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STEVE L. TAYLOR and MARCI W. SPECKHARD
Isolation of Histamine-ProducingBacteria From Frozen Tuna
Introduction
The bacterial spoilage of tuna andcertain other scombroid and nonscombroid fishes is sometimes accompanied by the formation of highlevels of histamine in the edible tissuesof the fish (Hillig, 1956; Tomiyasuand Zenitani, 1957; Arnold andBrown, 1978; Frank et aI., 1981). Theformation of histamine is particularlynoteworthy because the presence ofhigh levels of histamine in spoiled fishhas been associated with outbreaks offood poisoning also known as scombroid fish poisoning (Merson et aI.,1974; Arnold and Brown, 1978; Lerkeet aI., 1978; Kim, 1979). Fresh scombroid fish possess virtually no histamine (Hardy and Smith, 1976; Franket al., 1981). However, the scombroidfish have high levels of free histidinein their muscle tissues (Lukton andOlcott, 1958). Histamine is generatedfrom histidine during spoilage by bac-
The authors are with the Food Research Institute, Departments of Food Microbiology andToxicology and Food Science, University ofWisconsin, Madison, WI 53706.
ABSTRACT-A method wasdevelopedforthe isolation of histamine-producing bacteriafrom frozen tuna. The method was equally effective for the recovery of Proteus morganii,Klebsiella pneumoniae, and Enterobacteraerogenes, and was effective in recoveringthese bacteria when fewer than 10' organisms/g were present in the frozen tuna. Thekey feature of the isolation method was a24-hour incubation in trypticase-soy brothwhich allowed recovery of freeze-injuredbacteria.
Histamine-producing bacteria were isolated
April-May-June 1983,45(4-6)
teria that possess the reqUisite enzyme, histidine decarboxylase (Arnold and Brown, 1978; Omura et al.,1978; Yoshinaga and Frank, 1982).
Many different bacterial species areknown to possess histidine decarboxylase (Arnold and Brown, 1978; Tayloret aI., 1978; Taylor et aI., 1979;Yoshinaga and Frank, 1982). However, only Proteus morganii (Kawabata et al., 1956; Sakabe, 1973), Klebsiella pneumoniae (Taylor et aI.,1979), and Hajnia alvei (Havelka,1967) have been isolated from fish incriminated in scombroid fish poisoning incidents. In some species, i.e., K.pneumoniae, the ability to producehistamine is limited to only certainstrains (Taylor et aI., 1979). Behlingand Taylor (1982) indicated that thehistamine-producing bacteria couldbe divided into two categories: Thosespecies capable of producing largequantities of histamine (>100 mg/100 ml) in tuna fish infusion broth(TFIB) during a short incubation period «24 hours) at temperaturesabove 15 DC and those species capableof producing somewhat lesser quantities of histamine (>25 mg/l00 ml) in
from three of ten frozen tuna by use of thisisolation technique. Gills, intestines, andmuscle tissues were surveyed. A P. morganiistrain capable of producing 535 mg of histamine/IOO ml within 24 hours at 3rC in tunafish infusion broth (TF/B) was isolated fromone sample of gills. Two other gill samplesyielded Citrobacter freundii isolates capable ofproducing 24-35 mg ofhistamine/100 ml in 24hours at 3rC in TFIB. No histamineproducing isolates were obtained from any ofthe intestine or muscle samples.
TFIB after prolonged incubation(~48 hours) at temperatures of 30 DCor above. P. morganii, K. pneumoniae, and E. aerogenes appear tobelong to the category of prolifichistamine producers, while the testedstrains of H. alvei, Citrobacter jreundii, and Escherichia coli were slowproducers of histamine (Taylor et al.,1978; Behling and Taylor, 1982). Certainly, other bacteria may also be prolific histamine producers. The recentisolation of Clostridium perjringensfrom decomposing skipjack tuna andits identification as a prolific histamine producer (Yoshinaga andFrank, 1982) would seem to underscore this point.
Histamine-producing bacteria havebeen isolated from the skin, gills, intestines, and muscle tissues of spoilingfish (Kimata, 1961; Lerke et aI., 1978;Omura et aI., 1978; Yoshinaga andFrank, 1982), and are often considered part of the normal microflora offish (Yoshinaga and Frank, 1982).However, the circumstances underwhich tuna would acquire bacteriasuch as P. morganii, K. pneumoniae,and C. perjringens which are not partof the normal microflora of seawaterhas never been adequately explained.
