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

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.     

Isolation and characterization of histamine-producing bacteria from fermented fish products

Histamine is mainly produced by microorganisms that are found in fermented foods, and is frequently involved in food poisoning. Two histamine-producing bacteria were isolated from fermented fish products, anchovy sauce, and sand lance sauce by using a histidine decarboxylating medium. The species were identified as Bacillus licheniformis A7 and B. coagulans SL5. Multiplex PCR analysis showed the presence of the conserved histidine decarboxylase (hdc) gene in the chromosome of these bacteria. B. licheniformis A7 and B. coagulans SL5 produced the maximum amount of histamine (22.3±3.5 and 15.1±1.5 mg/L, respectively). As such, they were determined to be potential histamine-producing bacteria among the tested cultures.     

Klebsiella pneumoniae Produces No Histamine: Raoultella planticola and Raoultella ornithinolytica Strains Are Histamine Producers

Histamine fish poisoning is caused by histamine-producing bacteria (HPB). Klebsiella pneumoniae and Klebsiella oxytoca are the best-known HPB in fish. However, 22 strains of HPB from fish first identified as K. pneumoniae or K. oxytoca by commercialized systems were later correctly identified as Raoultella planticola (formerly Klebsiella planticola) by additional tests. Similarly, five strains of Raoultella ornithinolytica (formerly Klebsiella ornithinolytica) were isolated from fish as new HPB. R. planticola and R. ornithinolytica strains were equal in their histamine-producing capabilities and were determined to possess the hdc genes, encoding histidine decarboxylase. On the other hand, a collection of 61 strains of K. pneumoniae and 18 strains of K. oxytoca produced no histamine.     

High Frequency of Histamine-Producing Bacteria in the Enological Environment and Instability of the Histidine Decarboxylase Production Phenotype

Lactic acid bacteria contribute to wine transformation during malolactic fermentation. They generally improve the sensorial properties of wine, but some strains produce histamine, a toxic substance that causes health issues. Histamine-producing strains belong to species of the genera Oenococcus, Lactobacillus, and Pediococcus. All carry an hdcA gene coding for a histidine decarboxylase that converts histidine into histamine. For this study, a method based on quantitative PCR and targeting hdcA was developed to enumerate these bacteria in wine. This method was efficient for determining populations of 1 to 10⁷ CFU per ml. An analysis of 264 samples collected from 116 wineries of the same region during malolactic fermentation revealed that these bacteria were present in almost all wines and at important levels, exceeding 10³ CFU per ml in 70% of the samples. Histamine occurred at an often important level in wines containing populations of the above-mentioned bacteria. Fifty-four colonies of histamine producers isolated from four wines were characterized at the genetic level. All were strains of Oenococcus oeni that grouped into eight strain types by randomly amplified polymorphic DNA analysis. Some strains were isolated from wines collected in distant wineries. Moreover, hdcA was detected on a large and possibly unstable plasmid in these strains of O. oeni. Taken together, the results suggest that the risk of histamine production exists in almost all wines and is important when the population of histamine-producing bacteria exceeds 10³ per ml. Strains of O. oeni producing histamine are frequent in wine during malolactic fermentation, but they may lose this capacity during subcultures in the laboratory.     

Klebsiella pneumoniae produces no histamine: Raoultella planticola and Raoultella ornithinolytica strains are histamine producers

Histamine fish poisoning is caused by histamine-producing bacteria (HPB). Klebsiella pneumoniae and Klebsiella oxytoca are the best-known HPB in fish. However, 22 strains of HPB from fish first identified as K. pneumoniae or K. oxytoca by commercialized systems were later correctly identified as Raoultella planticola (formerly Klebsiella planticola) by additional tests. Similarly, five strains of Raoultella ornithinolytica (formerly Klebsiella ornithinolytica) were isolated from fish as new HPB. R. planticola and R. ornithinolytica strains were equal in their histamine-producing capabilities and were determined to possess the hdc genes, encoding histidine decarboxylase. On the other hand, a collection of 61 strains of K. pneumoniae and 18 strains of K. oxytoca produced no histamine.     

