1. Nonfermenters (Those that are Gram-negative bacilli)

2. Nonfermenters Nonfermenters are found in nature as inhabitants of soil and water and as harmless parasites on the mucous membranes of man and other animals. Nonfermenters can cause disease when they colonize and subsequently infect immunocompromised individuals or when they gain access to a normally sterile body site through trauma.

3. Nonfermenters Nonfermenters only comprise a small percentage of the total clinical isolates, but they require more effort for identification. Classification No family designation Includes many genera whose names are continually changing By definition they do not ferment glucose Morphology and cultural characteristics

4. Nonfermenters Gram-negative bacilli (g-b) or coccobacilli (g-cb) Most are nonfastidious and are isolated by the same means used to isolate the Enterobacteriaceae. Some grow on a Mac plate and others do not On CBA plates, morphology, size, hemolytic activity, and pigmentation may provide valuable information for identification

5. Gram stain - Acinetobacter

6. P. aeruginosa – green pigment

7. P. aeriginosa

8. Nonfermenters They are obligate aerobes and most will not grow or will grow poorly under anaerobic conditions. Some require 48-72 hours for growth Most grow best at 37 0 C, but a few grow better at RT (Pseudomonas fluorescens and Pseudomonas putida) Biochemistry and identification Initial clues that an organism is a NF include Lack of glucose fermentation (TSI= K/K) May be oxidase +

9. Nonfermenters May fail to grow on a Mac plate May be unusually resistant to antibiotics Additional testing – must identify the method or scheme to be used The frequency of isolation of the different NF is used to select tests that will best be able to identify the most commonly isolated organisms (those that you looked at in the lab). What is the most commonly isolated NF?

10. Nonfermenters The CDC scheme of identification separates organisms into 8 groups based on: Growth versus no growth on Mac Oxidase test results O/F results Further testing might include: Motility (by polar flagella) Nitrate reduction or denitrification Urease production Esculin hydrolysis Indole – use Ehrlichs rather than Kovacs reagent because Ehrlichs is more sensitive

11. Nonfermenters Rapid decarboxylation reactions Pigment production growth in cetramide Phenylalanine deaminase Growth at 42 0 C Virulence factors that are extracellular products (Pseudomonas aeruginosa) Expression is under control of two component signal transduction, quorum sensing systems. When the bacteria detects a critical concentration of an autoinducer released by the organism, a signal transduction cascade will trigger the expression of these products:

12. Quorum sensing

13. Nonfermenters Elastolytic proteases Elastin is a constituent of lung tissue and blood vessels. The damage caused by the elastotytic proteases causes an inflammatory reaction that compromises the host and aids in the dissemination of the organism. Alkaline proteases These proteases may degrade complement and IgA, thus hindering the immune response. Exotoxin A (iron limitation also contributes to inducing its expression) which is the most toxic product produced by Pseudomonas aeruginosa. It is cytotoxic for eukaryotic tissue culture cells and lethal for many mammals (LD 50 in mice= 60-80 ng.). The mechanism of action is to interfere with protein synthesis by ADP-ribosylation of elongation factor 2. The liver is a prime target for this toxin.

14. Nonfermenters Exotoxin S – ADP-ribosylates vimentin, a structural component of the host cell, and GTP-binding proteins Phospholipase C – a hemolysin that may be involved in the breakdown of phospahtidyl choline, a major surfactant of the lung, leading to pulmonary collapse. Leukocidin Pyocyanin- a secreted pigment that is toxic due to its involvement in the generation of reactive oxygen intermediates (superoxide radical and hydrogen peroxide)

15. Nonfermenters Virulence factors – (P. aeruginosa ) cell surface: Both pilin and non-pilus adhesions are important for attachment LPS – endotoxin Iron capturing ability Flagella Alginate synthesis Forms a viscous gel around the bacteria May function as an adhesion and may also function to prevent phagocytosis Antimicrobic resistance – due to outer membrane changes

16. Polar flagella of P. aeruginosa

17. Nonfermenters Virulence factors – Burkholderia Produce exotoxins Clinical significance Pseudomonas aeruginosa (most frequently isolated NF) Is a major cause of nosocomial infections Commonly found in burn wounds Is associated with chronic pulmonary disease in patients with cystic fibrosis (these patients have a dysfunction of the exocrine glands result in secretion of abnormal viscid mucous) where very mucoid isolates are usually isolated.

18. Mucoid strains of P. aeruginosa in cystic fibrosis patient

19. Nonfermenters Cause 70% of otitis externa (swimmer’s ear) Dermatitis associated with contaminated whirlpool baths Stenotrophomonas maltophilia (second most frequently isolated NF) Is part of the transient NF of hospital patients and causes a wide variety of nosocomial infections Is sxt sensitive

20. Nonfermenters Acinetobacter Is found in soil and water and as part of the skin NF Is a common colonizer and less commonly a cause of nosocomial infections Chryseobacterium meningosepticum Occasionally found causing meningitis and septicemia Moraxella M. lacunata causes conjunctivitis and keratitis in the malnourished alcoholic population Burkholderia – two species are true pathogens

21. Nonfermenters B. pseudomallei Causes melioidosis, a disease seen primarily in southeast Asia where it is a normal inhabitant of soil and water. The disease is acquired through contamination of wounds or via inhalation or ingestion. The disease may range from unapparent, to chronic or acute pulmonary infection, to overwhelming septicemia with multiple abscesses in many organs B. mallei Causes glanders in equines. Humans occasionally acquire the disease by contact with infected nasal secretions of equines, through abrasions and occasionally through inhalation. Used to be a problem in the military when horses where used.

22. Nonfermenters The disease may manifest as a chronic pulmonary disease, as a form characterized by multiple abscesses of the skin, subcutaneous tissue, and lymphatics (Farcy), or as an acute, fatal septicemia. Antimicrobial susceptibility Antimicrobial suseptibility testing is essential since these organisms tend to be even more resistant than the Enterobacteriaceae.