H2S Positive Bacteria: Introduction, List, Detecting Tests and Media Used

Introduction of H2S Positive Bacteria

H2S positive bacteria Proteus vulgaris and Salmonella Typhi from left to right respectively as shown above picture. H₂S positive bacteria have the ability to produce hydrogen sulfide from substrates such as sulfur-containing amino acids or inorganic sulfur compounds. The sulfur is used as the final hydrogen acceptor leading to the formation of H₂S. Sulfur-containing amino acids ( e.g. methionine, cysteine, cysteine, or an inorganic compound such as sodium thiosulphate, etc.) should be present in the medium to detect the presence of H2S. It is being gassed, will escape from the test medium. Therefore, the indicators such as heavy metal ions are incorporated into the test medium, which supports the growth of bacteria. Organisms that are tested for the production of hydrogen sulfide must contain an enzyme system that releases sulfide from the sulfur source. Sulfides combine with the hydrogen ion to form H₂S and H₂Scombines with heavy metals to form insoluble black precipitate as shown above picture.

List of H2S Positive Bacteria

Proteus vulgaris
Proteus mirabilis
Citrobacter freundii
Salmonella species ( except Salmonella Paratyphi A)
Salmonella arizonae
Edwardsiella tarda
Edwardsiella hoshinae
Erysiphilothrix rhusiopathiae
Brucella abortus
Brucella suis
Campylobacter sputorum
Shewanella putrefaciens

List of Media Used for Detecting Production of H2S

TSI agar
SIM medium
Kligler iron agar
SS agar
XLD agar
DCA
Hekton enteric agar (HEA)
Lysine iron agar
Bismuth sulfite agar
Citrate sulfide agar
Lead acetate agar
Peptone water, lead acetate paper inserts: The lead acetate procedure is more sensitive than any other method for detecting H₂S production. It detects even traces of H₂S. H₂S is a colorless gas that on contact with lead acetate produces lead sulfide, a black precipitate, indicated by a visible black colored reaction on the Lead acetate paper strip.

Keynotes on H2S Positive Bacteria

Sources of sulfur are sulfite, peptone, and sodium thiosulfate.
Some H₂S indicators are ferrous sulfate, ferric ammonium citrate, lead acetate, and peptonized iron.
Sources of sulfur and H₂S indicators are different for each medium.

Introduction of TSI test

This single TSI test is useful for the following features-

To know the carbohydrate utilization ability of the organisms
Gas formation by the organisms
Hydrogen sulfide production by the bacteria

TSI stands for triple sugar iron and it is an agar medium recommended for use in the differentiation of Enterobacteriaceae by their ability to ferment glucose, lactose, and sucrose, and their ability to produce hydrogen sulfide.

Principle of TSI Test

Triple sugar iron agar contains casein and meat peptones, phenol red as the pH indicator, 0.1% glucose, 1% lactose, and 1% sucrose for fermentation. Ferric or ferrous ions and sodium thiosulphate are present to detect hydrogen sulfide production. Bacteria that are non-lactose fermenting initially produce a yellow slant due to the production of acid from the glucose. The small amount of glucose is rapidly depleted. Oxidation metabolism continues in the slant after the low concentration of glucose has been depleted, producing an alkaline pH from the aerobic breakdown of peptone; the slant turns red. There is no oxygen penetration into the butt and no oxidative metabolism; the butt remains acid and yellow. Thus, a non-lactose fermenting bacterium yields an alkaline (K) slant over an acid (A) butt ( K/A; red slant; yellow butt). Lactose/sucrose bacteria continue to produce a large amount of acid in the slant and in the butt so the reaction in both remains acid (A/A; yellow slant; yellow butt). If the slant and butt remain neutral, the organism is not capable of fermenting glucose or other sugars (K/K; red slant; red butt) bubble, fracturing, or displacement of the medium indicates gas production by the organism due to sugar fermentation. Blackening of the medium denotes hydrogen sulfide production by the action of the bacteria with sodium thiosulfate and which is detected by the reduction of the ferric ions to produce a black precipitate.

