Proteus: General Characteristics, Morphology, Pathogenecity, Laboratory Diagnosis and Keynotes

Proteus: Growth on Various media, Gram Stain and Biochemical Reactions

Proteus General Characteristic

  1. Proteus species are Gram-negative rods and facultative anaerobes.
  2. They are members of Enterobacteriaceae.
  3. They are motile due to peritrichous flagella, non encapsulated, non-spore-forming.
  4. They are most commonly found in the human intestinal tract as part of normal human intestinal flora as well as saprophytic and are widely distributed in nature.
  5. They are non-lactose fermenting (NLF).
  6. The main species of medical importance are P. mirabilis and P. vulgaris. They are opportunist pathogens and may cause many types of infection.

Morphology of Proteus

They are actively motile, non-capsulated, Gram-negative bacilli measuring 1-3µm x 0.5 µm in size. Motility is not as easily observed at 35-37 °C at room temperature ( 20-28°C). Culture on blood agar shows a characteristics’ fishy or seminal odor’ with swarming growth as shown above picture.

MacConkey agar: Proteus produces individual non-lactose fermenting colonies after overnight incubation at 35-37°C and swarming is prevented due to bile salt present in the medium.

CLED agar: Blue-gray translucent colonies and absence of swarming are due to electrolyte deficiency.

XLD agar: Proteus produces individual non-lactose fermenting colonies after overnight incubation at 35-37°C and swarming is prevented due to bile salt present in the medium

Antigenic Structure of Proteus

The Proteus possess thermostable,  somatic (O), and thermolabile flagellar (H) antigens upon which, several serotypes have been recognized. WeIl and Felix ( 1966) observed flagellated strains of Proteus grew on agar as a thin surface film (named Hauch) whereas non flagellated variant strains without surface film ( Ohne Hauch). They also noted that certain non-motile strains of Proteus vulgaris, called X strains were agglutinated by sera of typhus patients. The sharing of these polysaccharide antigens of Proteus with some rickettsiae forms the basis of the Weil-Felix reaction for the diagnosis of some rickettsial infections. Total three non-motile strains, among them two of Proteus vulgaris  OX2, OX19, and one Proteus mirabilis OX K are applicable as the antigens for this test.

Pathogenicity of Proteus

They can cause –

  • Urinary infection
  • Abdominal and wound infections
  • Septicemia
  • Infection of ear
  • Respiratory infections
  • Nosocomial infections

Laboratory Diagnosis of Proteus

Specimens: It depends on the site of infection and common samples are urine, pus, and ear discharge. Midstream urine (MSU) in UTI whereas pus in pyogenic lesions.

Specimens collection: The sample should be collected in a sterile container under all aseptic conditions and transported to the laboratory immediately.

Gram stain: Gram-negative rods which are non-capsulated and non-sporing ( evidence).

Culture: Swarming effect over blood agar plate as a consequence of the organisms active motility and NLF colonies on Macconkey agar whereas Blue-gray translucent colonies on CLED agar with fishy or seminal odor.

Dienes phenomenon: When two different strains of Proteus species inoculate at different places of the same non-inhibitory medium (blood agar), swarming of the two strains remains separated by a narrow visible furrow. However, in the case of two identical strains of Proteus, swarming of two coalesce without signs of demarcation. Such a condition is called the Dienes phenomenon.

Hanging drop preparation: Actively motile rods observation

Biochemical Reactions

Catalase Test: Positive

Nitrate Test: Positive

Oxidase Test: Negative

Beta-galactosidase (ONPG) Test: Negative

Methyl red (MR) Test: The methyl red test is used to identify bacteria to produce pyruvic acid from glucose metabolism. Both  Proteus vulgaris and mirabilis are methyl red test positive.

Voges–Proskauer (VP) Test: It is a test used to detect acetoin in bacterial broth culture. A red-brown color indicates a positive result, while a yellow-brown color indicates a negative result. Both P. vulgaris and  P. mirabilis are VP negative.

Triple Sugar Iron (TSI) Test: This test is used to determine the ability of bacteria to ferment sugars and to produce hydrogen sulfide ( H2S)  and gas formation.

Proteus mirabilis: Red slope/yellow butt with  H2S production i.e. black and  presence of gas formation

Proteus vulagris: Red slope/yellow butt with  H2S production i.e. black and gas formation -different strains give different results

SIM Test: This single panel test is useful for detecting  H2S production, indole detection, and motility test.

Proteus mirabilis:  H2S production, indole negative and motile

Proteus vulagris:  H2S production, indole positive and motile

Citrate Utilization test: Ability of an organism to use citrate as the sole source of carbon and energy.

Proteus mirabilis: Generally positive but a minority of strains give a negative result.

