Lecture 12 – Non-fermenting Bacilli (NFB) PDF

BIOL 2010 Lecture 12 – Non-fermenting Bacilli (NFB) BAILEY AND SCOT T’S – CHAPTER 20, 21 DIAGNOSTIC MICROBIOLOGY – CHAPTER 21 In this lecture Explain the characteristics and pathogenesis of commonly encountered clinically significant microorganisms, including the most frequently isolated specie...

BIOL 2010 Lecture 12 – Non-fermenting Bacilli (NFB) BAILEY AND SCOT T’S – CHAPTER 20, 21 DIAGNOSTIC MICROBIOLOGY – CHAPTER 21 In this lecture Explain the characteristics and pathogenesis of commonly encountered clinically significant microorganisms, including the most frequently isolated species by body system Gram negative Rod - Non-fermenting bacilli Pseudomonas aeruginosa Acinetobacter baumanii Stenotrophomonas maltophilia Discuss bacterial virulence, as related to these organisms General characteristics of NFB Pseudomonas aeruginosa – most common NFB Acinetobacter baumanii – 2nd most common NFB Stenotrophomonas maltophilia – 3rd most common NFB Characteristics All GNB All grow on MAC Called non-fermenting bacteria (NFB) as they do NOT ferment Glucose All have increased resistance to antimicrobials Ubiquitous in nature and hospital environment Pseudomonas spp. – General characteristics Account for the largest percentage of all non-fermenters isolated from clinical specimens Called Pseudomonads – most pathogenic member is Pseudomonas aeruginosa Most members are Gram-negative bacilli or coccobacillus Strictly aerobic Motile with polar flagellum Oxidase and Catalase positive Usually oxidize carbohydrates Usually grow on MAC agar Pseudomonas aeruginosa Aerobic Most commonly isolated NFB (Non-Fermenting Bacteria) Ubiquitous in our environment Found in plants, soil, water, sewage Found in aqueous environments (showers, taps, pools, contact lens solution) Isolated from humidifiers in hospitals May colonize intestines of animals and humans Have been isolated form infusion fluids, disinfectants, cosmetics and food Pseudomonas aeruginosa - Infections Can be normal flora in gut P. aeruginosa is an opportunistic pathogen, and affects high risk population Patients with severe immunodeficiencies Patients with medical devices and mechanically ventilated Cystic Fibrosis Patients Burn Victims Nosocomial Infections - Widespread in hospitals infecting patients Accounts for 5-15% of nosocomial infections Leading cause of nosocomial respiratory tract infections Transmitted from patient-to-patient and direct patient contact with environmental reservoirs (sinks, taps, respiratory equipment, disinfectants, food) Pseudomonas aeruginosa - Infections Identified as a causative agent of a great variety of infections Meningitis Wound Infections (Especially Burn Wounds) Bacteremia UTI’s (nosocomial) Endocarditis Keratitis (inflammation of the cornea) Pneumonia Chronic Respiratory Infections in Cystic Fibrosis (CF) patients Otitis Externa (Swimmer’s Ear) in people participating in water sports Folliculitis in users of contaminated/underchlorinated hot tubs/pools Hot tub disease Pseudomonas aeruginosa – Virulence factors Lipopolysaccharide - Antiphagocytic activity, cytotoxicity Pili - Adhesion Flagella - Motility, adhesion Type III secretion system - Cytotoxic activity Phospholipases - Cytotoxicity Proteases - Cytotoxicity, proteolytic activity Exotoxin A - Cytotoxicity Capsule - Antiphagocytic activity Deoxyribonuclease (DNase) - Breaks down DNA Elastase - Cytotoxicity, proteolytic activity Lecithinase - Cytotoxicity, proteolytic activity Hemolysins - Cytotoxicity Pseudomonas aeruginosa – cellular morphology Slender Gram Negative Bacilli Pseudomonas aeruginosa – cultural characteristics Aerobe Non-fastidious Can grow at 42C Often used as QC for 42C incubator Pigment producer (More visible in clear agar) MOST common = pyocyanin Less common: Red  Called pyorubin Brown-Black  pyomelanin Yellow  pyoverdin Pseudomonas aeruginosa – cultural characteristics Pseudomonas aeruginosa – cultural characteristics Odor – fruity, grape-like Growth on MAC Non-lactose fermenter Pigment may be present Growth on BAP (typical colonies) Colonies are large with an irregular shape (Swarming growth pattern) Colonies display a metallic sheen Beta Hemolytic Mucoid Pseudomonas aeruginosa Mucoid Pseudomonas aeruginosa Alginate production Mucoid exopolysaccharide produced by P. aeruginosa Repeating polymer of mannuronic and glucuronic acid Forms the matrix of the Pseudomonas biofilm which anchors the cells to their environment In medical situations, it protects the bacteria from the host defenses such as lymphocytes, phagocytes, the ciliary action of the respiratory tract, antibodies and complement Mucoid strains most often isolated from patients with cystic fibrosis (usually found in lung tissue) Pseudomonas aeruginosa - ID Motile (single polar flagella) Oxidase - Positive Catalase - Positive Nitrate - Positive (usually beyond nitrite to nitrogen gas) Arginine Dihydrolase (ADH) positive Citrate - Positive OF Test - Non-Fermenter (Oxidizer of Carbohydrates) TSI - Alk/Alk or K/K Growth at 42C - Positive Pseudomonas aeruginosa - Treatment Innately resistant to many antimicrobial agents such as: Penicillin, ampicillin, first- and second-generation cephalosporins, trimethoprim- sulfamethoxazole (SXT), chloramphenicol, and