Nonfermenting Gram Negative Bacteria.pptx12

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Nonfermentering Gram Negative Bacteria Dr.T.V.Rao MD

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Growing Importance of Non-Fermenting Gram Negative Bacteria Non fermenting Gram Negative Bacteria are complex group of Bacteria with few defined characteristics, Many times discarded in Diagnostic Microbiology as Contaminants. The emerging challenges associated with Antibiotic resistance is a concern to Physicians, All Medical Microbiologists should update the Knowledge and improve the Diagnostic facilities in the Laboratories for better care of the patients

Nonfermenters can Cause Medically Important Infections

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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.

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Nonfermenters are Ill defined 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

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Most Common Non-Fermentative Gram-Negative Bacteria Pseudomonas aeruginosa (most common) Acinetobacter species (second most common) Stenotrophomonas maltophilia (third most common)

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Other Clinically Prevalent Gram-Negative Non Fermenters Pseudomonas stutzeri Burkholderia cepacia Burkholderia pseudomallei Moraxella Achromobacter xylosoxidans

Non-Fermentative Gram-Negative Bacteria

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Nonfermentative for glucose (TSI = alkaline over no reaction) Oxidative for glucose (Hugh-Leifson O-F glucose positive) Asaccharolytic for glucose (HughLeifson O-F glucose negative) Cytochrome oxidase positive or negative

Hugh-Leifson OF versus TSI Medium TSI AGAR SLANT Total protein = 2.6 g% Total carbohydrate = 2.1 g% Protein to carbohydrate (w/w) = 1.2 OF BROTH MEDIUM Total protein = 0.2 g% Total carbohydrate = 1.0 g% Protein to carbohydrate (w/w) = 0.2

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Growth of Gram-Negative Non-Fermenters on TSI Agar Slants  Non-fermentative gramnegative bacteria grow abundantly within 16-18 hours of inoculation on the surface of TSI agar slants.  Non-fermentative gramnegative bacteria neither grow in nor acidify the deep of TSI slants.

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Growth of Oxidative Non-Fermenters in Hugh-Leifson Broth Growth with acidification of broth in Hugh-Leifson tube not sealed by mineral oil (oxidative tube) No growth in Hugh-Leifson tube sealed by a layer of mineral oil (fermentative tube) Substrates utilized: glucose, lactose, maltose, xylose, Mannitol, sucrose, adonitil, dulcitol

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Growth of Asaccharolytic Non-Fermenters in Hugh-Leifson OF Broth

 Growth without acidification but with Alkalinization of broth in HughLeifson tube not sealed by mineral oil (oxidative tube)  No growth in HughLeifson tube sealed by a layer of mineral oil (fermentative tube)

The CDC scheme of identification

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 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

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Classification of Pseudomonads1 Burkholderia cepacia (family Burkholderiaceae) rRNA Group II Cytochrome oxidase +, OF glu +, motile, polymyxin B resistant Burkholderia pseudomallei (family Burkholderiaceae) rRNA Group II Cytochrome oxidase +, OF glu +, motile, polymyxin B resistant Stenotrophomas maltophilia (family Xanthomonadaceae) rRNA Group V Cytochrome oxidase –, OF glu +, OF maltose ++, motile, polymyxin B susceptible 1Pseudomonads

are separated into five taxonomically distinct ribosomal RNA homology groups.

Classification of NonPseudomonads

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Acinetobacter baumannii (family Moraxellaceae) Cytochrome oxidase –, OF glu +, OF lactose ++, non-motile Moraxella (family Moraxellaceae) Cytochrome oxidase +, OF glu – (asaccharolytic), non-motile Achromobacter xylosoxidans (family Alcaligenaceae) Cytochrome oxidase +, OF glu +, OF xylose +, motile

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Biochemical Tests to be performed for Identification Rapid decarboxylation reactions Pigment production growth in cetramide Phenylalanine deaminase Growth at 420 C

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Virulence Factors 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 auto inducer released by the organism, a signal transduction cascade will trigger the expression of these products:

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Pseudomonas aeruginosa  Pseudomonas aeruginosa is an

opportunistic pathogen, meaning that it exploits some break in the host defenses to initiate an infection. In fact, Pseudomonas aeruginosa is the epitome of an opportunistic pathogen of humans. The bacterium almost never infects uncompromised tissues, yet there is hardly any tissue that it cannot infect if the tissue defenses are compromised in some manner

Characters of Pseudomonas aeruginosa

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 Pseudomonas aeruginosa is a Gram-negative rod measuring 0.5 to 0.8 µm by 1.5 to 3.0 µm. Almost all strains are motile by means of a single polar flagellum. The bacterium is ubiquitous in soil and water, and on surfaces in contact with soil or water. Its metabolism is respiratory and never fermentative, but it will grow in the absence of O2 if NO3 is available as a respiratory

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Colony characters differ  P. aeruginosa isolates may produce

three colony types. Natural isolates from soil or water typically produce a small, rough colony. Clinical

samples, in general, yield one or another of two smooth colony types. One type has a friedegg appearance which is large, smooth, with flat edges and an elevated appearance. Another type, frequently obtained from respiratory and urinary tract secretions, has a mucoid appearance, which is attributed to the production of alginate slime. The smooth and mucoid colonies are presumed to play a role in colonization and virulence.

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Virulence Factors 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 (LD50 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.

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Virulence Factors  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)

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Virulence Factors 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

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Stenotrophomonas maltophilia: Natural Habitats Widely distributed including moist hospital environments (respiratory therapy equipment) Colonizer of human respiratory tract in a hospital setting

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Stenotrophomonas maltophilia: Modes of Infection  Colonization of hospital patients by environmental sources  Introduction of organisms into normally sterile sites by medical instrumentation (similar to Acinetobacter)

Stenotrophomonas maltophilia

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Stenotrophomonas maltophilia (second most frequently isolated NF) Is part of the transient Normal flora of hospital patients and causes a wide variety of nosocomial infections

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Stenotrophomonas maltophilia: Microbiological Properties  Short to medium-size, straight gram-negative rods  Glucose oxidizer (OF glu +) with occasional negative strains (~15%)  Strong maltose oxidizer (OF mal +) (100%) (more intense than OF glu + reaction)  Colonies on sheep blood agar rough and lavender-green with ammonia odor

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Stenotrophomonas maltophilia  Stenotrophomonas maltophilia is an aerobic, nonfermentative, Gram-negative bacterium. It is an uncommon bacterium and human infection is difficult to treat. Initially classified as Pseudomonas maltophilia, S. maltophilia was also grouped in the genus Xanthomonas before eventually becoming the type species of the genus Stenotrophomonas in 1993.[  S. maltophilia are slightly smaller (0.7–1.8 × 0.4– 0.7 micrometers) than other members of the genus. They are motile due to polar flagella and grow well on MacConkey agar producing pigmented colonies. S. maltophilia are catalase positive, oxidase negative (which distinguishes them from most other members of the genus) and have a positive reaction for extracellular DNase.

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Stenotrophomonas maltophilia: Microbiological Properties  Cytochrome oxidase negative  Positive for DNase (unlike most other glucoseoxidizing gram-negative bacilli)  Positive for lysine decarboxylase (unlike most other glucoseoxidizing gram-negative bacilli)  Resistant to most antibiotics except trimethoprimsulfamethoxazole

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S. Maltophilia Pathogenesis  S. maltophilia frequently colonizes breathing tubes such as

endotracheal or tracheostomy tubes, the respiratory tract and indwelling urinary catheters. Infection is usually facilitated by the presence of prosthetic material (plastic or metal), and the most effective treatment is removal of the prosthetic material (usually a central venous catheters or other device). The growth of S. maltophilia in microbiological cultures of respiratory or urinary specimens is therefore sometimes difficult to interpret and not a proof of infection. If, however, it is grown from sites which would be normally sterile (e.g., blood), then it usually represents true infection.