Identification and histamine formation of Tetragenococcus isolated from Thai fermented food products

Forty-one tetrad-forming halophilic lactic acid bacteria were isolated from 7 kinds of fermented foods in Thailand. All the isolates were identified as the genus Tetragenococcus by their phenotypic characteristics. On the basis of 16S rRNA gene restriction analysis using MboI and AluI and 16S rRNA gene sequence analyses, 41 isolates could be divided into two groups (groups A and B). All 22 isolates in Group A were identified as T. halophilus. 16S rRNA gene sequences of the representative isolates, SP37-2 and KS87-1 exhibited 99.4–99.5 % similarity to that of T. halophilus ATCC 33315^T. Nineteen isolates in Group B were identified as T. muriaticus. 16S rRNA gene sequences of the representative isolates, KM1-5 and KS87-14, showed 99.0–99.6 % similarity to that of T. muriaticus JCM 10006^T. Histamine formation was determined by using HPLC and the histidine decarboxylase (hdc) gene of the newly isolated histamine-producing strain was partially sequenced. The strain KS87-14 prolifically formed histamine 10 times higher than the reported T. muriaticus JCM 10006^T. The positive detection of KS87-14 was achieved by using hdcA gene-specific primers JV16HC and JV17HC.     

Histamine production by Klebsiella pneumoniae and an incident of scombroid fish poisoning.

A histamine-producing strain of Klebsiella pneumoniae was isolated from a sample of tuna sashimi implicated in an outbreak of scombroid fish poisoning. None of the other nine gram-negative bacterial strains isolated from the tuna sashimi was capable of equivalent histamine production. Bacterial histamine production was monitored in a tuna fish infusion broth (TFIB), and the implicated K. pneumoniae was capable of producing 442 mg of histamine per 100 g of tuna in TFIB in 7 h under controlled incubation conditions. Only 12 of 50 other K. pneumoniae strains, representing 5 distinct biochemical types, which had been originally isolated from foods, were able to produce such levels of histamine in TFIB. No correlation was found between histamine production and other biochemical characteristics or antibiotic resistance. Of the 12 histamine-producing strains, 11 belonged to type 2, which is characterized as indole negative with positive reactions in the urea and Voges-Proskauer tests. However, only 50% of the type 2 strains examined produced high levels of histamine in TFIB. Additionally, the implicated K. pneumoniae strain and one other strain belonged to type 1, which is characterized by positive reactions in the indole, urea, and Voges-Proskauer tests.     

Multiplex PCR for colony direct detection of Gram-positive histamine- and tyramine-producing bacteria

Formation of biogenic amines (BA) may occur in fermented foods and beverages due to the amino acid decarboxylase activities of Gram-positive bacteria. These compounds may cause food poisoning and therefore could imply food exportation problems. A set of consensual primers based on histidine decarboxylase gene (hdc) sequences of different bacteria was designed for the detection of histamine-producing Gram-positive bacteria. A multiplex PCR based on these hdc primers and recently designed primers targeting the tyrosine decarboxylase (tyrdc) gene was created. A third set of primers targeting the 16S rRNA gene of eubacteria was also used as an internal control. This multiplex PCR was performed on extracted DNA as well as directly on cell colonies. The results obtained show that this new molecular tool allowed for the detection of Gram-positive histamine- and/or tyramine-producing bacteria. The use of this molecular tool for early and rapid detection of Gram-positive BA-producing bacteria is of interest in evaluating the potential of cultured indigenous strains to produce biogenic amines in a fermented food product as well as to validate the innocuity of potential starter strains in the food industry.     

Histamine production by Klebsiella pneumoniae and an incident of scombroid fish poisoning.

A histamine-producing strain of Klebsiella pneumoniae was isolated from a sample of tuna sashimi implicated in an outbreak of scombroid fish poisoning. None of the other nine gram-negative bacterial strains isolated from the tuna sashimi was capable of equivalent histamine production. Bacterial histamine production was monitored in a tuna fish infusion broth (TFIB), and the implicated K. pneumoniae was capable of producing 442 mg of histamine per 100 g of tuna in TFIB in 7 h under controlled incubation conditions. Only 12 of 50 other K. pneumoniae strains, representing 5 distinct biochemical types, which had been originally isolated from foods, were able to produce such levels of histamine in TFIB. No correlation was found between histamine production and other biochemical characteristics or antibiotic resistance. Of the 12 histamine-producing strains, 11 belonged to type 2, which is characterized as indole negative with positive reactions in the urea and Voges-Proskauer tests. However, only 50% of the type 2 strains examined produced high levels of histamine in TFIB. Additionally, the implicated K. pneumoniae strain and one other strain belonged to type 1, which is characterized by positive reactions in the indole, urea, and Voges-Proskauer tests.     

