(
Received March 1 1;accepted for publication May 29, 1968.)This work was supported by the Health Research Council of the City of New York
(
U 1534),
theUpjohn Company, and The John A. Hartford Foundation (348-2603). KS. is supported by a career
development award, National Institutes of Health 5 K3 HD-22, 493-04.
ADDRESS: (KS.) College of Physicians and Surgeons of Columbia University, 630 \V. 168th Street,
New York, New York 10032.
PEDIATRICS, Vol. 42, No. 6, December 1968
957
PENICILLIN
RESISTANT
ALPHA
STREPTOCOCCI
IN
PHARYNX
OF
PATIENTS
GIVEN
ORAL
PENICILLIN
Katherine Sprunt, M.D., Winifred Redman, B.A., and Grace Leidy, M.A.
Babies Hospital and Department of Pediatrics Columbia University College of
Physicians and Surgeons, New York
ABSTRACT. Patients who have received oral
peni-cillin max’ carry in the oropharynx alpha
hemolyt-ic streptococci, of which varying proportions are
resistant to one or more units of penicillin per
milliliter. Since the use of oral penicillin is
in-creasing and penicillin is the drug of choice for
prevention of endocarditis before oral or
pharyn-geal trauma, semiquantitative methods have been
used to document
(
1)
the frequency ofa-strepto-cocci resistant to 1 or more units of penicillin per
milliliter in patients receiving prophylactic
peni-cillin orally and intramuscularly, (2) the
fre-quency and rate with which significant numbers
of a-streptococci appear after exposure to oral
penicillin, (:3) the proportion and degree of
re-sistance of a-streptococci which emerge resistant,
and (4) the rate at which the numbers of resistant
organisms are reduced to a presumably
insignifi-cant level when penicillin is withdrawn. The
re-suIts show that there is a statistically significant
increase (P < .001) in the proportion of
a-strep-tococci resistant to at least 1 U per milliliter of
penicillin in the pharynx of patients receiving their
)rophylaCtic penicillin in oral form (75%) in
corn-panison with those on intramuscular penicillin
pro-phylaxis
(
15l)
. Similarly, 17 of 22 patients onoral penicillin therapy developed flora containing
resistant streptococci. Six such patients carried
a-streptococcal populations, 7 to 50% of which were
resistant to 1 but not 5 U. Eleen carried
strepto-cocci, 1 to 100% of which were resistant to 5 or
more units per milliliter. Of 12 patients followed
closely, 8 developed resistant streptococci within
the first week of therapy, one after 2 days.
Resistant streptococci decreased rapidly when
therapy was discontinued. Eleven of 12 patients showed less than 20% of their streptococcal popula-tion resistant to 1 unit at 15 days and less than
10% at 30 days. Low levels (< 10%) persisted in
six patients followed for intervals longer than :30
days and up to 144 days. Pediatrics 42:957, 1968,
ORAL SURGERY, PENICILLIN RESISTANCE,
STREPTO-COCCUS, BACTERIAL ENDOCARDITIS, RHEUMATIC
FE-VER.
P
ATIENTS who have received oral penicil-un may carry alpha()
hemolytic strep-tococci in the mouth and pharynx, varying proportions of which are resistant to 1 ormore units of penicillin per milliliter. This fact has been recognized since the early
days of use of oral penicillin1 and is being
increasingly emphasized now that more
efficiently absorbed preparations of oral
penicillin are available.25 Since penicillin is
the generally accepted antibiotic of choice for prevention of endocarditis at the time of oral surgery, dental manipulation, or tonsil-lectomy, it is essential to document more
fully (1) the frequency and nate with which significant numbers of resistant
a-streptococci appear after exposure to oral
penicillin preparations,
(
2) the proportion and degree of resistance of organismswhich emerge resistant, and
(
3)
the rate atwhich the numbers of resistant organisms
are reduced to a presumably insignificant
level when the antibiotic is withdrawn. For
this reason, semiquantitative studies of throat cultures from children and a few adults receiving oral penicillin were carried out. The results show that with both
958
month or more may be required after ther-apy is discontinued before these organisms decrease to less than 10% of the total a-streptococcal population. The penicillin re-sistant streptococci noted were, with very
few exceptions, sensitive to low concentra-tions of erythnomycin and lincomycin.
Media
MATERIALS AND METHODS
Trypticase soy agar (BBL) #{176}containing
5%
defibninated horse blood was used rou-finely. Mitis-Salivarius agar with tellunitef was employed when necessary to facilitatecounting streptococcal colonies in those
cul-tunes in which the overgrowth of
gram-neg-ative entenic bacilli was a problem. Trypti-case soy broth
(
BBL) #{176}containing 1% horseblood was used for the growth of single col-ony isolates.
