Development of a real-time PCR method coupled with a selective pre-enrichment step for quantification of Morganella morganii and Morganella psychrotolerans in fish products.

Histamine fish poisoning is common and due to toxic concentrations of histamine often produced by Gram-negative bacteria in fin-fish products with a high content of the free amino acid histidine. The genus Morganella includes two species previously reported to cause incidents of histamine fish poisoning. Morganella morganii and Morganella psychrotolerans are both strong producer of histamine. However, little is known about the occurrence and critical stages for fish contamination with these bacteria. To elucidate contamination routes of Morganella, specific real-time quantitative PCR (RTi qPCR) methods for quantification of M. morganii and M. psychrotolerans have been developed. Selective primers amplified a 110 bp region of the vasD gene for M. psychrotolerans and a 171 bp region of the galactokinase gene for M. morganii. These primer-sets showed high specificity as demonstrated by using purified DNA from 23 other histamine producing bacteria and 26 isolates with no or limited histamine production. The efficiency of the qPCR reactions on artificially contaminated fish samples were 100.8% and 96.3% respectively. The limit of quantification (LOQ) without enrichment was 4 log CFU/g. A quantitative enrichment step with a selective medium was included and improved the sensitivity of the methods to a LOQ of below 50 CFU/g in seafood. RTi qPCR methods with or without enrichment were evaluated for enumeration of Morganella species in naturally contaminated fresh fish and lightly preserved seafood from Denmark. These new methods will contribute to a better understanding of the occurrence and histamine production by Morganella species in fish products, information that is essential to reduce the unacceptably high frequency of histamine fish poisoning.

Growth, inactivation and histamine formation of Morganella psychrotolerans and Morganella morganii - development and evaluation of predictive models.

Mathematical models for growth, heat inactivation and histamine formation by Morganella psychrotolerans and Morganella morganii were studied to evaluate the importance of these bacteria in seafood. Curves for growth and histamine formation by M. psychrotolerans in broth and seafood were generated at constant and changing storage temperatures (n=12). Observed and predicted times to formation of 100, 500 and 2000 ppm histamine were used for evaluation of an existing M. psychrotolerans histamine formation model [Emborg, J., Dalgaard, P., 2008-this issue-this issue. Modelling and predicting the growth and histamine formation by Morganella psychrotolerans. International Journal of Food Microbiology. doi:10.1016/j.ijfoodmicro.2008.08.016] Growth rates for M. psychrotolerans and M. morganii were determined at different constant temperatures from 0 degrees C to 42.5 degrees C whereas heat inactivation was studied between 37.5 degrees C and 60 degrees C. A M. morganii growth and histamine formation model was developed by combining these new data (growth rate model) and data from the existing literature (maximum population density and yield factor for histamine formation). The developed M. morganii model was evaluated by comparison of predicted growth and histamine formation with data from the existing literature. Observed and predicted growth rates for M. psychrotolerans, at constant temperatures, were similar with bias- and accuracy factor values of 1.15 and 1.45, respectively (n=11). On average times to formation of critical concentrations of histamine by M. psychrotolerans were acceptably predicted but the model was not highly accurate. Nevertheless, predictions seemed useful to support decisions concerning safe shelf-life in relation to formulation, storage and distribution of chilled seafood. Parameters for the effect of temperature on growth and inactivation of M. psychrotolerans and M. morganii differed markedly with Tmin of -8.3 to -5.9 degrees C vs. 0.3 to 2.8 degrees C, Topt of 26.0 to 27.0 degrees C vs. 35.9 to 37.2 degrees C and Tmax 32.0 to 33.3 degrees C vs. 44.0 to 47.4 degrees C, D(50 degrees C) of 5.3 min vs. 13.1 min and z-values of 6.8 degrees C and 7.2 degrees C. At temperatures above approximately 15 degrees C M. morganii grew faster than M. psychrotolerans. Bias- and accuracy factor-values of 1.41 and 2.44 (n=93) showed the predicted growth of M. morganii to be faster than previously observed in fresh fish and broth. In agreement with this, predicted times to formation of critical histamine concentrations by M. morganii were on average shorter than observed in fresh fish. A combined model was suggested to predict histamine formation by both psychrotolerant and mesophilic Morganella during storage of fresh fish between 0 degrees C and 37 degrees C.

Modelling the effect of temperature, carbon dioxide, water activity and pH on growth and histamine formation by Morganella psychrotolerans.

A mathematical model was developed to predict growth and histamine formation by Morganella psychrotolerans depending on temperature (0-20 degrees C), atmosphere (0-100% CO2), NaCl (0.0-6.0%) and pH (5.4-6.5). Data from experiments with both sterile tuna meat and Luria Bertani broth was used to develop the mathematical growth and histamine formation model. The expanded Logistic model with a growth dampening parameter (m) of 0.7 was used as primary growth model. A primary model for histamine formation during storage was obtained by combining the expanded Logistic growth model with a yield factor (YHis/CFU). 120 maximum specific growth rate (micromax)-values were generated for M. psychrotolerans and used to model the combined effect of the studied environmental parameters. A simple cardinal parameter type secondary model was used to model the effect of the four parameters on micro(max). The maximum population density (log Nmax) was correlated with log (YHis/CFU) and a simple constrained polynomial (quadratic) secondary model was developed for the effect of the environmental conditions on these model parameters. The developed model describes the effect of initial cell concentrations, storage conditions and product characteristics on histamine formation. This is a significant progress compared to previously available models for the effect of storage temperature only.