Proteus vulagris: different strains give different results

Urea hydrolyzation test: Positive

Note: Rapidly hydrolyze urea i.e. within 4 hours which is an important early screening test in differentiating salmonellae and shigellae from Proteus.

Phenyl pyruvic acid (PPA) Test: Deamination of phenylalanine to PPA. Both are always positive.

IMViC Test: The indole test is used to determine the ability of bacteria to convert tryptophan into indole. P. mirabilis can be differentiated from P. vulgaris by the indole test in which P. mirabilis is negative whereas P. vulgaris positive.

Agglutination test: Strain may be agglutinated with polyvalent antisera to conform it.

API test:  By inoculating microorganisms to a strip. After inoculation metabolic cause color change, to complete the identification of the strains either identified to the genus only or that have multiple genera consist of profile number.

Vitek system:  It is a new automatic system for identification and susceptibility testing for most clinically important bacteria. It compromises: -A fitter/sealer: enable -inoculation of the card within a few minutes.  An inoculator /leader – A computer and printer.

Antibiotic susceptibility test (AST): AST is important as Proteus bacilli are resistant to many of the common antibiotics.

Keynotes of Proteus

  • P. mirabilis has fascinated scientists for over 125 years for its ability to differentiate from short swimmer cells into elongated swarm cells that express hundreds to thousands of flagella.
  • Swarming is distinct from swimming motility in that it refers to multicellular flagellum-mediated migration across a surface rather than movement in a liquid medium or through soft agar.
  • Multiple drug-resistant (MDR) strains to carry R plasmids have become very important in nosocomial infections.
  • Swarming phenomenon swarming is described as the formation of concentric zones of bacterial growth, able to cover the whole surface of a solid culture medium. P. mirabilis and  P. vulgaris are known for their swarming ability over blood agar as shown above image.
  • The distinctive characters of the genus are PPA, urease, and  H2S positive.
  • Indole helps to differentiate P. vulgaris ( positive) from P. mirabilis ( negative).
  • Dienes phenomenon or typing is using successfully to determine the relationship between strains of Proteus species in studies of cross-infection.
  • Swarming growth is a problem In the laboratory when mixed growth is obtained in which Proteus bacilli are present with other bacteria and thus in such scenario swarming inhibition is mandatory. Several methods have been used to inhibit warming are-
    √Increasing agar concentration up to 6%
    √incorporating alcohol 5-6%
    √Chloral hydrate
    √Sodium azide
    √Sulphonamide
    √Surface active agents and
    √boric acid.

Related Videos on Proteus

#Proteus swarming growth on blood agar and its identification using various biochemical tests e.g. TSI test, MIU test, Citrate Utilization test as shown below-

#Proteus growth on blood agar and MacConkey agar-

#Proteus swarming growth covering other bacteria

#Swarm and vegetative cells of Proteus spp.

#Swarming growth of Proteus having swimmer and swarmer cells:
Swarming growth of Proteus having swimmer and swarmer cells can be determined using Gram’s staining-
i.e. swimmer cells- small-near the center of the growth plate
while swarmer cells- large-away the center of growth plate as shown just above the video.

# Dienes Phenomenon of typing of Proteus

#Motile bacteria under hanging drop Preparation as shown below-Testing motility of bacteria under hanging drop preparation is a recommended method.
Bacteria are motile due to having flagella.
The objective of this observation includes-
Active motile or true motility
Brownian Movement
Motile bacteria
and non-motile bacteria
Motile bacteria are-

  1. E. coli
  2. Salmonella
  3. Proteus
  4. Pseudomonas
  5. Enterobacter
  6. Citrobacter

Non-motile bacteria are

#CLED agar with a typical colony of bacteria, Staphylococcus aureus and Proteus species as shown below-

 

Further Readings

  1. https://jb.asm.org/content/195/6/1305
  2. https://www.annualreviews.org/doi/pdf/10.1146/annurev.mi.32.100178.000533
  3. Williams FD, Schwarzhoff RH. 1978. Nature of the swarming phenomenon in Proteus. Annu. Rev. Microbiol. 32:101–122.
  4. Armbruster CE, Mobley HLT. 2012. Merging mythology and morphology: the multifaceted lifestyle of Proteus mirabilis. Nat. Rev. Microbiol. 10:743–754
  5. Morgenstein RM, Szostek B, Rather PN. 2010. Regulation of gene expression during swarmer cell differentiation in Proteus mirabilis. FEMS Microbiol. Rev. 34:753–763.
  6. Rather PN. 2005. Swarmer cell differentiation in Proteus mirabilis. Environ. Microbiol. 7:1065–1073.
  7. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  8. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  9. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
  10. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
  11. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  12.  Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
  13.  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition 2007, Publisher Elsevier
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