Culture Methods
All cultures were taken by one
individ-ualt
(
K.S.)
with Falcon “Swubes” to assure a uniform culture process. The swabs were placed immediately in 1 ml of trypticase soybroth for transportation to the laboratory where they were twirled rapidly in the
broth, pressed as dry as possible by rolling against the side of the tube, and then dis-carded. The residual broth was designated the “undiluted” culture. Serial tenfold dilu-tions of the culture in trypticase soy broth were made and 0.3 ml aliquots of the
dilu-tions selected were spread evenly over the
surface of 100 X 13 mm blood agar plates (with and without antibiotics) and
incu-bated in an upright position. After 48 hours incubation at 36#{176}C,the numbers of alpha hemolytic streptococcal colonies were
re-corded.
Definition of 2-Hemolytic Streptococci
The term a-hemolytic streptococcus as used here refers to a-hemolytic mitis and
salivarius streptococci as well as pneumo-cocci which were not distinguishable by the
screening techniques used. Random testing of resistant streptococci revealed no
resis-tant pneumococci. Enterococci were cx-cluded.
Antibiotic Sensitivity Test on
Original Culture
An 0.3 ml aliquot of the 1ft1, 102, and
rn-s
dilutions of each culture was seeded on agar containing 0.1, 0.5, 1, and 5 U ofpenicillin G per milliliter. Aliquots of the 102, ]J-3, and 10 dilutions were plated on
antibiotic-free agar as controls; the 10
dilution was included when a-streptococci were very low in number. Sensitivity to
erythromycin (Ilotycin Gluceptate) given
intravenously and lincomycin (Lincocin)
were tested in parallel with penicillin.
In-itially the concentrations used were the
same for all three antibiotics; subsequent-ly, 0.01 and 0.05 g erythromycin and 0.1
and 0.5 lL lincomycin per milliliter were used. Higher concentrations of all three antibiotics were included when indicated. The numbers of a-streptococcal colonies were recorded after 48 hours’ incubation and the percent of colonies formed on the
varied antibiotic-containing agar were cal-culated. Since one plate only was used per dilution, the percent is approximate. In
or-der to limit bias, the exact figure (as per-cent) is recorded in the tables, as well as the actual number of streptococcal colonies in the control plate from which it was cal-culated.
Controls of Penicillin Activity
Crystalline penicillin G (100,000 U per
milliliter), lincomycin, and erythromycin (10,000 [L per milliliter) were prepared each month and small aliquots were stored frozen at
-
10#{176}C. Antibiotic-containing agar and control plates were prepared weekly and stored at 4#{176}C.Each batch of penicillin-containing agar plates waschecked for activity with “standard” a and
#{176}Baltimore Biological Laboratory, Inc., 2201
Aisquith Street, Baltimore, Maryland 21218.
f
Difco Laboratories, Detroit, Michigan 48201.Except those from other institutions obtained from patients receiving prophylactic antibiotic.
§ Falcon Plastics, 5500 West 83rd Street, Los
streptococci of known penicillin sensitivity. As a further control, the “standard” strepto-cocci were occasionally examined for sen-sitivity on agar containing freshly dissolved
weighed penicillin G (50 mg at a potency
of 400 U per milligram
)
.1!
A 2mm loopful of an overnight subculture of test strains was used as inoculum. Young (6 hour)subcul-tures were occasionally tested in parallel, but no significant differences in sensitivity were noted.
As a control of the agar medium selected
(
trypticase soy agar containing 5% horse blood), 24 independent single colonies of a-streptococci, selected for their measurable differences in degree of sensitivity topeni-cillin, were simultaneously tested for their sensitivity on three types of agar containing penicillin. A 2 mm loopful of an 18-hour
blood broth subculture was streaked on trypticase soy agar, with and without added horse blood and on 10% horse serum containing agar medium used by Kuttner6 for the growth of p-streptococci. No
differ-ences in the individual sensitivity pattern of the selected populations were noted with the various agar media.
RESULTS
Two approaches to the problem of
peni-cillin resistant a-streptococci as a compli-cation of the use of oral penicillin were considered. The first compared the propor-tion of penicillin-resistant a-streptococci in the pharynx of rheumatic fever patients re-ceiving prophylactic penicillin orally with the proportion of resistant streptococci to be found in patients on intramuscular pro-phylaxis or those not receiving penicillin. The second explored the frequency of re-sistant streptococci before, during, and after therapeutic oral penicillin.
Through-out the paper, unless otherwise specified, “resistant a-streptococci” refers to strepto-coccal populations at least 1% of which pro-duce microscopically detectable colonies on
agar containing 1 unit of penicillin per
milli-I! Kindly supplied by Charles Pfizer and
Com-pany, Inc., 2:35 E. 42nd Street, New York, New
York 10017.
liter. The term a-streptococci as used here does not include enterococci.
Oral Penicillin Prophylaxis-.-Proportion
of Patients with Resistant a-Streptococci
The data in Table I show that there is a
significant increase in the proportion of a-streptococci resistant to at least 1 unit per
milliliter of penicillin in the pharynx of pa-tients receiving their prophylactic penicillin in oral form in comparison with those on
intramuscular penicillin prophylaxis or not receiving penicillin. None of 53 control sub-jects and only 7 of 47 patients receiving monthly injections of long acting
benza-thine penicillin in recommended dosages carried streptococcal populations with as high a proportion as 1% resistant to 1 U per milliliter. These results were in marked contrast to those of the 72 patients on oral prophylaxis, 75% of whom were carrying
penicillin-resistant streptococci when cul-tuned. Significant differences between the groups are manifest even at the level of 0.5
units per millimeter.
