Histamine Production by Klebsiella pneumoniae and an

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1979, p /79/ /05$02.00/0 Vol. 37, No. 2 Histamine Production by Klebsiella pneumoniae and an Incident of Scombroid Fish Poisoningt STEVE L. TAYLOR,::* LINDA S. GUTHERTZ, MATTHEW LEATHERWOOD, AND ELLEN R. LIEBER Department of Nutrition, Food Hygiene Division, Letterman Army Institute of Research, Presidio ofsan Francisco, California Received for publication 27 November 1978 A histamine-producing strain of Klebsiella pneumoniae was isolated from a sample of tuna sashimi implicated in an outbreak of scombroid fish poisoning. None of the other nine gram-negative bacterial strains isolated from the tuna sashimi was capable of equivalent histamine production. Bacterial histamine production was monitored in a tuna fish infusion broth (TFIB), and the implicated K. pneumoniae was capable of producing 442 mg of histamine per 100 g of tuna in TFIB in 7 h under controlled incubation conditions. Only 12 of 50 other K. pneumoniae strains, representing 5 distinct biochemical types, which had been originally isolated from foods, were able to produce such levels of histamine in TFIB. No correlation was found between histamine production and other biochemical characteristics or antibiotic resistance. Of the 12 histamine-producing strains, 11 belonged to type 2, which is characterized as indole negative with positive reactions in the urea and Voges-Proskauer tests. However, only 50% of the type 2 strains examined produced high levels of histamine in TFIB. Additionally, the implicated K. pneumoniae strain and one other strain belonged to type 1, which is characterized by positive reactions in the indole, urea, and Voges- Proskauer tests. Numerous food poisoning incidents have been associated with the consumption of fish containing high levels of histamine (8, 12). Fish of the scombroid type, such as tuna and mackerel, are usually implicated, although other seafoods, particularly mahi-mahi (dolphinfish), are occasionally involved (2). The high levels of histamine in the incriminated fish are formed via microbial decarboxylation of histidine (11). A considerable number of food-borne microbial species have been reported to possess the enzyme, histidine decarboxylase, which is necessary for histamine formation in foods (9, 11, 13, 17). However, in the few instances of microbiological examinations of seafood actually incriminated in food poisoning, only Proteus morganii (10, 14) and Hafnia alvei (5) have been implicated as causative organisms in the formation of toxicologically significant levels of histamine. In September 1977, an incident of scombroid fish poisoning occurred in San Francisco. The incriminated food was sashimi, consisting of raw tuna fish. The diagnosis of scombroid fish poit Address reprint requests to: Commander, Letterman Army Institute of Research, Medical Research Library, Presidio of San Francisco, CA : Present address: Food Research Institute, University of Wisconsin, Madison, WI soning was based on the high levels of histamine in the implicated tuna sashimi (up to 919 mg/100 g, depending on the particular sample, with higher levels found on the surface portion of the filet), the similarities in the symptomology of affected individuals to other known outbreaks, and the effectiveness of anti-histamine therapy (P. A. Lerke, S. B. Werner, S. L. Taylor, and L. S. Guthertz, West. J. Med., in press). The aerobic plate count (21) and presumptive coliform plate count (21) were 7.0 x 107 and 8.3 x 105 organisms per g, respectively. Further microbiological examination of the sashimi resulted in the isolation and identification of a strain of Klebsiella pneumoniae as the primary histamine-forming microorganism in this tuna fish sample. An investigation of the histamine-producing capabilities of this isolated K. pneumoniae strain and others will be reported. (This paper was presented in part at the 78th Annual Meeting of the American Society for Microbiology, Las Vegas, Nev., May, 1978.) MATERIALS AND METHODS Tuna fish. A sample of tuna sashimi that had been seized in connection with an outbreak of scombroid fish poisoning in San Francisco, Calif., in September, 1977, was obtained from the San Francisco Depart- 274

