Aeromonas: Introduction, Pathogenecity, Lab Diagnosis and Treatment

Introduction of Aeromonas

Aeromoas: Greek; aer/aeros =air, gas; feminine gender noun monas = unit, monad

Aeromonas=gas(-producing) monad

Aeromonas was originally placed in the family Vibrionanceae  (1965 by veron), which also included the genera Vibrio, Aeromonas  & Plesiomomas.

Phylogenetic investigations indicated that the genus Aeromonas is not closely related to vibrios.

So, it is removed from the family Vibrionanceae and transfer to the new family Aeromanadaceae (1986 by Colwell,  MacDonell, and De Ley ) based upon 5s & 16s rRNA gene sequencing.

Total species = 24 species

Genus

Gram-negative, oxidase- and catalase-positive, facultatively anaerobic rods.

Motile: single polar flagellum.

Nitrates reduction test: positive.

Anaerogenic fermentation of carbohydrates.

Acid is rarely produced from carbohydrates and non-carbohydrate compounds.

Resistant: vibriostatic agent O/129(2,4 diamino 6,7 diisopropylteridine

Nutritional requirements: simple

No salt supplementation for growth

Many extracellular enzymes: proteases, lipases, nucleases, sulphatases, lecithinase, chitinase, amylase, and stapholysin.

Pathogenic: for man and fish

The G+C content of DNA: 57-63%

Type species: A. hydrophila ATCC 7966

Species

Total species = 24 species

Clinically significant 

• hydrophila (1943)
• salmonicida (1953)
• media (1983)
• caviae (1984)
• veronii (1988)
• schubertii (1989)
• jandaei (1992)
• trota (1992)
• bestiarum (1996)
• popoffii (1997)
• tecta (2008)

Clinically non-significant:

• sobria (1981)
• eucrenophila (1988)
• encheleia (1995)
• simiae (2004)
• molluscorum (2004)
• bivalvium (2007)
• aquariorum (2008)
• allosaccharophila (1992)
• culicicola (2002)
• sharmana (2006)

Classification

• Family : Aeromonadaceae
• It includes 3-genera: Aeromonas, Oceanimonas and Tolumonas
• Initially, it is classified into two major groups based upon optimal temperature for growth, disease spectrum, and biochemical characteristics.
• 1.Psychrophilic strains

• It inhabits freshwater environments
• The primary pathogen of fish i.e. salmon
• Non-motile, indole negative
• Brown diffusible pigment in tyrosine agar
• Grow best at temp. ranging between 22 & 28 °C
• Single species: salmonicida

2.Mesophilic strains

• Infect human and fish
• Motile, indole positive
• Fail to produce brown pigment
• Further divided into hydrophila, A. sobria, and A. caviae
• Grow optimally between 35-37°C, ranging from 4-42°C

Habitat

• Aeromonads are common inhabitants of the microbial biosphere.
• They can be isolated from virtually any fresh water source.
• During colder seasons, they are often recovered from poikilothermic animals and freshwater environments.
• Also recovered from marine environments

Morphology and cell structure

• Size: 0.3-1.0 μm x 1.0-3.5 µm
• The outermost layer contains S-layer (paracrystalline surface layer)
• A single 49-53 KDa surface array protein (SAP) forms S-layer
• Capsule present in several species
• Pilli: two types.

a.Rigid pilli : diameter-9nm ,mol. Mass- 17-21 KDa

b.Flexible  pilli : diameter-7nm ,mol. Mass- 19-23KDa

• Fatty acid analysis: aeromonads can be distinguished from Vibrio species by their lack of 3-hydroxy lauric acid and from members of the Enterobacteriaceae by their failure to produce significant amounts of cyclopropane fatty acids

Cultural characteristics & growth requirements

• On non-selective agar media: buff-colored, smooth, convex colonies 3-5mm in diameter after overnight incubation at 35-37°C.
• salmonicida strains produce a brown water-soluble pigment on tyrosine-containing agar.
• Most aeromonads grow in media containing up to 4% NaCl and over a pH range of 5-9.
• Alkaline peptone agar: also used to enrich
• Ferment sucrose, lactose, or both.
• Sheep blood agar: beta-hemolysis, some strains produce a double zone of hemolysis.
• Blood agar: used for cytochrome oxidase and tryptophanase deamination (both positive)
• CIN (cefsulodin-irgasan-novobiocin ) agar: bull’s eye like a colony