Histamine-Producing Pathway Encoded on an Unstable Plasmid in Lactobacillus hilgardii 0006

Histamine production from histidine in fermented food products by lactic acid bacteria results in food spoilage and is harmful to consumers. We have isolated a histamine-producing lactic acid bacterium, Lactobacillus hilgardii strain IOEB 0006, which could retain or lose the ability to produce histamine depending on culture conditions. The hdcA gene, coding for the histidine decarboxylase of L. hilgardii IOEB 0006, was located on an 80-kb plasmid that proved to be unstable. Sequencing of the hdcA locus disclosed a four-gene cluster encoding the histidine decarboxylase, a protein of unknown function, a histidyl-tRNA synthetase, and a protein, which we named HdcP, showing similarities to integral membrane transporters driving substrate/product exchange. The gene coding for HdcP was cloned downstream of a sequence specifying a histidine tag and expressed in Lactococcus lactis. The recombinant HdcP could drive the uptake of histidine into the cell and the exchange of histidine and histamine. The combination of HdcP and the histidine decarboxylase forms a typical bacterial decarboxylation pathway that may generate metabolic energy or be involved in the acid stress response. Analyses of sequences present in databases suggest that the other two proteins have dispensable functions. These results describe for the first time the genes encoding a histamine-producing pathway and provide clues to the parsimonious distribution and the instability of histamine-producing lactic acid bacteria.     

Histidine Decarboxylases and Their Role in Accumulation of Histamine in Tuna and Dried Saury

Histamine-producing bacteria (HPB) such as Photobacterium phosphoreum and Raoultella planticola possess histidine decarboxylase (HDC), which converts histidine into histamine. Histamine fish poisoning (HFP) is attributable to the ingestion of fish containing high levels of histamine produced by HPB. Because freezing greatly decreases the histamine-producing ability of HPB, especially of P. phosphoreum, it has been speculated that HFP is caused by HDC itself from HPB cells autolyzing during frozen storage, even when HPB survive frozen storage. Here we constructed recombinant HDCs of P. phosphoreum, Photobacterium damselae, R. planticola, and Morganella morganii and investigated the ability of HDCs to produce sufficient histamine to cause HFP. To elucidate the character of these HDCs, we examined the specific activity of each recombinant HDC at various temperatures, pH levels, and NaCl concentrations. Further, we also investigated the stability of each HDC under different conditions (in reaction buffer, tuna, and dried saury) at various temperatures. P. damselae HDC readily produced sufficient histamine to cause HFP in fish samples. We consider that if HDC is implicated as an independent cause of HFP in frozen-thawed fish, the most likely causative agent is HDC of P. damselae.     

Comparison of differential plating media and two chromatography techniques for the detection of histamine production in bacteria