2 VOL. 37, 1979 ment of Public Health. Bacteriological analysis of tuna fish. Aerobic gram-negative bacteria were isolated as described by Guthertz et al. (7). A 25-g portion of the tuna sashimi was weighed into a sterile 1-liter blender container, and 225 ml of sterile phosphate-buffered water was added. The sample was blended for 2 min at high speed. Gram-negative organisms were isolated by inoculating 10 ml of the food homogenate into 10 ml of double-strength GN broth. After incubating at 35 C for 24 h, the sample was inoculated onto Salmonella- Shigella, bismuth sulfite, brilliant green sulfadiazine, Hektoen Enteric, MacConkey, and eosin-methylene blue agar plates. After incubation of these agar plates at 37 C for 24 h, representative colony types were subcultured to eosin-methylene blue agar to assure purity. Identification of the gram-negative isolates was made by using the API 20 E Enterobacteriaceae system (Analytab Products, Inc.). The Analytical Profile Index was used to assign species designations to isolates identified by this procedure. Isolates were maintained on nutrient agar (Difco) slants until tested. Other K. pneumoniae strains. The additional K. pneumoniae strains were obtained from the culture collection of the Food Hygiene Division of Letterman Army Institute of Research. These strains were originally isolated from samples of either comminuted turkey (6, 7) or comminuted beef (J. F. Foster, L. S. Guthertz, R. C. Hunderfund, and J. L. Fowler, J. Food Protect., in press). The original isolations and identifications were made by the method of Guthertz et al. (7). All strain identifications were confirmed before use in later studies. Isolates were maintained on nutrient agar (Difco) slants until tested. The K. pneumoniae strains were arbitrarily grouped into five different types based on biochemical differences determined by using the API 20 E system. Media. Trypticase soy broth-histidine medium (TSBH) was prepared by addition of 30 g of Trypticase soy broth (Baltimore Biological Laboratory), 1 g of histidine, and 5 mg of pyridoxal hydrochloride to 1 liter of deionized water. The ph of TSBH was adjusted to ph 6.8 before autoclaving for 15 min at 121 C. Tuna fish infusion broth (TFIB) was prepared from fresh tuna muscle by homogenization with 2 volumes (wt/ vol) of water, steaming at 100 C for 1 h, filtration, supplementation with 1% glucose, and sterilization at 1210C for 15 min. The final ph of TFIB was ph 5.7. Preparation of inoculum. Tubes containing 5 ml of TSBH were inoculated from the maintenance slants and incubated at 32 C for 24 h. A 0.2-ml aliquot of this 24-h culture was transferred into a second tube containing 5 ml of TSBH and allowed to incubate for 18 h at 320C. This overnight culture served as the inoculum for the histamine production experiments. Histamine production. A 2-ml amount of the overnight culture was inoculated into 70 ml of TFIB in a 300-ml nepheloculture flask (Bellco). Flasks were incubated at 32 C in a shaking incubator (New Brunswick Scientific) at 100 rpm. Immediately after inoculation of the TFIB, 2 ml was withdrawn for an initial aerobic plate count, and 3 ml was removed for histamine analysis. After a 7-h incubation period, 5 ml was again withdrawn for an aerobic plate count and histamine analysis. An additional aliquot was removed HISTAMINE-PRODUCING K PNEUMONIAE 275 for histamine analysis after completion of a 24-h incubation. Histamine analysis. All samples withdrawn for histamine analysis were diluted 10-fold in methanol. A 5-ml aliquot was homogenized in 50 ml of methanol for 1 min in a Waring blender at high speed. The remainder of the analytical procedure has been described previously (20). Histamine was measured fluorometrically (18), with histamine levels calculated by comparsion of the sample fluorescence intensity to that of an external standard treated identically. The calculated histamine levels were corrected based on percent recovery of an internal standard as previously described (20). The histamine levels were also corrected for the small amount of histamine in TFIB by subtracting the histamine concentration of an uninoculated medium sample. Aerobic plate counts. Aerobic plate counts were conducted with dilutions of culture prepared in phosphate buffer and pour plating with standard methods agar (Baltimore Biological Laboratory). Plates were incubated at 32 C for 72 h (1). Antibiotic resistance. Antibiotic resistance was determined by the disk diffusion method of Bauer et al. (3). RESULTS Identification of gram-negative bacteria from tuna sashimi. Ten isolates of gram-negative bacteria were isolated and identified from the sample of sashimi implicated in an incident of scombroid fish poisoning that occurred in San Francisco in September The isolates represented eight different species, including Acinetobacter calcoaceticus, Citrobacter freundii, Enterobacter agglomerans, Enterobacter