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Anaphylactic reaction to penicillin (or penicillin-like substance) in a soft drink
Anaphylactic penicillin-like Konrad
Wither,
reaction to penicillin (or substance) in a soft drink
D.M.Sc., and Robert E. Reisman,
M.D. BufSaIo, N. Y.
An ucute anaphylactic reaction occurred in a patient known to be highly sensitive to penicillin following ingestion of the soft drink Wink. Bacteriologic studies showed the presence of penicillin or a penicillin-like substance in the Wink, suggesting it as the cause of the anaphylaxis. The source of this contaminant could not be identified.
Extreme allergic sensitivity to penicillin has been well documented. Patients with this exquisite degree of allergy react to small quantities of penicillin, often from an unexpected source.’ For example, allergic reactions have been described following exposure to airborne particles, such as in a hospital or a penicillin manufacturing plant, and following the ingestion of milk that contained small quantities of penicillin.2 We recently had the opportunity to study the prob-
lem of anaphylaxis following ingestion of a soft drink in an individual known to be highly sensitive to penicillin. Laboratory studies demonstrated the presence of penicillin or a penicillin-like substance in the soft drink, suggesting it as the cause of the anaphylaxis. METHODS AND MATERIALS Case report Mrs. F. R., a 52-yr-old woman, has been the subject of two previous reports, becauseof her extreme sensitivity to penicillin.“, 1 The first report described her anaphylactic symptoms following inhalation of small quantities of penicillin while working as an attendantat a local hospital. Clinical and immunologic investigations confirmed her extreme sensitivity to penicillin. The secondreport described an anaphylactic reaction following the ingestion of milk. Bacteriologic and immunologic assays suggestedthat the milk contained about 10 U/ml penicillin. Following the documentation of this exquisite penicillin sensitivity, the patient remained in good health without a From the Departmentsof Microbiology and Medicine, State University of New York at Buffalo; the Allergy ResearchLaboratory, Buffalo General Hospital; and the Division of Microbiology and Immunology, Erie County Laboratory. Received for publication Nov. 21, 1979. Accepted for publication Feb. 6, 1980. Reprint requeststo: Robert E. Reisman, M.D., Suite 1102,General Medical Towers, 50 High St., Buffalo, NY 14203. 0091-6749/80/080155+03$00.30/0
0 1980 The C. V. Mosby
serious anaphylactic reaction for over 10 yr. During this time she continued to work but avoided contact with penicillin. She had no further trouble following ingestion of milk.
In early December 1978 she drank approximately 90 ml carbonated grapefruit juice soft drink, Wink (Canada Dry
Corp., Lot No. 1690000231).This bottle was opened and its contents distributed among several membersof the family. She had nothing to eat with the Wink. Dinner had been
eaten approximately 2 hr previously. About 5 min after ingestion of the Wink her eyes and palms began to itch, followed by generalized urticaria. There were no respiratory
symptoms. She went to the emergency room of a local hospital, where she gradually improved after two injections of epinephrine subcutaneously. She had ingested Wink previously with no difficulty. A
subsequentintradermal skin test with grapefruit extract, 1: 10, was negative. A prick test with 1 U/ml freshly diluted aqueous penicillin G resulted in a 10 x 18 mm wheal-andflare reaction within 10 min. Control tests were negative.
Analysis
of Wink for penicillin
content
Examination of the suspect Wink. Approximately 100 ml remaining soft drink was provided by the patient in the original 1-pt 12-02 bottle. According to the label the soft drink contained carbonated water, nutritive sweetener (sugar or corn sweetener), citric acid, natural flavoring, sodium benzoate as preservative (less than 1120 of l%), ester gum, and cellulose gum. The soft drink was first examined for the presence of bacteria and fungi and then centrifuged in sterile tubes at 3,000 rpm for 30 min, aliquoted in 3- to 5-ml sterile tubes, and kept frozen at -70” C. There was no growth of either bacteria or fungi after 6 wk of incubation on a variety of media and at various temperatures The presence of penicillin activity was examined by the agar diffusion method, using a standard strain of Staphylococcus aureus (ATT No. 25923) and diagnostic strains of staphylococci sensitive or resistant to penicillin (IO-unit Co.