Morganella psychrotolerans sp. nov., a histamine-producing bacterium isolated from various seafoods.

Mesophilic Morganella morganii (n=6) and psychrotolerant M. morganii-like isolates from various seafoods (n=13), as well as clinical M. morganii isolates (n=3), were characterized by using a polyphasic approach including multi-locus sequencing. Based on the phylogenetic analysis, the 22 strains were divided into two distinct groups comprising mesophilic and psychrotolerant isolates, respectively. This classification was supported by DNA-DNA hybridization studies, whereby a psychrotolerant isolate (strain U2/3(T)) showed 41.0 and 17.8 % relatedness to the type strains of the mesophilic species Morganella morganii subsp. morganii (strain LMG 7874(T)) and Morganella morganii subsp. sibonii (strain DSM 14850(T)), respectively. Analysis of the 16S rRNA gene sequences showed a similarity of 98.6 % between mesophilic and psychrotolerant isolates. However, fragments of seven protein-encoding housekeeping genes (atpD, dnaN, gyrB, hdc, infB, rpoB and tuf) all showed less than 90.9 % sequence similarity between the two groups. The psychrotolerant isolates grew at 0-2 degrees C and also differed from the mesophilic M. morganii isolates with respect to growth at 37 degrees C and in 8.5 % (w/v) NaCl and fermentation of d-galactose. The psychrotolerant strains appear to represent a novel species, for which the name Morganella psychrotolerans sp. nov. is proposed. The type strain is U2/3(T) (=LMG 23374(T)=DSM 17886(T)).

Formation of histamine and biogenic amines in cold-smoked tuna: an investigation of psychrotolerant bacteria from samples implicated in cases of histamine fish poisoning.

Two outbreaks and a single case of histamine fish poisoning associated with cold-smoked tuna (CST) were reported in Denmark during 2004. The bacteria most likely responsible for histamine formation in CST implicated in histamine fish poisoning was identified for the first time in this study. Product characteristics and profiles of biogenic amines in the implicated products were also recorded. In the single poisoning case, psychrotolerant Morganella morganii-like bacteria most likely was responsible for the histamine production in CST with 2.2% +/- 0.6% NaCl in the water phase (WPS). In outbreak 1, Photobacterium phosphoreum most likely formed the histamine in CST with 1.3% +/- 0.1% WPS. In outbreak 2, which involved 10 persons, the bacteria responsible for histamine formation could not be determined. The measured concentrations of WPS were very low compared with those of randomly collected commercial samples of CST and cold-smoked blue marlin (4.1 to 12.7% WPS). Challenge tests at 5 degrees C with psychrotolerant M. morganii and P. phosphoreum in CST with 4.4% WPS revealed growth and toxic histamine formation by the psychrotolerant M. morganii-like bacteria but not by P. phosphoreum. In a storage trial with naturally contaminated CST containing 6.9% WPS, lactic acid bacteria dominated the microbiota, and no significant histamine formation was observed during the shelf life of about 40 days at 5 degrees C and of about 16 days at 10 degrees C. To prevent toxic histamine formation, CST should be produced with >5% WPS and distributed with a declared 5 degrees C shelf life of 3 to 4 weeks or less.

Significant histamine formation in tuna (Thunnus albacares) at 2 degrees C--effect of vacuum- and modified atmosphere-packaging on psychrotolerant bacteria.

Occurrence and importance of psychrotolerant histamine producing bacteria in chilled fresh tuna were demonstrated in the present study. The objective was to evaluate microbial formation of histamine and biogenic amines in chilled fresh tuna from the Indian Ocean and stored either vacuum-packed (VP) or modified atmosphere-packed (MAP). Firstly, biogenic amines and the dominating microbiota were determined in VP tuna involved in an outbreak of histamine fish poisoning in Denmark. Secondly, the microbiota of fresh MAP tuna was evaluated at the time of processing in Sri Lanka and chemical, microbial and sensory changes were evaluated during storage at 1-3 degrees C. To explain the results obtained with naturally contaminated tuna the effect of VP and MAP on biogenic amine formation by psychrotolerant bacteria was evaluated in challenge tests at 2 degrees C and 10 degrees C. The VP tuna that caused histamine fish poisoning had a histamine concentration of >7000 mg/kg and this high concentration was most likely produced by psychrotolerant Morganella morganii-like bacteria or by Photobacterium phosphoreum. Similar psychrotolerant M. morganii-like bacteria dominated the spoilage microbiota of fresh MAP tuna with 60% CO2/40% N2 and formed >5000 mg/kg of histamine after 24 days at 1.7 degrees C. These psychrotolerant bacteria were biochemically similar to M. morganii subsp. morganii and their 16S rDNA (1495 bp) showed >98% sequence similarity to the type strain of this species. Toxic concentrations of histamine were produced at 2.1 degrees C in inoculated VP tuna by both the psychrotolerant M. morganii-like bacteria (7400+/-1050 mg/kg) and P. phosphoreum (4250+/-2050 mg/kg). Interestingly, MAP with 40% CO2/60% O2, in challenge tests, had a strong inhibitory effect on growth and histamine formation by both the psychrotolerant M. morganii-like bacteria and P. phosphoreum. In agreement with this, no formation of histamine was found in naturally contaminated fresh MAP tuna with 40% CO2/60% O2 during 28 days of storage at 1.0 degrees C. To reduce current problems with histamine fish poisoning due to VP tuna it is suggested, for lean tuna loins, to replace vacuum packaging with MAP containing approximately 40% CO2 and approximately 60% O2.