Food Sci. Biotechnol. Vol. 12, No. 4, pp. 451 ~ 460 (2003) MINIREVIEW Food Science = Biotechnology Identification of the Main Bacteria Contributing to Histamine Formation in Seafood to Ensure Product Safety Shin-Hee Kim, Jorge Barros-Velizquez', Begofia Ben-Gigirey', Jong-Bang Eun’, Sang Ho Jun’, Cheng-i Wei‘ and Hagjung An* Deparment of Nutrition and Food Science, 328 Spidle Hall, Aubumn University; Auburn, AL 36849, USA 'Dpro. De Quimica, Nutviciény Bromatologia, Escuela Politéenica Superion Universidad de Santiago, E-27002 Lugo, Spain *Department of Food Science ane Technology, Chonnam National University, Gwang.ju 500-757, Korea ‘Collegeof Environmental Engineering, Kangwon National University. Chunchon 200-701, Korea “allege of Human Environmental Sciences, ONahoma State University, Sillater, OK 74078, USA Abstract Histamine is the main causative agent of scombroid poisoning and a chemical hazard regulated by the FDA acconding to the HACCP guideline. Although the mechanism of histamine formation is well understood, outbreaks of scombroid poisoning continue to occur in association with the consumption of seafoods. Since accumulation of histamine in seafood products requires the proliferation of histidine decarboxylase-possessing bacteria, itis possible to control and prevent histamine formation during handling and storage of fish. Despite ‘numerous reports showed that many types of bacterial species are capable of forming histamine, the two groups of bacteria, the enteric and marine bacteria, were identified as histamine formers in fish. Among them, Morganella ‘morganii has been shown as the main contributor to histamine accumulation in fish because of its capability to produce high levels of histamine, The presence of M. ‘morganii in fresh fish and its contamination route were demonstrated by using a newly developed molecular technique. The PCR analysis showed that M. morganii is endogenous to fish and may cause eross-contamination in the processing plants. For the efficient control of histamine formation, it is proposed that M. morganii and other prolife histamine formers in fish should be monitored prior to histamine accumulation. Such approach would bbe more effective in preventing histamine formation in fish and fishery products and ensuring product safety Keywords: scombroid poisoning, histami bacteria, Morganella morganit, PCR assay producing Introduction Histamine is identified by the FDA as a major chemical Universty, Auburn, AL 36849, USA, Tel -334 843296 Fass 1 SM-8H 3268 smal: Hojung An@aburnedu 451 hhazard of seafood products. It is the causative agent of scombroid poisoning, and is formed by time/temperature abuse of fish muscle (1), The outbreak of scombroid poisoning associated with the consumption of seafood hhas consistently been reported through the foodborne disease outbreak surveillance of the Centers for Disease Control and Prevention (2). Furthermore, the FDA has established the 50 ppm (5 mg/100 g) guideline for histamine ‘and intensified its inspection efforts (3). Seafood products containing above this level of histamine may not be used for human consumption, and are subjected to recalls. Seafood products, which have been recalled by the FDA ‘because of high histamine contents, include tuna and blue ‘mautin loins, chunk light tuna, and imported canned anchovy (@). Histamine formation continues to be a concem for both seafood product safety and economic losses. Histamine is formed by bacteria containing the enzyme, histidine decarboxylase (5). Because of the implication of histamine with seafood safety, great efforts have been ‘made to identify all types of bacteria possessing histidine decarboxylase and are capable of forming histamine, which Jed to the identification of numerous bacterial species as potential histamine formers (6-8). However, recent studies have shown that, rather, only a few bacterial species are important histamine formers because of their potential t0 accumulate toxicological levels of histamine in seafood products. Therefore, it may be feasible to focus on these small groups of bacterial species for their occurrence, distribution and proliferation during handling and storage Of fish in order to prevent the occurrence of scombroid poisoning. The information could assist in identifying better Critical control points to minimize contamination of prolific histamine formers, and prevent histamine formation to develop an effective Hazard Analysis and Critical Control Point (HACCP) system for the seafood industry. To understand the basic mechanism of histamine