Vol. 66, No. 2, pp. 155-157
156 Wither and Rellsman
J. ALLERGY
CLIN. IMMUNOL. AUGUST 1980
I. Inhibition of growth of a standard strain of S. aureus (ATCC No. 25923) by penicillin and by Wink
TABLE
Penicillin
Concentration (U/ml)
31
Undiluted
27 23
I:2
27 22
0.16 0.08 0.04
I7 I4
I :4 I:8
I6 <8
<8
II. Effect of penicillinase
activity
Reagent
disks). Petri dishes (IO-cm diameter) containing 5% Mueller-Hinton agar were used. Four wells, approximately 8 mm in diameter, were cut in each plate and the bottoms of the wells covered with fluid agar (to prevent sample diffusion under the medium). The surfaces of the places were spread to the edge of the wells with a 4-hr-old culture of the standard or laboratory strains of S. uureus. Two-tenths milliliter of various concentrations of penicillin or various dilutions of Wink were placed in the wells. The plates were sealed with paraffin and incubated at 3.5” C overnight, and the zones of growth i:nhibition were measured. Because the Wink contained sodium benzoate as a preservative, various concentrations (1% to 0.025%) of sodium benzoate were examined in a manner identical to that for the Wink. The activity of both the penicillin and the Wink was also examined by mixing various concentrations with equal volumes of 50,000 U penicillinase (BBL, Division of Becton & Dickinson Co., Cockeysville, Md.). These samples were examined against the standard strain of S. uureus (ATCC No. 24923) as described above. Both the Wink and the penicillin inhibited the growth of the standard S. rrureus (Table I). When compared with the penicillin, Wink showed a penicillin-like activity equal to 0.63 U/ml penicillin. The activity of both the penicillin and the Wink could be abolished by penicillinase, documenting a penicillin-like activllty in the Wink (Table II). At least 20 various laboratory strains of S. crureus sen-
Dilution
Zone inhibition (mm)
I .25
inhibitory
of the growth of a standard strain of Sfaphy/ococcus aureus (ATT No. 25923) by 10 U penicillin (p) and undiluted Wink (W).
Zone inhibition hnm)
0.63 0.3 I
TABLE
FIG. 1. Inhibition
Wink
Penicillin Penicillin and penicillinase Wink Wink and penicillinase
of penicillin
on growthand Wink
Concentration blml)
1.25 2.5 50,000
Undiluted 2 x concentrated 5o,cMlo
Zone inhibition (mm) 32 <8 27
<8
sitive or resistant to penicillin were examined using the disk diffusion method and undiluted Wink. One example is represented in Fig. 1. All penicillin-sensitive strains were inhibited by the Wink in varying degrees, and all resistant strains showed lack of growth inhibition. Experiments using various concentrations of sodium benzoate as the inhibitory substance showed no growth inhibition of the standard S. uureus. Experiments with other Wink sclmples. Two additional bottles of Wink of the same lot number were purchased in two widely separated stores and examined for growthinhibitory behavior. The Wink purchased in the same store as the first bottle, which had caused the anaphylactic reaction, when used undiluted showed a zone (24 mm) of growth inhibition. This inhibition, however, was not complete; within the zone of growth inhibition, colonies (10%) of the S. mweus were observed. The Wink purchased in the distant store did not show any penicillin-like activity. ConJirmcrtory studies. To verify our results two samples of Wink were sent for examination to the National Center for Antibiotic Analysis (NCAA), Bureau of Drugs, Food and Drug Administration, Washington, D. C. The samples were examined both chemically and biologically by the NCAA in a double-blind fashion. The sample of Wink that caused the anaphylactic reaction was found to have penicillin-like activity similar to that of penicillin V. The concentration was estimated to be 0.29 U/ml. This activity could be abolished by penicillinase. The other Wink sample
VOLUME NUMBER
66 2
did not contain detectable penicillin-like activity. These NCAA results confirmed our findings. The estimated concentrations of the antibiotic activity using different methods were very close.
