A Sensitive Visual Method for the Detection of Hydrogen Sulfide Producing Bacteria

Summary

Here, we present a protocol to detect hydrogen sulfide producing bacteria with a modified protocol used for bismuth sulfide (BS) precipitation. The key advantages of this method are that it is easy to evaluate and does not require specialized equipment.

Abstract

Hydrogen sulfide (H₂S) is a toxic gas produced by bacteria in the proteolysis of sulfur-containing amino acids and proteins that plays an important role in human health. The H₂S production test is one of the important bacterial biochemical identification tests. The traditional methods are not only tedious and time-consuming but also prone to inhibition of bacterial growth due to the toxic effect of heavy metal salts in sulfur-containing medium, which often leads to negative results. Here, we established a simple and sensitive method to detect H₂S in bacteria. This method is a modified version of bismuth sulfide (BS) precipitation that uses 96-well transparent microtiter plates. Bacterial culture was combined with bismuth solution containing L-cysteine and cultivated for 20 min, at the end of which a black precipitate was observed.The visual detection limit for H₂S was 0.2 mM. Based on the visual color change, the simple, high-throughput, and rapid detection of H₂S producing bacteria can be achieved. In summary, this method can be used to identify H₂S production in bacteria.

Introduction

Hydrogen sulfide producing bacteria can utilize sulfur-containing amino acids and proteins to produce hydrogen sulfide (H₂S). The production of H₂S occurs usually in gram-negative Enterobacteriaceae family bacteria and also in members of Citrobacter spp., Proteus spp., Edwardsiella spp., and Shewanella spp.¹. These bacteria have the ability to reduce sulphate into hydrogen sulfide (H₂S) in order to obtain energy. Hydrogen sulfide has been implicated in the development of bacterial drug resistance. H₂S protects bacteria from the toxicity of reactive oxygen species (ROS), thus antagonizing the antibacterial effect of antibiotics^2,3. H₂S also has an important physiological effect in maintaining homeostasis. At supraphysiological levels, H₂S has been shown to be profoundly toxic to the body. In the human body, H₂S has another role as a gas signaling molecule that is involved in a variety of physiological and pathological processes. H₂S can regulate the systolic function of the heart and plays an important physiological role in relaxing blood vessels, inhibiting vascular remodeling, and protecting the myocardium^4,5. H₂S also plays an important role in regulating the nervous system and digestive tract^6,7. It has been found that, when exposed to bactericidal antibiotics, bacteria produce lethal reactive oxygen species (ROS) leading to cell death^8,9,10,11.

As a common biochemical test in microbiological laboratory courses, the hydrogen sulfide test is an important experiment in the identification of bacteria, especially bacteria of the family Enterobacteriaceae. At present, the hydrogen sulfide test is usually performed on a large number of sulfur-containing amino acids and lead acetate medium inoculated with the bacteria to be tested. After a period of incubation (2-3 days), the results are judged by observing whether the culture medium or lead acetate paper strip is blackened because of lead acetate production¹¹. However, these traditional methods are not only tedious and time-consuming but also prone to inhibition of bacterial growth due to the toxic effect of heavy metal salts in sulfur-containing medium, which often leads to negative results. A bismuth-based method has been established for the detection of H₂S^12,13. H₂S can react with bismuth, forming black bismuth sulfide precipitation. In order to conduct a reform for this biochemical test, a simple and quick method with no side effects on bacterial growth needs to be established. Here, we set up a simple method for the detection of hydrogen sulfide producing bacteria grown in an in vitro environment using bismuth sulfide as a substrate in a 96-well microtiter plate format.

Protocol

1. Bacterial strains

NOTE: For this experiment, nine standard strains were used, including Salmonella paratyphi A, Salmonella paratyphi B, Fusobacterium nucleatum, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa PAO1, Aeromonas hydrophila YJ-1, Proteus vuigaris, and Klebsiella pneumoniae (Table 1). Salmonella paratyphi A, Fusobacterium nucleatum, Pseudomonas aeruginosa, and Proteus vuigaris can produce H₂S, as outlined in previous literature¹.

1. Preparation of bacterial culture
   1. Transfer one bacterial colony of Salmonella paratyphi A, Salmonella paratyphi B, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa PAO1, Aeromonas hydrophila YJ-1, and Klebsiella pneumoniae from a Luria-Bertani (LB) agar plate to 100 mL of LB medium and culture at 37 °C for 12-16 h until the bacterial concentration is about 1 x 10⁹ cells/mL (as indicated by OD₆₀₀ = 1).
   2. Transfer one bacterial colony of Fusobacterium nucleatum and Proteus vuigaris from trypticase soy broth (TSB) agar plates to 100 mL of TSB medium and culture at 37 °C anaerobically for 24 h until the bacterial concentration is about 1 x 10⁹ cells/mL (as indicated by OD₆₀₀ = 1).