formation and the properties of bacteria attributed to histamine ‘accumulation in fish, we focused the review on the bacterial species capable of forming histamine, their capacity 10 form histamine, and their distribution and occurrence in452 fish. The progress in developing isolation and detection ‘methods for histamine formers and a novel molecular approach {0 detect contamination route of the prolific histamine formers are also discussed, |dentiication of histamine-forming bacteria and histamine production in fish Unlike natural toxins, ie., ciguatera, saxitoxin, or tetrodotoxin, which may be present in fish or shellfish at the time of capture, histamine is formed postmortem by mishandling of fish (1), Although histamine formation often occurs together with fish decomposition, the two processes do not always coincide with each other. and histamine may be present in fish at toxicological levels without any signs of decomposition, It was reported that toxic levels of histamine can be generated within 6 to 12 hr ater catch, if the fish are left on deck at temperatures above 25°C and without proper chilling (9). Because of the parallel relationship between histamine formation and fish decomposition, many types of spoilage bacteria associated with decomposition have been suspected as histamine formers. Occurrence of prolific histamine-producing bacteria in fish during storage at elevated temperature A number of bacterial species have been isolated from temperature- abused fish under controlled storage conditions. Most of these species belong to enteric and marine bacteria (Tables 1 and 2). Histamine-producing bacteria vary considerably in their optimal conditions for growth and histamine formation, such as optimum temperatures and lower temperature limits. Most of the prolific histamine formers detected are mesophilic bacteria (7). Although they may exist as minor bacterial species in fresh fish, the prolific histamine formers at elevated temperatures (©15C) can proliferate, synthesize histidine decarboxylase, and lead t histamine accumulation in the muscles (6). ‘Thus, temperature is considered ax the most important factor among the many environmental parameters affecting bacterial histamine formation (9, 25). FDA (1) also stated that histamine formation mostly results from time and femperature-abuse of fish during handling, processing, and distribution, and thus, is accelerated when fish are exposed to ambient temperature (20-25°C). Enteric bacteria are most frequently isolated as histamine formers from temperature-abused fish. Morganella morgan, Klebsiella pneumoniae, Klebsiella oxytoca, Raoultella (formerly Klebsiella) planticola, and Raowltella omithinolytica are idemified as prolific histamine- forming bacteria, producing >1,000 ppm histamine in culture broth (7, 8, 11) (Table 1). Other enteric bacteria, such as Hafnia alvei, Citrobacter freundii, Enterobacter spp. Escherichia coli, Proteus mirabilis and Serratia spp. have been isolated as weak histamine formers, producing -<500 ppm histamine in culture broth (M. morganit was identified as the most prolific histamine former from temperature-abused skipjack tuna and jack mackerel (12). H. alvei, Proteus spp. and Klebsiella spp. SH. Kim et al were isolated as weak histamine formers. Out of the 55 histamine-producing isolates obtained from temperat abused sardine, SI were enteric bacteria, and these were M. morganii, Proteus vulgaris, P. mirabilis, and Providencia startii (11). R. planticola and R. ornithinolytica wer isolated from tuna, bonito, and sardines, and they produced 2,610-5.250 ppm of histamine in culture broth (17). However, several strains of K. pneumoniae, previously Known as an important histamine-producing. bacterium, seldom produced histamine (17). M. morganii and K, ‘oxytoca were the most active histamine formers found in tuna (Thunnus thynnus) (8). Other histamine-producing species identified were Plesiomonas shigelloides, Citrobacter Jfreundii, Enterobacter cloacae, Enterobacter aerogenes, Enterobacter intermedium, Pseudomonas fluorescens, H. alvei, P-mirabiis, P vulgaris, Serratia marcescens, Serratia plymuthica, and Serratia fonticola, For those histamine- former isolates from tuna during canning, M. morganii, K_ oxytoca, and K. pneumoniae were prolific producers (22.000 ppm), while F. cloacae and E. aerogenes were able to form 500 t© 1,000 ppm of histamine (22, 27) Other isolates, such as C.freunuti, P mirabilis, P vulgaris, E. agglomerans, and S. liquefaciens, produced low levels of histamine (<250 ppm). F. cloacae was a weak