Requirements for TSI Test

Test organism
Triple sugar iron agar
Bunsen burner and inoculating needle
BOD incubator
Test tubes rack
QC strains for quality control

Test Procedure

Warm medium to room temperature and examines for cracks and do not use if cracks appear.
Touch the center of a well-isolated colony by using a sterile inoculating needle.
Stab to within 3-5 mm from the bottom of the tube.
Place cap loosely on tube
Incubate aerobically at 35-37°C for 18-24 hours.

Observation and Interpretation

Examine the reaction in the slant and the butt also observe for gas and hydrogen sulfide production.

Yellow: Acid reaction
Red: Alkaline reaction
Blackening of the medium: H₂S production
Bubbles, cracks, or displacement of the medium: Gas production

Interpretation of carbohydrate

A/A: Glucose and lactose or sucrose fermented
K/A: Only glucose fermented or non-lactose fermenter
K/K: No carbohydrate fermented or non-glucose fermenter

Result

Tube No 1

K/K
No H₂S production
No gas formation

Tube No 2

R/A
H₂S positive
No gas formation

Tube No 3

A/A
Gas formation positive
No H₂S formation

Limitations of TSI Test

Do not read the TSI test before 18 hours, since false readings of acid in the plant may result.
H₂S production may on be inhibited on the TSI test for organisms that utilize sucrose and suppress the enzyme mechanism that results in the production of H₂S.
Sulfide indole motility (SIM) agar is more sensitive in the detection of H₂Sthan either TSI or KIA.

Keynotes

Kligler’s Iron Agar(KIA) test only differs from the Triple Sugar Iron agar (TSI) test due to lacking sucrose in its composition.
Do not attempt to interpret sugar fermentation reactions after 24 hours. Refrigerate tubes if reading will be delayed.
If desired, extend incubation only to detect H₂Sproduction. Campylobacter may take 3 days for the production of H₂S.

Introduction of SIM test

SIM (Sulfide, Indole, Motility ) medium is useful for the differentiation of gram-negative enteric bacilli. SIM test helps to isolate the organisms on the basis of sulfide production, indole formation, and motility.

Principle of SIM Test

The medium having the constituents ferrous ammonium sulfate and sodium thiosulfate, which together serve as indicators for the production of hydrogen sulfide (H₂S). Hydrogen sulfide production detects when ferrous sulfide, a black precipitate, is produced as a result of ferrous ammonium sulfate reacting with hydrogen sulfide gas. Casein peptone of this medium is rich in tryptophan. Organisms having the enzyme tryptophanase degrade tryptophan to indole. Indole detection is achieved after the addition of Kovac’s reagent following incubation of the inoculated medium. Indole combines with p-dimethylaminobenzaldehyde and produces a red band at the top of the medium. A negative indole test produces no color change after the addition of Kovac’s reagent i.e. Yellow color of Kovac’s reagent. A lower concentration of agar added to the medium provides a semi-solid structure allowing for the detection of bacterial motility. Motile organisms diffuse from the stab line and produce turbidity or cloudiness throughout the medium. The growth of non-motile bacteria is restricted along the stab line and leaves the surrounding medium clear. Another constituent, animal tissue of this medium which provides amino acids and nutrients necessary for bacterial growth.

Composition of the Medium

Ingredients for 100 ml of distilled water-

Pancreatic Digest of Casein: 2.0gm

Peptic Digest of Animal Tissue: 0.61gm

Ferrous Ammonium Sulfate: 0.02gm

Sodium Thiosulfate: 0.02gm

Agar: 0.35gm

Final pH 7.3 +/- 0.2 at 25°C.

Requirements for SIM Test

Test organisms
SIM medium
Inoculating wire and
Bunsen burner
Incubator

Quality Control Strains

Escherichia coli ATCC 25922
Salmonella enterica ATCC 14028

Procedure of SIM Test

Take pure colonies from an 18-24-hour old culture on a solid medium.
Inoculate the SIM Medium by stabbing the center of the medium to a depth of half an inch.
Incubate the inoculated medium aerobically at 37°C for 18-24 hours.
Observe for hydrogen sulfide production and motility of test organism.
Only apply Kovac’s reagent (three drops ) after reading the result of H₂S and motility reaction to the surface of the medium.
Observe for the development of a pink to red color.