The microflora of fish is often a reflection of the microflora of theiraquatic environment (Shewan andHobbs, 1967). Pseudomonas, Achromobacter, Flavobacterium, Vibrio,Micrococcus, Bacillus, and coryneforms comprise the typical microfloraof freshly caught marine fish (Shewanand Hobbs, 1967; Shewan, 1971; Seraet aI., 1974), although the microfloraof freshly caught tuna or other scombroid fish has not been studied.Enterobacteriaceae such as P. morganii and K. pneumoniae are rarely ifever found on freshly caught marinefish (Shewan and Hobbs, 1967). C.perjringens has been found in marinesediments from Puget Sound (Matches et al., 1974) and from fish on several occasions (Matches et aI., 1974;Yoshinaga and Frank, 1982). However, the possibility remains that histamine-producing bacteria may not bepart of the normal microflora ofscombroid fish but may instead represent post-catching contaminants. P.
35
morganii has been isolated from canned tuna associated with episodes ofscombroid fish poisoning in WestGermany, and the contamination almost certainly occurred during restaurant handling procedures (Yamani etal., 1981). A similar situation mayhave occurred in an outbreak ofscombroid fish poisoning involvingconsumption of tuna sashimi in SanFrancisco where K. pneumoniae wasisolated from kitchen scraps obtainedfrom a Japanese restaurant (Lerke etaI., 1978).
If the occurrence of histamineproducing bacteria in scombroid fishis the result of post-catching contamination, then the incidence of recoveryof such bacteria from fish off-loadedat canneries should be sporadic anddependent on the level of sanitationon the boats. If histamine-producingbacteria are part of the normal microflora of tuna, then such bacteriashould be isolated frequently fromtuna if an adequate isolation procedure was employed. Studies on the incidence of recovery of histamine-producing bacteria from such fish havenot been reported. Fish usually arrivefrozen, and studies would perhaps becompromised by freeze injury of thehistamine-producing bacteria. Thisstudy was undertaken to develop aneffective method for the recovery ofhistamine-producing bacteria fromfrozen tuna and to apply that technique in assessing the incid~nce ofrecovery of histamine-producmg bacteria from tuna.
Materials and Methods
Fish
Frozen skipjack tuna, Katsuwonuspelamis, each weighing between 2.2and 3.8 kg (5 and 8.5 pounds) we~e
obtained from a major tuna packer mthe western United States. The tunawere originally obtained from catchesoff the coasts of South America, thewestern United States, Japan, thePhilippines, and Nauru. The .tunawere shipped on dry ice to Madiso~,
Wis., and were held frozen untilanalysis. All tuna arrived froze~ .andwithout overt signs of decompositiOn.
36
Bacterial Media
Trypticase soy broth (TSB) andtrypticase soy agar (TSA) were prepared according to label directions.Niven agar (NA) was prepared according to the procedure of Niven etal. (1981). The preparation of TFlBhas been described previously (Omuraet al., 1978). Trypticase soy brothhistidine medium (TSBH) was TSBwith 0.1 percent histidine added(Taylor and Woychik, 1982).
Recovery, Isolation,and Identification ofHistamine-Producing Bacteria
The tuna were thawed, and samplesof muscle (from an area adjacent tothe intestinal cavity and near the gills),gills, and intestine (the entire intestinewas used) were aseptically removed.The samples were blended in anOsterizer' blender without any addition of liquid. Portions (I g) of theblended muscle and gill samples andportions (l ml) of the homogenizedintestinal samples were placed in 9 mlof TSB. After incubation at 35°C for24 hours, serial dilutions of the TSBwere plated onto TSA or NA. TheNiven agar plates were overlayed withadditional NA. The plates were incubated aerobically at 35°C for 24hours in the case of the TSA platesand up to 5 days in the case of the NAplates. The NA plates were checkeddaily for the presence of purple colonies or colonies with purple halosindicative of histamine-producingbacteria (Niven et al., 1981). Thesecolonies were picked and streakedonto TSA to obtain isolates. Theseisolates were then restreaked on NAplates to confirm that they producedpurple colonies. Additional isolateswere randomly picked from the TSAplates. These isolates were streakedonto NA plates, and any isolates producing purple colonies were confirmed as before and retained.