histamine former although it was frequently isolated from tuna, Other types of bacteria, such as marine bacteria and gram-positive bacteria, have also been isolated from fish during storage a elevated temperatures (215°C). Vibrio parahaemolyticus and Vibrio alginolsticus were isolated from Pacific mackerel during storage at 15°C and 25%C (7). Most of these strains produced <500 ppm histamine in culture broth, Ediwandsiella tarda, Actinobacillus ureae, and Eikenella corrodens were isolated from albacore stored at 25°C (6). They were both minor species and weak histamine formers, Yoshinaga and Frank (14) reported that 92% of the isolates obtained from temperature abused (28°C for 24 hr) skipjack tuna were facultative or obligate anaerobes. Clostridium perfringens and Vibrio alginolyticus were frequently isolated, and other frequently isolated enteric bacteria were E. aerogenes, K. pneumoniae, and P. mirabilis Presence of histamine-forming bacteria in fish during refrigeration and frozen storage A main concem related to fish quality during refrigeration storage at 2-8°C is the ‘growth of psychrotrophic and psychrophilic histamine- producing bacteria. Hazardous levels of histamine (250 mg/100 g) in muscle are often found after the fish become unsuitable for human consumption with extended storage at the refrigeration temperatures (28, 29) Psychrotrophic enteric bacteria have been isolated from fish stored at broad ranges of temperatures (4-25°C) (6) ‘Among the species identified, Hania alvei was the most frequently isolated enteric bacterium from the refrigerated fish (30, 31). The optimum growth temperature for H. alvei is 37°C (32), and the minimum temperatures for growth are 0.2 t0 3.7°C (33), Although prevalent, H. alveidentfcation of the Main Histamine-forming Bacteria ‘Table 1, Enteric bacteria isolated from fish stored and their histamine-predlucing capabilites 453 istamine former ourcets) eae oy nce Histamine former Source(s) Money Refere Bluefin tuna 3.351 (852-4416) 8 Mackerel Not reported Wo Mackerel 3.308 (3.222-3.413) 1 Sardine 2,600-3,870 u Morganella morgan Skipjack tuna Not reported 2 Mahi-mahi 51,650 (9,990-12.070) B Anchovy 2.123 (1.437.2.895) 5 Albacore tuna, 1630 3.582.3.672) 6 Yellowfin tuna Not reported 16 Bluefin tuna 398 8 Skipjock tuna Not reported 2 Klebsiella pneumoniae Skipjack tuna Not repored 14 Anchovy 795 (185-95) 15 Bonito 216 16 Raouleellaplanticola Tuna 2,610-5,200 17 Raouletella ornthinoltica Tuna 28105,250 7 ‘Skipjack tuna Not Reported 2 Bluefin tuna 286 (59-1,328) 8 Hae alvei Mackerel ‘Not reported 18 Mackerel 148. 7 Albacore tuna, 168 (2644-497), 6 ‘Bluefin ton @ 16 Mackerel Not reported 10 Fone Mackere! 3.299 (3.272-3.M5) 7 ad Albacore tuna, 31197 (2.945.374), 6 Sardine 100-1,970 n Yellowin tuna Not reported 16 Providencia stuart Sardine 125-1.280 U Providencia regent ‘Sardine Ts Bluefin cana 459 (025-2,008) 8 Anchovy 18.29.0.99.1) Is Bonito m 16 Emembacter aerogenes sSiipeh tm Rerneened 7 Mackerel 24 7 Albacore tun 49.3 29.1-86.1) 6 Yellowfin tuna Not repored| 16 Enterobacter agglomerans ae ey 6 ‘Yallowtin wna Not reported 16 pe eee Bluefin wna 17084) 8 ‘Bluefin tuna 3507 2337-4518) 8 Klebsiella oxstoca Albacore tuna. 97:7 (140-348) 9 Anchovy 262 2» Bluefin tuna 11 8 ‘Sardine 1.300 u Proseus mirabilis Mahi-rmahi How B ‘Skipjuck tna Not reported 3 Mackere! Not reported Is Bluefin want 413 (02968) 5 Citrobacter freunalt ‘Mackerel Not re 10 Anchovy 99:49 (8.22-330.1) a Girobacier braaki ‘Albacore tuna DEVE) 19 Bluein tana 357 (O-4.075) ® Alhacore una. 82.9 (183.137) 6 ene Anchovy 27.08 (0-220) Is Bonito Bot 2 Bluefin wana 206 (189-219) 8 Serratia fonticola nee Need Ey Escherichia col Bivefin wna 122 (64-160) 8om SH, Kim etal ‘Table 1, Continued a ae Histamine (ppm — Histamine fo Source(s) Moen ae) Rete Bluefin tana 144 (4-594) 8 Anchovy 217 15 emanaeea Albacore tuna 71.7 (159-218) 9 Yellowfin wna [Not reported 16 ‘Serratia sylosus ‘Anchovy 15 Serratia marcescens Bluefin wna 8 ‘Serratia plymuthica Bluefin wna 86 G687) 8 Escherichia col Bluefin wna 122 (64-160) 8 Yersinia intermedia Bluefin tna 103) 8 ‘Table 2. Marine bacteria and gram-positive bacteria identified as histamine-producers in fish and their histamine producing capabilities istamine former sources Histamine (ppm) ference ee Soureets) Mean (range) Rel Bluefin tana cB m Pseudomonas pusida Tuna 4410) a Mackeret 159) 7 Preudomonas 3. Mackerel Notrepored 8 Tuna 30-1) 2 peer teal Yellowtin tuna Not reported 16 Phorobacterion phosphoreum Mackerel BOR ST-L 6) 2 Mackerel Te 7 ed “Albacore tuna 333,325.340) 6 Vibrio