Hydrogen sulfide (H2S)-producing bacteria (sulfate reducers, sulfite reducers, sulfur reducers, and other molecules with sulfur) : Importance in the deterioration of fish and meat products - Qualitative and quantitative culture; Molecular identification (PCR and sequencing)

Information 15-04-2018.

The hydrogen sulfide producing bacteria (H2S) comprise various groups of bacteria and archaea that obtain energy by reducing various compounds that possess sulfur in their molecule, including organic compounds (sulfur amino acids) and inorganic compounds with oxidized sulfur (such as sulfate, sulfite, thiosulfate, tetrathionates, or elemental sulfur) to H2S. Hydrogen sulfide is a colourless and toxic gas with a strong foul odor to rotten eggs. It is one of the compounds resulting from the decomposition of organic matter, so it is present in the environment. It is found in untreated air and water mainly as a result of natural emissions, and its concentration is increased due to industrial processes. H2S is particularly noticeable in some groundwater, depending on the rock mineralogy of origin and the microorganisms present. Drinking water, as well as a number of foods and beverages may contain sulfides. In food, H2S-producing bacteria stand out as specific bacteria of deterioration in some fish products and red meats, especially if they have been processed.

As mentioned previously, the bacteria producing H2S, constitute a broad and heterogeneous group, where the sulfate-reducing bacteria (SO42-) stand out, the main responsible for the formation of H2S in anaerobiosis. Frequently, the bacterial reducing sulphate activity is linked with the oxidation capacity of other oxidized sulfur compounds. However, this is not always the case, and in addition to these bacteria, sulfur-reducing bacteria (So), sulfite-reducing bacteria (SO32-) are known, and thiosulfate (SO2O32-) reducing non sulfate-reducing bacteria have been isolated. Next, the characteristics of the main types of bacteria are described.

Sulfate-reducing bacteria (SRB), are chemolithotropic bacteria that use sulfate as the final electron acceptor in the degradation of organic matter, a process called sulfate reduction, which results in the production of H2S. So far, more than 220 species of 60 genera of SRB have been described, belonging to five divisions or phyla within the bacteria (the spore formers Desufotomaculum, Desulfosporomusa and Desulfosporosinus within the division Firmicutes, Deltaproteobacteria; the Thermodesulfovibrio species within the division Nitrospira and two phyla represented by the species Thermodesulfobium narugense and Thermodesulfobacterium/Thermodesulfatator) and two divisions within the archaea (the genus Archaeoglobus of the phylum Euryarchaeota and the two genera Thermocladium and Caldivirga of the phylum Crenarchaeota, affiliated to the order Thermoproteales).

SRBs are strict anaerobic bacteria that use sulfate, the most oxidized form of sulfur, as the final acceptor of electrons, converting it into the most reduced form, sulfur. This non-assimilative reduction of sulfate is a large-scale process limited to SRBs. In addition to sulfate, SRBs can use other oxidized sulfur compounds as terminal electron acceptors, including sulphites and thiosulfate, reducing sulphate intermediates. In addition, some facultative sulfur-reducing bacteria, which use elemental sulfur as a respiratory substrate in the absence of other possible terminal electron acceptors such as sulfate, sulfite, thiosulfate, nitrite or nitrate, are also found among the SRBs. Thus, although most SRBs cannot grow by elemental sulfur reduction, some bacteria of the genera Desulfomicrobium and Desulfovibrio, use sulfur as an alternative electron acceptor.

SRB can have a heterotrophic, autotrophic, lithoautotrophic, or respiratory type metabolism under anaerobiosis. In addition, recently, the possible microaerophilic nature of some SRB species previously considered as strict anaerobes has been discovered. SRBs can employ more than one hundred compounds as electron donors (sugars, amino acids, monocarboxylic acids, dicarboxylic acids, alcohols and aromatics) and are the microorganisms that reduce the greatest number of different end-acceptor terminal electrons, including inorganic compounds of sulfur. Due to this, its ecological and metabolic function in nature is of great importance. SRBs are widely distributed in aquatic and terrestrial environments that become anoxic. They can grow in different physical-chemical conditions, living in the most extreme environments of our planet, such as saline, hot, cold and/or alkaline ecosystems. The best studied genus is Desulfovibrio, which is common in aquatic environments or in flooded soils with abundant organic matter and sufficient levels of sulphate.

