Pseudomonasan update

Dr.T.V.Rao MD

Dr.T.V.Rao MD 1

What are Pseudomonas • Pseudomonads are aerobic, saprophytic

and innately resistant bacteria causing opportunist infections in man, animals, plants and insects. The most important pseudomonad species responsible for human infections are Pseudomonas aeruginosa, Burkholderia pseudo mallei and members of the Burkholderia cepacia complex.Dr.T.V.Rao MD 2

Pseudomonas

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• A large group of aerobic, non sporing gram negative bacteria motile by polar flagella

• Found In nature water, soil, other moist environments

• Some of them are pathogenic to plants• Creation of new genera such as

Burkholderia. Stenotrophomnonas

Pseudomonas aeruginosa

• Gram-negative aerobe bacteria

• Commonly found in the environment– At any moist location

• Common cause of nosocomial infections

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- Widely distributed in soil and water

- Gram negative rods- Aerobic- Motile- Produce water-

soluble pigments• Opportunistic

pathogens

General Characteristics

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Pseudomonas • Aerobic,

opportunistic pathogen

• Gram-negative bacillus

• FlagellatedDr.T.V.Rao MD 6

Structure of Pseudomonas

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Pseudomonas aeruginosa

•Morphologyslender, Gram negative bacillussize-1.5 microns-3*1.5micronsmotile by polar flagellanon capsulated though some mucoid

strains may sometimes occursome are pilated

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Morphology

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• They are slender gram negative bacillus, 1.5 – 3 microbes x 0.5 microns

• Monoflgellar ?• Non capsulated but many strains have mucoid

slime layer• Isolates from Cystic fibrosis patients have

abundance of extracellular polysaccharides composed of alginate polymers

• Escape the defence mechanisms by loose capsule in which micro colonies of bacillus are enmeshed and protected from host defences.

Cultural characteristicsObligate aerobeWide range of temperature

5°c-42°c optimum 37°cOrdinary media – large, opaque, irregular, with

distinctive musty, mawkish, earthy smell

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P. aeruginosaForms round colonies with a fluorescent greenish color, sweet odor, and b-hemolysis.

Pyocyanin- nonfluorescent bluish pigment;

pyoverdin- fluorescent greenish pigment;

pyorubin, and pyomelanin

Some strains have a prominent capsule (alginate).

Identification of P. aeruginosa is usually based on oxidase test

and its colonial morphology: b-hemolysis, the presence of

characteristic pigments and sweet odor, and growth at 42 oC. Dr.T.V.Rao MD 11

Cultural Characters

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• Obligate aerobe, but grow anaerobically if nitrate is available

• Growth occurs at wide range of temperatures 6-42 c the optimum being 37 c

• Growth on ordinary media producing large opaque irregular colonies with distinctive musty mawkish or earthy smell.

• Iridescent patches with metallic sheen are seen in cultures on nutrient agar.

• In broth forms dense turbidity with surface pellicle.

Motile (by single or multiple polar flagella) gram-negative rods

Obligate (strict) aerobes (most strains)

Oxidase (usually) and catalase positive

Nonfermentative chemoheterotrophic respiratory metabolism

Characteristics of Pseudomonas aeruginosa

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Characteristics of Pseudomonas

• Minimal nutritional requirements .; Many organic compounds used as C and N sources, but only a few carbohydrates by oxidative metabolism

• Glucose used oxidatively• Lactose negative on MacConkey agar

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Nutrient agar-Cont..

• Nutrient agar- Colonies are

smooth,large,translucent,low convex,2-4mm in diameter.