The intramuscular prophylaxis patients
(
Table 1)
are considered in two groups:(
1)
the Babies Hospital group of children attending the Babies Hospital Cardiology Clinic where oral penicillin is rarely if everused therapeutically, (2) and the “other” group, most of whom are cared for in other
New York City clinics and whose past
his-tory with respect to recent oral penicillin therapy is usually unknown to us. Two pa-tients in this latter group were eliminated from the table because of known recent cx-posure to oral penicillin.
TABLE I
DIFFERENCE IN DEGREE OF PENICILLIN RESISTANCE OF PIIARYNGEAL a-STREPTOCOCCI FROM (ONTROLS* AND RHEUMATIC FEVER PATIENTS RECEIVING ORAL OR INTRAMUSCULAR PENICILLIN PROIIIYLAXIS
Penicillin Prophylazis
Patients
Number Patients with at Least 1
cr-sirej)-tococcul (‘E. U. in Preseme of (flits per Milliliter Penicillin
Source .\u
in-oer .1
48 11
--i 5
0 0
None (controls) Babies Hospital 53
Intramuscular
Babies Hospital 23 21 11 ‘i (9) 0
Other 24
47
24
45
13 5(1,) 1
7 (15 c) 1
19 (86) 11
All sources
Oral
Small clinic ‘2 ‘2 ‘21
Large clinic #{232}2 22 20 14 (64) 3
Other 8
---
‘28 7 (79) 13All sources 72 72 68 55 (75 ) 38 (38)
* Controls = adults and children with no recent penicillin exposure. t C.F.U. = colony forming units.
One culture not tested at these concentrations.
§
64%,
respectively, with resistant organisms) may be an indication of the reliability with which the prophylactic regimen was fol-lowed.The data derived from the patients on
oral penicillin prophylaxis show that they fall into three groups (Table II
)
accordingto the degree of resistance demonstrated by
their pharyngeal streptococci. The data of
five patients from each group are shown in Table II to illustrate the patterns within each group. Approximately half of those de-veloping resistant streptococci carried orga-nisms resistant to 1 U but sensitive to 5 U per milliliter. The other half carried strep-tococci appreciable proportions of which were resistant to 5, 10, and 20 U per millili-ter.
The doses of oral penicillin used varied from 125 mg once a day to 250 mg twice a day. The majority received 125 mg once or twice a day. Both phenoxymethyl penicillin
(Pen-Vee
)
and penicillin C were used.Neither dose size nor preparation appeared
to influence the results.
It is apparent from the data in Table II that the usual doses of oral penicillin nec-ommended to protect the patient from a-streptococcal endocarditis at the time of oral or pharyngeal trauma might prove in-adequate for more than one third of the pa-tients. However, all the populations of peni-cillin resistant streptococci selected from at least 10 such patients were uniformly sensi-tive to low concentrations of erythromycin
(
0.05
‘.g per milliliter) and lincomycin(0.5
lL per milliliter).Oral Penicillin Therapy-Proportion of
Patients in Whom Resistant Streptococci
Emerged During Therapy
Fifteen pediatric patients
(
14 hospital-ized)
who received only oral penicillin andan additional 7 children (6 hospitalized) who received only one or two parenteral
a-STREI’TOCOCCI FROM PATIENTS RECEIvING ORAL PENICILLIN PROI’ILYLAXIS GRouPED ACCORDING TO
PERCENT AND DEGREE OF PENICILLIN RESISTANCE
a-Strep Group
Total
Patients
in Group
Single
Potient
Examples
0 to 1 resistant to
1j_T ml 25 3
4
0
‘2
37 3
4 5
38
At least resistant to
1 U per ml bitt selisitive
to 5 tT per nil
At least l resistant to
5 or more -t:per ml
ARTICLES
* (‘FI = (T)lOIIy forniing units (a-streptococci).
therapy had cultures taken until at least some detectable streptococci initially sup-pressed or eliminated by therapy had reap-peared. Of these 22 patients, 17
(
77%) car-ned streptococcal populations at least 1% of which were resistant to 1 U per milliliter or more(
Table III)
.
The failure of two ofthe five patients who did not develop orga-nisms with this degree of resistance could
be due to the fact that one (J.B.
)
receivedampicillin in capsule form and the other
(A.W.
)
received ampicillin administered by indwelling stomach tube. Consequently, 17 of the 20 patients (85%) whose throat flora was presumably exposed to high con-centrations of penicillin at repeated inter-vals developed resistant organisms (i.e., at least 1% of the population resistant to 1 unit per milliliter or greater). Ten of the 20 patients had cultures taken before therapy was begun. Of these, three had resistantor-ganisms initially (8%, 7%, and 2% resistant to 1 U per milliliter). Of the remaining seven patients, four (57%) developed their
resistant organisms while under observation
(Table
III).