The histamine-producing capabilities of the presumptive histamine-pro-
'Mention of trade names or commercial firmsdoes not imply endorsement by the NaltonalMarine Fisheries Service, NOAA.
ducing isolates were determined inTFlB as previously described (Behlingand Taylor, 1982). Samples of the culture fluid were withdrawn after 0, 6,24 and 48 hours of incubation at37'oC. The level of histamine in thesamples was determined by a modification of the AOAC method (AOAC,1980) as described previously (Behlingand Taylor, 1982). Aerobic platecounts (APC) were also conducted onthese samples (Behling and Taylor,1982).
Isolates producing in excess of 20mg of histamine/l00 ml in 48 hours inTFlB were identified using the API 20E Enterobacteriaceae system (Guthertz et al., 1976; Taylor et al., 1979).
Evaluation ofRecovery Procedure
To determine if the recovery procedure was adequate for the isolation oflow numbers of histamine-producingbacteria from frozen tuna, threeknown histamine-producing bacterialstrains were inoculated into tuna. Thethree histamine-producing bacterialstrains, Klebsiella pneumoniae T2,Proteus morganii IIOSC2, and Enterobacter aerogenes 42 were obtained inearlier studies (Lerke et aI., 1978;Taylor et al., 1978). The isolates weretransferred from TSA slants into 5 mlof TSBH and allowed to incubate for24 hours at 37°C; 0.2 ml of each culture was transferred to another 5 mlof TSBH and incubated for 18 hoursat 37°C. The 18-hour culture was diluted with saline until the density wascomparable to McFarland standard#2 (McFarland, 1907). This dilutedculture was inoculated at a rate of 0.1ml of culture/g of tissue into blendedsamples of either intestines, gills, ormuscle tissue obtained from a singleskipjack tuna. The sample was blen?ed further to insure homogeneous distribution of the bacteria. An uninoculated control was also included. Theinoculated tissues were separated into10 g samples and stored in a freezer at- 15°C for up to 14 days. Sampleswere removed from the freezer at 1-,7-, and 14-day intervals. Inoculatedcontrol samples that were not subjected to frozen storage were obtained
April-May-June 1983,45(4-6)
Table 1.-laolallon of hlstamlne-producing bacteria from frozen skipjack tuna.
No. of tentative No. of confirmedhistamine-producing isolates histamine·producing isolates
Fishno. Niven agar plates TSA plates Niven agar plates TSA plates
1 4-M.3-G' 1-G 0 02 8·G 0 8-G 03 0 0 0 04 2·G 0 0 05 30G 2-M.5-1. l·G 1-G
3-G6 7-M.3·1, 4-M.2-G 0 l-G
3·G7 0 0 0 08 4-M 30M 0 09 5-1.3-G 7-1.5-G 0 0
10 l·M.l-G 30G 0 0
Total 47 35 9 2
1M = muscle, G = gills, I = intestines.
Table 2.-Hlatamlne production by the confirmed histamine-producing IsolatesIn TFIB.
Histamine concentrationLog (mg/l00 mil
Source of increaseBacterial species isolation in APC' Oh 6h 24 h 48h
Proteus morganii Tuna #2 1.09 6.3 476 535 665Gills
Cftrobacter 'reundii Tuna #5 0.88 NO' 0.4 4.1 23.9Gills
Cftrobacter 'reundii Tuna #6 1.14 NO 0.2 7.3 35.3Gills
1 Logarithmic increase in aerobic plate count after 24 hours of incubation in TFIB at35°C with an initial inoculum level of more than 101 bacteria/ml.'NO = not detectable.
immediately after inoculation foreach organism and type of tissue. The10 g frozen samples were transferredaseptically to 90 ml of TSB and allowed to incubate at 35°C for 24hours. This culture fluid was seriallydiluted, plated on TSA and NA, andincubated for 24 hours (TSA) or 48hours (NA) at 35°C. For inoculatedcontrol samples not subjected tofreezing, the procedure was similarexcept that the samples were not giventhe 24-hour recovery period in TSB.The histamine-producing isolateswere picked from the NA plates andtheir identities checked using the API20 E Enterobacteriaceae system.