The bacterial enzyme histidine decarboxylase (Hdc) catalyses the conversion of histidine into histamine. This amine is essential for the biosynthesis of iron chelators (siderophores) and is an important cause of food poisoning after consumption of fish contaminated with histamine-producing bacteria. In this work we compared different methods for detecting histamine secreted by different bacterial strains. The presence of histamine in the culture supernatant of Vibrio anguillarum, which produces Hdc and secretes the histamine-containing siderophore anguibactin, was detected by thin-layer chromatography. Similar results were obtained using the culture supernatant of the Acinetobacter baumannii 19606 prototype strain that secretes the histamine-containing siderophore acinetobactin. Conversely, histamine was not detected in the culture supernatant of an isogenic V. anguillarum Hdc mutant and the A. baumannii 8399 strain that secretes a catechol siderophore different from anguibactin and acinetobactin. These results were confirmed by capillary gas chromatography/mass spectrometry. However, all these strains tested positive for histamine secretion when cultured on differential plating media containing histidine and a pH indicator, which were specifically designed for the detection of histamine-producing bacteria. The pH increase of the medium surrounding the bacterial colonies was however drastically reduced when the histidine-containing medium was supplemented with peptone, beef extract, and glucose. The histidine-containing culture supernatants of the A. baumannii and V. anguillarum strains showed an increase of about two units of pH, turned purple upon the addition of cresol red, and contained high amounts of ammonia. Escherichia coli strains, which are Hdc negative and do not use histidine as a carbon, nitrogen, and energy source, gave negative results with the differential solid medium and produced only moderate amounts of ammonia when cultured in the presence of excess histidine. This study demonstrates that, although more laborious and requiring some expensive equipment, thin-layer and gas chromatography/mass spectrometry are more accurate than differential media for detecting bacterial histamine secretion. The results obtained with these analytical methods are not affected by byproducts such as ammonia, which are generated during the degradation of histidine and produce false positive results with the differential plating media.     

Histidine decarboxylases and their role in accumulation of histamine in tuna and dried saury

Histamine-producing bacteria (HPB) such as Photobacterium phosphoreum and Raoultella planticola possess histidine decarboxylase (HDC), which converts histidine into histamine. Histamine fish poisoning (HFP) is attributable to the ingestion of fish containing high levels of histamine produced by HPB. Because freezing greatly decreases the histamine-producing ability of HPB, especially of P. phosphoreum, it has been speculated that HFP is caused by HDC itself from HPB cells autolyzing during frozen storage, even when HPB survive frozen storage. Here we constructed recombinant HDCs of P. phosphoreum, Photobacterium damselae, R. planticola, and Morganella morganii and investigated the ability of HDCs to produce sufficient histamine to cause HFP. To elucidate the character of these HDCs, we examined the specific activity of each recombinant HDC at various temperatures, pH levels, and NaCl concentrations. Further, we also investigated the stability of each HDC under different conditions (in reaction buffer, tuna, and dried saury) at various temperatures. P. damselae HDC readily produced sufficient histamine to cause HFP in fish samples. We consider that if HDC is implicated as an independent cause of HFP in frozen-thawed fish, the most likely causative agent is HDC of P. damselae.     

Evaluation of three decarboxylating agar media to detect histamine and tyramine-producing bacteria in ripened sausages

Histidine- and tyrosine-decarboxylase activity of 175 strains of bacteria isolated from eight retail samples of Spanish ripened sausages was tested in three decarboxylating agars (Niven medium, Joosten and Northolt medium and modified decarboxylating agar of Maijala) and confirmed by an enzymic method (histamine) and thin-layer chromatography (tyramine). Enterobacteria and pseudomonads showed the highest percentage of positive responses to histamine and tyramine in the three decarboxylating agars, but only enterobacteria were subsequently confirmed as histamine-producing. Confirmed tyramine-producing strains were all identified as enterococci or lactic acid bacteria. The medium described by Joosten and Northolt was more sensitive and faster at detecting tyramine-producing micro-organisms. However, all three media failed to detect one histamine-positive strain of lactic acid bacteria used as a control.     

Which lactic acid bacteria are responsible for histamine production in wine?

AIMS: To quantify the ability of 136 lactic acid bacteria (LAB), isolated from wine, to produce histamine and to identify the bacteria responsible for histamine production in wine. METHODS AND RESULTS: A qualitative method based on pH changes in a plate assay was used to detect wine strains capable of producing high levels of histamine. Two quantitative, highly sensitive methods were used, an enzymatic method and HPLC, to quantify the histamine produced by LAB. Finally, an improved PCR test was carried out to detect the presence of histidine decarboxylase gene in these bacteria. The species exhibiting the highest frequency of histamine production is Oenococcus oeni. However, the concentration of histamine produced by this species is lower than that produced by strains belonging to species of Lactobacillus and Pediococcus. A correlation of 100% between presence of histidine decarboxylase gene and histamine production was observed. Wines containing histamine were analysed to isolate and characterize the LAB responsible for spoilage. CONCLUSIONS: Oenococcus was able to synthesize low concentrations of histamine in wines, while Pediococcus parvulus and Lactobacillus hilgardii have been detected as spoilage, high histamine-producing bacteria in wines. SIGNIFICANCE AND IMPACT OF THE STUDY: Information regarding histamine-producing LAB isolated from wines can contribute to prevent histamine formation during winemaking and storage.     

