576. 851 . 2 5 2 : 6 1 5 . 778 (penicillin)

COAGULASE-POSITIVE STAPHYLOCOCCI RESISTANT TO PENICILLIN

MARY BARBER From. the Departnwnt of Bacteriology, British Postgraduate

Medical School, London

(PLATE LI)

SCHNITZER et al. (1943) divided penicillin-resistant staphylococci into three types : naturally resistant organisms, those which have acquired resistance through contact with penicillin, and small-colony variants or G forms which have a non-specific resistance to various compounds. Some workers (see Spink et al., 1945) further subdivide the second group according to whether the organisms have acquired resistance in vitro as a, result of serial subculture in penicillin or in vivo as a result of penicillin treatment. The present paper is concerned mainly with naturally resistant organisms.

Isolation of resistant strains

Colonies from all plates yielding coagulase-positive staphylococci in the routine laboratory during the period of investigation were tested for penicillin sensitivity by the ditch-plate method. The concentration of penicillin in the penicillin-agar was 10 units per ml. Nearly always two and sometimes more colonies were picked from each plate and streaked on to a penicillin ditch plate, either directly or after overnight culture in broth. No strain was regarded as resistant unless it grew almost to the junction of the penicillin-agar and plain agar or further. In fact minor degrees of resistance were unusual, most organisms appearing to be either frankly resistant or as sensitive as the Oxford staphylococcus, which was included on every plate. Fig. 1 shows a typical result, one strain being frankly resistant. On this plate one strain also appears to be slightly more resistant than the other four, but it was classified as sensitive.

Coagulase-positive staphylococci were isolated from 200 patients and sometimes from several specimens from the same patient. Table I summarises the results. In the first series all organisms were also tested for sulphathiazole sensitivity by the method of Harper and Cawston (1945). In all, 25 penicillin- resistant strains were isolated from 200 patients. In 6 cases penicillin-resistant and penicillin-sensitive organisms were isolated from the same patient and in 4 from the same specimen. In many other cases penicillin-resistant staphylococci were isolated from a number of specimens from different sources from the same patient. It will be seen that 5 of the 25 patients with penicillin-resistant

J. PATE. BACT.-VOL. LIX 373

374 M. BARBER

Pyogenic infections . Conjunctivitis . Urine . Lactating breasts . Vagina . Nose . Skin . Air .

Total .

staphylococci had been given penicillin before the specimen was taken ; only 2 of these had received a full course of treatment.

5 3 4 4 4 3 1 1

--__

25

TABLE I

Incidence of penicillin-resistant Staph. aureus among 200 patients

Description of Staph. aureu8 strain

Coagulase-positive . . Penicillin-resistant * . Sulphathiazole-resistant t . Penicillin- and sulphathiszole-resistant .

No. of patients

Series I I Series I1 1 Total

100 15 25 11 I ... I ...

* Three patients of series I and two of series I1 had been given penicillin before the swab was taken t Ten patients had been given sulphathiazole before the swab was taken.

Source of penicillin-resistant strains

Table I1 gives the primary source of isolation of each resistant strain.

TABLE I1

Primary source of penicillin-resistant strains

I Source 1 No. of strains I

Pyogenic infections. Case 1 was one of carcinoma of the larynx. A penicillin- resistant Staph. aureus was isolated from an infected tracheotomy wound in association with a Lancefield group-A hremolytic streptococcus. The sputum contained a penicillin-sensitive staphylococcus, also in association with Str. pyogenes. Case 2 was one of bronchiectasis. A penicillin-resistant Staph. a u r e w was isolated in association with a Lancefield group-A hzemolytic strepto- COCCUS from the sputum in life and from the bronchi and a thrombus of one of the neck veins post mortem. This patient had had a full course of penicillin treatment before any specimens were taken. In case 3 there was osteitis following a fractured tibia and fibula. A specimen of pus gave a mixture of penicillin-sensitive and penicillin-resistant Staph. aurew, Str. pyogenes and Proteus. The cocci were isolated only after treating a broth emulsion of the original plate with ether and replating (see Pearce, 1946). Case 4 was one of cervical abscess in a child, whose pus gave a pure growth of penicillin-resistant staphylococci. Penicillin treatment (10,000 units 3-hourly) had been started three days before the specimen was taken. Case 5 was one of bilateral otitis media in 8 child. A penicillin-resistant Staph. aureus was isolated from both ears

