International Journal of Food Microbiology, 5 (1987) 137-146 Elsevier
A rapid method for detection of histamine-producing bacteria Niels Kristian Klausen and Hans Henrik Huss Ministry of Fisheries, Technical University, Lyngby, Denmark (Received 29 May 1987; accepted 20 August 1987)
A method for detecting histamine-producing bacteria is described. The method is based on automated conductance measurements in a histidine-containing medium (HDB) incubated at 25 o C, with a large and distinct increase in the conductance being indicative of the presence of histamine-producing bacteria. An initial low pH (5.5) is optimal for measuring the histidine decarboxylating activity of Morganella morganiL but clear results are obtained at higher pH values (6.0-7.0) as well. It is shown that the histidine decarboxylating activity of M. morganii is unaffected by the presence of non-histamine producing bacteria. Using this new method in the examination of mackerel spoiled at high temperatures, the results obtained indicated the presence of large amounts of histamine-producing organisms. These were subsequently isolated and identified as belonging to the family Enterobacteriaceae. Key words: Fish; Histamine; Conductance; Rapid method
Introduction A n u m b e r of s c o m b r o t o x i n p o i s o n i n g i n c i d e n t s h a v e o c c u r r e d in recent y e a r s (Taylor, 1986). This illness h a s been a s s o c i a t e d w i t h high levels of h i s t a m i n e , p r o d u c e d b y the a c t i o n of b a c t e r i a l d e c a r b o x y l a s e e n z y m e s o n the a m i n o a c i d histidine, which is f o u n d in the flesh of fish f r o m the families S c o m b e r e s o c i d a e a n d S c o m b r i d a e a n d in lesser a m o u n t s in o t h e r p e l a g i c fish species. A v a r i e t y of h i s t a m i n e - p r o d u c i n g b a c t e r i a has b e e n i s o l a t e d f r o m s p o i l e d fish c o n t a i n i n g high levels of histamine. N e a r l y all the o r g a n i s m s are G r a m - n e g a t i v e r o d s b e l o n g i n g to the family E n t e r o b a c t e r i a c e a e ( F r a n k , 1985). H o w e v e r as p o i n t e d o u t b y T a y l o r (1986) o n l y Morganella morganii, Enterobacter aerogenes a n d selected strains of Klebsiella pneumoniae are c a p a b l e of the r a p i d p r o d u c t i o n of large q u a n t i t i e s of histamine. T h e h i s t i d i n e - d e c a r b o x y l a t i n g b a c t e r i a r e s p o n s i b l e a r e difficult to d e t e c t b e c a u s e t h e y n o r m a l l y c o m p r i s e a m i n o r i t y o f the b a c t e r i a l f l o r a o n fresh fish ( O k u z u m i et
Correspondence address: N.K. Klausen, Technological Laboratory, Ministry of Fisheries, Building 221, Technical University, DK-2800 Lyngby, Denmark. 0168-1605/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)
138 al., 1984). Niven et al. (1981) devised a medium for the quantitative detection of histidine decarboxylating bacteria based on the colour change of bromocresol purple in response to a pH-shift in the medium. This pH-shift is dependent upon the production of the more alkaline histamine from histidine included in the agar. However in a review, Baranowski (1985) discusses a number of problems related to the use of Niven's medium. One is the low p H (5.3) which may inhibit the growth of some histamine-producers (Yoshinaga and Frank, 1982). Another is the possibility of false positive results. The basis for the technique being solely a pH-change which brings about a colour reaction, gives this medium a low specificity due to possible production of other alkaline bacterial products. The same objection applies to other media based on the same principle such as the Decarboxylase Medium Base (Difco). Baranowski (1985) mentions another screening method based on gas production by decarboxylating bacteria. This method may give false positive results, as there is no assurance that the gas produced is a result of histidine decarboxylase activity. Yamani and Untermann (1985) developed a liquid histidine decarboxylase medium to detect histidine decarboxylase activity in both pure and mixed cultures. A positive reaction in this medium is again based on a shift in pH from 5.3 to at least 5.6. The present work describes a new method for the detection of histamine-producing bacteria using a modification of the histidine decarboxylase media and automated conductance measurements for the detection of activity.