Histamine-producing pathway encoded on an unstable plasmid in Lactobacillus hilgardii 0006

Histamine production from histidine in fermented food products by lactic acid bacteria results in food spoilage and is harmful to consumers. We have isolated a histamine-producing lactic acid bacterium, Lactobacillus hilgardii strain IOEB 0006, which could retain or lose the ability to produce histamine depending on culture conditions. The hdcA gene, coding for the histidine decarboxylase of L. hilgardii IOEB 0006, was located on an 80-kb plasmid that proved to be unstable. Sequencing of the hdcA locus disclosed a four-gene cluster encoding the histidine decarboxylase, a protein of unknown function, a histidyl-tRNA synthetase, and a protein, which we named HdcP, showing similarities to integral membrane transporters driving substrate/product exchange. The gene coding for HdcP was cloned downstream of a sequence specifying a histidine tag and expressed in Lactococcus lactis. The recombinant HdcP could drive the uptake of histidine into the cell and the exchange of histidine and histamine. The combination of HdcP and the histidine decarboxylase forms a typical bacterial decarboxylation pathway that may generate metabolic energy or be involved in the acid stress response. Analyses of sequences present in databases suggest that the other two proteins have dispensable functions. These results describe for the first time the genes encoding a histamine-producing pathway and provide clues to the parsimonious distribution and the instability of histamine-producing lactic acid bacteria.     

Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp.

Aims:  The main aims of this study were to clone and express an outer membrane protein (OMP), OmpW, of Aeromonas hydrophila and to study its distribution in Aeromonas spp.
Methods and Results:  The gene encoding OmpW in A. hydrophila has been cloned and expressed in Escherichia coli . Primers were designed for amplification of full-length ompW gene and used for identification of this gene in different Aeromonas spp. Of the 42 Aeromonas strains tested, all the isolates were positive by polymerase chain reaction (PCR) except one strain of Aeromonas veronii biovar veronii (VTE338). None of the other gram-negative bacteria were positive by PCR with primers specific to ompW gene of A. hydrophila . Polyclonal antibodies were raised in rabbit against the purified recombinant protein and the reaction of these antibodies was confirmed by western blotting using the purified recombinant protein and 42 Aeromonas cultures grown at various salt concentrations.
Conclusions:  The ompW -based PCR method developed in this study was found to be 100% specific and 97% sensitive. Expression of OmpW protein of Aeromonas was found to be salt-dependant. Recombinant OmpW protein was found to be highly immunogenic in fish.
Significance and Impact of the Study:  To our knowledge, this is the first report on cloning and expression of OmpW protein of A. hydrophila . Full-length ompW gene amplification by PCR can be used for the detection of Aeromonas . Recombinant OmpW protein can be useful for vaccination of fish against Aeromonas spp.     

Development of PCR Primer Systems for Amplification of Nitrite Reductase Genes (nirK and nirS) To Detect Denitrifying Bacteria in Environmental Samples

A system was developed for the detection of denitrifying bacteria by the amplification of specific nitrite reductase gene fragments with PCR. Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirK and nirS) after comparative sequence analysis. Whenever amplification was tried with these primers, the known nir type of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of the nir type of five laboratory strains. The nirK gene could be amplified from Blastobacter denitrificans, Alcaligenes xylosoxidans, and Alcaligenes sp. (DSM 30128); the nirS gene was amplified from Alcaligenes eutrophus DSM 530 and from the denitrifying isolate IFAM 3698. For each of the two genes, at least one primer combination amplified successfully for all of the test strains. Specific amplification products were not obtained with nondenitrifying bacteria or with strains of the other nir type. The specificity of the amplified products was confirmed by subsequent sequencing. These results suggest the suitability of the method for the qualitative detection of denitrifying bacteria in environmental samples. This was shown by applying one generally amplifying primer combination for each nir gene developed in this study to total DNA preparations from aquatic habitats.