Burkholderia pseudomallei Burkholderia pseudomallei (also known as Pseudomonas pseudomallei) is a Gramnegative, bipolar, aerobic, motile rod-shaped bacterium. It infects humans and animals and causes the disease melioidosis. It is also capable of infecting plants.

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Burkholderia pseudomallei

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 B. pseudomallei is not fastidious and will grow on a large variety of culture media (blood agar, MacConkey agar, EMB, etc.). Ashdown's medium (or Burkholderia cepacia medium) may be used for selective isolation.] Cultures typically become positive in 24 to 48 hours (this rapid growth rate differentiates the organism from B. mallei, which typically takes a minimum of 72 hours to grow). Colonies are wrinkled, have a metallic appearance, and possess an earthy odour. On Gram staining, the organism is a Gram-negative rod with a characteristic "safety pin" appearance (bipolar staining). On sensitivity testing, the organism appears highly resistant (it is innately resistant to a large number of antibiotics including colistin and gentamicin) and that again differentiates it from B. mallei, which is in contrast, exquisitely sensitive to a large number of antibiotics.-

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Burkholderia pseudomallei  Colonies are wrinkled, have a metallic appearance, and possess an earthy odour. On Gram staining, the organism is a Gram-negative rod with a characteristic "safety pin" appearance (bipolar staining). On sensitivity testing, the organism appears highly resistant (it is innately resistant to a large number of antibiotics including colistin and gentamicin and that again differentiates it from B. mallei, which is in contrast, exquisitely sensitive to a large number of antibiotics.-

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Other Non Fermenters 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

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Pseudomonas stutzeri: Microbiology Cytochrome-oxidase positive gram-negative rods forming distinctive dry, wrinkled colonies (1-6 mm) on blood agar Key reactions: OF glucose + and OF lactose –, arginine dihydrolase –, ability to grow in 6.5% NaCl broth, gas from nitrate, and no growth with cetrimide (growth of P. aeruginosa cetrimide-resistant)

Burkholderia cepacia: Natural Habitats and Clinical Infections

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Soil and environmental water Unpasteurized dairy products Contaminated respiratory therapy equipment, disinfectants, medications, and mouthwash Nosocomial pathogen causing bacteremia (most often associated with indwelling vascular catheters and polymicrobial), respiratory infections (ventilator-associated pneumonia), septic arthritis, urinary tract infections

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Burkholderia cepacia: Microbiology  Bright pink colonies on MacConkey agar after 4 days of incubation due to lactose oxidation  Positive for lysine decarboxylation (genomovar I) (DNase negative, vs. Stenotrophomonas maltophilia that is DNase positive)1  Saccharolytic with OF glu + and OF xyl + (100%), OF lac + and OF suc + (91%) (acidify slant and deep of TSI slant after 4-7 days be oxidation of glucose, lactose, and sucrose)  ONPG + 1Among non-fermentative gram-negative bacteria, only B. cepacia and S. maltophilia lysine positive

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Burkholderia cepacia: Use of Selective Agar

Pseudomonas cepacia agar (PCA): selective containing crystal violet, polymyxin B, and bacitracin; differential with B. cepacia forming a pink-red color due to pyruvate metabolism. Utilized to recover B. cepacia from cystic fibrosis sputum Isolation from PCA by single colony pick and ID by Vitek-2 but ~15% misidentification Confirmation of Vitek-2 ID by manual identification (? Role of PCR for ID of genomovariants)

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Burkholderia cepacia: Clinical Infections Second leading cause of bacteremia and third most common cause of pneumonia in chronic granulomatous disease of childhood Chronic pneumonia in cystic fibrosis (3-7%) with rapid decline in lung function, transmissibility of infection via close personal contact (nosocomial spread), and poor outcome with lung transplantation “Cepacia Syndrome” Rapid and fatal clinical deterioration with necrotizing granulomatous pneumonia