P E N I C I L L I N - R E S I S T A N T STAPHYLOCOCCI 375

and from a stomach washout. The ear swabs also grew Str. pyogenes. A course of penicillin was started four days before any of the specimens were taken. The Str. pyogenes strains from all five cases were penicillin-sensitive.

The three cases of conjunctivitis were all in babies and in every case swabs gave a pure growth of a penicillin-resistant Staph. aureus. One of the three babies had had penicillin eyedrops a t birth.

Patients in whom primary isolation was from the urine. Two had mixed urinary infections. Case 3 was one of nephritis. Penicillin-resistant Staph. aureus, Staph. albus, Str. fcecalis and diphtheroids were isolated from a non- catheter specimen of urine. Str. pneumonicq Str. viridans, H . injhenzm, neisseriae and a few colonies of a penicillin-resistant Staph. aureus were isolated from the sputum. A nasal swab gave a heavy growth of Staph. aureus, some of which were penicillin-resistant while others were as sensitive as the Oxford staphylo- coccus. Case 4 was a puerperal patient. A heavy growth of penicillin-resistant Staph. aureus was obtained from both the urine and a vaginal swab.

Two puerperal cases gave a mixed growth from the vagina, including penicillin-resistant Staph. aureus. Case 3 was one of toxremia of pregnancy and nephritis from whom penicillin-resistant Staph. aureus was isolated from the vagina and penicillin- sensitive Staph. aureus from the nose. Case 4 was one of vulvo-vaginitis in a child. A vaginal swab was overgrown with Proteus, but after ether treatment and re-plating penicillin-resistant and penicillin-sensitive Staph. aureus and Str. viridans were isolated. This child had had two courses of intramuscular penicillin, the first of 4 and the second of 15 days before a swab was taken.

Patients in whom primary isolation was from the lactating breast. Case 1 gave a heavy growth of penicillin-resistant Staph. aureus from milk from both breasts. From case 2 penicillin-resistant Staph. aureus was isolated from both nipples, from a swab taken from an infecteti Csesarean wound and from a vaginal swab. Case 3 gave a growth of penicillin-resistant Staph. aureus from a cracked nipple. From case 4 a mixture of penicillin-resistant and penicillin-sensitive Staph. aureus was isolated from the milk of one breast and penicillin-sensitive Staph. aweus only from the milk of the other breast.

Patients in whom primary isolation was from the nose all gave heavy growths of penicillin-resistant Staph. aureus.

Conjunctiwitis.

Patients in whom primary isolation was from the vagina.

Nose.

Study of penicillin-resistant strains All strains found resistant to penicillin on ditch plates were

re-tested by the serial-dilution method in broth. The inoculum was 1 drop of a 1 : 100 dilution of an 18-24-hour broth culture- approximately 50,000 viable organisms. The results with this inoculum are shown in table 111.

If the inoculum is altered the results are quite different-an observation in striking contrast to the surprisingly constant results irrespective of inoculum size with penicillin-sensitive staphylococci. The effect of inoculum size is shown in table IV. The inocula used were two drops each of 0.02 ml. of 18-hour culture, undiluted for the large inoculum and diluted 1 : 10,000 for the small inoculum. The number of bacteria was determined by the rapid viable-count method of Miles and Misra (1938). It will be seen that with the penicillin- sensitive staphylococci-S.G. 9727-3 and the Oxford staphylococcus- the results were the same whether the inoculum was many million or less than 1000 bacteria. In contrast to this, with the resistant

