Some biochemical changes accompanying penicillin-G resistance in Staphylococcus aureus

298

BIOCHIMICA ET BIOPHYSICAACTA

BBA 95029

SOME BIOCHEMICAL CHANGES ACCOMPANYING P E N I C I L L I N - G RESISTANCE IN S T A P H Y L O C O C C U S

AUREUS

LENNART WAHLSTROMAND MARIANNE WAHLSTR(3M The Roya/ Pkatmaes~/ca/Ita~/tm~, ~

(Smcdcn)

(Received December x6th, x963)

SUMMARY z. Five penicillin-G resistant strains of St~bby/ocooms ~ m r ~ prepared in vitro and their parent strains have been hydrolyzed with HCIO, and the hydrolyzates separated on a co]11mn of Sephadex G-25. The fractions obtained after gel filtration were further analyzed, and the ~ e s , found in Fraction III, were separated by paper chromatography and quantitatively estimated. 2. The DNA, RNA, and nitrogen contents of the strains have been determined. 3- The results show that both the protein synthesis and the nucleic acid synthesis are changed when a sensitive strain acquires penieiillrt resistance. 4. It is proposed that the parent strain of a peni"cdlinase (penicillin amidohydrolase, EC 3.5.2.6) producer has a pre-existent pathway for the pyrimidine metabolism which is insusceptible to the action of l~mi~l!in. Sensitive strains that can establish a simil~x pathway on acquiring resistance can maintain the osmotic barrier intact whereas those which cannot show a leakage in the cell envelope.

INTRODUCTION The microbial resistance to penici]lln has recently been discussed by several authors z- s. Three groups of peni~!!in resistance are considered: in the first two groups, resistance is associated with pe~llinp_~e (pen~llin amidohydrolase, EC 3.5.2.6) and acylase activity, respectively, whereas in the third group, resistance is due to some still unknown mechanism. K s o x AND SmTn4 propose the resistance of these organisms to be of a more individusl nature than that of the penicillinase producing strains. The mutational ~ of penici'Um resistance need not necemmily be the only explanation for resistance: some vital compcment, ~ l d e to pen~nln action, may be the cause of s~a~tivity, thus giving rise to v a r y i q d q t ~ m of pmicillin resistances. The antigenic patterns of the ~ used in this study have barn tested earlier by means of the double-diHusion agar technique~7. It was found that ~ sensitive strains of S~a,ph. aur~_s ehan~ed their antigemdc patte~us on acquiring resistance/~ v/~o. This was, however, not found to be valid for a penidllina~ producing strain. In the present paper, the strains used earlier are studied more extensively in Bioahim. Biophys. Acta, 87 (I964) 298-304

PENICILLIN-G RESISTANCE I n

S~#~lococc~ aureus

~9

order to find out if some common biochemical changes could account for the r e s u ~ obtained previously. As the accumulation of uridine uncleotides is known to,occ~iin all peniclnlr~-inhibited Slap& aureus, and of cytidine nucleotides in some 6f v~ese strains, it was of interest to make a quantitative comparison of certain ~ l i t e s especially of the corresponding two bases, uracil and cytosine, contained inca:resistant strain and in its parent sensitive strain. The application of gel filtration to the separation of low-molecular components of rihonucleic acids has recently been studied by ZADRAZlLe* aL'. They found that good separation is achieved with a mixture of nucleosides and their bases, yielding groups of pyrimidine and purine derivatives. MATERIAL AND METHODS

Organisms Five coagulase-positive strains of Staph. aureus were studied. The strains I, 2, and 3 (1~o. 853o, 853 I, and 8532, corresponding to Cowans' types I, II, and III respectively) were obtained from the National Collection 0f Type Cultures, London. The two remaining strains, 4 and 5, with the clinical numbers 1429 and 25z, were isolated from otitis pus. For details of cultivation technique, preparation of penicillin resistants, phagetyping and penicillinase production (resistant strain 5) see previous paperse, ~.