Materials and Methods
Bacterial strains A strain of M. morganii was supplied by the Institute of Veterinary Microbiology and Hygiene, Royal Veterinary and Agricultural University, Denmark. M. rnorganii is known as a potent histamine-producing bacterium (Behling and Taylor, 1982; Arnold et al., 1980), and the strain used has previously proved to be a histamine producer. Two isolates, originating from spoiled fish, and unable to produce histamine were used in the mixed culture studies. They were Gram-negative, cytochrome-oxidase positive and catalase positive, motile rods. One of the isolates (tentatively identified as a Pseudomonas sp.) had an oxidative glucose metabolism and was unable to reduce trimethylamine oxide or to produce hydrogen sulfide. The other isolate (tentatively identified as Alteromonas putrefaciens) could reduce trimethylamine oxide and produce hydrogen sulphide but was unable to metabolize glucose.
Media Histidine decarboxylase broth (HDB) was prepared from 2 g peptone (Difco), 1 g Lab-Lemco (Oxoid), 5 g NaC1, 8.1 g L-histidine (Fluka 53320) and 5 mg pyridoxal •
139 HCI (Sigma P-9130) in 1 1 of distilled water, and the pH was adjusted to 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 or 7.5 with 1 M HCI or 1 M NaOH. HDB was tested as a medium for the detection of histamine-producing bacteria. Decarboxylase control broth (DCB) was prepared as HDB without L-histidine. Trypticase broth (TB, pH 7.0) was prepared from 30 g trypticase (BBL) in 1 1 water. It was used as a growth medium for bacterial cultures. TB supplemented with 1% g-histidine (Fluka 53320) (TBH) was tested as a medium for the detection of histamine-producing bacteria. Iron agar (IA) was prepared from 3 g Lab-Lemco (Oxoid), 3 g yeast extract (Difco), 20 g peptone (Difco), 5 g NaC1, 0.3 g ferricitrate (BDH 28381), 0.3 g sodium thiosulphate, and 12 g agar (Difco) in 1 1 of water, and the p H was adjusted to 7.4 with HC1 (Jensen and Schulz, 1980). Peptone physiological saline (PPS) was prepared from 9 g NaC1 and 1 g peptone (Difco) in 1 1 of water and was used for preparing decimal dilutions except for bacterial cultures for inoculation into the Malthus test tubes where TB was used. All media were autoclaved (121 ° C, 15 min) before use. Determination of aerobic bacterial count
Decimal dilutions were prepared in PPS and counts were done using pour plates of IA incubated at 25 ° C for 3 days. Characterisation of bacterial isolates
Gram reaction was tested by the KOH-method (Gregersen, 1978). Cell morphology and motility were determined using phase-contrast microscopy of actively growing cultures in TB. Cytochrome oxidase was tested as described by Kovacs (1956) and catalase by suspending cell material in 3% hydrogen peroxide. Glucose metabolism was investigated by the O / F - t e s t of Hugh and Leifson (1953). Histamine producing isolates were further characterized by the (Enterotube) II Rochesystem supplied by F. Hoffmann-La Roche and Co. Ltd., Basel, Switzerland. Conductance measurements
The conductance measurements were carried out in a 64 channel Malthus Growth Analyzer with Exorset 100 computer (Malthus Instruments Ltd., Stoke-onTrent, U.K.). 9 ml of the various media as indicated were inoculated with 1 ml of sample in a test tube and two platinum electrodes fitted to a screw cap were placed in the medium and connected to the Malthus apparatus. The conductance was measured automatically at 12 min intervals during incubation in a water bath at 25°C. In the graphs the conductance changes are presented as a function of incubation time. All conductance measurements were carried out in duplicate but only the results of one of the duplicates are shown, except in Fig. 4. The results in Fig. 4 are representative of the reproducibility of the conductance assays.
Histamine analys& Determination of the content of histamine in H D B after incubation was carried out following the addition of 400 mg sulphosalicylic acid dihydrate (Merck) to the tubes containing 10 ml of medium and centrifugation at 17000 x g at 4 ° C for 20 rain. The content of histamine in the supernatant was determined in a K o n t r o n Automatic Aminoacid Analyzer, using post-column derivatisation with o-phthaldehyde (Sigma P-1378) and fluorometric detection as described by Klausen and Lund (1986).
Pure culture studies TB was inoculated with M. morganii and incubated at 25 ° C for 24 h. Decimal dilutions were prepared in TB and added to HDB, DCB, T B H and TB (9 ml medium + I ml sample). The conductance values were measured during incubation at 25 ° C for 24 h. The content of histamine in the H D B samples after incubation was determined. The effect of p H was studied by adding 1 ml of a 10 -1 dilution of a 24 h culture of M. morganii in TCB to 9 ml of H D B with initial p H values of 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 or 7.5. Conductances were measured during incubation at 25 ° C for 24 h.