cloacae, H. alvei, K. pneumoniae, Proteus rettgeri, and Proteus vulgaris. Two biochemically distinct types of K. pneumoniae and P. vulgaris were identified. Production of histamine in TFIB by gram-negative isolates. Among the 10 gramnegative isolates, the strain of K. pneumoniae type 1, characterized by positive reactions in the indole, urea, and Voges-Proskauer tests in the API 20 E system, formed by far the most histamine in TFIB (Table 1). This strain of K. pneumoniae produced 19,900 nmol of histamine per ml in 7 h of incubation as compared with less than 50 nmol/ml for the other isolated strains. After 24 h of incubation, the histamine production in TFIB by certain of the other strains, such as E. agglomerans, E. cloacae, and H. alvei, had increased considerably, whereas the histamine concentration formed by K. pneumoniae type 1 had dropped slightly to 13,400 nmol/ml. However, the amount of histamine produced by the K. pneumoniae strain remained considerably higher than that found with any of the other organisms. Production of histamine in TFIB by other

3 276 TAYLOR ET AL. TABLE 1. Histamine production in TFIB by gramnegative bacteria isolated from tuna sashimi involved in a scombroid fish poisoning episode Logarithmic production Species increase in (nmol/ml) at: APCa (7 h) 7h 24h A. calcoaceticus 1.79 <1 3 C. freundii 2.09 <1 8 E. agglomerans E. cloacae 1.75 <1 66 H. alvei K. pneumoniae type 1' ,900 13,300 K. pneumoniae type 2b 1.44 <1 <1 P. rettgeri 1.88 <1 <1 P. vulgaris type <1 <1 P. vulgaris type a APC, Aerobic plate count. b Types of K. pneumoniae are defined in Table 2. K. pneumoniae strains. Fifty additional strains of K. pneumoniae were tested to assess the frequency of histamine-producing capability within this species. As shown in Table 2, 12 of the 50 additional K. pneumoniae strains produced histamine in TFIB in amounts approximately equivalent to that formed by the original strain isolated from tuna sashimi. These 12 histamine-producing strains included 11 of 22 type 2 strains and 1 of 10 type 1 strains. None of the tested type 3, 4, or 5 strains produced this level of histamine in TFIB. Type 2 is characterized by positive reactions in the urea and Voges- Proskauer tests and a negative indole reaction within the API 20 E system. Although most of the histamine-producing strains belonged to type 2, no definite correlation between histamine formation and any other biochemical characteristic was noted. Also, no correlation was observed between antibiotic resistance and histamine production. Of the 12 histamine-producing strains, 9 formed substantially more histamine after 24 h in TFIB than they formed after a 7-h incubation. The other three strains produced little additional histamine upon further incubation. The original strain isolated from tuna sashimi seems to be unique in its formation of a large amount of histamine in 7 h in TFIB followed by a decrease in the histamine level with incubation for an additional 17 h. Curiously, some strains of K. pneumoniae seem to be capable of generating an intermediate level of histamine in TFIB. One of 10 type 1 strains, 2 of 22 type 2 strains, the 1 type 3 strain, 1 of 2 type 4 strains, and 6 of 15 type 5 strains were capable of producing histamine in the range of 50 to 1,000 nmol/ml in TFIB after either 7- or 24-h incubation periods. The remaining 27 strains were incapable of producing as much as 50 nmol of histamine per ml in TFIB. APPL. ENVIRON. MICROBIOL. DISCUSSION K. pneumoniae may have been the causative organism in the outbreak of scombroid fish poisoning associated with consumption of sashimi. The isolation from the tuna sashimi sample of a K. pneumoniae strain capable of such a high level of histamine formation should serve as strong circumstantial evidence of the organism's involvement. The identified K. pneumoniae strain seems to be capable of forming deleterious histamine levels in tuna fish. The implicated strain formed 19,900 nmol of histamine per ml in TFIB in 7 h of incubation. This level of histamine is equivalent to 442 mg of histamine/100 g of tuna. Although the precise level of histamine required to precipitate an outbreak of scombroid fish poisoning is unknown, Simidu and Hibiki (19) have suggested that the minimum histamine concentration necessary to elicit scombroid fish poisoning is 60 mg/100 g of fish. Ienistea (9) claims that consumption of as little as 40 mg of histamine in a single meal will cause adverse reactions in the most sensitive individuals. Based on these criteria, the implicated K. pneumoniae strain would seem to be capable of sufficient histamine formation in