CLINICAL BACTERIOLOGY GRAM NEGATIVE BACILLI & COCCOBACILLI (MAC CONKEY POSITIVE, OXIDASE POSITIVE) OUTLINE • Introduction • Acinetobacter, Stenotrophomonas, and Pseudomonas o Acinetobacter species o Stenotrophomonas maltophilia o Pseudomonas species • Vibrio, Aeromonas, Plesiomonas and Chromobacterium o Vibrio spp. o Aeromonas spp. o Plesiomonas shigelloides o Chromobacterium spp . INTRODUCTION • The nonfermenters or oxidative, gram negative bacilli are opportunistic pathogens that can colonize and cause infections in debilitated and immunocompromised hosts. . These organisms are usually found in natural water sources as well as in contaminants in medical devices, respiratory care equipment, water baths, and sinks. In addition some nonfermenters can colonize the mucous membranes and skin of humans especially when present in the hospital environment. Some non-fermenters have been implicated as contaminants in pharmaceutical compounds and intravenous solutions. These organisms are associated with nosocomial or hospital acquired infections. Classified under this category are members of family Pseudomanadaceae in which the most frequently isolated species is Pseudomonas aeruginosa, which is an important opportunistic pathogen. This organism accounts for 75-80% of all nonfermenters isolated from clinical specimens. • Vibrio organisms on the other hand are natural inhabitants of sea water, and all species except V. cholerae and V. mimicus requires increased sodium chloride for growth. All species of Vibrio can grow in the presence of increased sodium chloride. In fact, many require salt to grow. This preference for increased salt is known as halophilic and is attributed to the organism's need for increased sodium ions. Most species of Vibrio are associated with infections caused by contaminated water or seafood. Vibrio species have been isolated from the gastrointestinal tract as causes of gastritis and also have been found in blood and wound infections. Acinetobacter, Stenotrophomonas and Pseudomonas Acinetobacter species • Morphology o Acinetobacter spp. are plump coccobacilli that tend to resist alcohol decolorization o They may be mistaken for Neisseria spp.\ • Colony Characteristics o Blood Agar Plate ▪ Smooth, opaque, raised, creamy, and smaller than Enterobacteriaceae ▪ some genospecies are beta-hemolytic o MacConkey Agar Plate ▪ Non lactose fermenter ▪ Colonies exhibit a purplish hue that may cause the organism to be mistaken for a Lactose fermenter • Biochemical Testing • Pathogenesis o Clinical isolates are often colonizers. o True infections are usually nosocomial, occur during warm seasons o most commonly involve the genitourinary tract, respiratory tract, wounds, soft tissues, and bacteremia Stenotrophomonas maltophilia • Morphology o Aerobic, Non-fermentative, Gram-Negative Bacterium. o Uncommon bacterium and human infection is difficult to treat. o Initially classified as Pseudomonas maltophilia. o Thrives in wet and moist conditions. Mainly affects immunocompromised patients. • Characteristics o Slightly smaller (0.7 –1.8 × 0.4– 0.7 micrometers) than other members of the genus. o Motile (due to polar flagella). o Grow well on MacConkey agar producing pigmented colonies. o Catalase-positive, oxidase-negative (distinguishes them from most other members of genus). o Positive reaction for extracellular Dnase.
• Colony Characteristics o BAP - Large, smooth, glistening colonies with uneven edges and lavender-green to light purple pigment; greenish discoloration underneath growth; ammonia smell. o Mac Conkey - Colonies of Stenotrophomonas maltophilia on Mac Conkey Agar; Incubation period is 3 days at 30°-35°C. BAP Mac Conkey • Tests for Identification • Pathogenesis o Can lead to NOSOCOMIAL INFECTION o Ubiquitous in aqueous environments, soil and plants, including water, urine, or respiratory secretions. o CATHETERS AND IV LINES a source of infection. o Most cases of infection tends to occur through use of hospital appliances such as catheters. Pseudomonas species • Characteristics o Gram negative non-fermentative Bacilli o Strictly aerobic o Motile – single flagella at polar site o Grows well at 37-42 degree celcius. o Pseudomonas aeruginosa as most notable and common isolate among patients. o Can form tissue biofilms related to quorum sensing. • Tests for Identification o Non lactose fermenter on Mac Conkey Agar. o Exhibits Beta-Hemolysis on Blood Agar Plate. o Positive results for Oxidase test. o Triple Sugar Iron – K/NC o Blue and Green pigment production on Muehller-Hinton Agar. • Pathogenesis o Opportunistic pathogen that can cause hospital acquired infections and community acquired infections like: o Skin (folliculitis) o External ear canal (otitis externa) – Diver’s ear o Bone (osteomyelitis) o Heart (endocarditis) in IV drug abusers o Respiratory tract (patients with cystic fibrosis) o Bloodstream (bacteremia) o May also cause UTI in some cases. Vibrio, Aeromonas, Plesiomonas and Chromobacterium Vibrio spp. GENERAL CHARACTERISTICS • Gram negative and Facultative anaerobic • Curved or comma-shaped rods • Motile except Vibrio metschnikovii • Catalase and oxidase positive except Vibrio metschnikovii • All Vibrio spp. require sodium for growth and ferment glucose. • Found in Brackish or marine water • Transmission to humans is by ingestion of contaminated water, fresh produce, meat, dairy products, seafood or exposure of disrupted skin and mucosal surfaces to contaminated water.