sp. 103) ir 351247) B Vibrio alginolsticus Euan . Vibrio parahaemolvicus 125258230) 7 Vibrio fuviais Bluefin wana or 3 Plesiomonas shielloides Bluefin wn 2320612) 8 Panioea agglomerans ‘Albacore tuna 528 » Bluefin tuna 2110-42) 8 acetate Albacore tuna 884 G88.151) 6 ‘Albacore tna 203 6 creer Mackerel 252 (248.256) 7 Aeromonas caviae Bivefin una ba 8 ‘Aeromonas hdrophila Mackerel 145 (72.9-182) 9 Clasirilum perfringens ‘Spanish mackerel ‘Not reported ie Staphylococcus epidermidis Salted anchovy, 360.5 (1.6-1986) 20 ‘Staphslococcus capitis Salted anchovy’ 3087 20 Bailes pails Salted anchovy 124 (TATA) 20 Tetragenococcus muriaricus Fish sauce 668 35 1,000 ppm histamine in culture broth. All other isolates were weak histamine formers (<500 ppm), ‘The other types of psychrotrophic and/or psychrophilic histamine formers that had been reported, ie. Photobuceriuan pp. Pseudomonas spp. brio spp. Plsionionas shigelloides, and Aeromonas spp., are marine bacteria (Table 2). They are mainly isolated from fresh and refiigerated fish (7, 18, 24). Photobacterium phosphoreum (N-group bacteria) was frequently isolated from mackerel during the cold storage (24, 35), It is teported that this bacterium might be primarily responsible for histamine production in fish muscle during storage at refrigeration temperature. This bacterial species produced 898 ppm histamine in Moller'sMentfication ofthe Main Histamine forming Bacteria basal medium supplemented with 1% histidine in 16 days of storage in ice (24). Another species, Photobacterium damselae, was isolated from albacore and identified as a weak histamine former, producing histamine ranging from 89.5 to 340 ppm in culture broth (6). The upper limit growth temperature for P. damselae is 22°C GI, 36). thus, they were mostly isolated from fish during storage at refrigeration temperature (6). Vibrio alginofvicus constituted about 90% of histamine formers in mahi-mahi during the storage in ice, but most of the isolates did not produce detectable levels of histamine in culture broth (13). All these isolates were weak histamine formers (<100 ppm) Plesiomonas shigelloides, frequently isolated from the marine environment, produced 8 to 340 ppm histamine in culture broth (8). In general, the identified marine bacteria fare weak histamine formers, producing <500 ppm histamine in culture broth (6, 13). ‘The histamine-producing capability of the bacterial species varies with the source of the strain isolated. Not all isolates produce histamine in culture broth (13, 23). Pseudomonas fluorescens/putida and Pseudomonas putrefaciens isolated from Spanish mackerel were weak histamine formers (18), however, those strains isolated from ripened Spanish semi-preserved anchovies produced no histamine in culture broth (15). Thus, psychrotrophie and psychrophilic bacteria contributed insignificantly to histamine accumulation in fish compared fo mesophilic prolific histamine formers. Enteric bacteria exist as a minor bacterial flora in fish stored at (°C (31, 37). The prevalent bacterial species found in fish during cold storage are Pseudomonas spp Alteromonas putrefaciens, Flavobacterium spp.. Shewanella spp. and Acinetobacter spp. These bacterial species are rarely confirmed as histamine formers. The levels of histamine produced by the psychrotrophic A. purrefaciens (16 isolates) from mahi-mahi stored at O"C were all negligible (<1 mg/100 ml) (13). Histamine-forming bacteria are hardly detected in fish during the frozen storage. Prolific histamine formers were not isolated from albacore during frozen storage at ~18°C or -25°C (38). Only 10 of the 25 isolates were able to produce low levels of histamine (25 ppm) in tryptic soy broth supplemented with 2% histidine. Thus. the growth of prolific histamine formers and their histamine formation in many diferent types of fish can be effectively controlled by storage of the ish at temperatures below (°C. Indeed, histamine formation in fish was effectively controlled by storing the fish at refrigeration temperature and below (39). Rapid chilling of fish to 4°C or below immediately ater catch is recommended to ensure product safety (I). Morganella morganii_as the main contributor to histamine formation in fish Although numerous bacterial species can form histamine (Tables | and 2), only a Few of them can accumulate histamine to toxicological levels in fish. M. morganil, K. pneumoniae, and H. alvei have ‘often been isolated from the fish incriminated in scombroid 455 poisoning (5). Among them, M. morvanii is identified ‘most often as the prolific histamine