Result Interpretation of SIM Test

Positive H₂S test: blackening of the medium
A negative H₂S test: absence of blackening
Positive motility test: a diffuse zone of growth flaring from the line of inoculation
Negative motility test: restricted growth along the stab line
Indole positive test: a pink to red color ring is formed at the top of the medium after the addition of Kovac’s reagent
Indole negative test: A yellow color denotes a negative indole test after the addition of Kovac’s reagent
Escherichia coli ATCC 25922: Growth; Motility: positive, H₂S: negative, and Indole: positive (It turns pink after addition of Kovac’s reagent)
Salmonella enterica ATCC 14028: Growth; Motility: positive, H₂S: positive, and Indole: negative

Limitations of SIM Test

Caps should be loose during incubation otherwise erroneous results may occur
The inoculum should take from a solid medium because a liquid or broth suspension will delay the initiation of growth and may cause erroneous results.
When inoculating semi-solid media, it is important that the inoculating needle be removed along the exact same line used to inoculate the medium. A fanning motion may result in growth along the stab line that may result in a false-positive interpretation.
Take motility and hydrogen sulfide (H₂S) reaction results prior to the addition of Kovac’s reagent.
Weakly motile organisms or organisms that possess damaged flagella (due to heating, shaking, or other trauma) often result in false-negative motility tests, and therefore, motility results should be confirmed by performing a hanging drop motility test.
Some microorganisms like Yersinia enterocolitica demonstrates motility best at 25°C.
Obligate aerobes, such as Pseudomonas aeruginosa , will produce a spreading film on the surface of the medium and will not extend from the line of inoculation where oxygen is depleted.

Introduction of Salmonella-Shigella (SS) Agar

Salmonella-Shigella (SS) Agar is recommended for use as a selective and differential medium for the isolation of Salmonella and some Shigella species from clinical and non-clinical specimens( suspected foodstuffs).

Composition of Salmonella-Shigella (SS) Agar

(Himedia)
Ingredients Gms / Litre

Proteose peptone 5.0
Lactose 10.0
Bile salts mixture 8.5
Sodium citrate 8.5
Sodium thiosulphate 8.5
Ferric citrate 1.0
Brilliant green 0.00033
Neutral red 0.025
Agar 13.5
Distilled water: 1000 ml

Final pH ( at 25°C) 7.0±0.2

Principle of Salmonella-Shigella (SS) Agar

The basis for differentiation on Salmonella-Shigella (SS) agar depends on the fermentation of lactose and the absorption of neutral red as the bile salts precipitate in the acidic condition. Neutral red turns red in the presence of an acidic pH, thus showing fermentation has occurred. The inclusion of bile salts, sodium citrate, and brilliant green serve to inhibit gram-positive and coliform organisms. Salmonella, Shigella, and other non-lactose-fermenting organisms appear as transparent or translucent colorless colonies on SS Agar. Sodium thiosulfate is added to the medium as a hydrogen sulfide source, and ferric citrate is added as an indicator for hydrogen sulfide production.

Preparation of Salmonella-Shigella (SS) Agar

Suspend 60 grams of the powder of Salmonella-Shigella (SS) agar in 1 liter purified/distilled or deionized water.
Mix thoroughly and heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
Avoid overheating and do not autoclave.
Leave for cooling to 45-50°C.
Mix well before dispensing.
Pour into each plate and leave plates on the sterile surface until the agar has solidified.
Store the plates in a refrigerator at 2-8°C.

Storage and Shelf life of Salmonella-Shigella (SS) Agar

Store at 2-8ºC and away from direct light.
Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination.
The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.
Prepared culture media can be kept for at least a week in refrigeration.

Test Requirements for Salmonella-Shigella (SS) Agar

Test specimens
Salmonella-Shigella (SS) Agar plates
Inoculating loop
Bunsen burner
Incubator
Control strains ( For negative control-Enterococcus faecalis ATCC 29212, and Escherichia coli ATCC 25922 (Partial to complete inhibition) while positive control-Salmonella enterica subsp. enterica serotype Typhimurium ATCC 14028, Shigella flexneri 12022 ATCC )

Test procedure of Salmonella-Shigella (SS) Agar

Allow the plates to warm at room temperature, and the agar surface to dry before inoculating.
Take Salmonella-Shigella (SS) Agar plates.
Heavily inoculate and streak the specimen as soon as possible after collection. If the specimen to be cultured is on a swab, roll the swab over a small area of the agar surface. Streak for isolation with a sterile loop.
Incubate aerobically plates at 35 ± 2°C for 18-24 hours.