Produce sweetish aromatic odor

Greenish blue pigment diffuses

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Growing on • Blood agarSimilar to nutrient agar Many are haemolytic• Mac conkey agarColourless,non lactose fermenters• Cetrimide agar selective media

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Pigment Production

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Some strains produce diffusible pigments: • Pyocyanin (blue); fluorescein

(yellow); pyorubin (red) P. aeruginosa produces characteristic

grape-like odor and blue-green pus & colonies

Broad antibiotic resistance

• Toxic extracellular products in culture filtrates

• Exotoxin A and S• Exotoxin A acts as

NADase resembling Diphtheria toxin

• Proteases,elastatese hemolysins and enterotoxin

• Slime layer and Biofilms

Toxins and enzymes in pseudomonas

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Biochemical reactions

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• Oxidative and Non fermentative• Glucose is utilized oxidatively• Indole, MR and VP and H2 S tests

are negative• Catalase, Oxidase, and Arginine

tests are positive

Typing and Importance

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• Important cause of Hospital Infections• Important for epidemiological purpose• Serotyping• Bacteriocins typing• Pyocyanin• Aeruginosa typing• Restriction endonuclease typing with pulsed

gel electrophoresis

Resistance

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• Killed at 55oc in on 1 hour• High resistance to chemical agents• Resistance to quaternary ammonium

compounds.Chlorxylenol• Resistant to Hexachlorophenes• Grows also in antiseptic bottles• Dettol as Cetrimide as selective medium• Sensitive to acids silver salts, beta glutaraldehyde

Major Pathogenicity

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• Blue pus• Causing the nosocomial infection• Suppurative otitis • Localised and generalised infections• Urinary tract infection after catheterization• Iatrogenic meningitis• Post tracheostomy pulmonary infections

Pathogenesis and Immunity

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• P. aeruginosa can infect almost any external site or organ.

• P. aeruginosa is invasive and toxigenic. It attaches to and colonizes the mucous membrane or skin, invade locally, and produces systemic diseases and septicemia.

• P. aeruginosa is resistant to many antibiotics. It becomes dominant when more susceptible bacteria of the normal flora are suppressed.

• Septicaemia• Endocarditis• Ecthyma gangrenous• Infantile diarrhoea• Shanghai fever• Disabling eye

infections• Survive with minimal

nutrients

Clinical Presentations

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P. aeruginosa infections• P. aeruginosa infections are of

particular concern for Cystic fibrosis patients

• Burn patients• Hospitalised patients

–Case mortality rate for patients infected with P. aeruginosa approaches 50%

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Pseudomonas aeruginosa an important opportunistic pathogen

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• Pseudomonas aeruginosa is an opportunistic pathogen, meaning that it exploits some break in the host defences to initiate an infection. In fact, Pseudomonas aeruginosa is the epitome of an opportunistic pathogen of humans. The bacterium almost never infects uncompromised tissues, yet there is hardly any tissue that it cannot infect if the tissue defences are compromised in some manner

P. aeruginosa is an opportunistic pathogen

• Extremely broad host spectrum• Hardly any infections in the normal human host• Severe immunodeficiencies and medical devices

predispose the patients to P. aeruginosa infections• Broad spectrum of clinical symptoms

– Urinary tract infections – Pulmonary infections– Soft tissue infections– Sepsis– Bone and joint infections– Endocarditis

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P,aeroginosa is an opportunistic pathogen

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• P,aeroginosa is an opportunistic pathogen. It rarely causes disease in healthy persons. In most cases of infection, the integrity of a physical barrier to infection (eg, skin, mucous membrane) is lost or an underlying immune deficiency (eg, neutropenia, immunosuppression) is present. Adding to its pathogenicity, this bacterium has minimal nutritional requirements and can tolerate a wide variety of physical conditions

WHO are MORE SUSCEPTIBLE TO INFECTION

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• This bacterium is of particular concern to individuals with cystic fibrosis who are highly susceptible to pseudomonas lung infections. Pseudomonas aeruginosa is also of grave concern to cancer and burn patients as well as those people who are immunocompromised. The case fatality rate for individuals infected with Pseudomonas aeruginosa approaches 50 percent.

Nosocomial infections• Fourth most common isolated nosocomial

pathogen accounting for approx. • 10 % of all hospital acquired infections.• Patient-to-patient spread and direct patient

contact with environmental reservoirs– disinfectants, – respiratory equipment, – food, – sinks, taps

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Pseudomonas prominent hospital acquired infections

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• It causes urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteraemia, bone and joint infections, gastrointestinal infections and a variety of systemic infections, particularly in patients with severe burns and in cancer and AIDS patients who are immunosuppressed.