J. PATH. BACT.-VOL LIX 2 8

376 M . BARBER

No. of times more resistant than the

Oxford staphylococcus

strains S.T. 13 and S.G. 9727-1, an inoculum of 13-15 million organisms grew in 200 units per ml. of penicillin, whereas when the inoculum was less than 1000 the maximum concentration of penicillin permitting growth was 0.25 unit per ml. The two strains of B. subtilis recom-

TABLE I11

Degree of penicillin resistunce with an inoculum of 1 drop of a 1 : 100 dilution of an 18-24-hour broth culture (about 50,000 viable organLsms)

No. of strains

- -

X 64 x 32 x 16 x 8 x 4

1 6

10 7 1

I --I Total . 25

TABLE IV

Injluence of inoculum size in penicillin-sensitivity testa with penicillin- sensitive and penicillin-resistant organisms

Culture

Staph. ST 13 . Staph. SG 9727-1 . Staph. SG 9727-3 . Oxford Staph. . B. subtilis 6276 .

B. subtilis 6346 .

Maximum concentration of penicillin (u/ml.) permitting growth in 24 hrs.

Large inoculum

Inoculum

13 million 15 ,,

Q 11

12.8 ,, 600,000

2 million

Penicillin concentration

zoo+ 200 + 0.03 0.03 50

10

Small inoculum

Inoculum

830 970 940

1280 420

200

Penicillin concentration

0.25 0.25 0.03 0.03

No growth

0.03 in 0.008

mended by Duthie (1944) for penicillinase production were included in the experiment and showed the same phenomenon as the penicillin- resistant staphylococci. Strain 6276 appeared to be more sensitive to penicillin than the Oxford staphylococcus when an inoculum of 420 organisms was used, whereas an inoculum of 600,000 bacteria grew in 50 units per ml. of penicillin. I n another experiment the large inocula were prepared from thrice-washed bacterial cells re- suspended in a quantity of broth equal to the original volume. The results were the same.

All the resistant strains and 12 penicillin-sensitive strains of staphylococci were tested for their capacity to destroy penicillin,

PENICILLIN-RESISTANT STAPHYLOCOCCI 377

The preliminary test to determine simply presence or absence of penicillinase was carried out as follows. Equal quantities of bacterial culture and 4 units per ml. of penicillin were mixed, giving a final concentration of 2 units per ml. of penicillin ; each mixture was then tested for penicillin content by the cylinder-plate method, using the technique described by Hayes (1945). Controls were put up consisting of broth and penicillin only. All 12 sensitive strains mixed with penicillin gave an average diameter of inhibition approximately equal to that of the control. All the 25 resistant strains destroyed the penicillin, so that a circle of inhibition was completely absent. Fig. 2 shows a typical result when 2 resistant and 3 sensitive strains were tested. Fig. 3 shows the comparative results of a resistant strain of Staph. aweus (l), Salm. typhi (2), Bact. coli (3), the Oxford staphylococcus (4), and an uninoculated control (5). The cylinders have been removed in order to show that the two penicillin-destroying cultures and Salm. typhi have grown at the bottom of the cylinders. Fig. 4 shows two of the present series of in-vivo penicillin-resistant strains of Staph. aureus (1 and 2), an in-vitro acquired resistant culture of the Oxford staphylococcus (3), an ordinary culture of the Oxford staphylococcus (4) and an uninoculated control. This demonstrates the difference between in-vitro acquired resistance and natural resistance. All 3 cultures have grown, but the natural resisters have destroyed the penicillin, while the acquired resistant strain has not affected it.

Further tests for penicillinase production were carried out on the in-vivo-resistant organisms. Mixtures were made so that the final concentration of organisms was 5 per cent. and of penicillin 10, 5, 2 and 1 unit per ml. These were tested immediately and after 24 hours' incubation a t 37" C. When the tests were put up immediately all the resistant strains had destroyed a t least half the penicillin. After 24 hours' incubation so much penicillin had been destroyed that the tubes with 10 units per ml. gave no area of inhibition. In fact most strains were able to destroy considerably more penicillin than this. There was growth at the bottom of the cup with all strains even when the penicillin had not been destroyed. Fig. 5 shows an experiment in which strain S.T. 13 was mixed as above with penicillin of final concentration 20 (l), 10 (2), 5 (3), and 2 (4) units per ml. ; 5 is an uninoculated control of two units per ml. of penicillin. It will be seen that there was growth at the bottom of all the cups (which had been removed) except the control.

