J. clin. Pathi. (1956), 9, 59.

ATYPICAL ANAEROBIC FORMS OF STREPTOCOCCUSPYOGENES ASSOCIATED WITH TETRACYCLINE

RESISTANCEBY

E. J. L. LOWBURY AND L. HURSTFromii the Medical Researclh Couincil Butrnis Uniit, Birniiglt,'ha, Accident Hospital

(RECEIVED FOR PUBLICATION JULY 4, 1955)

The emergence of Streptococcus pyogenies resis-tant to the tetracycline antibiotics has been notedoccasionally during the treatment of streptococcalinfection of burns with oral chlortetracycline(Lowbury and Cason, 1954). These resistant strainsgrew well under aerobic or anaerobic conditions.More recently, however, we have isolated from anumber of burned patients streptococci whichfailed to grow aerobically on ordinary culturemedia (horse blood agar and nutrient broth), butgrew well in anaerobic culture on the same media;most of these streptococci were resistant to tetra-cycline compounds. A surprising feature aboutthese organisms is their ability to grow aerobicallyon a medium containing a higher concentrationof agar used to prevent the swarming of Proteusin routine culture from wounds (Hayward andMiles, 1943). These atypical forms, which for con-venience we shall refer to as AN forms, are ofsome practical importance, since they appear tobe related to the emergence of tetracycline resist-ance and would be missed in aerobic culture onordinary blood agar.The incidence and some characteristics of AN

forms in burns are described in this paper.

Materials and MethodsMoist swabs from burned patients were examined

by methods similar to those reported elsewhere (Jack-son, Lowbury, and Topley, 1951). These included in-oculation of swabs and of cooked meat broth cultureson concentrated agar plates," containing 1000 de-fibrinated horse blood and 400 New Zealand agar* in aLemco broth base, the medium being poured over alayer of Lemco agar. We shall refer to media contain-ing 4%' agar as "concentrated agar" (with blood, orwith serum); media containing 1.500 agar will be called

ordinary blood agar" or ordinary serum agar."The agar used in all the experiments and almost all the routine

work was oxoid agar-agar powder (New Zealand). Some cultureplates with Davis standard agar (New Zealand) were used at thebeginning of the study.

Small haemolytic colonies wvere examined for mor-phology in Gram-stained films, and subcultured forgrouping on two ordinary blood agar plates (contain-ing 1.500 New Zealand agar and 100 horse blood).These plates were incubated, one aerobically and theother anaerobically, at 37° C. overnight; the growthwas noted and grouping was carried out by Lancefield'smethod with antisera of groups A, C, D. and G(Wellcome).

Strains of Group A streptococci were typed for usby Dr. W. H. H. Jebb. of the Oxford PLiblic HealthLaboratory.

Fermentation reactions were tested in Hiss's serumwater containing 100 of glucose, laevulose, galactose.lactose, maltose, sucrose, sorbitol. mannitol, salicin.raffinose, and inulin.

Tests for soluble haemolysin were made by the addi-tion of 0.5 ml. of six-hour 20'1,o serum broth culturesto 0.5 ml. of 500 horse red blood corpuscles in physio-logical saline and incubating at 37' C. for 90 minutes.Anaerobic cultures were set up in a McIntosh and

Fildes jar, and incubated overnight at 37° C.Antibiotic sensitivity was tested by a ditch plate

technique. the agar ditches containing 10 ,g. ml.chlortetracycline (aureomycin, Lederle) and of erythro-mycin ( ilotycin," Lilly), and 10 units/ml. of peni-cillin in the agar. Eight strains of the AN form werealso tested on ditch plates with oxytetracycline (terra-mycin, Pfizer) and with tetracycline (" achromycin,"Lederle). The minimal inhibitorv concentration ofthese antibiotics against a number of resistant strainswas determined by a tube dilution test in nutrientbroth.

Other details of technique used in the experimentalstudy of these streptococci are described later with theexperiments.

AN Forms of Strep. Pyogenes in BurnsIn October, 1954, cultures on concentrated agar

(with blood) from a pedicle graft with purulentexudate showed a heavy growth of minute colonieswith zones of faint haemolysis after overnightincubation. The patient, a child who had receivedextensive burns of the trunk two years previously,

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FIG. 1.-Strep. pyogenes growing on concentrate dagar (with blood).Growth on the right side is of an ordinary aerobic strain, thaton the left of an AN strqptococcus.

had been treated with oral chlortetracycline forstreptococcal infection during earlier periods inhosp'tal, the duration of chlortetracycline therapytotalling at least 49 days. Nose and throat swabstaken on admission to hospital in September,1954, did not yield Strep. pyogenes on ordinaryblood agar incubated anaerobically.