2. H₂S detection assay

1. Hydrogen sulfide production test
   1. Mix 100 µL of bacterial culture with 100 µL of newly prepared bismuth solution (pH 8.0; 10 mM bismuth (III) chloride, 0.4 M triethanolamine-HCl, 20 mM pyridoxal 5-phosphate monohydrate, 20 mM EDTA, and 40 mM L-cysteine) in the 96-well microtiter plates and culture for 20 min at 37 °C. For each bacterial strain, perform the analysis in triplicate.
   2. After 20 min, check for color change. If the color of the solution changes from light yellow to black, this indicates that the bacteria is able to produce H₂S. Repeat this measurement 3x.
2. Sensitivity of the method
   1. Determine the sensitivity of the method using different concentrations of sodium hydrosulfide (NaHS): 2 mM, 1 mM, 0.8 mM, 0.6 mM, 0.4 mM, 0.2 mM, 0.1 mM, and 0 mM, mixed with BS solution ¹⁴.
   2. Determine the presence of HS⁻/S²⁻ by observing the formation of a black BS precipitate. Score the color of the wells using a visual scale from no color production (-) to darkest black color production (++++++).

Representative Results

Detection of hydrogen sulfide producing bacteria
The performance of the H₂S test was investigated using pure cultures of selected bacterial strains, as listed in Table 1. The results indicated that Salmonella paratyphi B, Fusobacterium nucleatum, Enterococcus faecalis, Pseudomonas aeruginosa, and Proteus vuigaris can produce H₂S with black BS precipitate, while Salmonella paratyphi A, Staphylococcus aureus, Aeromonas hydrophila, and Klebsiella pneumoniae failed to show any black precipitate. The most rapid H₂S production was seen for Fusobacterium nucleatum, reaching maximum color production (Figure 1).

Sensitivity of the method
The sensitivity was determined by mixing different concentrations of sodium hydrosulfide (NaHS) with BS solution. Visual inspection revealed that the color depth of the solution deepened with an increasing ion concentration of HS⁻/S²⁻ (Figure 2). The detection limit of H₂S for the method is 0.2 mM.

Figure 1: Detection of hydrogen sulfide producing bacteria. The production of H₂S in Fusobacterium nucleatum was detected by the black BS precipitate formation. Please click here to view a larger version of this figure.

Figure 2: Sensitivity of the bismuth sulfide (BS) method. The sensitivity of the BS method for detection of H₂S was recorded as no color production (-) to darkest black color production (+++++). From left to right, the NaHS concentrations are 2 mM, 1 mM, 0.8 mM, 0.6 mM, 0.4 mM, 0.2 mM, 0.1 mM, and 0 mM. Please click here to view a larger version of this figure.

Species		H2S production a
Salmonella paratyphi A		_
Salmonella paratyphi B		+++
Fusobacterium nucleatum		++++
Enterococcus faecalis		+
Staphylococcus aureus		_
Pseudomonas aeruginosa		++
Aeromonas hydrophila		_
Proteus vuigaris		+
Klebsiella pneumoniae		_

Table 1: Visual assessment of H₂S production. The production of hydrogen sulfide (H₂S) by different bacterial strains was measured using the visual method in a 96-well microtiter plate. a: Recorded as black bismuth sulfide (BS) precipitation from no color production (-) to black color production (+).

Discussion

The hydrogen sulfide production test is one of the conventional phenotypic tests for the identification and differentiation of bacterial strains. Many bacterial species can produce hydrogen sulfide in their natural environment, such as aquatic water. These bacterial species include Salmonella sp., Citrobacter sp., Proteus sp., Pseudomonas sp., some strains of Klebsiella sp., Escherichia coli, and some species of anaerobic Clostridia^15,16. However, the sensitivity of the traditional H₂S test method is low, and the method is time-consuming^17,18. The traditional H₂S test medium contains PbAc, which has toxic effects on the growth of bacteria, and the culture is puncture-inoculated into semi-solid agar. The oxygen content in the lower part of the medium is low, so the aerobic bacteria grow poorly. Therefore, it often leads to false-negative results.

In this method, we used bismuth (III) instead of lead or ferrous ions. When a bacterial isolate producing H₂S is exposed to bismuth (III) chloride, a substitution reaction takes place. In this reaction, the chloride ion and sulfide ion exchange positions, producing hydrogen chloric acid and bismuth (III) sulfide; this bismuth (III) sulfide product precipitates out of solution as a black solid. The color depth of the black precipitate can be used to some extent to determine the quantity of H₂S produced by the bacterial strain. Based on the BS precipitation and the high reproducibility, reliability, and simplicity, themethod shown in the study was established as a hydrogen sulfide test for the detection of H₂S producing bacteria . The critical step of this method is that the bismuth solution should be prepared fresh. Compared to the traditional method, there is no heavy metal salt toxicity in the growth of bacteria, and it can also save time for the detection of hydrogen sulfide producing aerobic bacteria.

In this paper, based on the reaction of bismuth (III) chloride and H₂S, which produced visualblack BS precipitation, a simple, sensitive, inexpensive, and high-throughput method for the detection of H₂S producing bacteria is shown. This method is useful for the rapid detection of contaminated samples.

Materials

Bismuth (III)chloride	Shanghai Macklin Biochemical Co., Ltd	7787-60-2
EDTA	Nanjing Chemical Reagent Co., Ltd	60-00-4
Enterococcus faecalis	ATCC	19433
Fusobacterium nucleatum	ATCC	25586
Klebsiella pneumoniae	ATCC	43816
L-cysteine	Amresco	52-90-4
Proteus vuigaris	CMCC	49027
Salmonella paratyphi A	CMCC	50001
Salmonella paratyphi B	CMCC	50094
Staphylococcus aureus	ATCC	25923
Triethanolamine-HCl	Shanghai Aladdin Biochemical Technology Co., Ltd.	637-39-8