Patient W.C. is of interest in that he went through a course of high dose oral penicillin therapy (200,000 units every 6 hours)
without developing a significantproportion of resistant organisms
(Table
III)
and then several months later yielded cultures with 16% and 18% of the strepto-cocci resistant to 1 U per milliliter follow-ing lower prophylactic dosage(
200,000 U twice daily) . Patient R. Bu. was culturedafter 0, 2, 7, 9, and 13 days of oral penicillin
treatment. There was a gradual increase in the proportion of streptococci resistant to 0.5 U per milliliter up to 100% at 13 days.
No colonies resistant to 1 U per milliliter were detected until day 13.
Rate of Emergence of Resistant
a-Streptococci
Table IV presents a record of results from 11 pediatric patients and 1 adult
(J.A.) who had cultures taken before and
at least twice during the first week of oral
TABLE II
%
(‘F. U. in Presence qf Units per if illiliter PenicillinC.F.U.* Control
Logio Vuinber
Dil. (0.3 ml) O5 1 5 10
T
.1
-3 61 60 16 0 -
--4 5’2 12 13 0 -
--4 180 4 2 0
--3 107 ‘21 ‘24 0 -
--4 100 23 19 0 -
-1 -4 151 - 78 14 1-2 21
‘2 -3 304 15 6 5 6 6
3 -4 23 -
-
100 10 104 -2 87 110 161) 110 100 82
962 STREPTOCOCCI penicillin therapy. The earliest
develop-ment of resistance to at least 1 U per millili-ter by more than 1% of the streptococcal population observed in patients shown to
be free of detectable resistant organisms at the start of therapy was 2 days, in D.O., a 5-month-old infant. The next earliest was at
4
days(
C.S.)
.
Resistant organisms were present in the pharynx of S.H. at 6 days,when the first culture was available after start of therapy.
Four of the patients
(
S.P., Q.O., J.A., andD.S.
)
carried a low proportion of organisms resistant to 1 U per milliliter before therapywas begun. By the second day of therapy
the proportion of resistant organisms in-creased markedly in Patients S.P. and Q.O.
The streptococcal population of both pa-tients decreased tenfold within 24 hours of onset of therapy; the five to tenfold increase on the second day was due largely to the ap-pearance of organisms resistant to at least 1 U per milliliter. A different pattern was
ob-served in Patient J.A. There was no marked drop in the number of streptococci and by the second day of treatment the proportion resistant to 1 U per milliliter increased to 22% and remained at this level throughout the
TABLE III
PERCENT a-STREPTococCI RESISTANT TO PENICILLIN (UNITS PER MILLILITER) IN PATIENTS RECEIVING ORAL PENICILLIN THERAPY
Pthen 1._If. or 1.V. Treatment Oral Trcalment Dose Product (rn:) C.F.U.-ConLrol Cultureal Days Oral Log1o Number Treatment Dii. (o.. ml)
% C.F.U.
. .
in Pre8ence of Units.
..
per Milliliter Penicillin
0. 1 (I..5 I J) i()
----
-- ---- -
-Few Few Few Few
56 S IS .08
S.”. TE.’ %;e;3; hr Ampi. #{176}#{176} 5Oi -__;_-__ 16
-:--_
-2 ::---FewI
- ---Few
B.S. Ampi. Unknown 10 3 131 46
J.G. Ampi. 5OO! 4 1 0 Fewi - Fewi Few Few
D.A.’ PenV. 5O’ 9 3 70 90 1Q8 117 134 4i
Few Few - -10 Li 0 0 O iO R.V. D.S.t Q.O.t DO.’ R. Bo. P.N. Once Once ()iwe Once Pen V. Pen V. Pen V. Pen G. Oxa. PenG. Oxa. 5o# 944 I31 10 0 7 -3 4 -4 Few 34 O 100 Few --140 43 -100 Few Few ---- -19 17 41 8 ---70 -20 I5O ‘2.505 63# 5OOJ 5O 5O9J 14 0 4 4 170 5 100 7 60 1
60 60 60
0.4
----104 10 i
- Few 0
80 40 0
1 .04 l
7
- -- --- ---
----S ISO 101 100 95
1 Few Few Few Few
31 108 109 80
.
3 43 111 60 37
Sand t=Cultures tested before oral treatment began: ‘resistant colonies not detected; t resistant coloniea detected (1sm) or greater).
: is colony forming units or less.
I 1:1 dilution.
I Single colonies selected-resistant.
#{182}Every 6 hours.
.
I (itient 1._If. or IV.
Treatment
Oral T,ealment
Dose I redact (‘n:i)
Culture at
Days Oral
C.F.U.-Co’itrol
V
Log,, ?santher
Dil. (0.3 ml)
‘ C.F.U. in Presence of lnz1s per Milliliter I’enicillin
-
-______
0. 1 Ii. . I ‘ . ii)
0
20
CR. Once Pen V. 9.SO 5 4 18 28 SQ 44 0
LL.Twice
s.p.t
PenV.