Subcultures of the minute colonies grew aerobic-ally and anaerobically on concentrated blood agar,but on ordinary blood agar and in nutrient broththey grew under anaerobic conditions only. Thecolonies on anaerobic blood agar had the typicalappearance of haemolytic streptococci, and micro-scopic examination showed short chains of Gram-positive cocci. They proved to be streptococci ofLancefield's group A, which were sensitive topenicillin and to erythromycin but resistant to allthe tetracycline antibiotics.

Similar colonies were soon noticed on culturesfrom other patients, and it was usually possible todistinguish the AN form from ordinary forms ofStrep. pyogenes on concentrated blood agar by itscolonial appearance (Fig. 1). Occasionally therewas no haemolysis in cultures on this medium.

TABLE ISTREP. PYOGENES ISOLATED FROM BURN SWABS

BETWEEN OCTOBER, 1954, AND MARCH, 1955

Sensitivity Tests withGrowth Form Chlortetracycline* Total

of Strep. pyogenesSensitive Resistant

Normal aerobic forms .. 177 9 186AN forms .. .. 12 58 70

Total .. 189 67 256

* Results of ditch plate sensitivity tests.

The Incidence of AN Forms in Burns.-Table Igives the numbers of swabs from which typical andAN forms of group A haemolytic streptococciwere isolated between October 12, 1954, andMarch 3, 1955. This shows that no fewer than 70out of the 256 strains isolated in that period hadthe colonial appearance and the anaerobic growthrequirements of the AN form; the majority ofthese (58/70) were resistant to chlortetracycine.Of the 186 normal aerobic forms, 177 were sensi-tive to chlortetracycline. TheAN strains, which werealso tested with tetracycline and oxytetracycline,showed the same sensitivity to these antibiotics astowards chlortetracycline. Tube dilution sensi-tivity tests showed that the minimal inhibitoryconcentration of the three tetracycline antibioticsagainst five of the resistant strains was 50-100tg. /ml.

Tetracyclne Therapy and the Presence of ANFormsn-Oral tetracycline or oxytetracycline wasadministered as a specific treatment for burns onwhich Strep. pyogenes had been found. Treatmentwas usually started before the antibiotic sensitivitywas known. Most of these streptococci wereaerobic and tetracycline-sensitive, in which casethey were regularly eliminated from burns afterthree to six days of treatment.As would be expected from their normal in-

sensitivity to tetracyclines, AN forms of strepto-cocci present at the beginning of treatment withtetracyclines were found to persist in burnsthroughout the course of treatment. In other casesthey were found to appear during or after such acourse. Of the 19 patients in whose burns ANforms were found, eight showed the organismsduring or shortly after a course of oxytetracyclineor tetracycline. In two cases both sensitive andresistant AN forms were found, one of themyielding the sensitive organisms before, and resis-tant organisms after, a course of tetracyclinetherapy.The association of the presence of AN forms

and tetracycline therapy in 19 patients is shown inTable II.Erythromycin was always effective in eliminat-

ing AN forms from burns; a higher dosage (2 g.,in divided doses, daily, for adults) than that pre-viously found effective (Lowbury and Cason, 1954)was used in these patients.

Pathogenic Role of AN Forms.-Out of 23 skingrafting operations in the presence of AN strepto-cocci, 20 showed less than 80% "take" (TableIII). This is similar to the proportion of suchfailures in the presence of aerobic Strep. pyogenes,

I

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61ATYPICAL ANAEROBIC FORMS OF STREPTOCOCCUS PYOGENES

TABLE I1TETRACYCLINE THERAPY AND THE INCIDENCE OF AN FORMS IN BURNS

Streptococcus pyogenes Isolated from Burns*

AN Form

Tetracycline Sensitive Tetracycline Resistant ITetracycline Sensitive Tetracycline Resistant

TypeDate Agglu- Precipi-

tination tationl~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

None

18.10.54None

.,

,,

10.1.559.2.55None

12.2.55None

,,,.

