Biochemical Test of Gram-Negative Bacteria: Introduction, Common Tests, List of Organisms, Keynotes, and Some Related Footages

Introduction of Biochemical Test of Gram-Negative Bacteria

Gram-negative bacteria are the most common etiological agents which are responsible for bacterial infections as well as viral co-infections. Clinical Microbiology Laboratory generally becomes busy due to those isolates. The need for ‘Biochemical Test of Gram-Negative Bacteria’ is mandatory to identify and isolate those pathogens and hence a group of common clinically relevant Gram-Negative bacterial biochemical reactions is included in a chart.

Common Tests

• Triple sugar iron (TSI) agar Test: This single TSI test is useful for the following features-

1. To know the carbohydrate utilization ability of the organisms
2. Gas formation by the organisms
3. Hydrogen sulfide (H₂S) production by the bacteria

• Oxidase test
• SIM (Sulfide,Indole,Motility) Test: It helps to isolate the organisms on the basis of sulfide production, indole formation, and motility.
• Citrate or citrate utilization test: A citrate test is used to determine the ability of an organism to utilize sodium citrate as its only carbon source.
• Urease Test or Urea hydrolization Test or Urea Test: It is important for certain bacteria that possess the enzyme urease that hydrolyzes urea releasing ammonia into the medium which produces a change in the pH of the medium that can be detected by the color change in the indicator dye.
• Lactose fermentation and sucrose fermentation test in OF basal medium

List of Organisms

Biochemical tests of clinically relevant bacteria are as follows-

1. Escherichia coli
2. Escherichia coli (AD group)
3. Shigella groups A, B, C
4. Shigella sonnei (group D)
5. Salmonella Most serotypes
6. Salmonella Typhi
7. Salmonella Paratyphi A
8. Citrobacter freundii
9. Citrobacter koseri
10. Klebsiella pneumoniae
11. Klebsiella oxytoca
12. Enterobacter aerogenes
13. Hafnia alvei
14. Serratia marcescens
15. Proteus mirabilis
16. Morganella morganii
17. Providencia rettgeri
18. Providencia alcalifaciens
19. Yersenia enterocolitica
20. Edwardsiella tarda
21. Vibrio cholerae
22. Alcaligenes spp.
23. Pseudomonas aeruginosa
24. Burkholderia Cepacia complex
25. Stenotrophomonas maltophilia
26. Acinetobacter baumannii-calcoaceticus complex (ABC)
27. Acinetobacter lwoffii

Table of Biochemical Reactions of Common Gram-Negative Glucose fermenting and non-fermenting Bacteria

Biochemical Reactions of Common Gram-Negative Glucose fermenting Bacteria

Biochemical reactions of clinically important common Gram-negative glucose-fermenting bacteria are as follows-

Species	TSI Slant/Butt	Oxidase	Gas	H2S	Motility	Indole	Citrate	Urease	Lactose	Sucrose
E. coli	Y/Y	–	+	–	+	+	–	–	+	+/-
E. coli (AD group)	R/Y	–	–	–	–	+	–	–	–	–
Shigella groups A, B, C	R/Y	–	–	–	–	+/-	–	–	–	–
S. sonnei	R/Y	–	–	–	–	–	–	–	–	–
Salmonella Most serotypes	R/Y	–	+	+	+	–	+	–	–	–
Salmonella Typhi	R/Y	–	–	+	+	–	–	–	–	–
S. Paratyphi A	R/Y	–	+	–	+	–	–	–	–	–
C. freundii	R or Y/Y	–	+	+/-	+	–	+	+/-	+/-	+/-
C. koseri	R or Y/Y	–	+	–	+	+	+	+/-	+/-	+/-
K. pneumoniae	Y/Y	–	++	–	–	–	+	+	+	+
K. oxytoca	Y/Y	–	++	–	–	+	+	+	+	+
E. aerogenes	Y/Y	–	++	–	+	–	+	–	+	+/-
E. cloacae	Y/Y	–	+	–	+	–	+	+/-	+	+/-
Hafnia alvei	R/Y	–	+		+	–	–	–	–	–
S. marcescens	Y/Y	–	+/-	–	+	–	+	–	–	+
P. mirabilis	R or Y/Y	–	+	+	+	–	+/-	++	–	+/-
P. vulgaris	Y/Y	–	+	+	+	+	–	++	–	+
M. morganii	R/Y	–	+	+/-	+	+	+/-	++	–	–
P. rettgeri	R or Y/Y	–	–	–	+	+	+	++	–	+/-
P. stuartii	R or Y/Y	–	–	–	+	+	+	+/-	–	+/-
P. alcalifaciens	R or Y/Y	–	+	–	+	+	+	–	–	+/-
Y. enterocolitica	Y/Y	–	–	–	-(+@22°C)	+/-	–	+/-	–	+
E. tarda	R/Y	–	–	+	+	+	–	–	–	–
V. cholerae	Y/Y	–	–	–	+	+	+/-	–	-+ slow	+