tetracyclines P. aeruginosa is usually susceptible to: Aminoglycosides Semi-synthetic penicillins such as piperacillin and ticarcillin, third- and fourth-generation cephalosporins (ceftazidime and cefepime, respectively) Carbapenems (except ertapenem) Fluoroquinolones. Treatment of severe infections requires combination therapy Ex. ceftazidime or cefepime, piperacillin, or a carbapenem (imipenem or meropenem) with an aminoglycoside (tobramycin or amikacin). Pseudomonas aeruginosa - Treatment Resistance can develop during treatment – hence susceptibility testing is crucial Multi-drug resistant P. aeruginosa Also called MDRP Infections with MDRP can be fatal in critically ill patients Common in hospital-acquired infections In short: P. aeruginosa is quite a deadly bug. ID of this organism from clinical sites is very important. Pseudomonas aeruginosa - Treatment Resistance can develop during treatment – hence susceptibility testing is crucial Multi-drug resistant P. aeruginosa Also called MDRP Infections with MDRP can be fatal in critically ill patients Common in hospital-acquired infections In short: P. aeruginosa is quite a deadly bug. ID of this organism from clinical sites is very important. Other Pseudomonas P. fluorescens and P. putida Low virulence Rare infections: UTI, Respiratory, Abscess, Wounds etc. Produce pyoverdine Do NOT grow at 42C P. stutzeri Wrinkled, leathery colonies on BAP (image) Can cause infections in immunocompromised: septicemia, meningitis, endocarditis, UTI etc. Acinetobacter - General Two species most commonly seen in clinical specimens Acinetobacter baumanii Acinetobacter iwolfii Ubiquitous in environment and hospital settings Colonize human hosts – skin, pharynx High resistance to antimicrobial agents – including carbapenems Multi-drug resistant strains are emerging (MDRA) Acinetobacter baumanii - Infections Opportunistic pathogens – target immunocompromised hosts 1-3% of healthcare associated infections Associated infections UTI Ventilator-associated pneumonia and sepsis – 22% mortality! Septicemia Meningitis Eye infections Acinetobacter baumanii – cellular morphology Gram Negative coccobacilli Can retain crystal violet – beware of false stain Sometimes they are VERY small – look like cocci Especially fluids like Blood Cultures ALWAYS remember to look at MAC growth to ensure it’s a NFB GPC and GNC should not grow on MAC Acinetobacter baumanii – colonial morphology Strict aerobes Very few growth requirements Grow happily on MAC – NLF Some species will produce purplish tinge and make colonies look LF (Ex. image) Grow on BAP Smooth, opaque, raised, creamy, and smaller than Enterobacterales; some genospecies are beta-hemolytic Acinetobacter baumanii – ID Oxidase negative Catalase positive Nonmotile TSI: K/NC or NC/NC Glucose OF: +/- Lysine Decarboxylase negative Acinetobacter baumanii – Treatment Often resistant to many antimicrobials such as Penicillins, first- and second-generation cephalosporins, fluoroquinolones, carbapenems Carbapenem resistance occurs due to carbapenemases and are called CRAB CRAB isolates are usually only susceptible to colistin and tigecycline A. baumannii demonstrates variable susceptibility to Aminoglycosides, β-lactam plus β-lactamase inhibitor combinations (e.g., ampicillin- sulbactam, piperacillin-tazobactam). Susceptibility testing required to determine susceptibility pattern. Stenotrophomonas maltophilia Not normal flora, but considered colonizer in hospitalized patients Infections occurs in immunocompromised hosts and primarily hospital-acquired Common infections: Endocarditis, wound infections, bacteremia Rare: meningitis and UTI Also isolated from CF patients Treatment Inherently resistant to: Cephalosporins, penicillins, carbapenems, aminoglycosides Common choice for treatment: SXT Other possible choices for treatment: combination drugs, tetracyclines MUST perform susceptibility testing Stenotrophomonas maltophilia Cellular morphology Gram Negative Bacilli Colonial morphology Colonies can have a strong ammonia smell On MAC: Non Lactose Fermenter with bluish tinge On BAP: Large, smooth, glistening colonies with uneven edges and lavender-green to light purple pigment; greenish discoloration underneath growth Stenotrophomonas maltophilia Oxidase negative Catalase positive Motility positive Dnase positive Esculin positive Gelatinase positive Lysine Decarboxylase positive Comparisons P. aeruginosa A baumanii S. maltophilia Cell. Morphology Slender GNB Small GNB (can be GNB coccoid, and resist decolorization) Col. Morph - MAC NLF (may have NLF (May have purple- NLF (may have bluish pigment) on MAC ish tinge) on Mac tinge) on MAC Metallic sheen on BAP NH, small, wet on BAP NH, small, wet on BAP Fruity Smell Ammonia odor Oxidase Positive Negative Negative Motility Positive Negative Positive TSI K/K K/K K/K OF test Oxidizer Oxidizer Oxidizer Knowledge Check 1 Which of the following may not grow on MacConkey agar? a. P. aeruginosa b. A. baumanii c. S. maltophilia d. H. influenzae Knowledge Check 1 Which factor contributes to the formation of biofilms by P. aeruginosa? A. Exotoxin A B. Pyoverdine C. Alginate D. Exoenzyme S Knowledge Check 1 The primary drug of choice for S. maltophilia is A. Trimethoprim-sulfamethoxazole B. Piperacillin C. Tobramycin D. Levofloxacin