Every year hundreds of tons of sardines are discarded due to the presence of histamine, a biogenic amine with the potential to cause intoxication in humans, which is formed by the degradation of fish musculature by microbial agents. There are numerous studies identifying microbes that form histamine in fish meet by plating methods, however, to our knowledge, no study has demonstrated which bacterial group(s) are of greatest importance for the formation of histamine in sardines by NGS so far. Thus, this study aimed to characterize the bacterial microbiota associated with histamine-contaminated sardines using NGS, as an attempt to identify the main causes behind it and help the industry to develop strategies to avoid it.
The regulations of the Brazilian Ministry of Agriculture determines that batches of fresh fish with histamine concentration >100 ppm must be discarded. Therefore, this study separated samples in two groups: contaminated, batches with histamine ≥ 200ppm; and non-contaminated, batches free of histamine (< 1ppm). These batches were from the same supplier, fishing areas and fishing/processing dates. Sample collection took place at the company GDC S/A, located in Itajaí-SC, Brazil. Five samples (10g) of each batch group were collected and submitted to NGS (quintuplicate). The sequences were processed and grouped into operational taxonomic units (OTUs) for identification and, finally, used to determine the relative abundance and for alpha and beta diversity analyses. Generalized univariate linear models were used to identify differences in the OTUs relative abundances (p<0.05) between contaminated and non-contaminated samples.
Eighteen OTUs were identified in total. Alpha diversity analysis revealed that contaminated samples showed higher bacterial richness but lower diversity than non-contamined. Beta diversity analysis confirmed that both groups showed different microbial composition (Figure 1). Bacteria with optimal growth under mesophilia and commonly associated with fish decomposition, such as Shewanella ssp. and Photobacterium ssp., were more abundant in the contaminated samples than in the non-contaminated ones (p<0.05), while psychotropic bacteria unrelated to spoilage, such as Psychrobacter ssp. and Pseudoalteromonas ssp. had higher relative abundance in samples without histamine (p<0.05). Therefore, the analyses of the microbiota structure and composition of both samples suggest that samples with histamine concentration ≥ 200ppm was handled differently from those without histamine, probably the cold chain was not correctly applied from the time of fishing to freezing and sending to the slaughter plant.