Gram Negative Rods

Gram Negative Rods Cytochrome oxidase (+) Family: Vibronaceae Family: Enterobacteriaceae Campylobacteriaceae Pseudomonaceae

3 Genera: A) Vibrio B) Aeromonas

(-)

Family: Vibronaceae

C) Plesiomonas

Biotypes V. cholerae 1) El tor – most frequent causative agent of cholera (Philippines) 2) Classical

rarely cause disease in man

Genus Vibrio Species: 1) V. cholerae 2) V. parahaemolyticus V. Cholerae - most important human pathogen Morphology: + short gram negative rod (0.5u X 1.5u to 3.0u) + curved/comma – shaped (Kommabacillus) + on stained smear – appear lying parallel to one another®”fish in the stream“ arrangement ==== + motile ® single, thick polar flagellum scintillating/darting Biochemical/Cultural Charac.: = facultative anaerobe = opt. temp. 18 – 370C = grow best at alkaline ph (extremely basophilic up to pH 9) = Sensitive to acid ph → kill organism = Non – lactose fermenter but ferment sucrose and mannose = Metabolism both respiratory and fermentative CM:

= large, smooth, flat yellow colonies with opaque center & transparent periphery TSI Acid/Acid (-) gas (-) H2S IMViC (+ - + +) = Possess lysine and ornithine decarboxylase

MEA (Meat Extract Agar)

= Translucent colony with iridescent green → red bronze color (viewed on oblique light) Selective CM: TCBS (Thiosulfate Citrate Bile Sucrose)

V. cholerae

V. cholerae 1)

El Tor

1) Voges – Proskawer test + 2) Agglutination to chicken rbc. + 3) Sensitivity 50u Polymixin B R 4) Choleraphage sensitivity R

Classical

S S

-

Disease – Asiatic Cholera: = food and waterborne disease = acquired fecal – oral route (contaminated food/H2O) = man only host infection = incubation period 2-3 days = serious disease charac. sudden onset of voluminous, non–bloody watery diarrhea (rice water stool) containing flakes of mucus, non-odorous accompanied with severe dehydration (hallmark of disease) = no fever, untreated cases 60% die = high attack rate – children = organism non – invasive (remains localized GIT) Determinant of Pathogenicity: 1) Cholera enterotoxin/Choleragin (heat labile) = resp. watery diarrhea = activate adenylcyclase of intestinal mucosal cell ↓ increase intracellular cyclic adenosine monophosphate (CAMP) ↓ hypersecretion of H2O & Cl into the intestinal lumen ↓ inhibition of + Na absorption ↓

diarrhea 2) Invasiveness of organism + ability to adhere or attached to microvilli of brush border of epithelial cells. Serological types – based on antigenic differences 1) Ogawa – A & B 2) Inaba – A & C 3) Hikojima – A, B, C

= virulence may be associated with prod. heat stable hemolysin (kanagawa) that hemolyze human red blood cell which is cytotoxic and cardiotoxic Disease: Gastroenteritis/Foodpoisoning = self-limiting disease = charac. nausea, vomiting, explosive, watery diarrhea (cholera – like) ↓ (without blood and mucus) = symptoms may persist for 10 days

Lab. Diagnosis: 1) Direct stool exam.– Alkaline Peptone broth incubated for 6 hours (darkfield microscopy → motility) 2) Stool culture – MEA / TCBS incubated 2 – 3 days 3) Direct Fluorescent Antibody Test Treatment: = Prompt replacement of fluid and electrolytes = Tetracycline/Furazolidone (shorten the course of disease and decrease) bacterial secretion Prevention: 1) Maintenance of adequate sewage treatment 2) H2O purification 3) Prompt detection & treatment of cases = vaccine no significant protection V. Parahaemolyticus = marine/saltwater organism = found estuaries throughout the world = major cause gastroenteritis involving seafood's Morphology: = closely related V. Cholerae Cultural/Biochemical charac.: = extremely halophilic (salt-loving, req. 2% NaCl) = oxidase (+) = non–lactose fermenter CM – TCBS = large, smooth, green colonies (non–sucrose fermenter) Determinants of Pathogenicity:

MOT = ingestion of raw or improperly cooked seafoods(shellfish) Lab. Diagnosis: 1) Rectal – Swab transport medium Cary – Blair/Amies ↓ TCBS & Alkaline Peptone broth Treatment: Chloramphenicol, Kanamycin, Tetracycline

Differentiation: V. Cholera

V. Parahaemolyticus

1. Growth 2% Na Cl 2. Sucrose fermentation + 3. Kanagawa test -

+ +

Genus Campylobacter Campylobacter jejuni = worldwide = most important human pathogen Morphology: = gram (-) spirally curved rods (seagull-wing shaped) = motile (single polar flagellum) ↓ darting motility = oxidase and catalase (+) = asaccharolytic (non-carbohydrate fermenter) = microaerophilic (CO2 ¯O2) = grows best at 42 – 430C (thermophilic) = susceptible to gastric acid

= H2S producer = fastidious Culture Media: Skirrous agar/Campy-BAP incubated with 10% CO2 5%O2 85%N2 ↓ colorless convex colonies watery and spreading Determinants of Pathogenicity: 1) heat – labile enterotoxin = activate adenylcyclase enzyme → increase CAMP → split of Cl → prevent absorption of Na+ → massive diarrhea (bloody mucoid stool) Disease: Enterocolitis = self – limiting disease lasting less than 7 days = one of the most common cause of infectious disease = symptoms includes: acute crampy abdominal pain, vomiting, bloody diarrhea with pus, malaise and fever = incubation period 1 – 7 days Reservoir and MOT: = organism exist as normal GI tract flora of many wild and domestic animals (dogs) = human infection is acquired through ingestion of raw or undercooked meat