Result Interpretation of Salmonella-Shigella (SS) Agar

If lactose fermentation occurs, the medium will turn red due to the acidic pH. Salmonella, Shigella, and other non-lactose fermenters appear as transparent or translucent colorless colonies on SS Agar. Colonies of Salmonella spp. may appear with or without black centers (depending on the species isolated).

Colony characteristics of SS agar

Typical colonial morphology on Salmonella-Shigella Agar after inoculation of Inoculum having 50-100 CFU is as follows:

Salmonella (good-luxuriant growth): Colorless, usually with a black center
Shigella flexneri (good growth): Colorless
E.coli (fair growth): pink or red
Enterobacter/Klebsiella (fair growth) :Pink
Proteus(fair-good growth): Colorless, usually with a black center
Pseudomonas (fair growth): Irregular
Gram-positive bacteria (Staphylococcus, Micrococcus): No growth
Enterococcus faecalis : (none-poor growth): colorless

Uses of Salmonella-Shigella (SS) Agar

SS agar is used as a selective and differential medium for the isolation of Salmonella and some Shigella species from clinical as well as non-clinical specimens.
This medium is not recommended for the primary isolation of Shigella because of inhibitory to most strains.
SS agar was also developed to aid in the differentiation of lactose and non-lactose-fermenters from clinical specimens, suspected foodstuffs, and other such specimens.

Limitations of Salmonella-Shigella (SS) Agar

Colony morphology is only presumptive identification and hence biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
The presence of brilliant green in this medium makes it highly selective and has been shown to inhibit the growth of some Shigella species. Thus, a non-selective but differential medium such as MacConkey Agar or HE Agar should also be streaked to increase the recovery of fastidious, or low numbers of, gram-negative bacteria.
The bile salts may crystallize over time which appears as small spider-like puff balls within the medium and even though they do not affect the performance of the medium.
Some strains of Shigella, such as Shigella sonnei and Shigella dysenteriae serovar 1, may ferment lactose relatively slowly, and colonies change to lactose-fermenting after cultivation for 2 or more days.
A few non-pathogenic organisms may also grow on Salmonella Shigella agar.

Keynotes on SS Agar

Other less inhibitory media used for the isolation, cultivation, and differentiation of gram-negative enteric bacteria are Desoxycholate Agar, MacConkey Agar, Eosin Methylene Blue (EMB) Agar, Xylose Lysine Deoxycholate (XLD Agar), and Hektoen Enteric Agar.
Despite its name, Salmonella-Shigella (SS) agar is not suitable for isolating shigellae as it is inhibitory to most strains.
Functions of SS agar ingredients are as follows-Lactose is the fermentable carbohydrate. Beef extract and proteose peptone provide nitrogen, vitamins, and amino acids. Ferric citrate and Sodium Thiosulfate: Sodium thiosulfate is added to the medium as a hydrogen sulfide source, and ferric citrate is added as an indicator for hydrogen sulfide production. Sodium thiosulphate and Sodium citrate are selective agents, providing an alkaline pH to inhibit Gram-positive bacteria and suppress coliforms. Bile salts: The bile salts inhibit the growth of gram-positive bacteria. Brilliant green and neutral Red both are pH indicators. Agar acts solidifying agent and sodium chloride is the source of electrolytes whereas water is the source of hydrogen and oxygen.

Precautions for Salmonella-Shigella (SS) Agar

This product may contain components of animal origin. Certified knowledge of the origin and/or sanitary state of the animals does not guarantee the absence of transmissible pathogenic agents and thus, it is recommended that these products be treated as potentially infectious, and handle observing the usual universal blood precautions. Do not ingest, inhale, or allow to come into contact with skin.
This product is for in vitro diagnostic use only and it is to be used only by adequately trained and qualified laboratory personnel. Observe approved biohazard precautions and aseptic techniques. All laboratory specimens should be considered infectious and handled according to “standard precautions.

XLD agar with Shigella

XLD stands for xylose lysine deoxycholate .