Fillration technique Sephadex G-e5 medium (Pharmacia, Uppsala, Sweden) with a water regain of

2.5 gig, was used throughout in this investigation. The column measured 8o cm in height and had a diameter of 3 cm. It was packed as described by FLODm', and eluated with o.o5 M sodium chloridelo. Fractions containing 4.5-5.5 ml were collected at a drop rate of 2 ml/min. All the experiments were performed at room temperature.

Analytical meawds The hydrolysis with HCIOt was carried out as follows: o.5oo g of lyophilized bacteria was heated with 72 % HCI04 in a glass-stoppered flask during I h in a boiling water bath. After neutralisation, the solutxon was centrifuged at 13 ooo Xg for Ih. The location of the fractions after gel filtration was followed with the aid of an Uvicord, LKB Ltd., at a wavelength of 2537 J~, giving the elution diagrams of the strains. The fractions of interest were collected and lyophilized. The pyrimidines were determir~ed by paper chromatography on Whatman No. I, according to HOTCHKISS11 (water saturated n-butanol) and WYATTli (isopropanolHC1). The chromatograms were developed by cutting them into I cm wide strips and extraction into 3.5 ml of water through shaking during I h. The ultraviolet absorption at 260o A was determined in a Beckman Model DB spectrophotometer. The nucleic acids were liberated from the bacterial cells by a modification of the method of JouEsxs: instead of shaking with ballotini glass beads for 3 h, ultrasonic treatment was used. The release of substances absorbing at 26oo A was followed at different time intervals, and was found to reach a maximum after 2-h treatment. The DNA and RNA determinations were carried out by the methods of BURTON1~ and CERIOTTIx5respectively, whilst nitrogen was estimated by the micro-Kjeldahl method.

Biochim. B~o'phys. Aaa, 87 (1964) 298-304

L. WAHLSTROM, M. WAHLSTROM

300

RESULTS

P)~imidine co~mt As seen in Fig. z, good separation of the pyrimidines is achieved by means of gel filtration. The pyrimidine contents of the different strains, obtained after sep-

I.? ~. 1.0 0.7 t

,m

o.4

r

.2 0.3

i

0.2 0.1 StreiR~

2 S 211---

40

I

I

go

lib

Tube Number

I

, . o

i

L

186

m

• 0.7 0.6.

u

0.4

0.2

0.1 , 0

~IL L 40

,

i

90 116 Tube Humber

185

Fig. I. Elution diagrams for gel-filtrsted HCIO4-hydrolyzates of two res~tant ~ (R) and their parent sensitive strsins (S). Fraction III, tube numbers 97-z35, c o n ~ the p y c i m i d ~ not entering the gel pores, Fraction II hlcludes ~veral UV-abm~mg c ~ _ ~ . ~ ~oB contains st lesst adenine. Fraction III only has been further mmlyr~o.

Iv

aration of Fraction III on paper chromatograms, are presemted in Table I. It is seen that the total uracil content varies greatly among the differmt sensitive stra~s of staphylococci. It is also the best represented pyr~'midine, except in the c~se oi the parent strain of the p e n ~ Im~ducer 5R in which cytosiae is d o m i m ~ g . It is further to be noted that all thesensitive s ~ hsve ~!mC~t ~ the same cytosine content, which is also true for thymine except for strain 4S, which shows a low thymine content. The acquirement of penicillin-G resistance, seems to affect the pyr/m~line vsl~s, generally musing a decrease. This is shown in Fig. z, which gives the increase and Biochim. Biophys. Acta, 87 (I964) e98-3o 4

St~J~hylococcus a w e u s

P E N I C I L L I N - G RESISTANCE IN

3oi

TABLE I CONTENTS OF TOTAL-NITROGEN, DNA, RNA AND PYRIMZDINES FOR SENSITIVE AND CORRESPONDING R E S I S T A N T S T A P H Y L O C O C C A L STRAINS, E X P R E S S E D AS ~0 O R m g / g O F L Y O P H I L I Z E D B A C T E R I A

