1. Objectives By the end of this lecture the student must be:  A) Identify the genus Pseudomonadaceae & Vibrionaceae B) describe the chemical tests for this genus C) Differentiate between different sps. D) List and match the symptoms, diagnosis and treatment for different sps.

2. Pseudomonas aeruginosa  Gram-negative bacilli belonging to Pseudomonadaceae  Motile by single polar flagellum, non spore  Non fastidious- Minimal nutritional requirements  Produces grape-like odor and blue-green pus and colonies  Two types of soluble pigments: 1. Pyoverdin : Yellow-green pigment & fluorescent 2. Pyocyanin : Blue-green pigment and non-fluorescent  Strict aerobic, O+/F-, Oxidase and catalase positive  Optimum temperature is 37 0 C & able to grow at 42 0 C  Resistant to dyes, weak antiseptics and many antibiotics 2

3. Epidemiology  The most important pathogenic is Ps. aeruginosa  Opportunistic pathogen  Important agent in causing nosocomial infections  Ubiquitous in moist environment, hospital, in soil and water  Colonized in the intestine in10% of human  Represents 10-20% of hospital-acquired infections  Transiently colonize respiratory & GIT of hospitalized patients: 1.Treated with broad-spectrum antibiotics 2.Exposed to respiratory therapy equipment 3.Hospitalized for long periods 3

4. Pathogenesis 1. Adhesins  Fimbriae, Pilli, Alginate (mucoid exo-polysaccharide)  Alginate slim forms biofilm that protect from antibodies, complement, phagocytosis & antibiotic 2.Invasins a. Proteases - Inactivate IFN and TNF i. Elastase Break down of elastin-containing tissues e.g. blood vessels, lung tissue, skin Cleaves collagen, IgG, IgA and complement) Produce hemorrhagic lesions associated with disseminated infection ii. Alkaline protease (lyses fibrin) b.Hemolysins (phospholipase & lecithinase) & Leukocidin 4

5. Pathogenesis 3. Toxins  Exotoxin A & S  Inhibit protein synthesis (Like diphtheria toxin)  Immunosuppressive  Causes dermatonecrosis in burn wounds, corneal damage in ocular infections, and tissue damage in chronic pulmonary infections  Pyocyanin – inhibits mitochondrial enzymes, impairs ciliary function, mediates tissue damage  LPS (endotoxin) 5

6. Clinical Finding 1.UTIs (Catheterized patients) 2.Wound, burn & other skin & soft tissues infections  Echtyma Gangrenosum 3.Pneumonia (Cystic Fibrosis) 4.Eye infection- Contaminated contact lens clearing fluids 5.Chronic otitis media & otitis externa (swimming pool) 6.Meningitis- following lumbar puncture 7.Systemic infections- Septicemia in debilitated patients 8.Pseudomonal Endocarditis 6

7. Echtyma Gangrenosum Causative agent: Ps. aeruginosa Under risk: immunocompromised, burn patients, and other critically ill patients Round or oval (1-15cm) severe invasive cutaneous ulceritic single or multiple lesion with halo of erthryma Pseudomonas exotoxin : Tissue destruction Elastase degrades elastin in blood vessels wall Phospholipase C degrades phospholipids in cell membranes Pyocyanin generates reactive oxygen species 7

8. Typing methods 1.Bacteriocin typing  Three types of bacteriocins are produced-R,F,S  Pyocin produced by test strain is employed to assess the growth inhibition of 13 indicators strains  Depending upon the growth inhibition of 13 indicators strains,105 types are recognized  Most popular method used 2.Phage typing 3. Serotyping -based on O & H,17 serotypes are recognized 8

9. Laboratory diagnosis  Specimen  Urine, pus, sputum, CSF, blood, skin swap  Microscopical Examination  Gram-negative rods and motile  Cultural Characteristics  On Nutrient agar:  Colonies are surrounded by bluish green coloration  On selective media "Cetermide"  Pigments are more obvious  On Blood agar:  -hemolytic colonies  On MacConkey agar  Pale yellow colonies i.e. non lactose fermenters  Ps. aeruginosa able to grow at 42 0 C for 3 days  Pyocin typing: Identification for epidemiological purpose 9

10. Vibrionaceae General characteristics Gram negative, curved, comma shaped bacilli Motile by single polar flagella Non spore, Non capsulated Facultative anaerobes, Fermentative Oxidase and catalase positive Most vibrios have relatively simple growth factor requirements and grow well in alkaline pH Natural inhabitants : Aquatic environment 10

11. Vibrio cholerae >200 serogroups based on O-antigen V. cholerae O1 (classical and El Tor biotypes) and O139 are primarily responsible for cholera outbreaks – V. El Tor biotype causes cholera-like but milder Recently, V. cholerae O75 and O141 strains has been associated with cholera-like diarrhea Some O1 strains do not produce cholera enterotoxin (atypical or nontoxigenic O1 V. cholerae ) Cholera is endemic in southern Asia (India, Pakistan and Bangladesh), Latin America 11

12. Pathogenesis Mode of Transmission – Consumption of contaminated water or food Incubation period: 1-4 days High infectious dose: >10 8 CFU V. cholerae attach to mucosa of small intestine V. cholerae multiply & secrete enterotoxin (cholargen) Toxin binds to specific receptor on the intestinal mucosal cell Toxin stimulates activity of cAMP, resulting in active secretion of chloride & secondary loss of Na and H 2 O The patient loss 20 Litre fluids/day V. cholerae cause same disease as ETEC but more severe 12

13. Diagnosis of V. cholerae Specimen: Rice watery stool or rectal swab Culture on Alkaline Peptone Water (APW) then subculture on selective differential medium Thiosulfate Citrate Bile Salts Sucrose agar Selective due to alkalinity pH9 & contains bile salts Differential because contains sucrose Sucrose fermenting V. cholerae  yellow colonies Sucrose non fermenting V. parahemolyticus appears as blue to green colonies Sucrose fermentation is gold standard in identification 13

14. Diagnosis of V. cholerae Any sucrose fermenting colonies were subjected to Gram stain and oxidase test Gram stain – Gram negative short rods, comma shaped, motile Biochemical reactions: – Oxidase positive – O+/F+ – Cholera red reaction –TCBS agar (Figure) (Thiosulfate-citrate-bile salts-sucrose agar) Serology: – Diagnosis confirmed & serotyping done by agglutination with specific antisera (O1, O139) 14

15. V. parahaemolyticus It is the cause of acute gastroenteritis following ingestion of contaminated sea-food such as raw fish Marine organism which live in high salt conc. So it requires  2% Na Cl in the isolation medium ◦ It is invasive affecting the colon ◦ V. cholerae is noninvasive, affecting the small intestine through secretion of an enterotoxin  Allied Vibrios are large group of organisms; some are saprophytic while others cause disease in animals 15