Cloning and sequencing of the histidine decarboxylase genes of gram-negative, histamine-producing bacteria and their application in detection and identification of these organisms in fish

Abstract

The use of molecular tools for early and rapid detection of gram-negative histamine-producing bacteria is important for preventing the accumulation of histamine in fish products. To date, no molecular detection or identification system for gram-negative histamine-producing bacteria has been developed. A molecular method that allows the rapid detection of gram-negative histamine producers by PCR and simultaneous differentiation by single-strand conformation polymorphism (SSCP) analysis using the amplification product of the histidine decarboxylase genes (hdc) was developed. A collection of 37 strains of histamine-producing bacteria (8 reference strains from culture collections and 29 isolates from fish) and 470 strains of non-histamine-producing bacteria isolated from fish were tested. Histamine production of bacteria was determined by paper chromatography and confirmed by high-performance liquid chromatography. Among 37 strains of histamine-producing bacteria, all histidine-decarboxylating gram-negative bacteria produced a PCR product, except for a strain of Citrobacter braakii. In contrast, none of the non-histamine-producing strains (470 strains) produced an amplification product. Specificity of the amplification was further confirmed by sequencing the 0.7-kbp amplification product. A phylogenetic tree of the isolates constructed using newly determined sequences of partial hdc was similar to the phylogenetic tree generated from 16S ribosomal DNA sequences. Histamine accumulation occurred when PCR amplification of hdc was positive in all of fish samples tested and the presence of powerful histamine producers was confirmed by subsequent SSCP identification. The potential application of the PCR-SSCP method as a rapid monitoring tool is discussed.

FIG. 1.

Alignment of partial HDC amino acid sequences of gram-negative histamine producers. Residues conserved in all sequences are shaded in black, and residues conserved in more than 50% of sequences are shaded in gray. The asterisk marks the lysine residue that binds PLP in the M. morganii holoenzyme (64).

FIG. 2.

Phylogenetic trees of 14 strains of gram-negative histamine producers based on 16S rRNA gene sequences (A) and partial HDC gene sequences (B). The trees were constructed by using the neighbor-joining method. The genetic distances were calculated using the Kimura's two-parameter method. The numbers on the nodes indicate the number of times (percentage) the species (shown on the right) grouped together in 1,000 bootstrap samples. Only values greater than 40% are shown.

FIG. 3.

Growth, histamine production, and detection of hdc by PCR for M. morganii (JCM1672^T) in histidine broth (A) and tuna homogenates inoculated with M. morganii at 30°C (B). Arrows indicate sampling times at which PCRs became positive for the first time. Symbols: ○, histamine; •, M. morganii, ▴, histamine producers.

FIG. 4.

SSCP band patterns of PCR products of the hdc amplified from reference strains (A) and fish samples held at 30°C (B). Identification was performed by comparing the band patterns of the fish samples with those of the reference strains (from species database, only those used for identification of the fish samples are shown in this figure). Lanes: m, 100-bp ladder size marker; a, E. amnigenus strain 23a06; b, M. morganii strain 4b19; c, M. morganii strain AP28; d, P. damselae strain 6a20; e, M. morganii strain 30a04; f, M. morganii strain 5c15. Fish samples correspond to the same samples as in Table 2. Lanes: m, 100-bp ladder size marker; 1, tuna 1 (16 h); 2, tuna 1 (20 h); 3, tuna 3 (16 h); 4, tuna 3 (20 h); 5, tuna 4 (16 h); 6, tuna 4 (20 h); 7, tuna 5 (16 h); 8, tuna 5 (20 h); 9, tuna 7 (20 h); 10, tuna 7 (24 h); 11, sardine 1 (12 h); 12, sardine 1 (16 h); 13, mackerel (20 h); 14, young yellowtail (24 h); 15, horse mackerel (16 h); 16, horse mackerel (20 h); 17, yellowtail (16 h); 18, yellowtail (20 h). Identical band patterns were indicated by arrows.