S = sensitive strain; 1~ ---- resistant strain. Strain

Nitrogen

DNA

RNA

Uracil

(%) IS I]~ 2S 21q 3S 3R 4S 4R 5S 51~

Cytosine

Tkymivte

(i, ,.ele, J

zo.57 8.47 IO.97 IO.29 I2.I 4 II.34 io.58 IO.20 zi.94 II.O 5

IO I. 3 I6 33 II 26 IO 9 I4 36

z2 2. 3 I3 I8 5 9 I2 zo 8 15

Re.~

~uoy* (~1,~)

7.3 1.8 4-3 3.3 4.3 1.7 5.I 2.7 5 "o I.7

3.9 0.45 3.4 3.3 3.7 I.I 3.4 3.8 3"7 2.9

3-7. -3 .2 3.3 3 .0 I.3 1.6 I.9 3 .0 4.I

2oo0 4 °00 40 5000 6000

* D a t a f r o m p r e v i o u s p a p e r ~. ** Too l o w t o b e e s t i m a t e d w i t h a c c u r a c y .

3°°I DNA r-

tO

200

gr

o

"~0 .0° 0 ._~

iii •

Thymine

-,

Cytosine

J~

"

Uracil

5

"

R-strains

100

_o s

1

&

2

3

Fig. 2. Relative values for the D N A - , R N A - , and pyrlmidine contents of resistent s t r ~ - ~ , M e t a b o l i t e c o n t e n t of the corresponding sensitive strains zoo. The arrangement of the str~;-.~ is based on increasing a m o u n t s of D N A . =

decrease of the pyrimidines relative to the corresponding sensitive strains. It is clearly seen that the amount of uracil is markedly lower in all the resistant strains than in the corresponding sensitive strains. Strains IR and 3R also show a marked decrease in the amount of cytosinecontaining compounds, whereas strain 5R exhibits only a slight decrease. The deBiochim. Biophys. Actao 87 (x964) 2 9 8 - 3 0 4

302

L. WAHLSTR(}M,M. WAHLSTROM

viation in the cytosine content of the two remaining resistant strains from that of the sensitive strains is within the limits of error of the method used. The almost parallel decrease in thymine, uracil and cytosine in strains IR and 3R, indicates a leakage in the cell envelope. On the other hand, the penicillinase producing strain 5R shows a significant increase in thymine content as compared with the parent strain, giving thymine as the best represented pyrimidine. In the strains ~R and 4R the thymine content is practically unchanged. The D N A and R N A contents

As seen from Table 1, the ratio RNA/DNA is generally rather low, only in two cases, viz. IS and 4S, being greater than I. The corresponding resistant strains differ from the other resistants in that they have lower RNA and DNA contents than their parent strains, as is readily seen from Fig. 2. Strains zS, 3S, and 5S have ratios of RNA/DNA < I; on acquiring resistance, both RN'A and DNA increase, but in such a way as to give still lower values for the ratio RNA/DNA. The nitrogen content

The values given in Table I show that the resistant strains have a lower nitrogen content than the corresponding sensitive strains, the decrease, however, being small with the exception of strain zR, whose nitrogen content is considerably lower than that of IS. DISCUSSION As seen in Fig. 2, there is a parallel decrease in all the pyrimidine compounds when strains IS and 3S acquire penicillin resistance. This may be taken as an indication of a leakage caused by damage to the permeability barrier, hence they might be considered as not being "perfect" resistants. SUGANUMAls, studying the effect of penicillin on staphylococcus with the aid of the electrone microscope, demonstrated defects in the cell wall through which cytoplasmic material could escape. This hypothesis wo~d also explain the poor growth ot these two resistants, since a deficiency of the building materials for I~NA would further affect protein synthesis. This is readily seen with IR, which shows a decrease in RNA and a considerable loss of nitrogen compounds. 3 R, however, shows an increase in I~NA; it is possible that this strain has the capacity to retain enough pyrimidine compounds for the synthesis of RNA, since the decrease is not quite as pronounced here as in strain IR. The degree of resistance is also considerably lower in strain 3R (see Table I). ~ e e m s to be in accordance with the assertion 1~that non-enzyme producing resistant variants are less exacting in their nutritional requirements, e.g. they are able to synthesize their own amino acids. It is reasonable to suPlmse that this capacity is necessary if the ceil cannot deal with an amino acid pool on account of disturbances in the permeability barrier, which, as here, seem to accompany the acquirement of resistance. On the other hand, Fig. z does not give any indication of a leakage of pyrimidinecontaining compounds in strains dR, 4R, and 5R. The cytosine content in the resistant strains is practically the same as in the parent sensitive ~ , whilst the penicillinase producer even shows an increase in the quantity of t h y m i n e - c o n ~ compounds. Thus the disturbances in the osmotic barrier, as revealed in the case of strains IR Biochira. Biophys. Acta, 87 (1964) 298-3o4