Mixed culture studies TB were inoculated with M. morganii and the isolates tentatively identified as Pseudomonas sp. or A. putrefaciens in separate tubes and incubated at 25 ° C for 24 h. Decimal dilutions of all cultures were prepared in TB and mixed as described in the results and added to H D B (9 ml medium + 1 ml sample). Conductances were measured during incubation at 2 5 ° C for 24 h. Bacterial counts of the individual cultures before use and after incubation were made to establish whether the non-histamine producers had grown in HDB. With the medium used (IA) it was possible to distinguish between the various bacteria used in the experiment.
Isolation of histamine producing organisms from spoiled mackerel Mackerel caught in the N o r t h Sea were iced in boxes and transported to the laboratory where they were filleted, vacuum packed and frozen ( - 24 ° C) within 24 h. When required, the fish were thawed, repacked in 300 g portions in air-permeable plastic bags and stored at 0 o C, 10 ° C and 25 ° C until spoilage was well advanced. Duplicate samples were taken from these fish and decimal dilutions were prepared in PPS, spread on IA-plates, and added to H D B (9 ml medium + 1 ml sample). The conductances were measured during incubation at 2 5 ° C for 24 h. R a n d o m l y selected isolates taken from the IA-plates with 10-100 colonies (10 .7 dilution) were inoculated into TB and incubated at 25 o C for 24 h. Each isolate was added to H D B in test tubes (9 ml medium + 1 ml sample) and conductances were measured during incubation at 25 ° C for 24 h.
Pure culture studies The conductance measurements in HDB, DCB, T B H and TB, all inoculated with 1 ml of a 10 - t or 10 -2 dilution of a 24 h broth culture of M. morganii are presented in Figs. 1 and 2. In DCB only a small increase in the conductance was seen during the incubation period, while M. morganii in H D B gave curves with a large and regular increase in the conductance. This indicates the possibility of differentiating between conductance changes caused by histamine production and other metabolic activity in the media and it was possible in H D B to determine the time when a significant increase in the conductance occurred. This point is defined as the detection time (DT) and is determined using the automated routine in the Malthus apparatus as described by Gibson (1985). The increases in conductance in T B H during the incubation period were comparable with those seen in H D B (Fig. 2). However, the increases in the conductance in TB, which can be regarded as background noise, were pronounced and they could be difficult to differentiate from the conductance increase caused by weak histamine production. The histamine content in H D B after growth of different inocula (decimal dilutions of M. morganiL 2 5 ° C for 24 h) presented as a function of the measured increase in conductance in Fig. 3. There was a good correlation (r = 0.998) between the content of histamine and the increase in conductance in the medium. The conductance measurements in H D B with different p H values and inoculated with M. morganii are presented in Fig. 4. H D B with p H 5.5-6.0 gives the most suitable curves, because the increase in conductance is pronounced with a short and well-defined detection time (DT). At lower p H (4.5-5.0) longer D T are obtained
t i l t DCB HDB DCB HOB - 1 - 1 - 2 - 2
Fig. 1. Conductance changes at 25 o C in histidine decarboxylase broth (HDB) and decarboxylase control broth (DCB) inoculated with a 10-1 and 10-2 dilution of a 24 h culture of M. morganii. Arrows indicate detection time (DT),
T B H TB
Fig. 2. Conductance changes at 25°C in trypticase casein broth (TCB) and TCB supplemented with histidine (TCBH) inoculated with a 10 -1 or 10 -2 dilution of a 24 h culture of M. rnorganii. Arrows indicate detection time (DT).
and at higher pH (6.5-7.5) the curves become flatter and the DT-determinations more uncertain. Mixed culture studies The conductance measurements in HDB (9 ml) inoculated with M. morganii (1 ml TB containing 10 3.6 cfu) mixed with different levels of the isolate tentatively
Fig. 3. Relation between conductance change and content of histamine after 24 h at 25 ° C in histidine decarboxylase broth (HDB) inoculated with various numbers of M. morganii.
Fig. 4. Conductance changes at 25 °C in histidine decarboxylase broth (HDB) adjusted to various pH values (duplicates) and inoculated with M. morganii107.8cfu/ml). identified as Pseudomonas sp. (0, 10 4, 10 5 or 106 cfu) are presented in Fig. 5, which also shows the c o n d u c t a n c e measurements in H D B inoculated with the last mentioned (108 cfu) alone. The changes in c o n d u c t a n c e in H D B inoculated with of M. morganii were identical and unaffected by the presence of the n o n - h i s t a m i n e p r o d u c i n g bacteria. N o changes in c o n d u c t a n c e were seen in H D B inoculated with the n o n - h i s t a m i n e - p r o d u c i n g bacteria alone. Similar results were obtained with different inocula of M. morganii ( 1 0 2 6 - 1 0 5 6 cfu) and in studies with M. morganii mixed with various n u m b e r s of the isolate tentatively identified as A. putrefaciens. Both of the non-histamine-producing bacteria grew well in H D B and final counts of 106-108 per ml were obtained.
