Microbiological profile and prevalence of histamine-producing bacteria in fresh sardines stored at different temperatures

Background and Aim: The European pilchard (Sardina pilchardus) is an important fish species for the Moroccan economy in terms of production and export. Biogenic amine histamine is a metabolite produced in the flesh of some fish species after death due to the decarboxylation of free histidine by histaminogenic bacteria. Failure to control the histamine risk in European pilchard may lead to public health and socioeconomic issues. This study aimed to estimate the prevalence of histaminogenic bacteria in association with histamine levels and the growth of microflora in Moroccan sardines (European pilchard). Materials and Methods: We conducted the study by monitoring Moroccan sardines of histamine content and microbiological profile (aerobic plate count [APC], coliforms, and thermo-tolerant coliforms [TTC]) during 6 days of storage at three different temperatures (0°C, 10°C, and ambient temperature [22°C]). The histamine assay was performed using a spectrofluorometric method, and the microbiological identification of histamine-producing bacteria was performed using a combination of biochemical and molecular tests. Results: The histamine content in European pilchard stored at 0°C was negligible. However, high concentrations were observed at 10°C and 22°C. The microbiological profile showed a positive association between microflora counts and histamine content according to storage time. At 0°C, a moderate increase in the APC, a decrease in coliforms, and an absence of TTC were observed. The rapid proliferation of all microflora was observed at 10°C, whereas at 22°C, the proliferation was almost exponential. Bacterial identification revealed the exclusive presence of species belonging to the Enterobacteriaceae family at varying frequencies depending on storage temperature. Morganella morganii and Proteus mirabilis had the highest histamine induction rates in L-histidine-supplemented broth, with 1600 and 255 parts per million (ppm), respectively, after 48-h incubation at 35°C. Klebsiella ozaenae could produce 136 ppm and Serratia plymuthica 115 ppm. Reverse transcription polymerase chain reaction showed positive results for the presence of genes associated with histidine decarboxylase. The hdc genes of M. morganii, P. mirabilis, and K. ozaenae were successfully amplified and exhibited strong similarity with the reference gene of M. morganii. Conclusion: This study describes for the first time the hdc gene in bacteria that form histamine in Moroccan sardines. The results also confirm that respect for the cold chain integrity is a crucial factor in histamine management. This information should help stakeholders in the implementation of sound strategies for managing the hazards associated with seafood and their products.