PenV.
5O#
94#
4 3
FewFew Few OO
S 110 80 65 50 - 0
V.A. Peii V. 5Of 7 4 17 95 43 17 0 0
c.s.* PenV. I5l 4 3 160 100 110 81 0
G.T. Oral Pen. for tooth extraction
:
48 30 3 20 0 0A.\V. Ampi.
NG tube
is 4 190 53 0 0 0 0
JR.’ Ampi.
capsule 19 4 .51 101 0 0 0 0
R. Ba.’ Pen V. 250” P3 3 33
I1
132 100 .6 - 0
TI.’ Pen V. 7 3 F17 93
‘a Four times aday.
tt Every 4 hours.
)) Two times a day.
Ampi. “ampicillin; Pen =penicilliti; Oxa. =oxacillin.
0 0
TABLE III (Continued)
first week of therapy. Patient D.S. was not
as closely followed. By the seventh day of
therapy, 20% of his streptococci were resis-tant to 5 U, and an unknown proportion ere resistant to 10 U per milliliter. Sixty
percent of his streptococci were resistant to
5 and 10 U per milliliter at 14 days, and an unknown proportion were resistant to 20
U. Four patients failed to develop orga-nisms resistant to at least 1 U per milliliter within the first week of treatment. Three of
these
(
M.E., R. Bu., andS.W.
)
showed re-sistant organisms at a later date(
Table IV). The fourth patient was discharged on the second day of therapy and there is doubt that treatment was continued at home.Cultures of all patients in Table IV, except J.A., showed an initial marked decline in
population density, eight to virtually detectable levels. Data from the cultures of C.S., presented in some detail in Table
v,ir
illustrate this finding. The results areexpressed as the number of a-streptococcal colonies rather than percent in order to
show typical data as collected.
The data in Tables IV and V show that
the rate of emergence of streptococci resis-tant to 1 U or more per milliliter is van-able, ranging from 2 days to more than 13
days. However, 8 of the 12 patients on
whom pretreatment cultures were taken and who were followed closely in the initial stages of therapy, carried resistant
strepto-cocci
(
at least 1 U per milliliter)
in the pharynx within the first week of exposure to oral penicillin.Degree of Resistance to Penicillin and
Stability of Resistance Trait
The degree of resistance to penicillin of the streptococci from patients on oral thera-peutic penicillin varies considerably as
11To save space, Tables V, VI, and VII are printed
964
shown in Table III. In 6 of the 17 patients
who developed resistant streptococci
(
C.R. through G.T., Table III),
resistance to 1 Ubut not 2 or 5 U per milliliter emerged in varying proportions of the populations
(
upto 100%). This demarkation point
(
1 U per milliliter) persisted for at least 8 days of therapy in C.S.’s cultures(
Table V) and has been shown to persist for long periods in individuals receiving prophylactic doses. Cultures of 8 of the remaining 11 patients(
Table III)
showed them to be carrying streptococcal populations, at least 10% of which were resistant to at least 5 U per mil-iJiter. Resistant colonies selected from con-trol and 5 U per milliliter plates of seven of these patients were resistant to at least 10U per milliliter and colonies from three of the four patients tested were resistant to 20 U per milliliter.
Colonies of a-hemolytic streptococci se-lected from cultures of 12 patients were studied in detail. From 9 to 50 colonies per patient were selected from antibiotic-free control plates as well as plates containing
varying concentrations of penicillin. These populations were seeded into broth, incu-bated overnight, and tested for their degree of resistance to penicillin by the streak plate method. In each instance the initial
resistance pattern was confirmed, and the proportions of colonies of various degrees of resistance found among those picked
from control plates were as expected from the quantitative data. Twelve single colony isolates from control and penicillin plates were chosen from a culture of each of two patients. After five sequential subcultures
(.05 ml as inoculum), they were examined for their degree of resistance. With a single exception, no change in the resistance pat-tern was noted.
Rate of Decline of Resistant Organisms
after Cessation of Oral Therapy
Twelve patients who developed
signifi-cant numbers of resistant streptococci were followed with at least one culture within 15
days of cessation of therapy (Fig. 1). Some of these patients received parenteral
peni-cillin therapy for the first few days before
they were shifted to oral therapy. Since no difference was observed in the rate of loss of their resistant populations as compared to the rate of loss of organisms in which re-sistance developed on oral therapy alone, no distinction is made between them. The
figure shows the rate of loss of the propor-lion of streptococci which produced cob-nies in the presence of 1 U per milliliter.