“Suicide  phenomenon” in Aeromonas species

• When certain strains of Aeromonas are grown in glucose-containing cultures, an accumulation of acetic acid and other acids occurs, as a result of suppression of the TCA cycle, and a reduction in the pH of the medium leads to inactivation of the electron transport system & ultimately cessation of cell growth and death.
• The suicide property(Wilcox et al 1992):

1. 1. A. sobria: suicidal/non-suicidal
2. A.hydrophila: suicidal at 37°C & non-suicidal at 30°C
3. A.caviae: suicidal at both 30°C & 37°C.

Differentiating  properties of  Aeromonas, Vibrio & Plesiomonas

Metabolism

• Aeromonads are chemo-organotrophs using a wide variety of sugars and carbon sources for energy.
• Glucose is metabolized both aerobically and fermentatively with or without the production of gas ( CO2, H2)

Epidemiology

Aeromonas hydrophila (motile bacteria ), found in water, is a causative agent of red leg disease of frogs from which it was originally isolated.

In 1988, California became the first state to make Aeromonas infections reportable.

Route of infection:

• Contaminated drinking water
• Ingested of food ( produce, dairy or meats)
• Through consumable products, bivalves such as oysters & mussels
• Recreational activities such as boating,fishing & diving
• From zoonotic origins
• Reptile & snake bites less commonly vertebrates bite, such as bears

Source of infection

Clinical infections & disease-associated syndrome

• Gastroenteritis
• Bloodborne infection: bacteremia & septicemia
• Skin &soft tissue infections: cellulitis
• Intra-abdominal infections: peritonitis
• Respiratory tract infections: pneumonia
• Urogenital tract infections
• Eye infections

Aeromonas species in
Medicinal leech(Hirudo medicinalis) therapy

• The use of medicinal leech Hirudio medicinalis for medical therapy following microvascular/plastic surgery to relieve venous congestion.

Unfortunately, Aeromonas species are present in the leech gut, where they aid in the breakdown of ingested red blood cells. As a result, an increasing no. of Aeromonas infections have been associated with leech application.

Pathogenicity

• Aeromonads are involved in both intestinal & extra-intestinal human infections.

Gastroenteritis

• Chief virulence factor: enterotoxin
• Common enterotoxin: cytolytic β-hemolysin or aerolysin

Two factors play important role in pathogenesis:

Bacterial flagella:

• polar flagellum: helps in the initial attachment of bacteria to gastrointestinal tract epithelium
• lateral flagellum: helps in cell adherence, biofilm formation, and colonization. Bacterial pili:

• short & rigid pilli :intestinal colonization
• long & wavy pilli: biofilm development

Gastroenteritis

• Route of infection: oral ingestion of contaminated food or water
• Ingested bacteria must then bypass the deleterious effects of gastric acidity & take up residence in the small or large intestines, competing successfully against autochthonous microorganisms.
• hydrophila to withstand pH 3.5
• Pathway:

Bacterial locomotion

↓

Attachment to the gastrointestinal epithelium

↓

Biofilm formation

↓

Colonization

↓

Elaboration of virulence factors

↓

Infection

• Diarrohea by elaboration of enterotoxigenic molecules causing enteritis or by the invasion of gastrointestinal epithelium producing dysentery or colitis.
• Cytolytic toxins with hemolytic activity
• Cytotonic enterotoxins
• Beta-hemolysin of hydrophila (Bernheimer’s aerolysin) : pore-forming toxin
• A second family of beta-hemolysins exhibits significant amino acid sequence homology to the Hly A hemolysins of cholerae .
• A third Aeromonas cytotoxic enterotoxin, pore-forming toxin with hemolytic activity- it induces fluid accumulation in ligated intestinal loops and stimulates proinflammatory response by increased cytokine production through elevated tumor necrosis factor, IL-1β & IL-6 levels.
• Vaculating toxin: A. veronii bv. Sobria, 60KDa nonhemolytic enterotoxin, acts as serine protease & causes apoptosis in Vero cells.
• Five diarrheal presentations are observed in patients in whom Aeromonas has been isolated from their stools:
• Acute, secretory diarrhea often accompanied by vomiting
• An acute, dysenteric form of diarrhea with blood and mucus
• Chronic diarrhea usually lasting more than 10 days
• A cholera-like disease including rice-water stools
• The nebulous syndrome commonly referred to as traveler’s diarrhea                      Wound infections 
• 