Virulence Factors (structural components)

• “Alginate”• Adherence proteins and “pili• Lipopolysaccharide (LPS)• Pyocyanin

Virulence factors

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• Respirators• Endotracheal

tubes• Can be Infected • All equipment's

to be sterilized

Infection of Equipment's

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• Pseudomonas aeruginosa is the most frequently encountered lung pathogen in patients with cystic fibrosis (CF). Following initial, often intermittent, episodes of infection, it becomes a permanently established component of the chronically infected lung in more than 80% of patients and confers an adverse prognosis

Pseudomonas and Cystic fibrosis

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Pigment production• Pyocyanin

Bluish green phenazine pigment Soluble in chloroform and waterNot produced by other species

• Pyoverdin(fluorescein)It is a greenish yellow pigment Insoluble in chloroform but soluble in waterProduced by many other species

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Pyocyanin Pyoverdin

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Cont..• PyorubinReddish brown pigment Insoluble in chloroform but soluble in

water• pyomelaninBrown to black pigmentProduction is uncommon

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Biochemical reactionsO/F test-

oxidativeCatalase-

positiveOxidase-positiveNitrate

reduction-positive Dr.T.V.Rao MD 38

Biochemical Tests• Indole test-negative• Methyl red test-

negative• Vp test-negative• Citrate test-positive• Urease test-negative

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Structural Components• Adherence to host cells mediated by pili and nonpilus

adhesins. • LPS (lipopolysaccharide) inhibiting antibiotic killing

and suppress neutrophil and lymphocyte activity• Alginate – mucoid exopolysaccharide that forms a

shiny biofilm protecting from antibodies, complement, phagocytosis, and antibiotics

• Procyanin – impairs ciliary function, mediates tissue damage through production of oxgen radicals

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Patho mechanisms• Adhesion

– Pile, flagella and fimbriae• Invasion

– Extracellular enzymes and toxins (proteases, elastase, phospholipases, rhamnolipids, Exotoxin A)

• Dissemination– Leukocidin inhibits neutrophils und leukocytes– LPS (Endotoxin)

• Protection– Capsule (Alginate)

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PathogenesisVirulence Factors (toxins and enzymes):

• Exotoxin A• Exoenzyme S• Endotoxins• Phospholipase C• Elastase and Alkaline Protease

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PATHOGENESIS• Important agent in causing nosocomial

infections• Most common infections are Urinary tract infections following

catheterizationAcute purulent meningitis following lumbar

puncturePost-tracheostomy pulmonary infectionSepticemia in debilitated patients

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Mechanism Of Pathogenesis

• Caused by exotoxins,proteases,elastases,haemolysins,lipases and enterotoxins

Exotoxin A-lethal toxinElastases-haemorrhagic lesions Enterotoxins-diarrhoeal diseaseSlime layer acts as a capsule and enhances

virulence

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Exotoxin A• Similar in structure to Diphtheria toxin • Inhibits protein synthesis by ADP-ribosylating

EF-2 (G-protein)• Causes Dermatonecrosis in burn wounds,

corneal damage in ocular infections, and tissue damage in chronic pulmonary infections.

• Also this toxin is immunosuppressive• ADP-ribosylates G-proteins including p21 RAS

interfering with host cell growthDr.T.V.Rao MD 45

Phospholipase C

• Heat labile hemolysins• Breaks down lipids and

lecithin causing tissue destruction

• Stimulates inflammatory response Dr.T.V.Rao MD 46

Lactase and Alkaline Protease

• Destruction of elastin-containing tissues (blood vessels, lung tissue, skin), collagen, immunoglobulin's, and complement factors

• Can produce hemorrhagic lesions (ecthyma gangrenous) associated with disseminated infection

• Inactivation of interferon and TNF-AlphaDr.T.V.Rao MD 47

Epidemiology• Ubiquitous in moist environmental sites

in the hospital as well as nature• No seasonal incidence• Can transiently colonize the respiratory

and GI tract of hospitalized patients• Minimal nutritional requirements and

can tolerate broad temperature spectrum

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Important cause of Hospital Acquired Infection

• Pseudomonas aeruginosa is an important cause of hospital-acquired infections, especially in intensive care units and in neutropenic patients. Infections range from topical to systemic and may be trivial or life-threatening.