It is a selective, differential, and indicator medium for the isolation of Salmonella and Shigella species.

Shigella species: red colonies

The above image is showing the growth of Shigella on XLD agar.

Introduction of Shigella

Shigella is a genus of the Enterobacteriaceae family of bacteria and causing an infectious disease called Shigellosis. Shigella is named after the Japanese microbiologist Kiyoshi Shiga who isolated the first member of the group in 1896 from epidemic dysentery in Japan which was then called Shigella Shiga and is now called S. dysenteriae. Most who are infected with Shigella develop diarrhea, fever, and stomach cramps starting a day or two after they are exposed to the bacteria. Shigellosis usually resolves in 5 to 7 days. Some people who are infected may have no symptoms at all, but may still pass the Shigella bacteria to others. The spread of these organisms can be stopped by frequent and careful handwashing with soap and taking other hygiene measures because of being a mode of infection feco-oral route.

Morphology

Shigellae are short, Gram-negative rods measuring about 1-3 µm X 0.5 µm. They are non-motile, non-encapsulated, non-sporing, and non-acid fast.

Culture characteristics

They are aerobes and facultative anaerobes and can grow on ordinary mediums like nutrient agar. The optimal temperature and pH for growth are 37°C and 7.4 respectively. But they can grow at a temperature range of 10 to 40°C.

MacConkey agar: Colonies are non-lactose fermenting (except S. sonnei) large, circular, convex, smooth, and translucent.

Deoxycholate citrate agar (DCA): Colonies are colorless (non-lactose fermenting) except in the case of S. sonnei which forms pink colonies due to late lactose fermentation.

Xylose lysine deoxycholate (XLD) agar: Colonies are red due to the organism has ability to decarboxylate lysine without black centers

Salmonella-Shigella(SS) agar: It is a highly selective medium for the isolation of Salmonella and Shigella. Colorless colonies with no blackening whereas Salmonella colonies are colorless with black centers due to their ability to produce hydrogen sulfide.

Heaktoen Enteric Agar (HEA): They give green to blue-green colonies.

Selenite F broth ( enrichment medium): Sodium selenite of this medium inhibits coliform bacilli while permitting salmonellae and shigellae to grow. This is recommended medium for the isolation of these organisms from feces.

Resistance

They are killed at 56°C in an hour and by 1 % phenol in 30 minutes. They remain viable in water and ice for 1-6 months. Chlorination or boiling of water and pasteurization of milk is effective and destroys the bacilli.

Antigenic Structures

They possess a large number of antigens and they are-

somatic (O) antigens
Capsular(K) antigen ( some strains only)
Fimbrial antigens.

Classification

Scientific classification

Domain: Bacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Enterobacterales

Family: Enterobacteriaceae

Genus: Shigella

There are four species or serogroups with multiple serotypes –

A. Shigella dysenterae : 12 Serotypes

B. Shigella flexneri: 6 serotypes

C. Shigella boydii: 18 serotypes and

D. Shigella sonnei : 1 sreotype

On Basis of nannitol Fermentation-

Non-mannitol-fermenters: Shigella dysenteriae
Mannitol-fermenters: Shigella flexneri, Shigella boydii and Shigella sonnei

Toxins

Endotoxin
Exotoxin
Verotoxin

Habitat and Transmission

Shigella species are found only in the human intestinal tract. Carriers of pathogenic strains can excrete the organism up to two weeks after infection and occasionally for longer periods. They are killed by drying and are transmitted by the fecal-oral route. The highest incidence of Shigellosis occurs in areas of poor sanitation and where water supplies are polluted. Factors Contributing Spread-Spread is always from a human resource and generally involves one of the five Fs i.e. food, fingers, feces, flies, and fomites. This is in contrast to salmonellae, which are often spread to humans from infected animals. Transmission-fecal-oral transmission is the main path of Shigella infection. Other modes of transmission include ingestion of contaminated food or water, contact with infected objects, or sexual contact. Outbreaks of Shigella infection are common in places where sanitation is poor.