EPIDEMIOLOGY PATHOGENESIS AND SPECTRUM OF DISEASE • As a notorious pathogen Vibrio cholera elaborates several toxins such as cholera toxin which is primarily responsible for key features of cholera. • Release of this toxin causes mucosal cells to hypersecrete water and electrolytes into the lumen of the gastrointestinal tract that result in profuse , watery diarrhea or the so called “ Rice watery stool” leading to dramatic fluid loss or severe dehydration. o Only bacteria to cause rice watery stool • V. cholera is divided into 3 subgroups : o V. cholerae O1 o V. cholerae O139 o V. cholerae non-O1 • Both O1 and non O139 are somatic antigens associated with the V. cholera envelope that are positive marker for strains capable of epidemic or pandemic spread of disease where areas non O1 and non O139 strains does not produce the toxin and hence do not produce cholera. LABORATORY DIAGNOSIS • Vibrio spp. should be collected and transported in Cary Blair medium. • Buffered glycerol saline is not acceptable because glycerol are toxic for vibrios. • Feces is preferable, but rectal swabs are acceptable during the acute phase of diarrheal illness. • V. cholera can be detected in stool using ELISA or commercially available latex agglutination test. • Stool specimens from patients with cholera are examined using dark field microscopy it will exhibit rapid darting or “shooting star motility” • Alkaline peptone water (pH 8.4) may be used as an enrichment broth obtaining growth of vibrios from stool. • Appearance: medium to large, smooth, opaque indecent with a greenish hue. V. cholera, V. mimicus and V. fluvialis can be beta hemolytic. NLF except V. vulnificus which may be LF. Aeromonas spp. • Gram-negative straight rods with rounded ends or coccobacillary facultative anaerobes that occur singly, in pairs, or in short chains • Oxidase and catalase positive and produce acid from oxidative and fermentative metabolism • Found in fresh water and brackish water or marine water with a low salt content • Mode of transmission is through ingestion of contaminated water or food like dairy, meat, produce LABORATORY DIAGNOSIS • 5% sheep blood agar, MacConkey agar and Aeromonas agar is used as a medium • Appearance: large, round, raised, opaque; most pathogenic strains are beta-hemolytic except A. caviae, which is usually nonhemolytic • Both NLF and LF • Incubate at 35°C in CO2 or ambient air for a minimum of 24 hrs • String test used to differentiate Vibrio spp. (+) from Aeromonas spp. and P. shigelloides (-)
CLINICAL SIGNIFICANCE • Aeromonas spp. produce various toxins and factors, but their specific role in virulence is uncertain. • Disease and infections includes: o Gastroenteritis, wound infections, bacteremia, and miscellaneous other infections, including endocarditis, meningitis, pneumonia, conjunctivitis, and osteomyelitis • Therapeutic options for soft tissue infections and septicemia: o ceftriaxone, cefotaxime, ceftazidime, imipenem, aztreonam, amoxicillinclavulanate, quinolones, and trimethoprim-sulfamethoxazole o Resistant to penicillin and cephalosporin Plesiomonas shigelloides • Microscopic characteristics : o pleomorphic o gram negative rods that occur: ▪ singly ▪ in pairs ▪ in short chains or ▪ in long filamentous form • Colonial appearance : o Shiny o Opaque o Smooth o Non hemolytic o Both NLF and LF(Non Lactose Fermenter; Lactose fermenter) BAP MacConkey Agar TEST FOR IDENTIFICATION • Biochemical tests: o Oxidase (+) o Gas from glucose (-) o Esculin hydrolysis (-) o Fermentation of sucrose (-) o Lysine Decarboxylase (+) o Arginine Dihydrolase (+) o Ornithine Decarboxylase (+) o Growth in 0% NaCl (+) o Growth in 6% NaCL (-) o TCBS growth (-) o String test (-) *to separate Vibrio spp. from Aeromonas spp. and P. shigelloides CLINICAL SIGNIFICANCE • P. shigelloides is similar to Vibrio spp. in terms of the types of infection they cause. • Gastroenteritis (most frequently in children) • Septicemia (in compromised adults and infants experiencing complicated delivery) • Note: The role of Plesiomonas shigelloides in intestinal infections is not always clear. Therefore, the significance of their isolation in stool specimens should be interpreted with caution Chromobacterium spp. • oxidase-positive,glucose fermenting, gram-negative bacilli capable of growth on MacConkey agar. • Chromobacterium violaceum is a facultative anaerobic, motile, gram negative rod or cocci. • Usually found in soil or water. • Transmission to humans is by ingestion of contaminated water, fresh produce, meat, dairy products, or seafood or by exposure of disrupted skin and mucosal surfaces to contaminated water. EPIDEMIOLOGY, PATHOGENESIS AND SPECTRUM OF DISEASES SPECIES VIRULENCE FACTORS • Environmental, soil & water of tropical & subtropical regions • Not part of human flora • Exposure of disrupted skin to contaminated soil or water • Endotoxin, adhesins, invasins and cytolytic proteins have been described. • Rare but dangerous infection. • Begins with cellulitis or lymphadenitis and can rapidly progress to systemic infection with abscess formation in various organs and septic shock Chromobacteriun violaceum • C. violaceum grows on most routine laboratory media. The colonies may be beta-hemolytic & have an almond like odor. • Most strains produce violacein, ethanol soluble violet pigment. • It grows well on 5% sheep blood, chocolate, and MacConkey agars; • They also grow well in the broth of blood culture systems and in thioglycollate or brain-heart infusion broths. • Negative lysine and ornithine reactions are useful criteria for distinguishing C. violaceum from Plesiomonas shigelloides. • Failure to ferment either maltose or mannitol also differentiates C. violaceum from Aeromonas spp. • C. violaceum is often resistant to β-lactams and colistin. ANTIMICROBIAL THERAPY & SUSCEPTIBILITY TESTING • Potentially active agents include cefotaxime, ceftazidime, imipenem, and aminoglycosides • Activity of penicillin is variable; activity of first and second generation of cephalosporin is poor • Grows on Mueller-Hinton agar, but interpretive standards do not exist