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Clinical Disease• Pulmonary Infections• Burn Wound Infections and other skin

and soft tissue infections (life threatening)

• UTI’s (especially catheterized)• External Otitis (malignant OE, swimmer’s

ear)• Eye Infections and corneal ulceration via

contaminated contact lens cleaning fluids• Pseudomonal Endocarditis

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Pulmonary Infections• Can range from asymptomatic colonization to severe

necrotizing bronchopneumonia• Colonization is seen in patients with cystic fibrosis,

chronic lung disease, and neutropenia• Mucoid strains are commonly isolated from chronic

pulmonary patients and are more difficult to eradicate

• Predisposing conditions include previous therapy with broad spectrum abx (disrupts normal protective bacteria population and use of respiratory therapy equipment (can introduce the organism to lower airways)

• Mortality rate can be as high as 70% for invasive bronchopneumonia Dr.T.V.Rao MD 51

Pseudomonas PNA

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Ecthyma Gangrenosum• Ecthyma gangrenosum is a well

recognized cutaneous manifestation of severe, invasive infection by Pseudomonas aeruginosa that is usually seen in immunocompromised, burn patients, and other critically ill patients

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Echtyma Gangrenosum

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Malignant Otitis Externa

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Pseudomonas Keratitis and Corneal Ulceration

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Endocarditis

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Cystic fibrosis• Most common life-threatening

inherited genetic disorder in the Caucasian population

• Mutation in the cystic fibrosis trans membrane conductance regulator (CFTR) gene

• one in every 25 carry the mutated recessive gene and more than 1 in 4000 live births suffer from CF.

• Dr.T.V.Rao MD 58

Chronic infection of the Cystic Fibrosis lung

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Pseudomonas and urinary tract infections

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• Pseudomonal UTIs are usually hospital-acquired and are associated with catheterization, instrumentation, and surgery. These infections can involve the urinary tract through an ascending infection or through bacteriuic spread. In addition, these infections are a frequent source of bacteraemia. No specific characteristics distinguish this type of infection from other forms of UTI.

Diagnosis of P.aeroginosa infection

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• Diagnosis of P,aeroginosa infection depends upon isolation and laboratory identification of the bacterium. It grows well on most laboratory media and commonly is isolated on blood agar or eosin-methylthionine blue agar. It is identified on the basis of its Gram morphology, inability to ferment lactose, a positive oxidase reaction, its fruity odour, and its ability to grow at 42°C. Fluorescence under ultraviolet light is helpful in early identification of P.s aeruginosa colonies. Fluorescence is also used to suggest the presence of P. aeruginosa in wounds.

Fluorescence of Pseudomonas• Fluorescence under

ultraviolet light is helpful in early identification of P.s aeruginosa colonies. Fluorescence is also used to suggest the presence of P. aeruginosa in wounds.

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Diagnosis of P. aeruginosa

• Isolation and lab identification of the pathogen• P. aeruginosa grows well on most laboratory media • Identified on the basis of its:

– Gram morphology, – inability to ferment lactose, – a positive oxidase reaction, – its characteristic odor, – its ability to grow at 42° C. – Fluorescence is helpful in early identification of P.

aeruginosa colonies and may also help identify its presence in wounds.

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What antibiotics to use

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• Aminoglycosides• Gentamycin, Amikacin,

Cephalosporins • Cefotaxime. Ceftazidime. Ofloxacin,• Piperacillin, Ticarcillin• Local application, colistin,

polymyxin

Treating pseudomonas infections

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• Combined antibiotic therapy is generally required to avoid resistance that develops rapidly when single drugs are employed. Avoid using inappropriate broad-spectrum antibiotics, which can suppress the normal flora and permit overgrowth of resistant pseudomonads.

Treatment of P. aeruginosa infections

• P. aeruginosa is frequently resistant to many commonly used antibiotics.

• To archive synergy a combination of e.g. gentamicin and carbenicillin is frequently used.