All the resistant strains were tested against sulphathiazole by the method of Harper and Cawston and against streptomycin on ditch plates. Seven were so resistant to sulphathiazole that they grew readily in a concentration of 1 : 20,000 or more ; the other 18 were completely inhibited by 1 : 50,000 or less. All showed a sensitivity to streptomycin approximately equal to that of the Oxford staphylococcus.

378 M . BARBER

Rabbits were injected intravenously with 0.5 ml. of 18-24-hour broth cultures from 6 penicillin-resistant and 3 penicillin-sensitive strains. All died and no appreciable difference in average survival times was noted (table V). Stuph. aureus was cultivated from the

TABLE V

Pathogenicity of penicillin-resistant and penicillin-sensitive staphylococci for rabbits

Penicillin-resistant strains

Strain

P. 8210-1 S.T. 13 N.B.

P. 8238 S.T. 366 M. 105

I Average .

Survival time of rabbit (hours)

18 60 44 32 22 90

44

Penicillin-sensitive strains

Survival time of rabbit (hours) Strain

P. 8210-2 P. 98 1 N.A. ... ... ...

60 20 32 ... ... ...

Avemge . 37

heart blood of 8 of the rabbits and all strains thus isolated showed a similar penicillin resistance or sensitivity to that of the parent strain.

Ten penicillin-resistant and ten penicillin-sensitive strains were tested for biochemical reactions in an attempt to differentiate them. This was not found possible. All 20 strains fermented glucose, maltose, sucrose and mannitol. None fermented dulcitol. All produced acid and clot in litmus milk. All gave the following reactions : M.R. +, V.P. +, M.B. reduction +, NH, +, H,S -, catalase +++. Slight acid was produced in salicin by 2 strains, 1 penicillin-sensitive and 1 penicillin-resistant, both isolated from the same vaginal swab. None of the remaining 18 fermented salicin. Indole was produced by only 5 strains-the two just mentioned which fermented salicin, another pair of resistant and sensitive strains from the same pus swab and one other resistant culture.

The colonial appearances of sensitive and resistht strains showed no constant differences. A single strain often gave rise to several different colonial forms if plates were incubated for 24 hours at 37°C. and then left on the bench for a few days, but sensitive and resistant strains produced similar variants.

Although the various cultures often showed a mixture of penicillin- resistant and penicillin-sensitive colonies, both types of colony on subculture bred true as regards penicillin sensitivity. Penicillin resistance appeared also to be a permanent characteristic. Resistant cultures have been plated out for as long as 9 months after primary isolation and the daughter colonies have a reaction to penicillin

PLATE LI FIQ. I.-Penicillin-sensitivity test showing 5 sensitive strains of Staph. aureu8 and 1

FIQS. 2-4.-Tests for penicillinam production : 4 u / d . of penicillin mixed with equal quantities of broth cultures or plain broth ( h a 1 concentration of penicillin = 2 ulml.).

FIQ. 2.-1 and 5, penicillin-resistant staphylococci; 2. 3, and 4, penicillin-

FIG. 3.-I, penicillin-resistant staphylococcus; 2, Salm. typhi ; 3, B&. ooli ;

resistant strain.

sensitive staphylococci.

4 Oxford staphylococcus ; 5, broth.

FIQ. 4.--1 and 2, in-vivo penicillin-resistant staphylococci ; 3, induced penicillin- resistant strain of Oxford staphylococcus ; 4, Oxford staphylococcus ; 5, broth.