DISCUSSION Clinical and laboratory studies indicated that the patient had had an anaphylactic reaction to penicillin or a penicillin-like substance contained in Wink. The patient was known to be highly sensitive to penicillin. Her current sensitivity was confirmed by the marked reaction following a prick test with only 1 U/ml aqueous penicillin. Examination of the suspect Wink by several methods indicated the presence of low concentrations of penicillin or a penicillin-like substance. This was not present in at least one other bottle of Wink examined. The patient failed to react to grapefruit extract used for intradermal testing and furthermore had been able to eat all other foods, including grapefruit, without difficulty. The use of penicillinase to destroy the penicillin activity and allow bacterial growth confirmed the presence of a penicillin-like substance in the Wink. The source of the penicillin contamination in the Wink could not be found. Both the local manager and
Penicillin
in a soft drink
157
the quality control manager of the Canada Dry Bottling Co. were unable to suggest a source of penicillin contamination. Penicillin is not used anywhere in the bottling process and would not be used even if there were some kind of contamination in the plant. Thus the source of this contaminant remains a mystery. The fact that the penicillin was also found in other bottles of Wink opened in the laboratory suggests that it was not added to the incriminated bottle after it was opened. The substance in the Wink inhibiting the growth of S. CIUI~UShas been referred to as a “penicillin-like substance, ” because its low activity and small sample size did not allow for true chemical identification of this active ingredient. REFERENCES 1. Siegel BB: Hidden contact with penicillin. Bull WHO 21:703, 1959. 2. Welch H: Problems of antibodies in food as the Food and Drug Administration sees them. Am J Public Health 47:701, 1957. 3. Reisman RE, Arbesman CE: Systemic allergic reactions due to inhalation of penicillin. JAMA 203:986, 1968. 4. Wither K, Reisman RE. Arbesman CE: Allergic reactions to penicillin in milk. JAMA 208:143, 1969.
Wither,
reaction to penicillin (or substance) in a soft drink
D.M.Sc., and Robert E. Reisman,
M.D. BufSaIo, N. Y.
An ucute anaphylactic reaction occurred in a patient known to be highly sensitive to penicillin following ingestion of the soft drink Wink. Bacteriologic studies showed the presence of penicillin or a penicillin-like substance in the Wink, suggesting it as the cause of the anaphylaxis. The source of this contaminant could not be identified.
Extreme allergic sensitivity to penicillin has been well documented. Patients with this exquisite degree of allergy react to small quantities of penicillin, often from an unexpected source.’ For example, allergic reactions have been described following exposure to airborne particles, such as in a hospital or a penicillin manufacturing plant, and following the ingestion of milk that contained small quantities of penicillin.2 We recently had the opportunity to study the prob-
lem of anaphylaxis following ingestion of a soft drink in an individual known to be highly sensitive to penicillin. Laboratory studies demonstrated the presence of penicillin or a penicillin-like substance in the soft drink, suggesting it as the cause of the anaphylaxis. METHODS AND MATERIALS Case report Mrs. F. R., a 52-yr-old woman, has been the subject of two previous reports, becauseof her extreme sensitivity to penicillin.“, 1 The first report described her anaphylactic symptoms following inhalation of small quantities of penicillin while working as an attendantat a local hospital. Clinical and immunologic investigations confirmed her extreme sensitivity to penicillin. The secondreport described an anaphylactic reaction following the ingestion of milk. Bacteriologic and immunologic assays suggestedthat the milk contained about 10 U/ml penicillin. Following the documentation of this exquisite penicillin sensitivity, the patient remained in good health without a From the Departmentsof Microbiology and Medicine, State University of New York at Buffalo; the Allergy ResearchLaboratory, Buffalo General Hospital; and the Division of Microbiology and Immunology, Erie County Laboratory. Received for publication Nov. 21, 1979. Accepted for publication Feb. 6, 1980. Reprint requeststo: Robert E. Reisman, M.D., Suite 1102,General Medical Towers, 50 High St., Buffalo, NY 14203. 0091-6749/80/080155+03$00.30/0
0 1980 The C. V. Mosby
serious anaphylactic reaction for over 10 yr. During this time she continued to work but avoided contact with penicillin. She had no further trouble following ingestion of milk.
