Pseudomonas & Other Non-fermentors

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Pseudomonas & other Non-fermentors . :

Pseudomonas & other Non-fermentors . Mr. Gunjal Prasad Niranjan M.Sc. Medical Microbiology, P.G. Dip. CRA, Assistant Professor, Dept. of Microbiology, PDVVPF’s Medical College, Ahmednagar

General Overview:

General Overview Opportunistic Pathogens of Plants, Animals, and Humans. Many Taxonomic Changes in Last Decade. Clinically Important Aerobic Gram-Negative Bacilli Include: Aerobic nonfermenters : 10-15% of clinical isolates Pseudomonas aeruginosa; Burkholderia cepacia; Stenotrophomonas maltophilia; Acinetobacter baumannii; Moraxella catarrhalis : Account for >75% of all clinical isolates of aerobic nonfermenters. Facultative anaerobes and microaerophiles: 70-80% of clinical isolates

General Characteristics of Nonfermenters:

General Characteristics of Nonfermenters Oxidative Gram-Negative Bacilli , including Pseudomonas spp., produce acid from glucose or other carbohydrates only in the presence of oxygen ( nonfermenters ). NOTE : Enterobacteriaceae , Aeromonas and Vibrio are fermentative and can utilize carbohydrates in the absence of oxygen. Pseudomonas aeruginosa oxidizes but does not ferment glucose . Alcaligenes faecalis neither ferments nor oxidizes glucose.

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Clinically Important Nonfermentative Gram-Negative Bacilli

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Pseudomonas aeruginosa (Family Pseudomonadaceae)

INTRODUCTION :

INTRODUCTION The genus Pseudomonas comprises. Aerobic (Strict), Gram Negative Bacilli (GNB), Non-fermentative, Non-sporing. Catalase test – Positive, Oxidase test – Positive. Motile = polar flagella. P. aeruginosa produces characteristic grape-like odor and blue-green pus & colonies. Most species produces soluble pigments , which diffuse through culture medium. Pyocyanin (blue); Fluorescein (yellow); Pyorubin (red)

INTRODUCTION :

INTRODUCTION Family – Pseudomonadaceae. Has 200 species. Human pathogens includes – Pseudomonas aeruginosa, P. maltophila, P. mallei, P. pseudomallei, P. cepacia, etc. Broad antibiotic resistance Most important amongst all is P. aeruginosa.

INTRODUCTION :

INTRODUCTION Recently P. mallei, P. pseudomallei, P. cepacia are included into a new genus Burkholderia, which belongs to family Pseudomonadaceae. Therefore now the names are - B. mallei, B. pseudomallei, B. cepacia. P. maltophila has been named as Stenotrophomonas maltophilia.

Common in the environment:

Common in the environment Water Air Soil Hospital environment – Basin, Bed, Pillows, Door handlers, Disinfectant solutions, Soaps, Skin of health care workers. Can transiently colonizes the gastrointestinal tract or respiratory tract of hospitalized patients, particularly those treated with broad spectrum antibiotics, exposed to respiratory therapy equipments or hospitalized for extended period. Survive where most organisms cannot; e.g., “oil-eating” bacteria are Pseudomonas.

Medically important species of Pseudomonas:

Pigment production Greenish yellow pigment No pigment Pseudomonas aeruginosa Burkholderia mallei Burkholderia pseudomallei Medically important species of Pseudomonas

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SPECIES MOT. OXIDASE PYOCYANIN FLUORESCIN GROWTH AT 42 0 C OXIDATION OF LYSINE DECARBOXYLASE ARGININE DIHYROLASE GLU LAC MANN. MALT Ps. aeruginosa + + + + + + - _ _ _ + Ps. stutzeri + + _ _ + + _ _ + _ + Ps. putida + + _ + _ + _ _ _ _ + Steno. maltophilia + _ _ _ + +/-- + _ + + _ B. mallei _ _ _ _ + + _ _ _ _ + B. pseudomallei + + _ _ + + + + + _ + B. cepacia + + _ _ + + + + + + -

Pseudomonas aeruginosa :

Pseudomonas aeruginosa MORPHOLOGY – Slender, GNB. 1.5-3 μ m X 0.5 μ m. Non- capsulated. Non-sporing. Actively Motile with Polar flagellum. Occasionally strains may have 2 or 3 polar flagella. Most strains have Pilli/Fimbriae. Although non-capsulated mucoid strains also occurs rarely.

CULTURAL CHARACTERISTICS :

CULTURAL CHARACTERISTICS Strict Aerobe. Grows well on ordinary media. Opt. temp -37 0 C. Range – 05 0 C to 42 0 C. NUTRIENT AGAR – Colonies are – Smooth, large, translucent, low convex, 2-4 mm. Growth gives sweetish aromatic odour (Due to production of 2 amino- aceto - phenone). Usually greenish-blue pigment produced, which diffuses into the medium.

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BLOOD AGAR – Colony characteristics are similar to colonies on NA. Many strains are haemolytic on BA .