Lab. Diagnosis: 1) Gram staining (demonst. gull-wing shaped of organism) 2) Darkfield microscopy for motility 3) Microaerophilic culture on special media (Skirrous) incubated at 420C with 5% O2 10% CO2 85% N2 Rx.: Aminoglycosides, Tetracycline & Chloramphenicol Genus Helicobacter Species: Helicobater pylori (Campylobacter pylori)

Morphology: = = = = =

curved or S – shaped gram negative organism motile (4 – 6 lopotrichous flagella) urease and oxidase (+) microaerophile killed at acid ph

Habitat: = gastric mucosa where it invade tissues and causes gastritis and ulcers = grows at ph 6 –7 Disease: Anthral Gastritis/Chronic Gastritis Duodenal ulcers (no direct evidence that H. pylori) causes duodenal ulcers but there is strong evidence between the presence of H. pylori and duodenal ulcer MOT = fecal–oral route (contaminated food & H2O) Lab. Diagnosis: 1) Microaerophilic culture of biopsy material incub. 370C 2) Histologic Examination 3) Urease test 4) Culture – Skirrous agar (selective CM) 370C ↓ small, circular, translucent colonies after 7 days incub. Treatment: Bismuth + Metronidazole + Tetracycline Genus Pseudomonas Specie: Pseudomonas aeruginosa = gram negative, non-fermentative, bacilli that inhabit soil and water = important opportunistic pathogen among individual with impaired host defense = human infection severe and difficult to treat = major agent of nosocomial infection = exhibit broad resistant to many antimicrobials = biochemically inactive = considered to be the 3rd most frequently isolated agent of hospital-acquired pneumonia Morphology: = gram negative rod, motile – single polar flagellum = piliated and slime layer present Biochemical/Cultural Charac.: = strictly aerobic

= opt. temp. for growth 350C = beta–hemolytic BAP = Pseudomonas agar- special media which enhance pigment production: colonies appears turquioise blue due to (pyocyanin) pigment which is water soluble and fluorescent (pyoverdin) pigment which is greenish color Determinants of Pathogenicity: 1. Colonization to appropriate site by the pathogen = Pili (enable org. to attach to host cell) = Slime layer/glycocalex enhances adherence to tissue 2. Hemolysin (phospholipase and Glycolipids) = contribute to invasiveness of organism 3. Protease enzyme = resp. hemorrhagic skin lesions 4. Enterotoxins = resp. diarrhea 5. Elastase = impt. in corneal infection Clinical infections: = occurs in individual with altered host defenses = Meningitis, burned-wound infection, GUT, malignant disease, etc. Lab. Diagnosis: 1. Isolation & bacteriological demonstration of organism 2. Culture Rx: Aminoglycosides (Amikacin, Gentamycin, Tobramycin) Prevention: 1. Proper isolation procedure 2. Hyperimmune gamma – globulin = decrease mortality rate of infection 3. Vaccine – Pseudogen (heptovalent vaccine) Family Enterobacteriaceae = composed large groups of organism found in soil, H2O, decaying matter and large intestine of humans, animals and insects = habitat → Gastrointestinal tract of humans “Enteric bacilli”

Members of the Family: Opportunistic members Escherichia Klebsiella lactose fermenters-pink colonies Enterobacter Proteus Morganella Citrobacter Serratia colonies Providencia Edwardsiella

non-lactose fermenter-colorless

Intestinal pathogens Salmonella Shigella non-lactose fermenter-colorless colonies Yersenia

Gen. Morphology: = small, gram negative non-sporeforming rod = most are motile (peritrichous flagella) except Shigella and Klebsiella which are non-motile = all are noncapsulated, except Klebsiella = some are provided with fimbriae/pili Biochemical/Cultural Charac.: = facultative anaerobe = grows readily on ordinary media = ferment CHO under anaerobic or low O2 atmosphere = ferment glucose with the production of acid or both acid and gas = reduce nitrate to nitrite = oxidase (-) = culture medium for isolation: Differential: EMB, Mc Conkey Highly selective: Hektoen Enteric agar, SSA, XLA Resistance: = destroyed easily by heat and low concentration of common germicide and disinfectants = bactericidal to phenols, formaldehyde, glutaraldehyde = bacteriostatic to quaternary ammonium compound = chlorination of water impt. in controlling dissemination

of intestinal pathogen like agent of TF = relatively sensitive to drying Antigenic Structure: = plays an important role in epidemiological classification of Enterobacteriaceae O H major antigen used in serologic typing K of the organism

Determinants of Pathogenicity: 1) Endotoxin = LPS of the cell wall®toxicity resides in lipid A = heat - stable = effect of endotoxin causes: Fever, Fatal shock, Leucocytic alteration, Disseminated intravascular coagulation (DIC), Shwartzman reaction 2) Enterotoxin = heat-labile usually affect small intestine causing transduction of fluid into intestinal lumen→diarrhea 3) Shiga toxin and Verotoxins = interferes with protein synthesis of mammalian cells = unclear role in Shigellosis VTEC toxin - important cause of hemolytic diarrhea and hemolytic uremic syndrome (HUS) 4) Colonization factor = cellular surface structure which plays a role in the establishment of organism in the host Laboratory Diagnosis: = specimen includes sputum, tissue, pus, body fluids, rectal swab/feces A) Culture (commonly used media) Differential (EMB, Mc Conkey) Selective (Hektoen agar, SS agar) B) Biochemical test TSI, IMViC, LIA, Carbohydrate fermentation C) Serologic (used primarily for epidemiologic purposes) Treatment: = remains a major therapeutic problem = several factors contribute to the difficulty of treating these infection = one of the most important factor is:

A) presence of underlying disease of the

patient B) emergence of resistant organism due to indiscriminate use of antibiotics