5445 12 144

2'

2

TypeDate Agglu- Precipi- Date

tination tation

None 12.10.54

18.10.5427.10.54

9.10.54 4 4 11.11.54None _ _ 16.11.54

_ 20.10.54

3.10.54 5 27 44 0 _None _ _ 23.10.54

,.,, _ _ 12.12.54

0

0 5.2.55None*

_ *sI

19.12.54'0.I.55

{27:1.S

5.2.5518.2.5513.2.5522.2.551.3.55

28.2.5528.2.55

Type

Agglu- Precipi-tination tation

5 2744 0

4 4

_ I_5 2744 0

52744 05 12 44 12V

2 O05 27 0

5 27 44 0

52744 052744 0

52744 0

* Dates refer to the first isolation of the form of streptococcus indicated at the head of the column.

and is in marked contrast with the results of skingrafting burns free from streptococci (Lowburyand Cason, 1954).

In addition to its association with failure of skingrafts, the presence of AN forms in burns andon pedicle grafts was often associated with sup-puration and other local signs of infection.

Serological Types of the AN Forms.-The ANforms from 11 of the patients were typed (TableII). From 10 of these the streptococci showedthe agglutination pattern 5/27/44 or a relatedpattern (5/27, 5/44, 5/44/12). Two other agglu-tination types were found, 2 and 4. The ANstrain of type 2 was isolated from a patient (450)

TABLE Il1AN FORMS OF STREP. PYOGENES IN BURNS AND SKIN

GRAFT RESULTS

8o0' LessStrep. pyogenes or More than 80%

in Burns Take Take Total(Successful (Failed

Graft) Graft)

AN forms present 3 20 (87°,) 23Aerobic forms present* 2 16 (88%) 18Burns cleared of strepto-

cocci by antibiotictherapy* .. . 33 11 (25%) 44

* These data from a previous investigation (Lowbury and Cason,1954) are quoted here for comparison.

who yielded AN streptococci of agglutinationpattern 5/44/12 on another burn five days beforeand aerobic tetracycline-resistant organisms ofthe same type again one week later. Two patientshad AN streptococci of agglutination and precipi-tation type 4 on their burns. Only one otherpatient yielded AN streptococci which gave a pre-

cipitation reaction (type 12); aerobic tetracycline-sensitive streptococci from the same patient at an

earlier date were found to have the same agglutina-tion and precipitation type. A number of aerobictetracycline-sensitive streptococci of types 4 and5 / 27/44 were isolated from other patients in theward at the same time as those who yielded ANstreptococci of these types.From five patients typing results were obtained

on aerobic sensitive strains of Strep. pyogeneswhich were present on burns before the appear-ance of AN forms. Two of these patients (450and 330) had type 5/27/44 tetracycline-sensitiveaerobic strains initially, and this or a relatedagglutination pattern was found in the AN tetra-cycline-resistant form which appeared later (ineach case after a course of a tetracycline anti-biotic). In the three other patients (938, 349 and761) AN strains of type 5/27/44 appeared in theplace of unrelated aerobic strains (two of type 4and one of type 28); two of these patients had

Aerobic Form

Type

Agglu- Precipi-tination tation

Patient

873

299281938

434618349689361743995450979a555961330761979b457

TetracyclineTherapy beforeAN FormsAppeared

1952 and 1953

None

5 to 10. 11.54 (be-fore resistantAN forms ap-peared)

None5 to 17. 10.54None14 to 23.10.5426.11 to 4.12.54None5 to 20.1.5514 to 28.1.55None

I I to 24.2.5518 to 28.2.55None

Date

1952 and1953

None21.10.54

18.10.5417.10.54None

20.10.5412.10.5422.11.54None

29.12.5412.1.555.2.55

22.2.555.2.558.2.5518.2.55None

27.2.55

5 27 44

44

48 255 2744

5 12 4452744

3 135 4428

44

400

120

00

28

-I ~~~~~I-

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been treated with a tetracycline antibiotic beforethe AN form appeared.

In one case (995) during tetracycline therapy anaerobic-resistant streptococcus of type 5/44 was

isolated on the same day as an AN form of type5/27/44. In another case (450) the presence ofan AN-resistant form was followed by the isola-tion of an aerobic-resistant streptococcus of thesame typo 12 days after the end of tetracyclinetherapy; a tetracycline-sensitive AN strain wasisolated from the same patient.

Haemolysis and Fermentation Reactions of ANForms.-Soluble haemolysin was produced by allof the six strains tested. Three strains which were

tested showed a range of fermentation reactionssimilar to that obtained from aerobic strains. Acidwas produced from glucose, laevulose, galactose,lactose, maltose, and sucrose, and there was no

fermentation of mannitol, sorbitol, raffinose, andinulin. The reactions with salicin were variable.No difference could be detected in the morpho-

logy of Gram-stained films of AN forms and ofaerobic strains of Strep. pyogenes from burns.