Fre. 5.-A strain of penicillin-resistant staphylococcus mixed with 20 ( I ) , 10 (2) 5 (3) and 2 (4) u/ml. penicillin : 5 is a broth control containing a h a 1 conoentration of 2 u / d . of penicillin.

J. PATH. BLi(''P.-VOL. LIS pE:NICILLIN-ILESISTAST STAPIIYLOCOCCI

PLATE LI

PELVICILLILV-RESISTANT STAPHYLOCOCCI 379

If penicillin-resistant and penicillin-sensitive strains were isolated from the same patient, cultures were sent to Dr V. D. Allison and Dr Betty Hobbs, who kindly typed them for me. The results are shown in table VI. It will be seen that, except in the case of patient

TABLE VI Typing of penicillin-resistant and penicillin-sensitive strains of

staphylococci f rom the same patient

Patient no.

1

4

22

47

111

180

Specimen

Pus swab 8037 ,, ,> 8038

9 , ,, 8210 ,, ,, 8210 9 , 9 , 8210

Nose swab

Sputum 2731 ,, 1 ,

Vaginal swab 9727 ,, 9727

Breast milk 692 ,, ,, 693

Vaginal swab 9853 Throat swab 161

Colons no.

1 1

1 2 3

1 2 1

1 3

1 1

1 1

N.T. = not typal

Reaction to penicillin

Resistant Sensitive

Resistant Sensitive Resistant

Sensitive Resistant

Resistant Sensitive

Resistant Sensitive

Resistant Sensitive

~

3.

Phage reaction

3A N.T.

N.T. 42C N.T.

6/47 6/47 6/47

N.T. N.T.

6/47 42E

6/47 N.T.

Serological reaction

8 I11

1114 721 113537

II /4

I11 10667 10667

3537/B83 7211

I11 10153

I11 2411/B83

22, from whom resistant and sensitive strains showed an identical phage reaction and a closely related serological reaction, the resistant and sensitive strains isolated from the same patient were of different types.

DISCUSSION Fleming ( 1942) encountered occasional strains of staphylococci

which " were almost insensitive " to penicillin, although he found that the majority were about equally sensitive. Since then a number of authors have isolated penicillin-resistant strains of staphylococci from many sources. Table VII gives the number of penicillin-resistant strains of coagulase-positive staphylococci isolated by various authors before and after treatment with penicillin. Fleming (1942) and Hobby et ul. (1942) give no details. Rammelkamp and Maxon (1942) used a small inoculum-1000-30,000 organisms-for their tests and thus found that the 2 resistant strains isolated before the onset of treatment were killed by 0.17 and 0.35 units per ml., and the 4 isolated after treatment had begun were killed by from 0.35 to 2.85 units per ml. Kirby (1944) described the production of penicillinase by 7 " naturally " penicillin-resistant strains of Xtaph. aureus. He also tested 7 penicillin-sensitive strains but found they had no penicillinase activity. Anderson et al. (1944) studied cultures of Xtuph. aureus

.PATH. BACT.-VOL. LIX 2332

380 M . BARBER

1942 1942 1942 1944 1944 1944~1 1945 1945 1945 1945 1946

isolated from 32 cases of chronic osteomyelitis. They used the technique described by Rammelkamp and Maxon for penicillin- sensitivity tests, and therefore a small inoculum, so that of the 8

No. tested

? ?

29 ?

32 68

66 ?

85 ?

TABLE VII No. qf penicillin-resistant strains of coagulase-positive staphylococci isolated by various authors before and after treatment with penicillin

12 57 11

134

Author

... ...

... ... ? 8 ? 9.4 per cent.

Fleming . Hobby et al. . Rammelkamp and Maxon Kirby . . Anderson et al. . Spink et al. . Bondi and Dietz . Gots . Gallardo . Harley .