tuna fish to precipitate an outbreak of scombroid fish poisoning. However, the possible involvement of other organisms in this outbreak cannot be totally discounted. A count of K. pneumoniae type 1 organisms was not determined for the implicated tuna sashimi sample. Consequently, the relative proportion of histamine-producing organisms within the total bacterial load is unknown. Although the presumptive coliform plate count was rather high, other identified organisms in addition to K. pneumoniae, including E. cloacae, E. agglomerans, H. alvei, and C. freundii, would contribute to the presumptive coliform count. Because histamine may persist in food products, the possibility also exists that the high levels of histamine in the tuna sashimi were formed by some organism that subsequently died out. Because the microbiological analyses were performed with a kitchen scrap sample of tuna sashimi, the K. pneumoniae type 1 strain may have been a secondary contaminant. However, histamine-producing K. pneumoniae have recently been identified in various fish products and the intestinal contents of mackerel (D. A. Corlett, Jr., M. B. Jeffrey, and C. F. Niven, Jr., Abstr. Annu. Meet. Am. Soc. Microbiol., 1978, P38, p. 192). This study did not seek to identify histamine-producing, gram-positive organisms. The potential involvement of gram-positive organisms is less likely, although some Lactobacillus strains produce histamine (4, 13). Also, the use of alternative incubation conditions

4 VOL. 37, 1979 HISTAMINE-PRODUCING K PNEUMONIAE 277 TABLE 2. Histamine production, growth in TFIB, biochemical characteristics, and antibiotic resistance patterns of additional K. pneumoniae strains Bio- Strain Antibiotic re- Logarithmic production Bio- Strain Antibiotic re- Logarithmic production type code sistancea inrapc (7 e h) olm APC" type code sistance'a_icrese_n ol_l (7 h) ~~~~~APCb (7 h) - 7h 24h 7h 24h Type 1C A, CB A, CB ,380 25, A ,400 18, A, CB ,690 14, C, A, CB, ST 1.92 < A, CB ,300 18, C, A, CB, NF A, CB ,620 25, A, CB 1.90 < A, CB ,850 25, A, CB 2.07 <1 < A, CB ,100 14, A, CB 1.81 <1 < A, CB 1.81 <1 <1 Type 3' < A, CB 1.82 <1 < A, CB 1.76 <1 <1 Type 4f 99-4 A, CB 1.60 <1 < A, CB Type 2d < A, CB ,310 22,200 Type A, CB 1.72 <1 < CB < CB, NF 2.02 <1 < K, A, CB 1.66 < A, CB, T 1.90 <1 < A, CB, ST <1 31G1 A, CB, T, ST 2.12 <1 < CB, NF 1.88 < H11 A, CB, ST < CB 1.93 <1 < A, CB ,800 12, CB 1.85 < A, CB 1.58 <1 < A, CB < A, CB, CH 1.81 <1 < CB 1.93 < A, CB, NF 1.99 < CB 2.13 < A, CB 1.97 <1 < A, CB A, CB 1.73 <1 < A, CB A, CB ,460 15, A, CB CB ,600 12, A, CB < CB ,600 11,900 _ 89-3 CB, NF 2.02 <1 19 'Antibiotic resistance as tested against the following antibiotics: K, Kanamycin; C, cephalothin; A, ampicillin, CB, carbenicillin; PB, polymyxin B; T, tetracycline; G, gentamicin; NF, nitrofurantoin; N, neomycin; ST, sulfamethoxizole-trimethoprim, CH, chloramphenicol. h APC, Aerobic plate count. Indole positive, urea positive, Voges-Proskauer positive in API 20 E system. d Indole negative, urea positive, Voges-Proskauer positive in API 20 E system. 'Indole negative, urea positive, Voges-Proskauer negative in API 20 E system. f Indole negative, urea negative, Voges-Proskauer positive in API 20 E system. ' Indole positive, urea negative, Voges-Proskauer positive in API 20 E system. might have resulted in increased histamine production by one of the other identified organisms. Still, growth and histamine production in TFIB can be taken as a reasonable index of an organism's capacity for histamine formation in scombroid fish products. Although the aerobic nature of the incubation and the addition of glucose may affect the rate and extent of histamine formation in TFIB as compared to raw, solid tuna, the results should be at least a semiquantitative indication of an organism's histamineproducing capability. The causative involvement of the isolated K. pneumoniae type 1 strain in this outbreak of scombroid fish poisoning is still the most likely conclusion. The capability for production of high levels of histamine in TFIB was limited to less than 25% of the K. pneumoniae strains examined. Histamine-producing strains belong primarily to type 2, which is characterized as indole negative with positive reactions in the urea and Voges-Proskauer tests. However, only 50% of the examined type 2 strains were found to produce high levels of histamine in TFIB. Also, the strain implicated in the scombroid fish poisoning episode and one other histamine-producing strain belong to type 1, which displays positive reactions in the indole, urea, and Voges-Proskauer tests. The histamineproducing