former, and it plays the most significant role in histamine formation during the storage of fish (7). All isolates of M. morgan are invariably capable of producing >1,000 ppm histamine in culture broth. They have been isolated from spoiled fish, ice, skipjack tuna, bluefin tuna, albacore tuna, mahi-mahi, mackerel, sardines, and Spanish salted semi-preserved anchovies (Table 1). M. morganii was frequently isolated from fish during storage when total bacterial counts, reached 10” CFU/g (7). The occurrence of M, monganii was directly related to histamine accumulation in fish muscle (Table 3). The histamine content inereased rapidly with the proliferation of M, morganii, reaching 283 mg/ 100 g in 2 days in mackerel stored at 25°C (7). On the contrary, the isolation of M. morganii from albacore Was negligible: and, coincidently, low level of histamine, 67 ‘mg/100 g, was detected in fish muscle (6). A toxicological level of histamine (>50 mg/100 g) was detected only when. the fish appeared completely spoiled following prolonged storage under abusive conditions. Only weak histamine formers, such as H. alvei and Enterobacter spp.. were ‘commonly found in albacore. ‘Temperature plays an important role in affecting the growth and histamine production of M, morganti. The ‘optimal temperature range for this bacterium to produce histamine is 25-37°C (40, 41). M. morganii generally produced the highest level of histamine in fish and culture media in the stationary phase (26). It produced 5,400 ppm histamine when the bacterial counts reached 10” CFU/ml in culture broth (42). The minimum temperature for M. morganii to produce toxicological levels of histamine is 15°C (26, 41, 42). M. morganii showed the typical characteristics of mesophilic bacteria in that the bacterial growth as well as the histamine formation were inhibited by incubating the bacterial culture at below 4°C (26, 42, 43), This was also observed with M, morganti isolated from Pacific mackerel stored at 0 to 25°C (7). ‘The isolation rate of M, morganti decreased as the storage temperature decreased, regardless of the length of storage. No M, morganii was isolated at 4°C, coinciding with the ‘considerably reduced levels of histamine found in the fish muscle, However, when M. morgan that had been held at 25°C for 23 hr was transferred to S*C, histamine level continued to accumulate from 800 ppm to 2.700 ppm in culture broth following 5 days of incubation (42), ‘Apparently, the histidine decarboxylase that had already been produced by the bacteria could continue to exert its action to convert histidine to histamine even after the inactivation of the bacterial cells by low temperature storage (43). Therefore, any delay in proper storage of the fish would allow histamine to accumulate at high levels in the muscle even after the fish is chilled and stored at 4°C and below, if the fish were previously ‘exposed to high temperatures allowing the growth of M. ‘morganii and other prolific histamine-producing bacteria. ‘Studies were conducted in various fish species, such as456 SH, Kim etal Table 3. Histamine accumulation and identification of histamine-producing bacteria in mackerel and albacore during storage at 25°C (from ret. 6,7) = Hisamine isting Fish Storage day Maa Strains isolated No, isolated ‘om Mackerel T 576 Vibrio parahaemolticus 3 25820 Morganelta morganii 1 3191 Vibrio alginolsticus 1 882 2 2.860 M, morgan 0 3222-3413 V. parahaemolyticus 5 982-323, Proteus vulgaris 4 3272.3,345 Vealginolsticus 4 415.780) Enterobacter aerogenes 1 24 3 2.100 M. moranii a 3,208.3,527 P vulgaris 6 I8S-3AN Vealginolyicus 5 639122 V-parativemolsicus 5 Ms ‘Albacore 3 9 Hata alvet 5 263497 M, morganii 1 3875 Fdwardsiells tarda 1 393 4 81 Habvei n 244.351 P.wulgaris 3 2945-3474 M. morganti 2 3.841-3,940 Actinobacillus ureae 1 208 5 176 H.alvei 8 2538-429 Enterobacter cloacae 6 18.3137 E- aerogenes 2 891-130 M, morgan 1 3,687 Eikenella corrodens 1 142 6 on Halvei B 170-467 E. aerogenes 7 193366 M. morgant 3 3.582.3672 E- aerogenes 3 291.8611 mackerel, albacore, mahi-mahi, and salmon, stored at 30°C to 37°C for the growth of M. morganii and its histamine formation (44), Mackerel, albacore, and mahi- mahi were good substrates for histidine decarboxylation by M. morganii at temperatures above >15°C. Among the fish species studied, mackerel was the most susceptible to histamine accumulation by M, morganii, The highest level of histamine detected in 24 hr of storage was 461 mg/100 g in mackerel, followed by 343 mg/100 g in albacore, and 334 mg/100 g in mahi-mahi. Salmon was the