Pathogenesis

The species of this genus cause a serious illness known as dysentery /shigellosis, which is an acute diarrheal disease characterized by the passage of pus, blood, or mucous through the stool. Infection mainly occurs because of the ingestion of contaminated food or water. The incubation period is 12-48 hours but may vary between 1-7 days. Through the ingestion, the bacilli will reach the large intestine of humans. The multiplication occurs in the epithelial cells of the large intestine. Then the bacteria spreads to adjacent cells and to the lamina propria (which is a thin layer of loose connective tissue which lies beneath the epithelium) where the colonization occurs. After the growth and multiplication, it starts to produce toxins. The lamina propria and submucosa develop an acute inflammatory reaction with the formation of abscess on the mucosal surface along with capillary thrombosis by the production of toxins. The necrosed epithelium becomes soft and sloughed out and causing superficial ulcers and bleeding. The toxin produced by the shigella bacteria has both enterotoxin effect and neurotoxic effect. Thus their combined action leads to severe diarrhea, polyneuritis, coma, and meningitis.

Clinical Syndromes (Shigellosis)

Ranges from asymptomatic infection to severe bacillary dysentery
Two-stage disease: watery diarrhea changing to dysentery with frequent small stools with blood and mucus, tenesmus, cramps, fever
Early-stage:- Watery diarrhea attributed to the enterotoxin activity of Shiga toxin
Fever attributed to the neurotoxic activity of the toxin

Clinical Features

Following are the clinical features of Shigellosis-

abdominal pain,
tenesmus,
watery diarrhea, and/or dysentery (multiple scanty, bloody, mucoid stools)
abdominal tenderness,
fever,
vomiting,
dehydration,
and convulsions.

Epidemiology

Shigellosis is a major cause of the diarrheal disease (developing nations). The major cause of bacillary dysentery (severe second stage form of shigellosis). Leading cause of infant diarrhea and mortality (death) in developing countries. They occur naturally in higher primates. Spread from human to human via the fecal-oral route. Less frequently, transmission by ingestion of contaminated food or water. Outbreaks usually occur in close communities; Secondary transmission occurs frequently. Low infectious dose (10-100 bacilli) with 1-3 day incubation period. Carriage of the organism persists for approximately one month following convalescence.

Laboratory Diagnosis

Diagnosis depends upon isolating the organisms, Shigella from feces.

Specimen: Collection of fresh stool

Transport: Specimens should be transported immediately otherwise, use a transport medium like Sach’s buffered glycerol saline.

Note: Avoid the use of an alkaline transport medium because it is inhibitory for shigellae ( but useful for Vibrios).

Direct Microscopy of feces

Saline and iodine wet mount: Fields are suggestive for pus cells, red blood cells, and the absence of parasites.

Culture of specimens

MacConkey agar: Colonies are non-lactose fermenting (except S. sonnei) large, circular, convex, smooth, and translucent.

Deoxycholate citrate agar (DCA): Colonies are colorless (non-lactose fermenting) except in the case of S. sonnei which forms pink colonies due to late lactose fermentation.

Xylose lysine deoxycholate (XLD) agar: Colonies are red due to the organism has ability to decarboxylate lysine without black centers

Heaktoen Enteric Agar (HEA): They give green to blue-green colonies.

Shigella biochemical tests

Shigella species biochemical tests-

TSI test: Red/yellow, no production of hydrogen sulfide and gas formation

SIM test: The indole test is negative, non-motile, and has no production of hydrogen sulfide.

Urease test: The urea hydrolyzation test is negative.

Citrate test: The citrate utilization test is also negative as shown above image.

Slide Agglutination Test

Identification of Shigella is conformed by slide agglutination test using polyvalent and monovalent antisera. Then type-specific antisera belonging to subgroups A, B, or C is used for the agglutination test.

Colicin Typing

It is used for subgroup D (S. sonnei) strains.

Differences between Amoebic dysentery and bacillary dysentery

Amoebic dysentery

Trophozoites of Entamoeba histolytica
Cyst of E. histolytica
RBCs in clumps
Macrophages
pus cells
CL crystals

Bacillary dysentery

Non-motile bacteria
Shigella
Plenty pus cells
RBCs

Treatment

Tetracycline and chloramphenicol is the drug of choice against the Shigella dysenteriae. The treatment should be continued for 5-7 days.

Prevention

It may prevent by following ways-

Using pure water supply
Maintaining personal hygiene
Proper disposal of sewage
Controlling of insects ( flies).

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