PENICILLIN-G RESISTANCE IN Stt~bhy~oco¢c~ aureus

303

and 3R, seem not to be valid for the remaining strains, and in contrast to the former, they are considered as having become "'fully" resistant. Regarding the uracilcontaining compounds, however, a decrease occurs in all the strains upon acquiring resistance. It is known that under aerobic conditions uracil is normally synthesized by staphylococci, but for anaerobic growth uracil is a specific growth factor, and as such is irrepiacable by cytosine. FUSILLOAND WEISSTMhave pointed out in connection with aerobiasis-anaerobiasis and resistance-susceptibility, that "multiple potential pathways do exist in staphylococci and consequently, the concept of the pre-existence of potential resistant variants is substantiated". It is therefore credible to suppose that the organism can, to a limited extent, compensate for this deficiency in uracil metabolism. The parent strain of the penicillinase producer, 5S, differs from the other sensitive strains with respect to the percentage composition of the pyrimidines, having a higher content of cytosine-containing compounds than that of uracil. Does this mean that this strain is better equipped for acquiring resistance? It is interesting to note (see Table I) that strain 4R, resistant to 6ooo pg of penicillin/ml, has almost the same proportion of these pyrimidines as 5R, and strain 2R, resistant to 4ooo pg of penicillin/ml, seems to tend in the same direction. This may indicate that the metabolism of these pyrirnidines in 5S follows a pathway which differs from that followed by the other sensitive strains, and that this pathway is not susceptible to the action of penicillin. On acquiring resistance, this pathway is also followed by those organisms which can establish it, v/z. 2S and 4S. It does not seem impossible, as has been suggested by several authors, that penicillinase in some way would function via this pathway. Although all the resistant strains show a lower content of uracil compounds as compared with their parent strains, three of them show an increase in RNA and DNA content. This is in accordance with the results of BELJANSKIID, and of GILLISSEN2°, who reported an increase in DNA on acquiring penicillin resistance. The shift in the ratio RNA/DN A to lower values has earlier been reported by GALEsl. This, however, is not valid for IR and 4R, the former showing an increase in RNA/DNA ratio whilst in the latter it is almost unchanged. In addition, both RNA and DNA contents are decreased. The fact that the RNA values are much lower than what could be expected on the basis of the uracil values, may indicate that all of the cell's RNA has not been fully extracted. Furthermore, it is possible that both DBrA and RNA are more easily extracted from the resistant than from the sensitive strains. It is further known that the cells will maintain their weight of DNA per cell constant, and thus difference in cell size will alfect the values. With this in mind, further disoussion seems to be indicated. Although the raw materials necessary for the synthesis of RNA and DNA decrease, it is nevertheless observed that the occurrence of these substances in fact increases. A possible explanation for this phenomenon is that certain compounds containing the corresponding pyrimidines act as feedback regulators, and a decrease of these would allow an increase in the corresponding compound. GORINI AND KAL~AN" have found that uracil, known to exist freely in staphylococcisa, controls the synthesis of carbamylphosphate in Esdwrichia coll. This compound holds a key Biochim. Biophys. Acta, 87 (x964) 298-304