Nine patients carried significant proportions
of streptococci resistant to 5 U, and five patients had organisms resistant to 20 U per milliliter. With a single exception (10% present after 8 days
)
, no streptococci resistant to 20 U per milliliter were found 1 week after therapy ceased. Uniformly the organisms resistant to 5 U decreased atleast as rapidly as those resistant to 1 U per milliliter. The figure shows that the
propor-tion of streptococci resistant to 1 U per mib-liliter dropped to 20% or below within 2 weeks after therapy in all but 1 patient. Again, with a single exception, in all
indi-viduals tested 1 month after therapy was discontinued, the proportion of resistant
or-ganisms was 10% or less, usually much less. The one exception is a child
(
D.S.)
whose mother admitted feeding him “the rest ofthe medicine” after he was thought to be off medication; 15% of his population was still resistant to 1 U per milliliter at what
was thought to I)e 30 days following treat-ment.
Eight individuals were followed for more than 31 days. Ore had 3% of his a-strep-tococci resistant to 1 U per milliliter at 31
days, but none was detectable after 80 days
after therapy. Another had .02% resistant
to 1 U at 38 days and none at 57 days. The six others yielded 1.0% at 39 days, .01% at 49 days, 0.5% at 57 days, 0.7% at 68 days,
1.0% at 85 days, and 2% at 144 days. The course of the eighth individual (adult), fol-lowed every few weeks for over 4 months, is shown in Table VI. The results from these few individuals suggest that low pro-portions of organisms resistant to at least 1
% (F. U. in Presence of at Least 1 Unit Penicillin Per Milliliter After l)ays of Treatment
4 56
(‘oinment
81
rare col.
0’)
0*
7
20 0* 0
0 50
0
28
0
No follow-up
No follow-up
l)ay 0 equivalent to 1ay 144 ‘rable VI
60% (lay 14
‘20% at 26 dayst
0.6% day 13
0.7% at next culture (lay 68
No follow-up
* a-streptococci either very low in numl)er or absent at 10I (lilUtiOlt.
t Days 7-22 no a-streptococci detecte(l at 10 dilution. Received paretiteral penicillin an(l kallaIllycill On
(mv 10 for 3 days. Oral penicillin treatment resuitied on day 13.
Sensitivity of Penicillin Resistant
a-Streptococci to Erythromycin and
Lincomycin
Aliquots of the cultures of most of the patients discussed were also cultured on agan containing particular concentrations of erythromycin and lincomycin.
Prelimi-nary work with randomly selected cultures
containing penicillin-sensitive streptococci
showed that from 20 to 100% of many of
the streptococcal populations grew on agar containing 0.01 .tg per milliliter of
erythro-mycin, and none grew to colony size on
agar containing 0.05 .tg per milliliter.
Simi-larly, up to 100% of many of the
strepto-coccal populations grew in the presence of 0.1 ‘.g per milliliter of lincomycin and not over 1% to 5% grew in the presence of 0.5
g per milliliter. For convenience, orga-nisms resistant to no more than .05 p-g of erythromycin and 0.5 pg of lincomycin were considered “sensitive” to the antibi-otic discussed.
A total of 49 initial cultures (28 patients)
containing streptococci resistant to at least 1 U of penicillin per milliliter and 43
cul-tunes
(
33
patients)
lacking penicillin-resis-tant streptococci were examined for sensi-tivity of the streptococci to erythromycin and lincomycin. Virtually all the streptococ-cal populations were sensitive to 0.05 .tg permilliliter of erythromycin and to 0.5 p.g per milliliter of lincomycin, i.e., were “sensitive”
organisms. The rare exceptions are shown in Table VII.
Patients R.J. and J.W.
(
Table VII)
bothcarried relatively small numbers of
strepto-cocci resistant to 1, but. not 5, g per millili-ter of lincornycin. Single colonies of these organisms were not studied, but there is no reason to believe from the data shown that
cross resistance with penicillin existed. Patient D.S. (whose past exposure to antibiotics is unknown) carried organisms
resistant to both erythromycin and
linco-mycin in his pretherapy culture. Study of 17 single colonies selected from control
plates and plates containing 1.0
,.gerythro-mycin and 0.5 .g lincomycin pen milliliter
showed that those selected from control plates were either sensitive to all three anti-biotics or resistant to 1 U of penicillin per
TABLE IV
RATE OF EMERGEN(E OF a-STREI’TOCOCCI RESISTANT TO PENICILLIN (AT LEAST I U PER MILLILITER)
Patient
0 1 2 2
1)0. 0 0 100*
C.S. 0 0* 0* 0*
5.11. 0* - - 0*
F.M. 0 - : 0 0
S.P. 7 5 50
Q.0. ‘2 - 95
J.A. ‘2 7 22 ‘26
D.S. 8 - -
-ME. 0 0 0
-R.Bu. 0 0
S.W. 0 0 -
-TI. 0 0 0*
-No follow-up
See Table V
No follow-up
milliliter and sensitive to erythromycin and
lincomycin. Organisms selected from the
plates containing 1.0 and 5.0 g of erythro-mycin and 0.5 .g of lincomycin were
sensi-tive to 1 U of penicillin per milliliter. One hundred percent of these populations picked from erythromycin and
lincomycin-contain-ing plates grew on 5 pg of erythromycin and 0.5 pg of lincomycin, and 50% or less grew
on 1 and 5 tg of lincomycin per milliliter. Sensitivity to higher concentrations of eryth-romycin was not tested. In this instance, these results suggest some degree of cross resistance between erythromycin and
linco-mycin but no cross resistance between
pen-icillin and the other two antibiotics. Treat-ment with penicillin increased the degree
and incidence of penicillin-resistant orga-nisms and promptly removed the organisms resistant to the other antibiotics. The latter did not recur within the period of observa-tion.