• No vaccines so far

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Pseudomonas aeruginosa a resistant pathogen

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• Pseudomonas aeruginosa is frequently resistant to many commonly used antibiotics. Although many strains are susceptible to gentamicin, tobramycin, colistin, and amikacin, resistant forms have developed. The combination of gentamicin and carbenicillin is frequently used to treat severe Pseudomonas infections. Several types of vaccines are being tested, but none is currently available for general use.

Antibiotic Resistance• Inherently resistant to many antibiotics• Can mutate to more resistant strains

during therapy• Penetration of abx highly dependent on

outer membrane pores which can be altered

• Production of B-lactamases• Combination of active antibiotics

generally required for successful therapy

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P. aeruginosaPrevention and Control

Pseudomonas spp. normally inhabit soil, water, and vegetation and can be isolated from the skin, throat, and stool of healthy persons.

Spread is mainly via contaminated sterile equipment's and cross-contamination of patients by medical personnel.

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Prevention and Control

• High risk population: patients receiving broad-spectrum antibiotics, with leukaemia, burns, cystic fibrosis, and immunosuppression.

• Methods for control of infection are similar to those for other nosocomial pathogens. Special attention should be paid to sinks, water baths, showers, hot tubs, and other wet areas.Dr.T.V.Rao MD 70

BURKHOLDERIA• Although most of the

presently 30 known Burkholderia species are saprophytes, B. pseudomallei, B. mallei and the B. cepacia complex are important human or animal pathogens

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Burkholderia pseudomallei

• Burkholderia pseudomallei is the causative agent of melioidosis, a serious tropical infection of man and animals endemic in South-East Asia and Northern Australia

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B. pseudomallei • The environmental saprophyte

B. pseudomallei causes melioidosis, a life-threatening tropical infection of man and animals in Southeast Asia and Northern Australia. In endemic areas the organism is found in soil and surface water, particularly rice paddy fields and monsoon drains. Isolation rates are highest during the rainy season and in still rather than flowing water.

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Other related organisms • A closely related species, B. mallei,

causes glanders, a potentially fatal infectious disease of horses, mules and donkeys. Laboratory-acquired infection with either organism is a serious risk (hazard category 3) and both are regarded as potential bioterrorism agents.

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B. cepacia• B. cepacia, the cause of

soft rot of onions, is also an important human pathogen causing life-threatening respiratory infection in immunocompromised patients, particularly those treated in intensive care or with chronic granulomatous disease. Dr.T.V.Rao MD 75

B.cepacia and Cystic fibrosis

• In cystic fibrosis, anxiety over B. cepacia is based on the innate multiresistance of the organism to antibiotics, its transmissibility by social contact, and the risk of cepacia syndrome, an acute, fatal necrotizing pneumonia, sometimes accompanied by bacteraemia, which occurs in 20-30% of infected patients.

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Virulence factors in B.cepacia

• B. cepacia complex isolates produce several putative virulence determinants, including proteases, catalases, haemolysin, exopolysaccharide, cable pili and other adhesins. In vitro the lipid A of these bacteria stimulates pro-inflammatory cytokines tenfold more than Ps. aeruginosa lipid A.

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Pathogenesis• Human infection is acquired mainly

percutaneously through skin abrasions or by inhalation of contaminated particles, especially during monsoon rains. Melioidosis commonly presents as pyrexia. The clinical manifestations are protean and range from dormant subclinical infection,.

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Diagnosis • Diagnosed by the presence of specific

antibodies, to acute pneumonia or chronic pulmonary infection that may resemble tuberculosis and other conditions, leading to a fulminating septicaemia with a mortality rate of 80-90%.. Suppurative parotitis is a characteristic presentation of melioidosis in children

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Treatment • Accurate and early diagnosis, including

culture of B. pseudomallei, and appropriate antibiotic therapy are key to successful management. The optimum treatment of severe melioidosis is unclear. Intravenous ceftazidime, followed by a combination of co-trimoxazole and doxycycline, is emerging as the treatment of choice.

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Treatment is a Urgent Concern

• Intravenous ceftazidime, followed by a combination of co-trimoxazole and doxycycline, is emerging as the treatment of choice. Imipenem or meropenem have sometimes been effective when treatment with ceftazidime has proved unsuccessful. Prolonged treatment is necessary to avoid relapse

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