2 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 andimprove the Diagnostic facilities in the Laboratories for better care of the patients
Nonfermenters can Cause Medically 3 Important InfectionsNonfermenters 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.
4Nonfermenters 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
5 Most Common Non-Fermentative Gram-Negative BacteriaPseudomonas aeruginosa (most common)Acinetobacter species (second most common)Stenotrophomonas maltophilia (third most common)
6 Other Clinically PrevalentGram-Negative Non Fermenters Pseudomonas stutzeri Burkholderia cepacia Burkholderia pseudomallei Moraxella Achromobacter xylosoxidans
Non-Fermentative 7 Gram-Negative BacteriaNonfermentative for glucose (TSI = alkaline over no reaction)Oxidative for glucose (Hugh-Leifson O-F glucose positive)Asaccharolytic for glucose (Hugh- Leifson O-F glucose negative)Cytochrome oxidase positive or negative
Hugh-Leifson OF versus 8 TSI MediumTSI AGAR SLANT Total protein = 2.6 g% Total carbohydrate = 2.1 g% Protein to carbohydrate (w/w) = 1.2OF BROTH MEDIUM Total protein = 0.2 g% Total carbohydrate = 1.0 g% Protein to carbohydrate (w/w) = 0.2
9 Growth of Gram-Negative Non-Fermenters on TSI Agar Slants Non-fermentative gram- negative bacteria grow abundantly within 16-18 hours of inoculation on the surface of TSI agar slants. Non-fermentative gram- negative bacteria neither grow in nor acidify the deep of TSI slants.
10Growth 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
11 Growth of Asaccharolytic Non-Fermenters in Hugh-Leifson OF Broth Growth without acidification but with Alkalinization 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)
12 The CDC scheme of identification 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
13 Classification of Pseudomonads1Burkholderia cepacia (family Burkholderiaceae) rRNA Group II Cytochrome oxidase +, OF glu +, motile, polymyxin B resistantBurkholderia pseudomallei (family Burkholderiaceae) rRNA Group II Cytochrome oxidase +, OF glu +, motile, polymyxin B resistantStenotrophomas maltophilia (family Xanthomonadaceae) rRNA Group V Cytochrome oxidase –, OF glu +, OF maltose ++, motile, polymyxin B susceptible1Pseudomonads are separated into five taxonomically distinct ribosomal RNA homology groups.
Classification of Non- 14 PseudomonadsAcinetobacter baumannii (family Moraxellaceae) Cytochrome oxidase –, OF glu +, OF lactose ++, non-motileMoraxella (family Moraxellaceae) Cytochrome oxidase +, OF glu – (asaccharolytic), non-motileAchromobacter xylosoxidans (family Alcaligenaceae) Cytochrome oxidase +, OF glu +, OF xylose +, motile
15Biochemical Tests to be performed for IdentificationRapid decarboxylation reactionsPigment productiongrowth in cetramidePhenylalanine deaminaseGrowth at 420 C
16 Virulence FactorsVirulence 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:
17 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
18Characters of Pseudomonas aeruginosa 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
19 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 fried- egg 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.
20 Virulence FactorsElastolytic 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.
21 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)
22 Virulence FactorsVirulence 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
23 Stenotrophomonas maltophilia: Natural HabitatsWidely distributed including moist hospital environments (respiratory therapy equipment)Colonizer of human respiratory tract in a hospital setting
24Stenotrophomonas 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 25Stenotrophomonas maltophilia (second most frequently isolated NF) Is part of the transient Normal flora of hospital patients and causes a wide variety of nosocomial infections
26 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
27 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.
28 Stenotrophomonas maltophilia: Microbiological Properties Cytochrome oxidase negative Positive for DNase (unlike most other glucose- oxidizing gram-negative bacilli) Positive for lysine decarboxylase (unlike most other glucose- oxidizing gram-negative bacilli) Resistant to most antibiotics except trimethoprim- sulfamethoxazole
29 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 30Burkholderia pseudomallei (also known as Pseudomonas pseudomallei) is a Gram- negative, bipolar, aerobic, motile rod-shaped bacterium. It infects humans and animals and causes the disease melioidosis. It is also capable of infecting plants.