On the other hand, the cultures of pa-tient R.Bo. suggest cross resistance between all three antibiotics. Selected colonies showed resistance to 10 U of penicillin, 0.5 [Lg of erythromycin, and 1 pg of lincomycin per milliliter. Similar cross resistance has been noted in a-streptococci of another pa-tient who was not included in the study. Selected colonies from her culture grew on
agar containing 5 U of penicillin, 50 pg of
erythromycin, and 1 tg of lincomycin per
milliliter. About
50%
of the population of one colony grew on 5 pg of lincomycin per milliliter. These in vitro results suggest thatlincomycin should have been an effective drug therapeutically for each patient if needed. In the first two patients
erythromy-cm should have been at least equally use-ful or preferred.
DISCUSSION
Krumweide in 1949,1 and more recently Garrod and Waterworth, Naiman and Bar-row,3 Tozer, et al., and Stirland and Shotts,5 reported that a high proportion of patients who received oral penicillin carried
penicillin-resistant a-streptococci in their pharynx1’3 or saliva2,4,5 and tooth-gum mar-gins;3 various types of patients used as
con-trol, some of whom received penicillin
in-tramuscularly, rarely were similarly
af-fected. Garrod and Waterworth2 and
Doyle, et al. have shown that these
resis-tant streptococci may cause endocarditis. While disease caused by such organisms can be successfully treated with large doses
of penicillin, in Garrod and Waterworth’s cases,2 routine preventive measures with
penicillin failed to protect the patients from endocarditis. Our data show that only 7 of
47 children with rheumatic fever who
re-ceived intramuscular penicillin prophylaxis developed what we have defined as “resis-tant” a-streptococcal populations
(
1% orgreater resistant to at least 1 U of penicillin per milliliter). Fifty-four of 72 patients who
received their prophylaxis orally developed this degree of resistance, and 38 of the 54 developed resistance to 5 U or more
(
Table I). Our data in conjunction with those in the literature indicate that there is little reason to use oral penicillin for prophylaxis. If oral rather than intramuscular prophy-laxis is to be used, sulfisoxazole(
Gantrisin) is at least as efficacious as penicillin,8 is cheap, relatively nontoxic, does not sensi-tize to penicillin, and does not favor devel-opment of penicillin-resistant flora in the oropharynx. Although resistance at the 1 Uper milliliter level is not alarming and under some circumstances might even be desirable,9 higher degrees of resistance may develop (Table II) and produce avoidable hazards.
The physician who uses oral penicillin in
individuals at risk from endocarditis should bear in mind that routine protective mea-sures with penicillin at the time of oral trauma may not be adequate if such trauma occurs within a month of the time of then-apy. The data presented show that small numbers of a-streptococci resistant to at
least 5 U of penicillin per milliliter were
present in one infant (D.O.) within 2 days
of initiation of treatment (Table III); such a degree of resistance was a frequent find-ing after 4 or 5 days of oral therapy. The rate of decrease of these resistant organisms
00
90
80
U)Z 70
60
Ow 0G.
o 40
G 30
. 20
(no
0
0 5 10 5 20 25
DAYS AFTER ORAL PENICILLIN
DISCONTIN UED
30 35 40
FIG. 1. Rate of decline of resistant a-streptococci following cessation of oral penicillin therapy.
by the end of the third week were
fol-lowed. Four had very low (< 1%) propor-tions of their streptococci resistant to 5 U
per milliliter by the end of the third week
(
10, 14, 18, and 24 days)
after therapy. The proportions were still high at the time ofthe last culture
(
at 8, 9, and 10 days after therapy) in three of the patients. The datasuggest that most children will not carry
significant proportions of these highly
resis-tant organisms a month after therapy.
In many instances the organisms resistant
to 1 U but not 5 U per milliliter decreased in frequency as rapidly as the more highly resistant ones. The low proportions of such
organisms
(
< 10%) which may persist for months in some individuals are ofdoubt-ful signfficance. It should be emphasized,
however, that children with these small
numbers of resistant organisms may de-velop large proportions of resistant a-streptococci within 2 days of reinstitution of oral therapy.