Experimental Study of AN FormsChange to Aerobic Mode of Growth.-Cultures

of AN forms on Dorset egg medium and on bloodagar allowed to stand for 48 hours or longer atroom temperature were often found to growaerobically on subculture to ordinary blood agaror nutrient broth. Further subcultures remainedaerobic. The change to an aerobic mode ofgrowth in these organisms was not accompaniedby any loss of resistance to the tetracyclineantibiotics.

Daily anaerobic subculture in nutrient broth ofAN forms from three patients was not accom-panied by any change to aerobiosis until 20 or

more transfers had been made. At this stage a

subculture from broth, which gave a confluentgrowth on anaerobic blood agar culture, yieldeda few isolated colonies on blood agar plates incu-bated aerobically, and these could be further sub-cultured under aerobic conditions.

Strains which had adopted the aerobic modeof growth sometimes showed small weakly haemo-lytic colonies similar to those of the AN form;but usually they were strongly haemolytic and in-distinguishable from a normal aerobic strain.

Emergence of Tetracycline Resitance in vitro.-During an attempt to induce resistance to tetra-cycline and oxytetracycline, a strain of sensitiveaerobic Strep. pyogenes was found to have becomeresistant to these antibiotics (but still aerobic) afterstanding for several days on Dorset egg medium.

This organism was isolated from the burn of apatient (979a) who yielded a resistant AN strepto-coccus of the same serological type on the sameday.

Effect of Tetracycline on Retention of ANCharacteristics.-Three tetracycline-resistant ANstrains of Strep. pyogenes were each subculturedto two blood agar plates, one of which containedoxytetracycline (12 ,ug./ml.). After incubation(anaerobic), the plates were allowed to stand forthree days and subcultured in duplicate onordinary blood agar plates. These subcultureswere incubated at 370 C., one aerobically and theother anaerobically.

All three strains were found to have changed tonormal aerobic growth after standing on bloodagar without the antibiotic. Subcultured fromoxytetracycline blood agar, however, the threestreptococci retained their AN characteristics. Asecond oxytetracycline plate inoculated with theseorganisms was allowed to stand for three days andagain showed AN forms on subculture. In furthersubcultures on oxytetracycline plates, however,aerobic colonies started to emerge.

Tests on Filtrates of AN Forms.-No growth,aerobic or anaerobic, was obtained from Seitifiltrates of anaerobic broth cultures of one ANstrain of Strep. pyogenes.The Phenomenon of Conditional Aerobiosis.-

Fig. 2 illustrates the peculiar growth characteristicsof the AN forms of Strep. pyogenes. Two platesare shown. Each is divided in two halves, the lefthalf containing concentrated agar (with blood),while the right half contains ordinary bloodagar. Strains (1) and (3) (aerobic strepto-cocci) grow equally well on the ordinary andon the concentrated agar medium under aerobicor anaerobic conditions. Cl. welchii grows wellon either medium anaerobically, but on neithermedium aerobically. Strain 2, the AN form,grows well anaerobically on either medium;aerobically it grows only on the concentrated agarmedium. This phenomenon is found also whenconcentrated agar is enriched with serum insteadof blood.The atypical streptococcus cannot be described

as " anaerobic " except in relation to particularmedia. "Conditional aerobiosis" describes thephenomenon more accurately.

Ponsible Mechanism of Conditional Aerobiosis.-It has long been known that the growth of somehaemolytic streptococci is favoured by anaerobicconditions (e.g., Rosenow, 1914; Oliver andPerkins, 1919). It is also well known that primary

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ATYPICAL ANAEROBIC FORMS OF STREPTOCOCCUS PYOGENES

isolation of Strep. pyogenes from sites of infectionis more frequent on anaerobic than on aerobicculture (Ministry of Health Mon. Bull., 1942;Williams and Miles, 1945).The demonstration by M'Leod and Gordon (1922)

that haemolytic streptococci often form peroxideon aerobic culture provides a possible explanationof their preference for anaerobic conditions. Todd(1930) showed that streptococci of the matt viru-lent type produce more hydrogen peroxide on

aerobic culture and are also more sensitive to itthan glossy avirulent variants; self sterilization,especially of the matt colonies, resulted from theaccumulation of hydrogen peroxide in cultures,which did not, however, occur until the end of thelogarithmic phase of growth.