Isolation of histamine producing organisms from spoiled mackerel N o change in c o n d u c t a n c e was noted in samples f r o m unspoiled fish and fish spoiled at 0 ° C (Table I), indicating that only small n u m b e r s ( < 1 0 2 / g ) of hista-
M morgan~r ~~Pseu~omonas sD. +
Fig. 5. Conductance change at 25 °C in histidine decarboxylase broth (HDB) (9 ml) inoculated with M. morganii (1 ml TB containing 103.6 cfu) and various numbers of the isolate tentatively identified as a Pseudornonas sp. (0, 104, 105 or 106 cfu) or with this organism alone (10 s cfu).
144 TABLE 1 Detection times (hours) determined by conductance measurements at 25 ° C in histidine decarboxylase broth (HDB) inoculated with 10-1 dilution of mackerel samples stored at various temperatures. Storage
Storage time (days)
temp" ( ° C )
SP * ND
* no change in conductance in HDB after 24 h. SP, fish spoiled. NT, not tested.
_,oo Incubation time
Fig. 6. Conductance changes at 25 ° C in histidine decarboxylase broth (HDB) inoculated with a 10 ~, 10 -3, 10 - s or 10-7 dilution of mackerel spoiled at 25 ° C for 48 h. Arrows indicate detection time (DT).
pS -200 100 Incubation time
Fig. 7. Examples of conductance changes at 25 ° C in histidine decarboxylase broth (HDB) inoculated with 1 ml 24 h broth culture of isolates from mackerel spoiled at 25 ° C.
145 mine-producing bacteria were present. Samples stored for 3 days or more at 10 ° C and for 2 days at 25 ° C were obviously spoiled. All these samples caused increase in conductance of H D B indicating the presence of histamine producing bacteria. The sample stored for 2 days at 25 ° C had a D T of 3 h for the 10-1 dilution as shown in Fig. 6. This indicated the presence of approximately 108-109 histamine producing organisms/g fish by extrapolating results presented in Fig. 1. Out of twenty five randomly selected isolates from IA plates incubated at 25 ° C for 3 days twenty isolates were able to produce a specific change in conductance of the medium as shown in the examples included in Fig. 7. Eight of these organisms were randomly selected and biochemically characterised. All were Gram-negative, catalase-positive, oxidase-negative, fermentative motile rods and, according to the (Enterotube) II Roche system, 3 isolates could be identified as M. morganii/Proteus mirabilis and 5 isolates as Enterobacter agglomerans. All were able to produce large amounts of histamine (2500-3400 ppm) during growth in HDB (24 h, 25 o C). Discussion
There is a need in microbiology today for rapid automated methods to be used in modern food quality assurance programmes. Methods based on conductance measurements have been developed for determinations of total bacterial counts (Firstenberg-Eden and Eden, 1984), and the same principle has been found useful in the estimation of bacteriological quality of fish (Gibson et al., 1984; Ogden, 1986; Huss et al., 1987) and for detection of Salmonella (Easter and Gibson, 1985). The present investigation has shown that histamine-producing bacteria can be detected within 24 h by means of conductance measurements. This is the case even when these organisms are present in low numbers only (102-103/g) and in mixed cultures. Some of the criticism of and reservations about existing methods for screening histidine decarboxylase activity are eliminated using conductance measurements. By comparative studies of the same substrate with and without added L-histidine it appears that the method is rather specific. Thus the risk of false positive results is diminished significantly. The present investigation has confirmed that a medium of low pH (5.5) is optimal for measuring the decarboxylase activity of M. rnorganii. However a clear and distinct change in conductance is also recorded if the pH of the medium is increased to 7.0. It is suggested therefore that screening of unknown samples for histidine decarboxylase activity should be carried out at pH 6.5-7.0. This would eliminate the risk of any growth inhibition of the organisms of interest and thus reduce the risk of false negative results. The Enterobacteriaceae isolated in this study produced only half the amounts of histamine as our test culture M. morganii. Considering the studies of psychrophilic luminous bacteria isolated from scombroid fish (Moril et al., 1986) showing that they produced only negligible amounts of histamine we concluded that at any temperature when growth of Enterobacteriaceae is possible (i.e. at 10 o C) they are likely to be significant, and probably the major, histamine producers in mackerel.
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