Biochemical Reactions of Common Gram-Negative Glucose fermenting Bacteria

Biochemical reactions of Bichemedically important common Gram-negative glucose-non- fermenting bacteria are as follows-

Species	TSI Slant/Butt	Oxidase	Gas	H2S	Motility	Indole	Citrate	Urease	Lactose	Sucrose
Alcaligenes spp.	R/R (NC)	+	–	–	+	–	+/-	–	–	–
P. aeruginosa	R/R	+	–	–	+	–	+	+/-	–	–
Burkholderia Cepacia complex	R/R	+	–	–	+	–	+	+/-	–	–
S. maltophilia	R/R	–	–	–	+	–	–	–	–	–
Acinetobacter baumannii-calcoaceticus complex (ABC)	R/R	–	–	–	–	–	+	+/-	–	–
	R/R	–	–	–	–	–	+/-	+/-	–	–

Keynotes on Biochemical Test of Gram-Negative Bacteria

• Alkalescens-Dispar (AD) group of Escherichia coli are biochemically inactive and non-motile strains of E. coli
• Shigella is classified into four major serological groups and they are -Group A- Shigella dysenteriae, Group B-Shigella flexneri, Group C- Shigella boydii, and Group D-Shigella sonnei.
• Shigella sonnei has only one serotype and it is responsible for most cases of dysentery in developed countries.
• Salmonella Typhi differes from Salmonella Paratyphi in TSI reactions on the basis of H₂S production (S. Typhi) and gas formation (S. paratyphi).
• Klebsiella oxytoca is indole postive and K. pneminiae negative.
• Proteus vulgaris is indole positive while P. mirabilis is indole negative.
• Y. enterocolitica is motile at room temperature (22°C).

Footages of Biochemical Test of Gram-Negative Bacteria

Escherichia coli reaction in TSI agar

E. coli biochemical reaction in SIM medium

E. coli biochemical reaction in urea agar

E. coli biochemical reaction in citrate agar

E. coli biochemical tests in TSI, SIM, Citrate and Urea agar Demonstration

Klebsiella pneumoniae biochemical tests in TSI, SIM, Citrate and Urea agar Demonstration

Klebsiella oxytoca biochemical tests in TSI, SIM, Citrate and Urea agar Demonstration

Proteus vulgaris biochemical tests in TSI, SIM, Citrate and Urea agar Demonstration

Proteus mirabilis biochemical tests in MIU, TSI and citrate agar demonstration

Salmonella Typhi Biochemical Tests in TSI, MIU and Citrate Agar Demonstration

Salmonella Paratyphi Biochemical Tests in TSI, SIM, Citrate, and Urea Agar Demonstration

Shigella sonnei biochemical tests in TSI, SIM, Citrate and Urea agar Demonstration

Shigella groups A,B and C biochemical tests in TSI, SIM, Citrate and Urea agar Demonstration

Acinetobacter baumannii-calcoaceticus complex (ABC) Biochemical Tests in TSI, SIM, Citrate, and Urea Agar Demonstration

Pseudomonas aeruginosa Biochemical Tests in TSI, SIM, Citrate, and Urea Agar Demonstration

Further Reading

• Cowan and Steel’s, manual for the identification of medical bacteria
• Topley and Wilson’s microbiology and microbial infection Topley and Wilson’s microbiology and microbial infection – Bacteriology-2-10th Edn.
• Manual of Clinical Microbiology   -Patrick R. Murray -8th Edn.
• Bailey and Scott’s  Diagnostic Microbiology -13th Edn
• Mackie & Mc Cartney  Practical Medical Microbiology- 14th  Edn.
• Diagnostic Microbiology -Connie R. Mahon & George Manuselis
• Koneman Color Atlas and Textbook of Diagnostic Microbiology-6th  Edn.
• Jawetz Melnick and Adelberg’s Medical Microbiology- 25th Edn.
• Lippincott’s –Illustrated- review-Microbiology-3rd Edn.
• Mandell’s Infectious Disease-7th Edn.
• https://www.microbiologyresearch.org/content/journal/jmm/10.1099/00222615-21-4-337
• https://www.sciencedirect.com/topics/immunology-and-microbiology/shigella
• Bergey’s Manual of Systemic Bacteriology – 2nd  Edn.
• Text Book of Microbiology -Anantanarayan & Paniker-9th Edn.