It seems to us that AN forms may fail to growaerobically either because exceptional quantitiesof hydrogen peroxide are produced at an earlystage of growth, or because these organisms are

exceptionally sensitive to hydrogen peroxide.Their ability to grow on concentrated blood orserum agar might, on this hypothesis, be due tothe production of hydrogen peroxide being sup-pressed on these media. We therefore testedgrowth of aerobic streptococci on ordinary serumagar and on concentrated agar (with serum) forhydrogen peroxide by the potato-benzidine test(see Todd, 1930). Growth of AN streptococci on

ordinary serum agar (anaerobic) and on concen-trated agar (with serum) (aerobic) was also testedfor production of hydrogen peroxide. Severaltetracycline-resistant strains derived from ANforms were included among the aerobic strepto-cocci.Table IV shows that no hydrogen peroxide

could be detected in any streptococcus growingaerobically on concentrated agar (with serum) or

anaerobically on ordinary serum agar; aerobiccultures on ordinary serum agar, however, yieldedabundant hydrogen peroxide (except in the case ofthree out of seven tetracycline-resistant organismsderived from AN forms). The AN forms weretherefore found to grow under conditons which

FIG. 2.-Two blood agar plates streaked with the different culturesunder discussion, the upper plate incubated anaerobically, thelower aerobically.

were unfavourable to the production of hydrogenperoxide, and did not grow under conditions whichfavoured its production.We could find no difference in the sensitivity of

aerobic streptococci and of AN forms to the

TABLE IVHYDROGEN PEROXIDE PRODUCTION ON SERUM AGAR MEDIA

1-5% Agar in Medium 4% Agar ip Medium

Organism No. of Aerobic Culture Anaerobic Culture Aerobic CultureStrains AeoiCutr AneoiCutr AeoiClueGrowth H20 Growth H202 Growth H202

Aerobic Strep. pyogenes.6 +++ +++ +++ 0 +++ 0ANforms.4 0 +++ 0 +++ 0Aerobic streptococci derived from AN 7 +++ +++ (4) +++ 0 +++ 0forms 0(3)

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bactericidal action of hydrogen peroxide. Todd(1930), however, reports that the difference insensitivity to hydrogen peroxide of matt and ofglossy strains refers to the bacteriostatic tests only.Such tests on the AN form were impracticablebecause of the instability of hydrogen peroxideunder the anaerobic conditions which would berequired. A bactericidal mechanism is, however,suggested by the fact that plates inoculated withAN streptococci, showing no growth after aerobicincubation, showed no growth after further incu-bation in an anaerobic jar. Further aerobicincubation of such plates also failed to showgrowth of streptococci.

DiscussionThe appearance of Strep. pyogenies which fails

to grow aerobically on ordinary blood agar is afinding which may be expected to become morecommon if tetracycline-resistant strains are allowedto accumulate in the population. It is importantthat these organisms should not be missed, as theyappear to be of clinical importance and cannotbe eliminated by tetracycline. All the strains wehave examined are sensitive to penicillin and toerythromycin. Systemic penicillin has been foundineffective for the treatment of establishedinfection of burns with Strep. pyogenes (Jack-son et al., 1951), presumably because of thepresence of penicillinase-producing organisms(especially Staph. aureus) in the same burn.Erythromycin is highly effective in eliminatingAN streptococci from the burn surface, but inview of the importance of preventing acquiredresistance in staphylococci it is, perhaps, desirableto try repeated applications of concentrated peni-cillin cream (10,000 units per g.) before prescribingerythromycin for this purpose.The capacity of concentrated agar to allow

aerobic growth of these AN forms is an un-expected virtue in a medium which has alreadyproved its value in preventing the swarming ofProteus. The mechanism of this conditional aero-biosis is uncertain, but our experiments show anassociation between conditions that allow thegrowth of these organisms and conditions that areunfavourable to the production of hydrogenperoxide by aerobic streptococci. It is possiblethat AN forms produce more peroxide or aremore sensitive to it than aerobic streptococci. Ifthis is the mechanism, it is perhaps surprising thatAN forms fail to grow aerobically on ordinaryblood agar, as the catalase in the blood preventsaccumulation of detectable hydrogen peroxide onthe surface culture of typical Strep. pyogenes.