Results
Confirmed histamine-producingbacteria were isolated from only threeof ten frozen skipjack tuna (Table 1).In all three cases, the histamine-producing isolates were obtained fromthe gills. No histamine-producingbacteria were isolated from any of themuscle or intestinal samples. A totalof 82 tentative histamine-producingisolates were initially obtained fromthe NA and TSA plates. However,only 11 of these tentative isolates wereconfirmed as histamine producers bytheir ability to produce in excess of 20mg of histamine/IOO ml in TFIBwithin 48 hours at 37°C. Most of theremaining 71 isolates did not producecharacteristic purple colonies onNiven agar plates. Instead, the colonies were often red or
Apri/·May·]une 1983,45(4·6)
pink. The inclusion of these isolates astentative histamine producers represented a conservative approach in theuse and interpretation of Niven agarplates along with a desire to insurethat no histamine producers would bemissed in the recovery procedure.
Of the eleven histamine-producingisolates obtained from the gills of frozen tuna, eight were identified as Proteus morganii and three were identified as Citrobacter freundii. The eightisolates of P. morganii were obtainedfrom the Niven agar plates of the gillsample from tuna #2 indicating thepossible presence of relatively highnumbers of this organism in that particular sample. The C. freundii strainfrom tuna #5 was isolated once eachfrom the TSA and NA plates, whilethe C. freundii strain from tuna #6was isolated only once from a TSAplate.
The P. morganii strain obtainedfrom tuna #2 was by far the most prolific histamine producer among thethree strains compared in Table 2.This strain was able to produce in excess of 535 mg of histamine/IOO ml inTFIB within 24 hours at 37°C. Bycomparison, the two C. freundiiisolates produced only 3-5 percent asmuch histamine as the 48-hour sampling period. The C. freundii isolatesalso showed a more pronounced lagperiod in terms of histamine production by comparison to the P. morganiistrain. Large inoculum levels wereused in these experiments to increase
the likelihood of bacterial histamineproduction and eliminate any possibleproblems associated with failure togrow rapidly in TFIB. All three bacterial strains grew in TFIB increasingabout 1 log in numbers within 24hours. The high initial histamine levelwith the P. morganii strain representscarryover from the 18-hour TSBHcultures.
The evaluation of this procedurefor the recovery of histamine-producing bacteria from frozen tuna tissues indicated that the methodworked quite well. When between 102
and 103 organisms/g were inoculatedinto muscle, gill, and intestinesamples from skipjack tuna and thesamples were frozen, histamine-producing bacteria were consistently recovered from the NA plates. Therecovery procedure using 24 hours incubation in TSB worked equally wellfor P. morganii 11OSC2, K. pneumoniae T2, and E. aerogenes 42, andfor all three tuna tissues studied.Histamine-producing isolates wereobtained just as easily from samplesstored frozen for 1, 7, and 14 days.Usually, 48-hour incubations werenecessary before purple colonies appeared on the NA plates. The purplecolonies were confirmed to be identical in biochemical profiles to theknown histamine-producing bacteriawith which the sample was originallyinoculated. Failure to use the TSB recovery step usually resulted in a lackof recovery of histamine-producing
37
bacteria from frozen tuna samples,while the histamine producers wereconsistently recovered from nonfrozen samples without the recovery step.
Discussion
A suitable method was developedfor the recovery of histamine-producing bacteria from frozen tuna.This method was effective for recovering rather low levels «103 organisms/g) of histamine-producing bacteria from frozen tuna. Without the24-hour recovery period in TSB, thehistamine-producing bacteria couldnot be isolated successfully from frozen tuna indicating that freezing sublethally injures these bacteria. Thismethod was shown to be effective forthe recovery of K. pneumoniae, P.morganii, and E. aerogenes. Its effectiveness for the recovery of otherhistamine-producing bacteria such asC. perjringens and the recently described psychrophilic, halophilicN-group bacteria (Okuzumi et al.,1981) cannot be evaluated since thesebacteria were not included in thisstudy.