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MAC CONKEY’S AGAR – Colonies are pale colour or colourless ( Non-lactose fermenting - NLF).

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CETRIMIDE AGAR – It is selective medium for the Ps. aeruginosa.

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PEPTONE WATER – It forms turbidity with surface pellicle. Pseudomonas being a strict aerobe tends to collect at the surface for more oxygen hence forming surface pellicle.

PIGMENT PRODUCTION :

PIGMENT PRODUCTION Pseudomonas aeruginosa produces number of pigments which diffuses into medium. PYOCYANIN – Bluish – Green phenazine pigment. Soluble in chloroform and water. Not produced by other species. Hence diagnostic for Ps. Aeruginosa.

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FLUORESCIN (PYOVERDIN) – It is a Greenish –Yellow. Insoluble in chloroform but soluble in water. May be produced by many other species. PYORUBIN – Reddish- Brown pigment. Insoluble in chloroform but soluble in water. PYOMELANIN – Brown – Black . Chemically unrelated to melanin. Production is uncommon.

BIOCHEMICAL REACTIONS :

BIOCHEMICAL REACTIONS P. aeruginosa derives energy from carbohydrates by oxidative (aerobic) breakdown rather than a fermentative (anaerobic) breakdown. Special OF medium of Hugh - Leifson is only used. Oxidase positive, Catalase – Positive, Indole, MR, VP, H2S – Negative. Utilizes Citrate as a sole source of Carbon by all members of genus.

ANTIGENIC STRUCTURE :

ANTIGENIC STRUCTURE O Antigens – It possess 19 distinct, group specific O antigens. All are heat stable. H Antigens – Two Heat-Labile H antigens have been recognized in P. aeruginosa.

TOXINS AND ENZYMES :

TOXINS AND ENZYMES 1. EXTRACELLULAR PRODUCTS – Pyocyanin inhibits mitochondrial enzymes in mammalian tissues and causes disruption and cessation of ciliated nasal epithelium. Thus, this favors colonization of these organisms in the nasal mucosa .

TOXINS AND ENZYMES :

TOXINS AND ENZYMES 2. EXTRACELLULAR ENZYMES AND HAEMOLYSINS – Protease – Any enzyme that catalyzes the splitting of proteins into smaller peptide fractions and amino acids by a process known as proteolysis. General protease, alkaline protease and elastase. Elastase - It destructs elastin containing tissues like blood vessels, lung tissue, skin, collagen, immunoglobulin and complement factors. Protease - Causes tissue destruction, inactivation of interferon and tumor necrosis factor α .

TOXINS AND ENZYMES :

Haemolysins – Any substance that can cause lysis (destruction) of erythrocytes (red blood cells) and the release of their hemoglobin. (Phospholipase C, Heat stable Rhamnilipid). These plays the key role in producing local lesions. Antibiotic resistance – Complicates antibiotic therapy. TOXINS AND ENZYMES

EXOTOXIN – :

EXOTOXIN – Two Exotoxins are produced – A and S. A – Polypeptide of – 66,000-72,000 Kd . Inhibits protein synthesis. Mechanism of Action of exotoxin A is similar to that of diphtheria toxin.

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Mechanism of Action of Exotoxin A

Toxin :

Frag A – Active part of toxin. Frag B - Binds toxin to cells. When released by bacterium toxin is inactive. Active site on frag A is masked. Activation is achieved by proteases present in infected tissues or in culture medium. Antibodies to B are protective, as act against binding of B fragment to host cells. Toxin

ENDOTOXIN – :

ENDOTOXIN – Lipopolysaccharide (LPS) Exhibiting many biological properties of enterobacterial LPS including “Pyrogenic action” etc. Capsule – Mucoid, exopolysaccharide, inhibits phagocytosis. Pyocyanin – Impairs ciliary functions, stimulates inflammatory response, mediate tissue damage through production of toxic oxygen radicals for e.g. - hydrogen peroxide, superoxide , hydroxyl radicals. Pilli – Attachment.

TYPING METHODS :

TYPING METHODS As P. aeruginosa is an imp. organism causing most of the Hospital –acquired infections. It is essential to type the strains for epidemiological purposes. BACTERIOCIN TYPIG (PYOCIN) – 3 types of bacteriocin are produced by 90% of P. aeruginosa strains Known as Pyocin. Know as R, F & S. The pyocin genes are located on the P. aeruginosa chromosome and their activities are inducible by mutagenic agents such as mitomycin C.

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Three types of pyocins are described. (i). R-type pyocins resemble non-flexible and contractile tails of bacteriophages. They provoke a depolarisation of the cytoplasmic membrane in relation with pore formation. (ii). F-type pyocins also resemble phage tails, but with a flexible and non-contractile rod-like structure. (iii). S-type pyocins are colicin-like, protease-sensitive proteins. They are constituted of two components. The large component carries the killing activity

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Pyocin producing strains are resistant to their own pyocin though they are sensitive to pyocin produced by others. Pyocin produced by the test strain is employed to assess the growth inhibition of 13 (1-8 and A-E) indicator strains of P. aeruginosa. Depending upon the growth inhibition of these 13 indicator strains , 105 types are recognized. Pyocin typing is the most common method for typing of P. aeruginosa.