strains of K. pneumoniae must possess the enzyme, histidine decarboxylase. However, some strains may also have histaminase activity which could affect the overall histamine production (9). The coincident presence of histaminase may explain the occurrence of strains of K. pneumoniae capable of only limited histamine production in TFIB. Because no biochemical characteristic or antibiotic resistance pattern was correlated uniformly with histamine production in TFIB, the identification of histamine-producing K. pneumoniae strains will require the performance of histamine production experiments. K. pneumoniae has never previously been implicated as the causative organism in scombroid

5 278 TAYLOR ET AL. fish poisoning. The widespread distribution of K. pneumoniae in the environment (16) and in various food products (6, 7, 15, 16) would indicate that the potential for involvement of K. pneumoniae in scombroid fish poisoning is relatively great. The identification of K. pneumoniae as a histamine-producing microorganism in various fish products and the presence of K. pneumoniae in intestinal contents of mackerel (D. A. Corlett, Jr., et al., Abstr. Annu. Meet. Am. Soc. Microbiol., P38, p. 192, 1978) could be very significant in terms of its potential for involvement in scombroid fish poisoning. Further work is needed to define the conditions necessary for histamine formation in scombroid fish products by K. pneumoniae strains. ITERATURE CITED 1. American Public Health Association Standard methods for the examination of dairy products, 13th ed. American Public Health Association, Washington, D.C. 2. Anonymous Scombroid poisoning from mahimahi. Calif. Morbid. no Bauer, A. W., W. M. M. Kirby, J. C. Sherris, and M. Turck Antibiotic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol. 45: Cheeseman, G. C., and R. Fuller A study by high voltage electrophoresis of the amino acid decarboxylases and arginine dihydrolase of bacteria isolated from the alimentary tract of pigs. J. Appl. Bacteriol. 29: Ferencik, M Formation of histamine during bacterial decarboxylation of histidine in the flesh of some marine fishes. J. Hyg. Epidemiol. Microbiol. Immunol. 14: Guthertz, L. S., J. T. Fruin, R. L. Okoluk, and J. L. Fowler Microbial quality of frozen comminuted turkey meat. J. Food Sci. 42: Guthertz, L. S., J. T. Fruin, D. Spicer, and J. L. Fowler Microbiology of fresh comminuted turkey meat. J. Milk Food Technol. 39: Hughes, J. M., M. A. Horwitz, M. H. Merson, W. H. Barker, Jr., and E. J. Gangarosa Foodborne APPL. ENVIRON. MICROBIOL. disease outbreaks of chemical etiology in the United States, Am. J. Epidemiol. 105: Ienistea, C Bacterial production and destruction of histamine in foods and food poisoning caused by histamine. Nahrung 15: Kawabata, T., K. Ishizaka, T. Miura, and T. Sasaki Studies on the food poisoning associated with putrefaction of marine products. VII. An outbreak of allergy-like food poisoning caused by sashimi of Parathunnus mebachi and the isolation of the causative bacteria. Bull. Jpn. Soc. Sci. Fish. 22: Kimata, M The histamine problem, p In G. Borgstrom (ed.), Fish as food, vol. I. Academic Press Inc., New York. 12. Merson, M. H., W. B. Baine, E. J. Gangarosa, and R. C. Swanson Scombroid fish poisoning. Outbreak traced to commercially canned tuna fish. J. Am. Med. Assoc. 228: Rodwell, A. W The occurrence and distribution of amino-acid decarboxylases within the genus Lactobacillus. J. Gen. Microbiol. 8: Sakabe, Y Studies on allergylike food poisoning. 1. Histamine production by Proteus morganii. J. Nara Med. Assoc. 24: Schiemann, D. A Occurrence of Klebsiella pneumoniae in dairy products. J. Milk Food Technol. 39: Seidler, R. J., M. D. Knittel, and C. Brown Potential pathogens in the enviromnent: cultural conditions and nucleic acid studies on Klebsiella pneumoniae from clinical and environmental sources. Appl. Microbiol. 29: Shimizu, T., H. Yago, and A. Oba Lipase and histamine production by gram-negative bacilli. Miyazaki Daigaku Nogakubu, Kenkyu Hokoku 19: Shore, P. A Fluorometric assay ofhistamine. Methods Enzymol. 17: Simidu, W., and S. Hibiki Studies on putrefaction of aquatic products. XXIII. On the critical concentration of poisoning for histamine. Bull. Jpn. Soc. Sci. Fish. 21: Taylor, S. L, E. R. Lieber, and M. Leatherwood A simplified method for histamine analysis of foods. J. Food Sci. 43: U. S. Department of Health, Education, and Welfare, Public Health Service, Food and Drug Administration, Division of Microbiology Bacteriological analytical manual for foods. Department of Health, Education and Welfare, Washington, D.C.