1 Coagulase-positive staphylococci resistant to penicillin

Date 1 Before penicillin treatment I After penicillin treatment

No. resistant I No. tested 1 No. resistant

8 slightly 5 8

resistant strains isolated before treatment 7 were inhibited by 0.17 and 1 by 0.35 unit per ml. of penicillin. After treatment 5 more strains were found to be penicillin-resistant and these strains were inhibited only by a concentration of 5.7 units per ml. or more. This was considered to be an acquired penicillin resistance, but it appeared to be unrelated to penicillin dosage. No correlation was noted between results of therapy and the initial variation in penicillin sensitivity, but the resistant strains occurring after treatment had started were regarded as the major cause of penicillin failures. Spink, Ferris and Vivino ( 1 9 4 4 ~ ) studied 68 strains of coagulase-positive staphylococci isolated from patients who had had no penicillin treatment. Their usual inoculum for sensitivity tests was small (100,000-300,000 bacteria) and with this they found that the 8 resistant strains were inhibited by 0.4-0.8 units per ml. They showed, however, that the size of inoculum greatly affected the results and 2 strains tested with a large inoculum grew in 5 units per ml. of penicillin, which was the highest concentration used. They also tested for sulphathiazole sensitivity and found that 19 of the 68 strains were sulphathiazole resistant, but only one of these was also penicillin-resistant. Spink, Hall and Ferris (1945) discussed the clinical significance of natural and acquired penicillin resistance in staphylococci. In spite of having shown that a large inoculum of a naturally resistant strain will grow in at least 5 units per ml. of penicillin, they state that the " sensitivity test as used in this laboratory has failed to reveal a strain of staphylococcus not previously exposed

PENICILLIN-RESISTANT STAPHYLOCOCCI 381

to penicillin that was not inhibited by 1 unit of penicillin per cubic centimeter of medium, and well over 100 strains have been tested ”. On this evidence they conclude that naturally penicillin-resistant strains of Staph. aureus are not likely to be a serious cause of failure in penicillin therapy. These authors also contrast cultures which have acquired resistance to penicillin in vitro with naturally resistant organisms and those which have developed resistance in vivo. Spink, Ferris and Vivino (1944b) trained 4 penicillin-sensitive strains of Staph. aureus to grow in from 4 to 50 units per ml. These strains with acquired resistance were found to show the morphological and cultural changes described by other workers and were more susceptible to the bactericidal action of human blood. They did not produce penicillinase. In contrast, naturally penicillin-resistant organisms and resistant bacteria occurring in vivo after penicillin treatment show none of these changes and all produce penicillinase. Bondi and Dietz (1945) found that 12 of 66 strains of Staph. aureus isolated from various sources were penicillin-resistant. They were all penicillinase producers and this was regarded as the cause of their resistance. Gots (19453 studied 57 strains of Staph. aureus which needed 1 unit per ml. of penicillin for inhibition and found that all destroyed penicillin, whereas of 42 sensitive strains none produced a penicillin inactivator. Like Bondi and Dietz he regarded the production of penicillinase as the cause of the resistance. Gots also considered that there was a difference between resistance acquired in vitro and in vivo “ inasmuch as we have yet to encounter a resistant strain, isolated directly from an infected process, which is incapable of producing an inactivator ”. Gallardo (1945) isolated 85 strains of Staph. aureus, of which 11 were penicillin-resistant before treatment ; 8 more were found to be sensitive before treatment but resistant after. He gives no details. Harley et al. (1946), studying war wounds in India, isolated a large number of penicillin-resistant Staph. aureus, and the incidence was much higher among penicillin-treated patients than among those who had not been treated with penicillin. In a group of 43 cases with late infected compound fractures admitted to the base hospital before penicillin treatment in the forward areas, 9.4 per cent. of the Staph. pyogenes strains isolated were resistant to penicillin. Of Staph. pyogenes strains in wounds with hospital infection 30 per cent. were penicillin-resistant. The degree of penicillin resistance varied enormously, ranging from strains twice as resistant as the Oxford staphylococcus to strains 12,000 times as resistant. One strain was claimed to have increased its resistance more than 40 times in 6 days’ treatment. The technique for testing resistance was the serial- dilution method, but the size of inoculum is not stated; perhaps it varied. Any strain was regarded as resistant if it was not inhibited by 0.04 unit per ml. of penicillin.