least susceptible to histamine formation, probably due to the low free histidine content in the muscle. However, ‘M. morganit managed to produce histamine in salmon at over 25 mg/100 g, a level well above the FDA guideline, ‘This data indicated that histamine production may not be restricted to only the fish species rich in free histidine if 'M, morganii is present in the fish. Salmon was previously reported to be involved in the incidents of scombroid poisoning (44). In general, the growth of M. morganii can be controlled by cold storage of the fish at 4°C or below (26). Histamine formation by M. morganii was completely inhibited by frozen storage of the fish. However, care should be taken, as M, morganii can survive through frozen storage, recover its cellular and enzyme activity, and produce toxicological levels of histamine in fish muscle when held at 25°C after thawing (44-46). Therefore, the importance of handling and storage conditions cannot be overlooked after thawing of fish in restaurants and seafood processing facilities. Methods for detection of histamine-producing bacteria Conventional culture methods Detection of histamine- producing bacteria is always challenging. The approaches have always relied on conventional culture methods. Most of the histamine-forming bacteria are enumerated of isolated by using differential media, such as Niven medium (47) Differential media are designed to rapidly differentiate many different species of histamine formers from the non-formers. Niven’ diferent medium was frst developed for quantitative detection of histamine formers based on the color change of bromocresol purple, an indicator in the medium (47). It tums purple when histidine in the medium is decarboxylated resulting in an increase in pH, Nivens medium was modified to optimize forthe enumeration of histamine-forming bacteria (34). The modified parameters were the increase of the medium pH from 5.3 to 6.3 and the optimization of plate incubation time (24,48, and 72) and temperature (30 and 37°C). Morganella morganii, Klebsiella pneumoniae, and Hafnia alvei were tested for their ability t0 change color on these media, and theirIdentification of the Main Histamine-forming Bacteria ‘growth on the medium was noticeably improved at pH 5.3 to 5.5 than at pH 6,3. Quantitative analysis of histamine- producing bacteria on these media, however, was limited asonly | £0 80 colonies developed on the medium allowed for effective enumeration of histamine-forming bacteria ‘The Nivens medium was further modified 10 other differential media, ie. the modified Niven’s medium, the JoostewNortholt medium, and the Maijara medium, 0 enhance the detection rate of histamine-forming lactic acid bacteria in cheese, meat, poultry, ripened sausages, and other products (48-50). Niven's medium has widely been used to enumerate or isolate histamine formers (11). However, its most serious limitation is the tendency to generate high rates of false results. It is mainly due to the formation of various alkaline compounds and fermentative activity of other bacterial species (21). To exclude non-histamine formers and reduce the occurrence of false positives during screening, selective media were used for prescreening prior to the use of differential media for histidine decarboxylase activity (19, 50). For the detection of histamine formers in albacore tuna, several target bacteria were isolated using selective media, ie., EMB agar for enteric bacteria, MRS agar for lactic acid bacteria, KF agar for streptococci PI agar for pseudomonads, and $110 agar for staphylococci (19), Selected bacterial isolates were screened from the color changes of the colonies on differential media, Among the selective media tested, EMB agar was most effective in selecting prolific histamine producers as demonstrated by the highest rate of true positives based on histamine analysis, Only a few of the presumptive isolates from KF, MRS, and S110 agar were true histamine producers as determined by histamine analysis. Although the detection of true histamine formers was greatly improved by using the two-step isolation procedure, such approach was encountered with limitations such as the intense labor, the lengthy assay time (>1 week), and the low sensitivity. Molecular detection of histamine-producing bacteria Molecular techniques have been applied to improve many of the limitations observed with the conventional culture methods for bacterial detection and analysis (51). The techniques have been applied successfully for rapid and sensitive detections of