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Burkholderia cepacia: Clinical Infections  Skin and soft tissue infections with burn or surgical wounds, in soldiers with prolonged foot immersion in water  Isolation from blood cultures of multiple patients over short period of time should be investigated for “pseudo bacteremia” (contaminated infusion or disinfectant fluid)

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Burkholderia cepacia: Microbiology Burkholderia cepacia complex (BCC): nine genomic species (genomovars) including B. cepacia (genomovars I) DNA PCR and microarray technology under development for laboratory identification Cytochrome-oxidase positive gramnegative rods forming smooth, round, opaque, and yellow colonies (genomovar I) on blood agar Wet, runny, and mucoid colonies when recovered for cystic fibrosis sputum (requires at least 3 days for appearance)

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Acinetobacter Species: Natural Habitats Widely distributed including the hospital environment Able to survive on moist and dry surfaces including human skin More frequently colonizing than infecting

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Acinetobacter Species: Modes of Infection Colonization of hospital patients by environmental sources Introduction of organisms into normally sterile sites by medical instrumentation (intravenous or urinary catheters, endotracheal tubes or tracheostomies, respiratory care equipment) in debilitated hospital patients (antibiotic treatment, surgery, intensive care units, surgery)

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Acinetobacter Species: Types of Infectious Disease  Nosocomial infections of the respiratory tract, urinary tract, and wounds (including catheter wounds) often with progression to bacteremia  Sporadic cases of ambulatory peritonealdialysis related peritonitis, endocarditis, meningitis, arthritis, and osteomyelitis

Acinetobacter Species: Microbiological Properties

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 Gram-negative coccobacillary rods occurring singly and in Neisseria-like pairs  Oxidize rather than ferment D-glucose (OF glucose +)  Neither oxidize nor ferment D-glucose (OF glucose –)  A. baumannii complex/OF glu + OF lac +, nonhemolytic  A. lwoffii/OF glu – OF lac –, non-hemolytic  A. haemolyticus/OF glu – OF lac –, β-hemolysis on sheep blood agar

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Acinetobacter: Genomospecies Twenty-one different Genomospecies based on DNA-DNA hybridization Genomospecies 1, 2, 3, and 13: A. calcoaceiticus-baumanii complex (A. baumanii1) Genomospecies 8/9: A. lwoffi2 Genomospecies 4: A. haemolyticus3 1Saccharolytic, non-hemolytic 2Non-Saccharolytic, non-hemolytic 3Non-Saccharolytic, β-hemolytic

Moraxella: Natural Habitats and Clinical Infections1

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Saprophytic on human skin and mucous membranes Most frequently isolated species by culture M. nonliquefaciens is a component of normal respiratory flora Ocular pathogens (conjunctivitis, keratitis) and unusual causes of invasvie infection (meningitis, bacteremia, endocarditis, and arthritis) 1Excludes Moraxella catarrhalis (identified in the laboratory using Neisseria protocols)

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Moraxella: Microbiology Cytochrome-oxidase positive gramnegative or gram-variable Neisseria-like diplococci, forming small (0.5-1mm) colonies on blood agar (24-48 hr), smooth, translucent to semi opaque in appearance, occasional strains show pitting of agar Non-motile, indole negative, and asaccharolytic Species identification generally not performed because given the similarity of pathogenic signficance of all species

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Pseudomonas stutzeri: Natural Habitats and Clinical Infections Environmental sources (especially aqueous) as with Pseudomonas aeruginosa Bacteremia and meningitis reported in immunosuppressed individuals Pneumonia in alcoholics Endophthalmitis following cataract surgery and bacteremia due to contaminated hemodialysis fluid (iatrogenic infections)

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Other Non Fermenters  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.  The disease may manifest as a chronic pulmonary disease, as a form characterized by multiple abscesses of the skin, subcutaneous tissue, and lymphatic's (Farcy), or as an acute, fatal septicemia.

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Created by Dr.T.V.Rao MD for „ e „ learning resources for Medical Microbiologists in the Developing World Email [email protected]