31 Burkholderia pseudomallei B. pseudomallei is not fastidious and will grow on a large variety of culture media (blood agar, MacConkey agar, EMB, etc.). Ashdowns 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.-
32 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.-
33 Other Non FermentersAcinetobacter Is found in soil and water and as part of the skin NF Is a common colonizer and less commonly a cause of nosocomial infectionsChryseobacterium meningosepticum Occasionally found causing meningitis and septicemiaMoraxella M. lacunata causes conjunctivitis and keratitis in the malnourished alcoholic population Burkholderia – two species are true pathogens
34 Pseudomonas stutzeri: MicrobiologyCytochrome-oxidase positive gram-negative rods forming distinctive dry, wrinkled colonies (1-6 mm) on blood agarKey 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: 35Natural Habitats and Clinical InfectionsSoil and environmental waterUnpasteurized dairy productsContaminated respiratory therapy equipment, disinfectants, medications, and mouthwashNosocomial pathogen causing bacteremia (most often associated with indwelling vascular catheters and polymicrobial), respiratory infections (ventilator-associated pneumonia), septic arthritis, urinary tract infections
36 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
37Burkholderia cepacia: Use of Selective AgarPseudomonas 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 sputumIsolation from PCA by single colony pick and ID by Vitek-2 but ~15% misidentificationConfirmation of Vitek-2 ID by manual identification (? Role of PCR for ID of genomovariants)
38 Burkholderia cepacia: Clinical InfectionsSecond leading cause of bacteremia and third most common cause of pneumonia in chronic granulomatous disease of childhoodChronic 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
39 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)
40 Burkholderia cepacia: MicrobiologyBurkholderia cepacia complex (BCC): nine genomic species (genomovars) including B. cepacia (genomovars I)DNA PCR and microarray technology under development for laboratory identificationCytochrome-oxidase positive gram- negative rods forming smooth, round, opaque, and yellow colonies (genomovar I) on blood agarWet, runny, and mucoid colonies when recovered for cystic fibrosis sputum (requires at least 3 days for appearance)
41 Acinetobacter Species: Natural HabitatsWidely distributed including the hospital environmentAble to survive on moist and dry surfaces including human skinMore frequently colonizing than infecting
42 Acinetobacter Species: Modes of InfectionColonization of hospital patients by environmental sourcesIntroduction 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)
43Acinetobacter 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 peritoneal- dialysis related peritonitis, endocarditis, meningitis, arthritis, and osteomyelitis
44 Acinetobacter Species: Microbiological Properties 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 +, non- hemolytic A. lwoffii/OF glu – OF lac –, non-hemolytic A. haemolyticus/OF glu – OF lac –, β-hemolysis on sheep blood agar
45 Acinetobacter: GenomospeciesTwenty-one different Genomospecies based on DNA-DNA hybridizationGenomospecies 1, 2, 3, and 13: A. calcoaceiticus-baumanii complex (A. baumanii1)Genomospecies 8/9: A. lwoffi2Genomospecies 4: A. haemolyticus31Saccharolytic, non-hemolytic2Non-Saccharolytic, non-hemolytic3Non-Saccharolytic, β-hemolytic
46Moraxella: Natural Habitats and Clinical Infections1Saprophytic on human skin and mucous membranesMost frequently isolated species by culture M. nonliquefaciens is a component of normal respiratory floraOcular pathogens (conjunctivitis, keratitis) and unusual causes of invasvie infection (meningitis, bacteremia, endocarditis, and arthritis)1Excludes Moraxella catarrhalis (identified in the laboratory using Neisseria protocols)
47 Moraxella: MicrobiologyCytochrome-oxidase positive gram- negative 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 agarNon-motile, indole negative, and asaccharolyticSpecies identification generally not performed because given the similarity of pathogenic signficance of all species
48 Pseudomonas stutzeri: Natural Habitats and Clinical InfectionsEnvironmental sources (especially aqueous) as with Pseudomonas aeruginosaBacteremia and meningitis reported in immunosuppressed individualsPneumonia in alcoholicsEndophthalmitis following cataract surgery and bacteremia due to contaminated hemodialysis fluid (iatrogenic infections)
49 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 lymphatics (Farcy), or as an acute, fatal septicemia.
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