histamine-producing bacteria in foods ‘The structures of histidine decarboxylase from the several bacterial species have been studied, and their peptide and gene sequences are available for designing primers and probes. The pyridoxal-5'-phosphate (PLP)-dependent histidine decarboxylase was isolated from Morganella ‘morganii, Klebsiella pneumoniae, Enterobacter aerogenes, and Vibrio anguillarum (52, 53). ‘The PLP-dependent enzyme isolated from M. morganii ha identical subunits with a molecular weight of 43,000. The gene contains 1.131 nucleotides and encodes for 377 amino acids (54), ‘The PLP-dependent enzymes from Klebsiella planticola and E. aerogenes have the same number of amino acids 457 (377) as M. morganii (55). These three PLP-Jependent enzymes show high identity in both nucleic acid (75%) and amino acid (80%) sequences. Therefore, it is speculated that there occurred no addition or deletion of nucleotides during their evolution from the parental gene of these PLP-dependent enzymes. The amino acid sequence of the histidine decarboxylase from V. anguillarum also shows 2 high homology with those of the three bacteria (56), The V. anguillarum gene sequence of the enzyme is 65.1%. 63.5%, and 61.4% identical to those of K. planticola, M. morganii, and B. aerogenes, respectively. The Pvr (pymuvoy))-dependent histidine decarboxylase group includes Lactobacillus 30a, Clostridium perfringens Lactobacillus buchneri, and Micrococcus spp. The characterization of PLP-dependent enzyme shows different from that of Pvr-dependent enzyme (57). The amino acid sequences of the PLLP-dependent enzymes show no homology with those of the Pvr-dependent enzymes (57). Polymerase chain reaction (PCR) technique was first used to detect histamine-producing lactic acid bacteria in cheese and dairy products (58). The PCR primers were designed based on the conserved sequences of the histidine decarboxylase gene of Lactobacillus 30A. The universal primers used were the forward primer, JV 16HC (AGA ‘TGG TAT TGT TIC TTA TG) and the reverse primer, JV ITHC (AGA CCA TAC ACC ATA ACC TT) comesponding, {0 169-190 and 515-536 of the hicA gene of Lactobacillus 30A, respectively. This primer set was suitable for the etection of histamine-forming lactic acid bacteria. The primers generated 370 bp PCR product with DNA extracted from L. buchneri, C. perfringens, and Leuconostoc oenos DNA/DNA dot-blot hybridization has been used for detection of numerous histamine formers in albacore tuna (59). Probes used for hybridization were prepared by extracting the total DNA from the histamine-producing bacteria, such as M. morganii, K. planticola, H. alvei, Stenotrophomonas maltophila, E. agglomerans, Acinetobacter baumanii, and Pseudomonas spp. The probes prepared from the total DNA of S, maltophilia and M. morganii showed no cross-reactivity with other histamine-producing bacteria tested. The sensitivity of this method was I CFU/g with the enrichment of albacore homogenate at 37°C for 18 hr. Yet, limitation still occurred with the use of genomic DNA as a probe in detecting a specific target species. The probe prepared from E. aerogenes cross-reacted with H. alvei. Therefore, the authors emphasized the need for designing more specilic probes for detection of specific histamine-forming bacteria. APCR assay applying the 16S rDNA target PCR primers has recently been developed specifically for the detection of M. morganii (60). Although several hie gene sequences of gram-negative histamine Formers are available (55), dhe information provided is not sufficient enough for the design of species-specific primers, Because the analysis of the 16S rDNA sequence allows for phylogenetic comparison with the available sequences of many bacterial species in the database (61), this gene was alternatively selected as458 a target sequence for species-specific primers. The 16S IDNA of M. morganii isolated from albacore was first cloned and sequenced (60). The gene sequence (1,503 bp) showed 95% identity to those of enteric bacteria, i.e, Enierobacter spp.. Klebsiella spp., Citrobacter spp.. Hagia ‘alvei, Proteus spp. and Providencia spp. The 16S rDNA sequence was further compared with those of other species, ‘and unique primers found for M. morganii were: the forward primer, MmF207 (S-CTC GCA CCA TCA GAT GAA CCC ATA T-3),; and the reverse primer, MmR1017 (S:CAA AGC ATC TCT GCT AAG TTC TCT GGA ‘TG-3). By using these primers, the PCR assay developed could detect as little as 9 CFU/mL of M. morganit in albacore homogenate with 6 hr enrichment in tryptic soy broth at 37°C. Origin