The use of Niven agar aided considerably in the isolation and identification of the histamine-producing bacteria, although the low pH of thismedium would be predicted to discourage growth by some histamineproducing bacteria, especially theanaerobes. The histamine-producingbacteria isolated from frozen tunagrew well on NA following the TSBrecovery step. Although only 11 of 82tentative histamine producers wereconfirmed to be histamine producersin TFIB, this should not be taken asan indication that NA yields a highnumber of false positives. Only eightisolates gave typical purple colonieson NA plates, and these were the P.morganii isolates from the gills oftuna #2. The remaining 74 isolateswould have been classified as questionable or negative reactions in mostsituations. However, we wanted to becertain that no histamine-producingisolates were missed, and thus manyof the colonies were picked andassayed to insure that no atypicalreactions would be found among his-
38
tamine producers on NA plates. Theresults indicate that NA will be veryuseful in future studies, and that onlytypical purple colonies should be retained fo~ further investigation.
Despite the use of these effectiverecovery, isolation, and identificationmethods, only one of ten frozen tunayielded a high-level histamine producer. Apparently, histamine-producing bacteria are not common components of the natural microflora oftuna. If histamine-producing Enterobacteriaceae are present, the level ofcontamination must be below \03organisms/g. If such low level contamination occurs in tuna, other bacteria would likely overgrow the histamine producers in temperature abusesituations. Although 10 tuna constitute a fairly small sample size, thetuna came from a variety of fishinglocales and fishing boats.
While this study provides evidencethat the histamine-producing Enterobacteriaceae are not part of the normal microflora of tuna, the possiblepresence of other types of histamineproducing bacteria cannot be ignored.C. perjringens and the psychrophilic,halophilic N-group bacteria (Yoshinaga and Frank, 1982; Okuzumi etaI., 1981) would not likely have beendetected in these experiments due tothe choice of aerobic and mesophilicincubation conditions and the use ofNiven agar.
P. morgan ii, K. pneumoniae, andH. alvei are the only species ofhistamine-producing bacteria thathave been isolated from tuna implicated in outbreaks of scombroid fishpoisoning (Kawabata et aI., 1956; Havelka, 1967; Sakabe, 1973; Taylor etaI., 1979) .. Yet, these species were notisolated from nine of the ten tuna included in the survey. The isolation ofenteric bacteria, including one strainof P. morgan ii, from gill samples ofseveral tU:1a may not reflect the occasional presence of these bacteria aspart of the normal microflora of thegills. Tuna are often moved using thegills as convenient "hand-holds." Thepossibility of contamination of gillsamples during post-catch handlingcannot be discounted. In fact, post-
catching contamination should beconsidered as the likely source ofhistamine-producing bacteria in manyoutbreaks of scombroid fish poisoning. Many outbreaks involve raw fishthat have been handled in restaurantsor homes increasing the likelihood forcontamination with histamine-producing enteric bacteria. Certainly,Yamani et al. (1981) provide strongevidence to indicate that restaurantshave been involved as sites of contamination in some outbreaks.
The isolation of histamine-producing enteric bacteria from the gillsemphasizes the possible importance ofthe gills as a reservoir for these bacteria. In several studies on the distribution of histamine in spoiled tuna(Lerke et a!., 1978; Frank et al.,1981), the muscle tissue adjacent tothe gills had the highest level of histamine. The bacteria responsible forhistamine formation in these studies(Lerke et a!., 1978; Frank et a!., 1981)could have arisen from either the gillsor the anterior portion of the intestinal tract. As mentioned above, thegills are much more likely to be contaminated with histamine-producingbacteria during handling than are theintestines or muscle tissues. Invasionof histamine-producing bacteria intothe muscle tissues should certainly beconsidered a secondary event.
This study provides the first clearevidence that histamine-producingEnterobacteriaceae are not part of thenormal microflora of tuna. Unfortunately, the isolation scheme was notoriented toward the possible isolationof C. perjringens and the N-groupbacteria. Future studies will have totake these bacteria into consideration.Also, the isolation of P. morganiifrom the gills of one tuna could havebeen due to either its presence as apart of the normal microflora or topost-catching contamination. Theonly way to determine if P. morganiiand other histamine-producing entericbacteria exist occasionally as constituents of the normal microflora of tunais to repeat our isolation studies onfresh-caught tuna on a tuna boatbefore any chance occurs for contamination via handling.
Apri/-May-June 1983,45(4-6)
Acknowledgments
This research and its publicationwere supported in part by the Collegeof Agricultural and Life Sciences,University of Wisconsin, Madison; bya National Marine Fisheries Servicegrant, NA80AA-D-00095; and bycontributions from the food industry.We are particularly grateful to theTuna Research Foundation and StarKist, Inc., for supplying the tuna.
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