Catalase in blood agar, however, cannot beassumed to prevent the accumulation of inhibitoryconcentrations of hydrogen peroxide inside thebacterial cells.The association of AN streptococci with tetra-

cycline resistance suggests that they emerged asa consequence of tetracycline therapy, either byselection of mutants or by adaptation. Three un-related serological types of Strep. pyogenes haveappeared with AN characteristics in six months,a fact which suggests that this transformation isnot rare, and proves that the outbreak of infectionwith AN forms did not arise by cross-infectionfrom a single mutant. It is of interest that theAN strains included one of precipitin type 12,which is known to have a special association withacute nephritis, and most of the other AN strainswere agglutinated by antisera to types 5, 27, and 44,which are related to type 12 (Dingle, Rammel-kamp, and Wannamaker, 1953; Wilmers, Cunliffe,and Williams, 1954). The successive isolation firstof sensitive aerobic and then of resistant ANstreptococci of the same type from the samepatient suggests that such changes were occurringin burns, particularly when the AN form appearedafter tetracycline treatment. Cross infection withAN forms was, however, common, since more thanhalf of the patients from whom they were isolatedhad not received tetracyclines.

In its association with acquired antibiotic resist-ance and its small colony size, the AN form showssome resemblance to the appearance of "G"colonies of staphylococci and other organisms(e.g., Hadley, Delves, and Klimek, 1931 ; Wise andSpink, 1954). Another point of resemblance is thereversion to the normal kind of growth (aerobic)on storage or repeated subculture away from theantibiotic. It seems probable that the reversionto aerobiosis is, in fact, a final stage in the estab-lishment of resistance to tetracycline. The ANform would, on this hypothesis, be an intermediatestage in the development of tetracycline resistancein which there is a temporary narrowing of theadaptability of the organism to environmentalchanges.

Tests in vitro did not show the emergence ofAN forms from stable Strep. pyogenes exposed totetracyclines; they did, however, suggest that thepresence of tetracyclines might delay the establish-ment of full aerobic growth in AN streptococci.

SummaryHaemolytic streptococci of group A (AN)

isolated from burns of 19 patients during thecourse of six months had the following character-

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ATYPICAL ANAEROBIC FORMS OF STREPTOCOCCUS PYOGENES

istics: (1) anaerobic but no aerobic growth in brothand on serum or horse blood agar (1.5% agar);(2) both aerobic and anaerobic growth on serumor blood agar containing 4% agar (concentratedagar media); (3) resistance to the tetracyclineantibiotics in most strains ; (4) sensitivity to peni-cillin and erythromycin; and (5) reversion toaerobiosis without loss of tetracycline-resistanceon storage.

Eight of the patients had received tetracyclineshortly before these AN forms appeared, andanother had received prolonged courses of tetra-cycline therapy in the previous two years. Threedifferent agglutination types were represented, butmost strains were of type 5/27/44 or a relatedpattern. Skin grafts usually failed in the presenceof these organisms.Hydrogen peroxide was produced in surface

cultures of aerobic streptococci on 1.5% but noton 4% agar. The bearing of this observation on

" conditional aerobiosis " of the AN form isdiscussed.

We are grateful to Dr. W. H. H. Jebb, of the OxfordPublic Health Laboratory, for typing our streptococci;we also wish to thank Mr. H. Lilly, A.I.M.L.T., andMiss Georgina Hogg for assistance, and Mr. C.Richardson, A.I.B.P., A.R.P.S., for the photographs.

REFERENCESDingle, J. H., Rammelkamp, C. H., and Wannamaker, L. W. (1953).

Lancet, 1, 736.Hadley, P., Delves, E., and Klimek, J. (1931). J. infect. Dis., 48, 1.Hayward, N. J., and Miles, A. A. (1943). Lancer, 2, 116.Jackson, D. M., Lowbury. E. J. L., and Topley, E. (1951). Ibid., 2,

705.

Lcwbury, E. J. L., and Cason, J. S. (1954). Brit. med. J., 2, 914.M'Leod, J. W., and Gordon, J. (1922). J. Path. Bact., 25, 139.Ministry of Health (1942). Monthly Bull. Emergen(y Pubi. Hlth Lab.

Service, 1, February, p. 11.Oliver, W. W., and Perkins, 0. C. (1919). J. infect. Dis., 24, 22.Rosenow, E. C. (1914). Ibid., 14, 61.Todd, E. W. (1930). Brit. J. exp. Path., 11, 368.Williams, R. E. O., and Miles, A. A. (1945). J. Path. Bact., 57, 27.Wilmers, M. J., Cunliffe, A. C., and Williams, R. E. 0. (1954).

Lancet, 2, 17.Wise, R. I., and Spink, W. W. (1954). J. clin. Invest., 33, 1611.

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