Sulfur-reducing bacteria are characterized by their ability to reduce elemental sulfur to sulfide, a process called sulforeduction. Several genera of archaea and chemoorganotrophic bacteria possess the ability to oxidize organic substrates (mainly small peptides, glucose and starch) anaerobically, using S° as the final electron acceptor. In archaea, this process of anaerobic respiration is observed mainly in the genera Thermococcus and Thermoproteus, and to a lesser degree in the genera Desulfurococcus, Thermofilum and Pyrococcus. Also, another group of chemolithotrophic archaea of the genera Acidians, Pyrodictium and Thermoproteus demonstrate the ability to grow autotrophically at the expense of CO2, H2 and S°.

Among the eubacteria, the anaerobic respiration of S° is carried out by bacteria belonging to the genera Desulfuromonas, Desulfurella and Campylobacter. These bacteria couple the oxidation of substrates such as acetate and ethanol to the reduction of elemental sulfur to hydrogen sulfide. The ability to reduce elemental sulfur also extends to chemoreganotrophic facultative aerobic bacteria belonging to the genera Proteus, Pseudomonas and Salmonella, which also exhibit the ability to reduce sulfur compounds such as thiosulfate, sulfite and dimethylsulfoxide.

Sulfite-reducing anaerobes constitute a group associated with Clostridium spp. and as such they are characterized as Gram-positive organisms, anaerobic, spore-forming. They are found in various environmental sources, such as soil, marine sediments, decomposing vegetation, human and animal intestines, feces and infected wounds of humans and animals, surface waters, as well as in food, especially when the hygiene conditions in the preparation They are deficient. They are deteriorating, since they produce bad smells and, very often, blacken the product when it has iron, forming a dark precipitate of iron sulfide. These microorganisms have the ability to reduce sulfites to sulfides from amino acids and sulfur compounds. These bacteria have been proposed as indicators of high-risk water pollution. The most important advantage is that their spores survive in water much longer than the organisms of the coliform group and are resistant to disinfection, to the point that they can be detected in some water samples after having received pre-disinfection, flocculation, sedimentation, filtration and terminal disinfection. They also have value as indicators of fecal contamination of a recent nature in food.

The detection of H2S-producing bacteria in the laboratory is mainly based on the detection of the production of hydrogen sulphide in culture. To detect the different types of bacteria, by means of isolation in culture, the source of sulfur, the indicators of sulfides and the incubation conditions of the culture are adjusted. The different species of H2S producing microorganisms use different inorganic compounds or sulfur amino acids as sulfur sources (such as protein digests - peptone -, sulfur amino acids - cysteine ​​or methionine - and thiosulfate). In the market, there are many culture media with different sulfur compounds, where gaseous H2S produced by the reduction of an inorganic sulfur source such as thiosulfate is detected, or by the reduction of the organic sulfur as provided by the functional group R-SH of the amino acid cysteine ​​present in peptones. H2S production is detected when the gas comes into contact with certain metals, such as lead, iron or bismuth, and forms sulphides with these metals (black). Sulfur indicators vary between different media: peptonized iron, ferrous sulfate, ferrous or ferric ammonium sulfate, ferric citrate, sodium thiosulfate, bismuth sulfite or lead acetate. The iron salts are widely used for the detection of members of the Enterobacteriaceae family, while the procedures with lead acetate are the most sensitive for the detection of minute amounts of H2S in other bacteria that are not of the Enterobacteriaceae family.

Tests carried out in IVAMI:

  • Detection and counting of bacteria producing H2S by isolation by culture in selective aerobic media.
  • Molecular identification of isolated colonies in culture at the species level (PCR and sequencing).

Recommended sample:

  • Sample of suspect or controlled food. A minimum of about 100 g in a sterile polypropylene (non-rigid plastic) container is recommended.
  • Sample of the water suspected or controlled. A minimum of 200 mL is recommended, introduced in a sterile polypropylene (non-rigid plastic) container.

Conservation and shipment of the sample:

  • Refrigerated (preferred) for less than 48 hours.
  • Frozen: more than 48 hours.

Delivery of results:

  • Detection and counting by isolation in aerobic and anaerobic cultures: 4 or 5 days
  • Detection and counting by isolation in aerobic and anaerobic culture and molecular identification of species: 5 to 8 days.

Cost of the test: