Pseudomonas aeruginosa gram negative bacilli

Gram Negative non fermenter oxidase positive organism

1. PSEUDOMONAS AERUGINOSA Presenter: Dr Payal Moonka Moderator : Dr Vidushi Topno SENIOR RESIDENT DEPT OF MICROBIOLOGY RIMS, RANCHI.

2. HISTORY In 1894, German botanist Walter Migula coined the term Pseudomonas for a genus he described as, “Cells with polar organs of motility. T he description of Pseudomonas as “false unit” does not make much sense, The Name Pseudomonas was simply created for the resemblance of the cells to those of the nanoflagellate Monas in both size and active motility.

3. TAXONOMY

4. Non-fermenting gram-negative bacilli Strict aerobes Most strains are motile by means of one or more polar flagella . Oxidase positive Pigment producing Major pathogen among the hospitalized patients and in patients with cystic fibrosis. Typical smell (fruity grape-like odour of aminoacetophenone)

5. Pseudomonas aeruginosa is a dominant pathogen in people with cystic fibrosis (CF) contributing to morbidity and mortality. They are ubiquitous mostly saprophytic , being found in water, soil or other moist environment. They can dwell well in the soap solutions with hexachlorophene, detergents and disinfectants. Cidex (2% aqueous alkaline solution of glutaraldehyde ) is suitably active against Pseudomonas aeruginosa

6. Environmental factors :- • Pseudomonas are commonly found in environment , like in soil and water •Most common type causing infection among humans are Pseudomonas aeruginosa At risk populations :- •Patients in healthcare settings especially those who -Are on ventilators -Have devices such as catheters -Have open wounds from surgery or burns EPIDEMIOLOGY

7. Phenotypic Classification of the Pseudomonads (Polar Flagellated NFBs) rRNA Group I Fluorescent Group Pseudomonas aeruginosa Pseudomonas fluorescens Pseudomonas putida Stutzeri Group Pseudomonas stutzeri Pseudomonas mendocina Alcaligenes Group Pseudomonas alcaligenes Pseudomonas pseudoalcaligenes Pseudomonas species group 1 Other Clinically Relevant Pseudomonas Pseudomonas andersonii Pseudomonas fulva Pseudomonas otitidis

8. Importantly, Pseudomonas aeruginosa is one of the MDR ESKAPE pathogens which stand for: Enterococcus faecium Staphylococcus aureus Klebsiella pneumoniae Acinetobacter baumannii Pseudomonas aeruginosa Enterobacter

9. Pseudomonas in Healthcare associated Infections P. aeruginosa is a common cause of nosocomial infections, manifesting as pneumonia, surgical site infections, urinary tract infections and bacteremia. It is estimated that P. aeruginosa has a prevalence of 7.1%–7.3% amongst all healthcare-associated infections. Healthcare-associated pneumonia (HAP) and ventilator-associated events (VAE) are a significant source of stress on the healthcare system, and they account for up to 22% of all healthcare-acquired infections. The global prevalence of VAE due to P. aeruginosa as 4.1%, and P. aeruginosa was the most common cause of VAE globally, accounting for 26% of cases. TAKEN FROM https://doi.org/10.1007/s40265-021-01635-6

10. P. aeruginosa is a common cause of nosocomial urinary tract infections (UTI), particularly catheter-associated UTI (CAUTI). P. aeruginosa accounts for approximately 10% of all CAUTIs, and up to 16% of UTIs in ICU patients . Nosocomial UTI secondary to P. aeruginosa is associated with high morbidity and mortality and bacteremia is a potential complication.Additionally, P. aeruginosa CAUTI is associated with high rates of antimicrobial resistance.

11. VIRULENCE FACTORS ALGINATE: Capsular polysaccharide that allows infecting bacteria to adhere to lung epithelial cell surfaces and form biofilms which, in turn, protect the bacteria from antibiotics and the body’s immune system. PILI: Surface appendages that allow adherence of organism to GM-1 ganglioside receptors on host epithelial cell surfaces. NEURAMINIDASE: Removes sialic acid residues from GM-1 ganglioside receptors, facilitating binding of pili. TAKEN FROM KONEMAN’S COLOR ATLAS AND TEXTBOOK

12. EXOTOXIN A Tissue destruction, inhibition of protein synthesis; interrupts cell activity and macrophage response ENTEROTOXIN Interrupts normal gastrointestinal activity, leading to diarrhea EXOENZYME S Inhibits protein synthesis PHOSPHOLIPASE C Destroys cytoplasmic membrane; destroys pulmonary surfactant; inactivates opsonins LIPOPOLYSACCHARIDE Produces endotoxin, causes sepsis syndrome: fever, shock, oliguria, leukopenia or leukocytosis, disseminated intravascular coagulation, metabolic abnormalities

13. ELASTASE Cleaves immunoglobulins and complement components, disrupts neutrophil activity LEUKOCIDIN Inhibits neutrophil and lymphocyte function PYOCYANIN Suppress other bacteria and disrupt respiratory ciliary activity; cause oxidative damage to tissues, particularly oxygenated tissues such as lung

14. TAKEN FROM https://doi.org/10.1007/s40265-021-01635-6

15. PIGMENT PRODUCTION

16. Pseudomonas aeruginosa causes a wide spectrum of infections with varying severity, examples include: TAKEN FROM https://doi.org/10.1007/s40265-021-01635-6 Pseudomonas folliculitis is characterized by a maculopapular pruritic rash, axillary lymphadenopathy, breast tenderness, and fever. It is associated with the use of hot tubs, jacuzzis, and pools. Puncture wounds in the foot, especially in children, can lead to infection with Pseudomonas . A classical scenario is a nail or glass injury through rubber shoes or soles. Local tenderness, purulent discharge, other signs of infection are found; however, delay in the presentation and diagnosis due to lack of symptoms initially may predispose to serious complications like osteomyelitis and septic arthritis.

17. In cystic fibrosis exacerbations, pseudomonal coverage is always needed. Chronic infection with Pseudomonas aeruginosa occurs in more than 60% of adults with cystic fibrosis and is linked to higher mortality. In patients with burns, Pseudomonas infections were found to decline and are replaced by gram-positive organisms and other emerging gram-negative organisms e.g., Aeromonas hydrophila . In pseudomonal burn infections, a blue-green purulent discharge may be found, among other signs of local and systemic inflammation, and it may progress to sepsis and septic shock.

18. Patients with diabetes are more likely to develop malignant otitis externa caused by Pseudomonas and present with otalgia and otorrhea. Examination frequently reveals granulation tissue inside the ear canal. Populations at-risk are extremely susceptible to pneumonia caused by Pseudomonas . Pseudomonas pneumonia should be considered in any patient who has signs and symptoms of pneumonia and who is immunocompromised. While patients with an organ transplant in general, are susceptible to all systemic pseudomonal infections, kidney transplant patients are at increased risk of recurrent pseudomonal urinary tract infections. Other examples include endophthalmitis, endocarditis, meningitis, and many others

19. PATHOPHYSIOLOGY P. aeruginosa can be traced everywhere including hospital environments and cause serious infection of almost any organ. Lipopolysaccharide induces inflammatory responses in the lung after bacterial infection, epithelial cells secrete cytokines and chemokines, thereby recruiting and activating innate immune cells and adaptive immune cells. The recruitment of neutrophils is a sign of inflammatory response activation. Although the activation of neutrophils is critical for host defense, excessively activated immune cell infiltration will cause severe tissue damage and aggravate bacterial infections. Therefore, studying the balance between the virulence factors secreted by bacteria and corresponding host immunity is important for the treatment of infections TAKEN FROM https://doi.org/10.1007/s40265-021-01635-6

20. Specimen Pus Wound swab Urine Sputum Blood CSF Endotracheal Aspirate Bronchoalveolar lavage Direct Smear - Slender gram-negative bacilli Motility of the bacteria can be seen by HANGING DROP METHOD LABORATORY DIAGNOSIS OF PSEUDOMONAS INFECTIONS

21. Nutrient Agar Colony Morphology: Color: Bluish-green or greenish-blue colonies. Appearance: Irregular, spreading colonies with a mucoid or slimy texture. Size: Medium to large-sized colonies. Texture: Slimy or viscous appearance due to the production of extracellular polysaccharides. CULTURE Growth Patterns Growth Rate: Moderate to fast growth. Pseudomonas aeruginosa typically exhibits rapid growth on nutrient agar, forming colonies within 24-48 hours. Colonial Characteristics: Individual colonies may appear rough or wrinkled, with irregular edges and a shiny surface.

22. SIX DISTINCT COLONY TYPES ON NUTRIENT AGAR Type 1 : most common and easily recognised , large , rough in appearance and often oval with the long axis in the line of inoculum streak. Type 2 : small , smooth , domed , described as coliform. Type 3 and Type 4 : also small and appear rough and rugose Type 5 : the copious exopolysaccharide produced may result in merging of colonial growth and may eventually drip onto the lid of the Petri dish . Type 6: Dwarf colony

23. Blood agar plate demonstrating the typical appearance of Pseudomonas aeruginosa after 24-hour incubation at 35°C. Colonies are large with a spreading periphery and are frequently beta hemolytic. Colonies in the heaviest area of growth appear to have a metallic sheen and a scaling appearance sometimes described as alligator skin morphology. Colonies appearing like this typically produce a sweet grape-like odor. Blood agar is a non-selective medium commonly used for the cultivation of a wide range of microorganisms, including Pseudomonas aeruginosa for the observation of hemolytic activity. Beta-hemolysis: Complete lysis of red blood cells around the colonies, indicating beta-hemolytic activity. BLOOD AGAR

24. MacConkey agar plate demonstrating growth of a mucoid variety of Pseudomonas aeruginosa typical of the strains isolated from the sputum of patients with cystic fibrosis. Colonies can be extremely mucoid and runny. several colony variants can be seen, which is the typical presentation for these mucoid variants of P. aeruginosa. In the heaviest growth area, there appears to be some pyocyanin pigment developing . TAKEN FROM KONEMAN’S COLOR ATLAS AND TEXTBOOK

25. Cetrimide Agar Cetrimide agar is a selective medium designed to isolate Pseudomonas species, including Pseudomonas aeruginosa , from mixed cultures: Colony Morphology Color: Bluish-green or greenish-blue colonies Appearance: Smooth or slightly mucoid colonies. Size: Variable, ranging from small to large-sized colonies. Texture: Glistening or shiny appearance, metallic sheen. Growth Patterns: Selective Inhibition: Cetrimide in the agar selectively inhibits the growth of other bacterial species, allowing for the selective isolation of Pseudomonas aeruginosa.

26. Biochemical Properties • Oxidase and catalase positive • Non-fermenter , utilises sugars oxidatively. Tested by OF test • ICUT test: -Indole test is negative -Citrate test: positive -Urease test: Negative -Triple sugar iron (TSI) test Alkaline slant/alkaline butt with no gas and no H2S.

27. The oxidative-fermentative (OF) test. Fermentative organisms produce acid in both the closed and open tubes (yellow); oxidative organisms produce acid only in the open tube. Asaccharolytic organisms that do not use carbohydrates produce no change in either tube. TAKEN FROM KONEMAN’S COLOR ATLAS AND TEXTBOOK

28. Typing Method • For Surveillance & epidemiological purpose Phenotypic methods Bacteriocin (pyocin) typing Antibiogram typing- It classifies the organism into different groups based on their resistance pattern to different antimicrobials. Since antimicrobial susceptibility testing is routinely done in any hospital, this typing system provides the first clue to a microbiologist about outbreaks occurring in a hospital. Serotyping - O and H antigens, 17 serotypes It refers to a typing method based on the antigenic property of an organism. This is the most widely used and the most reliable phenotypic method. •Molecular methods -Pulse field gel electrophoresis (PFGE) -Sequence based typing method

29. Antipseudomonal Agents - Penicillins : Piperacillin ,ticarcillin - Cephalosporins : Ceftazidime,cefoperazone,ceftolozane, cefepime - β-lactam/β-lactamase inhibitor combinations ( piperacillin-tazobactam and cefoperazone-sulbactam) - Carbapenems : Imipenem, meropenem and doripenem - Monobactam : Aztreonam - Aminoglycosides : Tobramycin, gentamicin, amikacin - Quinolones : Ciprofloxacin, levofloxacin - Polymyxins : Polymyxin B, colistin

30. Drug Resistance Possesses a number of drug resistant plasmids which confer resistance to several antibiotics. Many strains are producers of β lactamases , such as ESBL (extended spectrum β lactamases), Carbapenemases , and AmpC β lactamases. Many strains are resistant to aminoglycosides and quinolones.