In early December 1978 she drank approximately 90 ml carbonated grapefruit juice soft drink, Wink (Canada Dry
Corp., Lot No. 1690000231).This bottle was opened and its contents distributed among several membersof the family. She had nothing to eat with the Wink. Dinner had been
eaten approximately 2 hr previously. About 5 min after ingestion of the Wink her eyes and palms began to itch, followed by generalized urticaria. There were no respiratory
symptoms. She went to the emergency room of a local hospital, where she gradually improved after two injections of epinephrine subcutaneously. She had ingested Wink previously with no difficulty. A
subsequentintradermal skin test with grapefruit extract, 1: 10, was negative. A prick test with 1 U/ml freshly diluted aqueous penicillin G resulted in a 10 x 18 mm wheal-andflare reaction within 10 min. Control tests were negative.
Analysis
of Wink for penicillin
content
Examination of the suspect Wink. Approximately 100 ml remaining soft drink was provided by the patient in the original 1-pt 12-02 bottle. According to the label the soft drink contained carbonated water, nutritive sweetener (sugar or corn sweetener), citric acid, natural flavoring, sodium benzoate as preservative (less than 1120 of l%), ester gum, and cellulose gum. The soft drink was first examined for the presence of bacteria and fungi and then centrifuged in sterile tubes at 3,000 rpm for 30 min, aliquoted in 3- to 5-ml sterile tubes, and kept frozen at -70” C. There was no growth of either bacteria or fungi after 6 wk of incubation on a variety of media and at various temperatures The presence of penicillin activity was examined by the agar diffusion method, using a standard strain of Staphylococcus aureus (ATT No. 25923) and diagnostic strains of staphylococci sensitive or resistant to penicillin (IO-unit Co.
Vol. 66, No. 2, pp. 155-157
156 Wither and Rellsman
J. ALLERGY
CLIN. IMMUNOL. AUGUST 1980
I. Inhibition of growth of a standard strain of S. aureus (ATCC No. 25923) by penicillin and by Wink
TABLE
Penicillin
Concentration (U/ml)
31
Undiluted
27 23
I:2
27 22
0.16 0.08 0.04
I7 I4
I :4 I:8
I6 <8
<8
II. Effect of penicillinase
activity
Reagent
disks). Petri dishes (IO-cm diameter) containing 5% Mueller-Hinton agar were used. Four wells, approximately 8 mm in diameter, were cut in each plate and the bottoms of the wells covered with fluid agar (to prevent sample diffusion under the medium). The surfaces of the places were spread to the edge of the wells with a 4-hr-old culture of the standard or laboratory strains of S. uureus. Two-tenths milliliter of various concentrations of penicillin or various dilutions of Wink were placed in the wells. The plates were sealed with paraffin and incubated at 3.5” C overnight, and the zones of growth i:nhibition were measured. Because the Wink contained sodium benzoate as a preservative, various concentrations (1% to 0.025%) of sodium benzoate were examined in a manner identical to that for the Wink. The activity of both the penicillin and the Wink was also examined by mixing various concentrations with equal volumes of 50,000 U penicillinase (BBL, Division of Becton & Dickinson Co., Cockeysville, Md.). These samples were examined against the standard strain of S. uureus (ATCC No. 24923) as described above. Both the Wink and the penicillin inhibited the growth of the standard S. rrureus (Table I). When compared with the penicillin, Wink showed a penicillin-like activity equal to 0.63 U/ml penicillin. The activity of both the penicillin and the Wink could be abolished by penicillinase, documenting a penicillin-like activllty in the Wink (Table II). At least 20 various laboratory strains of S. crureus sen-
Dilution
Zone inhibition (mm)
I .25
inhibitory
of the growth of a standard strain of Sfaphy/ococcus aureus (ATT No. 25923) by 10 U penicillin (p) and undiluted Wink (W).