38 2 M . BARBER

CONCLUSIONS

Three questions are disputed in the literature. First, what is the degree of penicillin resistance of the resistant strains and is it enough to be a serious menace to treatment ? It is clear, I think, from my results that the degree of penicillin resistance recorded in the laboratory depends on the size of the inoculum used for the test of resistance. With the same organism a difference of more than 800-fold was obtained by varying the inoculum. This has also been shown by Luria (1946). As to treatment, it is obvious that a heavy infection with one of these resistant staphylococci would be difficult if not impossible to treat with penicillin.

Second, is the penicillin resistance of these naturally resistant strains solely the result of their capacity to destroy penicillin ? The experiments presented in table I V and fig. 5 make it clear that these organisms have a residual resistance to penicillin apart from their capacity to destroy it.

Third, do staphylococci readily acquire resistance to penicillin in vivo during treatment and if so does this type of acquired resistance differ from resistance acquired in vitro ? Penicillin-resistant staphylo- cocci are certainly more common in penicillin-treated than in untreated cases, but it does not follow that this results from originally sensitive strains developing resistance. From a number of patients I isolated both penicillin-resistant and penicillin-sensitive strains of Staph. aureus, sometimes from the same swab. Therefore, unless many colonies are tested on each occasion, it cannot be accepted that a strain of Staph. aureus has developed penicillin resistance during treatment. McKee and Houck (1943) passaged a single strain of Staph. aureus through mice treated with penicillin and after 20 such passages no increase in resistance could be demonstrated. Further work along these lines is needed, but this one experiment suggests that staphylococci do not readily acquire resistance to penicillin in vivo. In human infection with Staph. aureus, the original infection may be due to more than one strain, and reinfection with a further strain is also possible. It is a t least possible, therefore, that an increase in penicillin-resistant staphylococci after penicillin treatment may not depend on acquired resistance, but may be the result of a process of selection-naturally sensitive bacteria being quickly destroyed and resistant organisms surviving.

Further evidence in support of this suggestion has become available since the work here described was completed. A patient with carcinoma of the lung had sputum heavily infected with Staph. aureus. From a specimen examined soon after admission 31 colonies were tested for their reaction to penicillin by the ditch-plate method. All had a sensitivity approximately equal to that of the Oxford staphylococcus. Twelve days after penicillin treatment (30,000 units 3-hourly) had been begun a second specimen of sputum was in-

PENICILLIN-RESISTANT STAPHYLOCOCCI 383

vestigated. Cultivation again yielded a heavy growth of Staph. aureus ; 30 colonies were tested against penicillin and all were found to be highly resistant. Subcultures of 2 colonies from each specimen were sent to Dr V. D. Allison and Dr Betty Hobbs for typing. They found that both cultures from specimen 1 gave a phage reaction 29/52 and were serologically type I, whereas both cultures from specimen 2 gave no phage reaction and were serologically type 11115.

SUMMARY

1. Out of 200 patients yielding cultures of coagulase-positive Stuphylococcus aureus, penicillin-resistant strains were isolated from 25. In 6 cases the same patient and in 4 cases the same swab yielded both resistant and sensitive strains.

2. The degree of resistance as measured in vitro was shown to vary more than 800-fold according to the size of inoculum used.

3. All resistant strains produced a penicillin inactivator. 4. It was impossible to differentiate between resistant and

sensitive strains on grounds of morphology, cultural appearances, biochemical reactions or pathogenicity to rabbits.

5 . Resistant and sensitive strains isolated from the same patient were usually of different types.

My thanks are due to Dr V. D. Allison and Dr Betty Hobbs for typing many of the cultures.

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HARLEY, H. R. S., BATY, J. A., 1946.

HARPER, G. J., AND CAWSTON, 1945.

HAYES, W. . . . . . . . 1945. HOBBY, GLADYS L., MEYER, K., 1942.

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FERRIS, V.