and contamination route of prolific histamine formers Bacteria are rarely found in the muscle of fresh healthy fish (7). However, mishandling of fish allows bacteria to invade through the skin, gill, and intestine into the muscles and proliferate during storage. Bacteria may also be introduced to fish muscle through cress-contamination uring handling of fish on board and at processing plants or during distribution and handling by consumers (5, 9). ‘Dominant bacterial flora in fish may alter depending. on the handling and sanitary conditions of the boat, processing facilities, and markets, Histamine-producing bacteria are speculated to exist in the salt-water environment (1). Many researchers have suspected the presence of histamine-producing bacteria in the intestine and gill as a past of natural bacterial flora in fresh fish, since histamine is usually found in fish at high levels in tissues adjacent to the gills and intestines (8, 19, 62-64), The histamine content in uneviscerated mackerel was 10-times greater than that of eviscerated mackerel during storage at ambient temperature (63). Tuna burgers and salads prepared from the belly meat ‘were implicated in the outbreaks of scombroid poisoning, North Carolina (65). Although histamine formers are minor constituents and have not been widely isolated from fish, they could be isolated following enrichment of the test samples. [t was pointed out that the initial population of prolific histamine-producing bacteria. in fresh fish would be too low (<10? CFU/g) to be isolated by the conventional culture method (6, 7, 66). Histamine-producing bacteria identified in fresh mackerel and albacore were enteric and marine bacteria (6, 7). They ‘were distributed mostly in the gill and skin of fresh fish and produced histamine ranging from 20 to $00 ppm in ‘culture broth, These enteric bacteria included Hania alvei, Enterobacter cloacae, anxl Citrobacter braakii. H. alvei was a dominant bacterial species found on the skin and ill of fresh albacore, but all the strains were weak histamine formers (',000 ppm. histamine are rarely identified from fresh mackerel and. albacore. -M. morganii has been identified as the most important histamine former in fish, however, its presence in fresh, fish could not be detected using the conventional culture method. To overcome the low sensitivity of the culture method, the PCR assay system was used to detect M. ‘morganié in fish and processing plant to investigate and to determine its origin and the contamination source (67). Using the PCR assay, M. morganii was detected in fresh albacore, mackerel, and sardine and in the processing plant Fig. 1). M- morganii was frequently found in mackerel, and sardine but rarely in albacore, and the gill and skin ‘were the main harbor sites of the bacterium in fresh fish. Raw fish was the source of equipment contamination in the processing plant. These results indicated that histamine- forming bacteria are endogenous to the fish and are likely to proliferate if the growth conditions for the bacteria are met. Therefore, chilling and temperature control of the fish ry ” ® + e00bp _ errors... ig, 1. Detection of M. morganii in fish and environmental samples by using PCR or PCR coupled with Southern hybridization assay using Mf. morgani-specfic primers, MmE207 and MmR1017. (A) The PCR products were separated on agarose gel and visualized by staining with ethidium bromide. (B) The PCR products were transferred onto nylon membrane and hybridized with the PCR generated biotinylated probe. The amplified PCR product with the expected size of 809 bp is marked with arzow on the right. “The lanes are marked for: (1) DNA ladder in (A) or Mf, moreanit OSL36 in (By; (2)-(13) Mackerel (2. Skin: 3. Gill: 4, Cavity: 5, Skin; 6, Gill; 7, Skin: 8, Gill 9, Skin; 10. Gill: 11, Cavity Cavity; and 13, Skin): (14) 15, Gill: Cavity: 17, Gut: 18, Skin; 19, Gill; and 20, Cavity): 21) ladder in (A) of M. morganil OSL36 in (B); (22)-24) Sardine (22, Gut: 23, Skin; and 24, Gill; 25)-33) Albacore (25, Skin 26, Gill: 27, Cavity; 28, Gut, 29, Skin; 30, Skin: 31, Cavity Gut and’ 33, Cavity and (34-(40) various samples swabbed from conveyer belt in the plant during processing (from re. 67)Identification ofthe Main Histamine-forming Bacteria along with constant monitoring of the handling conditions from the time of harvest till consumption are absolute requirements to ensure safety of fish. References |. Food anu Drug Administration. Ch Scombrotoxin histamine) {ormation. pp. 73-90. 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