg/ml) at both pH 7.0 and 5.2. [3H]benzylpenicillin (10 p.g/ml) wasadded 10 min after exposure to methicillin (sequential competition).PBPs 1, 2, 2a, and 3 are labeled. Lanes 0 are controls to which only[3H]benzylpenicillin was added.

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516 HARTMAN AND TOMASZ

We compared a methicillin-sensitive strain of S. aureus toits isogenic methicillin-resistant transductant. In contrast toprevious studies, we used labeling of PBPs by live growingbacteria. This method should allow a more appropriatecomparison of PBP saturation with the MIC than a methodwith membrane preparations. None of the strains had detect-able P-lactamase activity. The comparison of the penicillintitration profiles of the two strains indicated minor differ-ences in the quantity (or affinities?) of PBPs 1, 3, and 4 (morein the sensitive strain) and a possible minor difference in theelectrophoretic mobilities of PBP-2 (slightly slower mobilityin the sensitive strain). However, the major differencebetween the PBP patterns of the two strains was onlydetectable at high concentrations of penicillin. At thesehigher concentrations, only the resistant strains showed anextra PBP (PBP-2a) running between PBP-2 and -3 at pH 7.0.An indication of the existence of PBP-2a is seen in thefluorograms published in our earlier communication (seeFig. 8, in reference 9). However, the complete separation ofthis PBP from PBP-2 required the improved technique usedin the present work. This PBP-2a was also detected in amethicillin-resistant clinical isolate (strain Col) which had nobeta-lactamase activity.The competition experiments with methicillin indicated

that PBP-2a had a low affinity for methicillin. The fact thatthe concentrations of both benzylpenicillin and methicillinneeded to achieve saturation of this PBP were close to thebiologically effective concentrations (MICs) suggests thatthe beta-lactam resistance in these strains may be related tothe acquisition of this low-affinity protein. This additionalPBP may allow continued cell wall synthesis in the presenceof antibiotics in the medium at elevated concentrations suchthat the other PBPs would be inactivated. This suggestion issimilar to the one already proposed by Fontana et al. (7) forthe possible function of low-affinity binding protein 5 detect-able in the beta-lactam-resistant strains of Streptococcusfaecalis and Streptococcus faecium.

The inability of methicillin-resistant staphylococci to ex-press their antibiotic resistance when grown at low pH (5.2)is a general property of all resistant isolates. In an earlierreport (9) we noted that there were no significant changes inthe molecular weight and drug affinities of the three high-molecular-weight PBPs when the cultures were grown ateither pH 7.0 or 5.2. We report now the resolution andseparation of an additional PBP (PBP-2a) present in culturesgrown at pH 7.0 but not in cultures grown at pH 5.2. Thiscorrelates well with the expression of resistance at the twopH values.One would expect that the bacterial cell wall synthesis

catalyzed in growing bacteria by a normal complement ofPBPs at pH 7.0 would be slowed or altered in the presence ofhigh concentrations (sub-MIC) of beta-lactams, since onlyPBP-2a would be available for the catalysis of the cell wallsynthetic reactions. It is likely that cell wall synthesizedunder these conditions would have abnormal structure. Weare presently investigating these possibilities. Further ex-periments will also be needed to determine whether thedisappearance of PBP-2a at pH 5.2 is due to its instability,nonfunctioning state, or inhibition of the biosynthesis of thisprotein in low pH cultures.

LITERATURE CITED1. Bonner, W. M., and R. A. Laskey. 1974. A film detection

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3. Buchanan, C. E., and J. L. Strominger. 1976. Altered penicillin-binding components in penicillin-resistant mutants of Bacillussubtilis. Proc. Natl. Acad. Sci. U.S.A. 73:1816-1820.

4. Centers for Disease Control. 1981. Methicillin-resistant Staphy-lococcus aureus-United States. Morbid. Mortal. Weekly Rep.30:557-559.

5. Dougherty, T. J., A. E. Koller, and A. Tomasz. 1980. Penicillin-binding proteins of penicillin-sensitive and intrinsically resistantNeisseria gonorrhoeae. Antimicrob. Agents Chemother.18:730-737.

6. Fairbanks, G. T., T. L. Steck, and D. H. F. Wallach. 1971.Electrophoretic analysis of the major polypeptides of the humanerythrocyte membrane. Biochemistry 10:2606-2617.

7. Fontana, R., R. Cerini, P. Longoni, A. Grossato, and P. Cane-pari. 1983. Identification of a streptococcal penicillin-bindingprotein that reacts very slowly with penicillin. J. Bacteriol.155:1343-1350.

8. Georgopapadakou, N. H., S. A. Smith, and D. P. Bonner. 1982.Penicillin-binding proteins in a Staphylococcus aureus strainresistant to specific P-lactam antibiotics. Antimicrob. AgentsChemother. 22:172-175.

9. Hartman, B., and A. Tomasz. 1981. Altered penicillin-bindingproteins in methicillin-resistant strains of Staphylococcus aur-eus. Antimicrob. Agents Chemother. 19:726-735.

10. Hayes, M. V., N. A. C. Curtis, A. W. Wyke, and J. B. Ward.1981. Decreased affinity of a penicillin binding protein for ,B-lactam antibiotics in a clinical isolate of Staphylococcus aureusresistant to methicillin. FEMS Microbiol. Lett. 10:119-122.

11. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

12. Mirelman, D., Y. Nuchamowitz, and E. Rubinstein. 1981. Insen-sitivity of peptidoglycan biosynthetic reactions to P-lactamantibiotics in a clinical isolate of Pseudomonas aeruginosa.Antimicrob. Agents Chemother. 19:687-695.

13. O'Callaghan, C. H., A. Morris, S. M. Kirby, and A. H. Shingler.1972. Novel method for detection of P-lactamases by using achromogenic cephalosporin substrate. Antimicrob. Agents Che-mother. 1:283-288.

14. Rosegay, A. 1981. High specific activity (Phenyl-3H) benzylpeni-cillin N-ethylpiperidine salt. J. Labelled Compd Radiopharm.18:1337-1340.

15. Sabath, L. D. 1977. Chemical and physical factors influencingmethicillin resistance of Staphylococcus aureus and Staphylo-coccus epidermidis. J. Antimicrob. Chemother. 3(Suppl. C):47-51.

16. Sabath, L. D., S. J. Wallace, and D. A. Gerstein. 1972. Suppres-sion of intrinsic resistance to methicillin and other penicillins inStaphylococcus aureus. Antimicrob. Agents Chemother. 2:350-355.

17. Saravolatz, L. D., D. J. Pohlod, and L. M. Arking. 1982.Community-acquired methicillin-resistant Staphylococcus aur-eus infections: a new source of nosocomial outbreaks. Ann.Intern. Med. 97:325-329.

18. Schwarz, U., K. Seeger, F. Wengenmayer, and H. Strecker.1981. Penicillin binding proteins of Escherichia coli identifiedwith a 1251-derivative of ampicillin. FEMS Microbiol. Lett.10:107-109.

19. Spratt, B. G. 1978. Escherichia coli resistance to 3-lactamantibiotics through a decrease in the affinity of a target forlethality. Nature (London) 274:713-715.

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21. Zighelboim, S., and A. Tomasz. 1980. Penicillin-binding proteinsof multiply antibiotic-resistant South African strains of Strepto-coccus pneumoniae. Antimicrob. Agents Chemother. 17:434-442.

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