Zone inhibition hnm)
0.63 0.3 I
TABLE
FIG. 1. Inhibition
Wink
Penicillin Penicillin and penicillinase Wink Wink and penicillinase
of penicillin
on growthand Wink
Concentration blml)
1.25 2.5 50,000
Undiluted 2 x concentrated 5o,cMlo
Zone inhibition (mm) 32 <8 27
<8
sitive or resistant to penicillin were examined using the disk diffusion method and undiluted Wink. One example is represented in Fig. 1. All penicillin-sensitive strains were inhibited by the Wink in varying degrees, and all resistant strains showed lack of growth inhibition. Experiments using various concentrations of sodium benzoate as the inhibitory substance showed no growth inhibition of the standard S. uureus. Experiments with other Wink sclmples. Two additional bottles of Wink of the same lot number were purchased in two widely separated stores and examined for growthinhibitory behavior. The Wink purchased in the same store as the first bottle, which had caused the anaphylactic reaction, when used undiluted showed a zone (24 mm) of growth inhibition. This inhibition, however, was not complete; within the zone of growth inhibition, colonies (10%) of the S. mweus were observed. The Wink purchased in the distant store did not show any penicillin-like activity. ConJirmcrtory studies. To verify our results two samples of Wink were sent for examination to the National Center for Antibiotic Analysis (NCAA), Bureau of Drugs, Food and Drug Administration, Washington, D. C. The samples were examined both chemically and biologically by the NCAA in a double-blind fashion. The sample of Wink that caused the anaphylactic reaction was found to have penicillin-like activity similar to that of penicillin V. The concentration was estimated to be 0.29 U/ml. This activity could be abolished by penicillinase. The other Wink sample
VOLUME NUMBER
66 2
did not contain detectable penicillin-like activity. These NCAA results confirmed our findings. The estimated concentrations of the antibiotic activity using different methods were very close.
DISCUSSION Clinical and laboratory studies indicated that the patient had had an anaphylactic reaction to penicillin or a penicillin-like substance contained in Wink. The patient was known to be highly sensitive to penicillin. Her current sensitivity was confirmed by the marked reaction following a prick test with only 1 U/ml aqueous penicillin. Examination of the suspect Wink by several methods indicated the presence of low concentrations of penicillin or a penicillin-like substance. This was not present in at least one other bottle of Wink examined. The patient failed to react to grapefruit extract used for intradermal testing and furthermore had been able to eat all other foods, including grapefruit, without difficulty. The use of penicillinase to destroy the penicillin activity and allow bacterial growth confirmed the presence of a penicillin-like substance in the Wink. The source of the penicillin contamination in the Wink could not be found. Both the local manager and
Penicillin
in a soft drink
157
the quality control manager of the Canada Dry Bottling Co. were unable to suggest a source of penicillin contamination. Penicillin is not used anywhere in the bottling process and would not be used even if there were some kind of contamination in the plant. Thus the source of this contaminant remains a mystery. The fact that the penicillin was also found in other bottles of Wink opened in the laboratory suggests that it was not added to the incriminated bottle after it was opened. The substance in the Wink inhibiting the growth of S. CIUI~UShas been referred to as a “penicillin-like substance, ” because its low activity and small sample size did not allow for true chemical identification of this active ingredient. REFERENCES 1. Siegel BB: Hidden contact with penicillin. Bull WHO 21:703, 1959. 2. Welch H: Problems of antibodies in food as the Food and Drug Administration sees them. Am J Public Health 47:701, 1957. 3. Reisman RE, Arbesman CE: Systemic allergic reactions due to inhalation of penicillin. JAMA 203:986, 1968. 4. Wither K, Reisman RE. Arbesman CE: Allergic reactions to penicillin in milk. JAMA 208:143, 1969.