Most patients given parenteral penicillin
in the usual doses do not develop resistant organisms during short-term therapy or long-term prophylaxis. A likely explanation may be found in the indirect evidence#{176} which suggests that the level of penicillin achieved on the pharyngeal mucosa during
parenteral administration is low, possibly in the 0.1 to 0.5 U per milliliter range even during massive dose intravenous therapy
(
20,000,000 U per day). In patients receiv-ing oral therapy or prophylaxis, it seemsprobable that sporadic local exposure of
pharyngeal flora to high concentrations of penicillin is thci significant factor in emer-gence of resistance of streptococci to peni-cillin. The data are consistent with the hy-pothesis that the mechanism of emergence
of resistance is spontaneous stepwise muta-tion with environmental selection of
resis-tant mutants.
Cross resistance between penicillin, eryth-romycin, and/or lincomycin of penicillin-resistant streptococci was rare in our ex-penience. However, two patients carried streptococci which showed some degree of resistance to all three antibiotics; this obser-vation raises the possibility of the existence of some factor functioning in a manner analogous to that of a resistance transfer factor.
SUMMARY
Patients who have received oral penicil-un may carry in the oropharynx alpha he-molytic streptococci, of which varying pro-portions are resistant to one or more units of penicillin per milliliter. Since the use of oral penicillin is increasing and penicillin is the drug of choice for prevention of en-docarditis before oral or pharyngeal
trau-ma, semiquantitative methods have been
used to document (1) the frequency of
penicillin per milliliter in patients receiv-ing prophylactic penicillin orally and in-tramuscularly, (2) the frequency and rate with which significant numbers of a-strep-tococci appear after exposure to oral peni-cillin, (3) the proportion and degree of
re-sistance of a-streptococci which emerge re-sistant, and (4) the rate at which the num-bers of resistant organisms are reduced to
a presumably insignificant level when pen-icillin is withdrawn. The results show that
there is a statistically significant increase (P < .001) in the proportion of x-strepto-cocci resistant to at least 1 unit per milli-liter of penicillin in the pharynx of patients receiving their prophylactic penicillin in oral form (75%) in comparison with those on intramuscular penicillin prophylaxis (15%). Similarly, 17 of 22 patients on oral
penicillin therapy developed flora contain-ing resistant streptococci. Six such patients
carried a-streptococcal populations, 7 to
50% of which were resistant to 1 but not 5 U. Eleven carried streptococci, 1 to 100% of which were resistant to 5 or more units per milliliter. Of 12 patients followed close-ly, 8 developed resistant streptococci with-in the first week of therapy, 1 after 2 days.
Resistant streptococci decreased rapidly
when therapy was discontinued. Eleven of 12 patients showed less than 20% of their streptococcal population resistant to 1 unit at 15 days and less than 10% at 30 days. Low levels (<10%) persisted in six pa-tients followed for intervals longer than 30 days and up to 144 days.
REFERENCES
1. Krumweide, E.: Penicillin resistance of
nonhe-molytic streptococci from rheumatic children
receiving prophylactic penicillin. PEDIATRICS,
4:634, 1949.
2. Garrod, L. P., and Waterworth, P. M. : The
risks of dental extraction during penicillin
treatment. Brit. Heart J., 24:39, 1962.
3. Naiman, R. A., and Barrow, J. C. :
Penicillin-re-sistant bacteria in the mouths and throats of
children receiving continuous prophvlaxis
against rheumatic fever. Ann. Intern. Med.,
58:768, 1963.
4. Tozer, R. A., Boutfiower, S., and Gillespie, W.
A. : Antibiotics for prevention of bacterial
en-docarditis during dental treatment. Lancet,
1:686, 1966.
5. Stirland, R. M., and Shotts, N. :
Antibiotic-resis-tant streptococci in the mouths of children
treated with penicillin. Lancet, 1 :405, 1967.
6. Kuttner, A. C. : Production of bacteriocines by
group A streptococci with special reference
to the nephritogenic types. J. Exp. Med.,
124:279, 1966.
7. Doyle, E. F., Spagnuolo, M., Taranta, A.,
Kutt-ner, A. G., and Markowitz, M. : The risk of
bacterial endocarditis during antirheumatic
prophylaxis. J.A.M.A., 201:807, 1967.
8. Wood, H. F., Feinstein, A. R., Taranta, A.,
Ep-stein, J. A., and Simpson, R.: Rheumatic
fever in children and adolescents. A
long-term epidemiologic study of subsequent
pro-phvlaxis, streptococcal infections, and clinical
sequelae. III. Comparative effectiveness of
three prophylaxis regimens in preventing
streptococcal infections and rheumatic
recur-rences. Ann.
mt.
Med. (Suppi. 5 ), 60:31,1964.
9. Sprunt, K., and Redman, W. : Evidence
suggest-ing importance of role of interbacterial
inhi-bition in maintaining balance of normal flora.
Ann.
mt.
Med., 68:579, 1968.Acknowledgment
We wish to express our gratitude to the
follow-ing doctors who made available to us cultures from
patients receiving penicillin prophylaxis for
rheu-matic fever: Drs. Wan Ngo Lim, Eugenie Doyle,
Mario Spagnuolo, Lucy Swift, and Grace Cheng
(from their clinics); Drs. Alexander Blum, David
Franklin, Jack Schiller, Jerry Jacobs, B. M.