~O 4

L. WAHLSTROM, M. WAHLSTROM

position in that it leads to precursors of both nucleic acids and proteins. Thus a deficiency of uracil would lead to a higher rate of synthesis o1 these macromolecules. The protein synthesis, however, is lower within all the resistant strains than in the corresponding sensitive ones: possibly the increase in the nucleic acid synthesis is a compensation for this in order to maintain a protein level sufficient for survival. From the presented data it is seen that disturbances in both nucleic acid synthesis and protein synthesis accompany acquisition of a high degree of penicillin resistance. "Fully" resistant variants, here exemplified by 2R and 4 R, have an unchanged cytosine metabolism but a defect in that of uracil, for which they are able to compeusate to some extent. A~ has been discussed, this compensation is proposed to involve a pathway similar to that followed by the parent strain of the penicillinase producer. Thus, 5S is suggested to have a preexisting pathway that is better suited for the acquirement of penicillin resistance. Sensitive strains which cannot establish a similar pathway on acquiring resistance are unable to maintain the osmotic barrier intact. This is in agreement with the vital role played by the pyrimidines uracil and cytosine in the formation of the bacterial cell wall *t. The strains IS and 3S are such examples, and the corresponding resistant strains can be regarded as not "perfect" resistants. The results obtained in previous serological studies, as mentioned in the INTRODUCTION, seem not to be surprising when account is taken of the great changes that seem to occur when a sensitive strain acquires penicillin resistance, as ehicidated in this paper. The penicillinase-producing strains also show changes, but as has been discussed, this does not involve changes as fmudamental as the use of different metabolic pathways. REFERENCES I A. R . ENGLISH, T. J. MCBI~DE AND H . T. HUANG, Proc. Soc. gxptl. Biol. Med., I o 4 (x96o) 547. t W . B. H U G O A N D A. D. R u s s ~ , J. Pkarm. PharmacM., x3 (I96I) 705t A. E . I/t~iLAUSHAARAND E . E. ~CltltlID, Arzseimittel-Forsrh., x2 (I962) xx57. • R. K N o x AND J. T. SiIITK0 Last*at 2 (x96I) 520. • P . D. COOPER, B a a e r / M . R ~ . , 20 (x956) 28. s B. NORKRANS AND L. WAm~TltOM0 A a a Pathol. Mi~obiol. StaNd., 56 (x962) 45 I. v L. WAJILSTROM A N D B. INIORKItANS,A a a Patlwl. Mioyobiol. Sea,&, in t h e p r e ~ . e S. ZADRAZIL, Z. SORMOV~ AND F. SORM, Collection Czedt. Chem. C o m ~ . , 26 (x96I) 2643. • P. FLODXN, J. Chromatog.o 5 ° (x96I) lO 3. 10 B. GELoa~rE, J. Chromatog,, 3 (x96o) 33 o. n R . D. HOTCHKISS, J . B/ot. CStem., 175 (1948) 315 • it G. R . WYATT, Biocltam. J., 48 (x95 x) 584 . 1* A. S. JONES, Biochim. Biopltys. Acta, xo (I953) 607. t• K . BURTON, Bioc~m. J., 62 (I956) 3x5 • it G. CE~IOTTI, J. Biol. Chem., 2I 4 (I955) 59xs A. SUGA~UM*, J. Is/ca. Di,umses, iIX (i962) 8. t~ A. BONDL J. KOm~eLUM A2qe M. DE ST. PHALL*t, J. Baaeriol., 68 (I954) 6x7. xe M. H . FUSmLO AND D. L. W E m S , A,tibiot. C~aot~rapy, 8 (x958) 2x. t. M. BELJANSKI, Ann. I~tsL Pasteur, 84 (I953) 4 °2. te G. GmLmSEN, Z. Hyg, Itt/eit~kratthh., x46 (I959) 97. m E. F. GALE, Bull. Jolms t l e ~ s Hosp., 83 (x948) xxg. st L. GOmNI AND S. M. KAL1LAN,Biochim. Biopitys. Acta, 69 (I963) 355. t , M. R . J. SALTON, J. ~ , Miefobiol., 5 (x95 x) 39x. u H . R . PERKINS, B~tsf/o~. R~)., 27 (t963) x8.

Biodlim. Biopliys. Aaa, 8 7 (I964) 298--304