Laboratory diagnosis

Isolation:

• Specimen: mainly stool
• Transport media: Cary-Blair, Amies, or modified Stuart’s medium, buffered glycerol in saline at RT
• Facultatively anaerobic, gram-negative rods, grows on basic laboratory media
• CIN(cefsulodin-irgasan-novobiocin agar): bull’s eye-like colony due to fermentation of D-mannitol.

Isolation:

• Specimen: mainly stool
• Transport media: Cary-Blair, Amies, or modified Stuart’s medium, buffered glycerol in saline at RT
• Facultatively anaerobic, gram-negative rods, grows on basic laboratory media
• CIN(cefsulodin-irgasan-novobiocin agar): bull’s eye-like colony due to fermentation of D-mannitol.

Laboratory identification

• Colony characters: large mucoid, raised, opaque, colonies with entire edges & a smooth, often mucoid surface after 24hrs incubation at 35°C.
• Aeromonas are oxidase positive
• Sheep blood agar: β-hemolysis(> 90%) except papoffi & A. trota (0%, 50%)
• Ampicillin blood agar(ABA): blood agar with 20µg/ml of ampicillin.(sensitive to trota)
• Indole positive except schubertii.
• Aeromonas agar: produced by lab-M
• XGA(xylose-galctosidase-agar)

Different  properties of Aeromonas species

Characters useful for identification of Aeromonas species from clinical samples

Separation of A. hydrophila from A.veronii bv. sobria :
fermentation of L-arabinose & hydrolysis of esculin

Other aeromonads isolated from clinical specimens:

Notably,

• veronii bv. Veronii strains are ornithine decarboxylase positive.
• trota strains are susceptible to ampicillin.
• schubertii doesnot produce gas from glucose
• jandaei, A. schubertii , A.popoffi & most strains of A. trota do not ferment sucrose

Molecular identification

• Restriction fragment length polymorphism(RFLP).
• Random amplified polymeric DNA(RAPD).
• Enterobacterial repetitive intergenic consensus (ERIC) sequences.
• Amplified fragment length polymorphism (AFLP).
• 16S rRNA gene(SSU) sequencing.

Antimicrobial susceptibility test

Resistance mechanism

• Three principal classes of beta-lactamase are recognized in Aeromonas species, namely class-C cephalosporins, a class-D penicillinase & a class-B Metallo-β-lactamase.
• hydrophila complex strains: expressing class B, C & D β-lactamase
• caviae strains: expressing class C & D β-lactamase
• veronii strains: expressing class C & D β-lactamase

Bibliography

1. Koneman’s color atlas and textbook of diagnostic microbiology, sixth edition, Washington Winn, jr.,Stephen Allen,Willam Janda,Elmer Koeman,Paul schreckenberger,Gail woods
2. Collier Leslie, Balows Albert & Sussman Max; Topley and Wilson’s Microbiology and Microbial Infections, 9th edition Vol-2; Arnold.
3. Mackie & McCartney practical medical microbiology, 14th edition, Churchill Livingstone.
4. Textbook of DIAGNOSTIC MICROBIOLOGY, Third edition, CONNIE R. MAHON, DONALD C.LEHMAN, GEORGE MANUSELIS.
5. A TEXTBOOK OF MICROBIOLOGY,Panikkar
6. A TEXTBOOK OF MICROBIOLOGY, P.CHAKRABORTY
7. Abbott, S. L., W. K. W. Cheung, and J. M. Janda.2003. The genus Aeromonas: biochemical characteristics, atypical reactions, and phenotypic identification schemes. J. Clin. Microbiol.41:2348-2357
8. Janda MJ, Abbott SL (2010) The genus Aeromonas: taxonomy, pathogenicity, and infection. Clin Microbiol Rev 23: 35–73