TYPING METHODS :

TYPING METHODS PHAGE TYPING, SEROTYPING & MOLECULAR METHODS ARE OTHER TYPING METHODS.

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Toxin A - ADP ribosylates EF2 functions as NADase similar to diphtheria toxin Slime layer is anti-phagocytic Pathogenesis

Virulence factors of P. aeruginosa:

Virulence factors of P. aeruginosa Lipopolysaccharide: Endotoxin , pyrogen. Capsule: Anti-phagocytosis , inhibits complement function. Proteases: Damage host proteins such as complement and IgA. Hemolysins

Virulence factors of P. aeruginosa:

Virulence factors of P. aeruginosa Exotoxin A: A Diptheria -like toxin - toxic for macrophages. Pyocyanin: Impairs ciliary activity. Fimbriae: Adherence factor. Other non-pilus adhesions.

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Transmission Endogenous infection Contact spread Virulence Toxin and Extracellular products Protease , Pyocyanin, Cytotoxin (leukocidin) Endotoxin, Haemolysin, Exotoxin A & Exotoxin S Pathogenesis Source Endogenous (skin of axilla, perineum or GIT) Exogenous Respirators, bed pans, lotions, ointments, eye drops, stock of distilled water, disinfectants (QAC)

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Community Suppurative Otitis Hospital Eye Keratitis and Endophthalmitis Ear Otitis externa and Otitis media Skin Burns infection, wound sepsis UTI Cystitis (catheterized) RTI Pneumonia (ventilation / tracheostomy) GIT Infantile diarrhea CNS Meningitis brain abscess (iatrogenic) Diseases (Pathogenicity) Nosocomial infection Ecthyma gangrenosum

Lab Diagnosis:

Lab Diagnosis Specimens Wound discharge, sputum, urine, blood, Blue Pus is characteristic of Pseudomonas. Microscopy Gram negative bacilli MAC NLF, blue green with distinct feathered edges NA Green diffusible pigment Cetrimide agar Selective media Identification tests Oxidase + AST Ceftazidime, ticarcillin, piperacillin, ciprofloxacin, Amikacin, gentamicin, Polymyxin B Bacteriophage typing Hospital outbreak investigation Aeroginocine (Pyocine) typing 11 Indicator strains

Burkholderia pseudo mallei:

Also know as Whitmore’s bacillus. No pigment, motile. Cause Melioidosis (Glanders like disease). Usually by ingestion of contaminated food or water. Symptoms of Glanders include the formation of nodular lesions in the lungs and ulceration of the mucous membranes in the upper respiratory tract. The acute form results in coughing, fever and the release of an infectious nasal discharge, followed by septicaemia and death within days. Burkholderia pseudo mallei

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In the chronic form , nasal and subcutaneous nodules develop, eventually ulcerating. Death can occur within months, while survivors act as carriers. It has been eradicated from North America, Australia and most of Europe through surveillance and destruction of affected animals. Glanders is endemic in Africa, Asia, the Middle East, Central and South America.

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Burkholderia mallei is able to infect humans and is therefore classed as a zoonotic agent. Transmission occurs by direct contact with infected animals and entry is through skin abrasions, nasal and oral mucosal surfaces, or by inhalation. The Mallein test is a sensitive and specific clinical test for glanders. Mallein - A protein fraction of the glanders organism ( Burkholderia mallei ), is given by eye-drop. In infected animals, the eyelid swells markedly in 1 or 2 days.

Burkholderia mallei:

No pigment, non motile. Burkholderia mallei Cause Glanders in horses, donkeys and mules. Rarely cause human disease (suppurative lesions ).

Acinetobacter baumannii (calcoaceticus) :

Acinetobacter baumannii (calcoaceticus) An important Nosocomial Pathogen. Known to cause infections in debilitated patients eg. Patients admitted to ICU, NICU, MICU,. CLINICAL MANIFESTATIONS :- RTI, UTI, Septicemia, Wound Infections. EPIDEMIOLOGY : - Natural environment, Mostly in moist surfaces in hospital ( e.g. respiratory therapy instruments) Dry surfaces (eg. Human skin) rare for GNB. Occasionally as normal flora in oropharynx.

Treatment , Prevention & Control :

Treatment , Prevention & Control Antibiotic resistance is common. Empirical treatment for acute infections :- β lactam + Aminoglycosides. Specific therapy according to antibiotic sensitivity report.

KEY WORDS:

KEY WORDS Pseudomonas aeruginosa Pigments Pyocyanin Fluorescein Toxin A Opportunistic Infections Greenish Pus Non-fermenter Oxidase positive Motile Hospital infection Antibiotic resistance