NON-FERMENTERS

1. NON-FERMENTERS PRESENTED BY—DR. ANINDYA DAS PGT, . DEPT. OF MICROBIOLOGY GMCH

2. INTRODUCTION Bacterial metabolism is a dynamic balance between anabolic reactions and catabolic reactions. Catabolic reactions, in addition to providing smaller building blocks for subsequent biosynthetic processes also provide energy in the form of ATP. Using this energy, bacterial cell wall,proteins nuclic acids,other structural and regulatory macromolecules are synthesised .

3. Bacteria that derive their energy from organic compounds are called chemoorganotrphs . Most of the bacteria encountered in clinical medicine derive energy from the utilization of carbohydrates by one of several metabolic pathways. Some free-living bacteria, such as the nitrogen-fixing groups or those capable of oxidizing sulfur or iron can derive energy from inorganic chemicals.They are called Chemolithotrophs .

4. Some bacteria, such as Moraxella , do not metabolize carbohydrates, but derive energy from degradation of other compounds,like amino acids, alcohols and organic acids. Utilization of carbohydrates by bacteria and the condition under which this utilization occurs are key characteristics for broadly characterizing bacteria. Many tests performed in Microbiology lab. for identification of bacteria are actually detection of the end products of bacterial metabolism.

5. Bacterial degradation of carbohydrate proceeds by three major pathways: Embden -Meyerhof- Parnas , Entner-Doudoroff and HMP pathway. Glucose is converted to pyruvic acid in each of these three pathway by a different set of degradation steps. Bacteria use one or more of these pathways for glucose metabolism depending on their enzymatic composition and the presence or lack of oxygen .

6. Utilization of glucose under anaerobic condition is termed fermentation. It occurs via glycolysis or EMP pathway with the intermediate product being pyruvic acid. Pyruvate is then oxidized by giving up its H+ ion to Na-lactate to form lactic acid or to other organic salts to form mixed acids. These acids are the end products of fermentation process accounting for the drop in Ph.

7. Bacteria that possess appropriate enzyme system can further degrade mixed acids into alcohols,carbon-di-oxide,or other compound. Due to the absence of dehydrogenase enzyme some bacteria cannot ferment glucose. They oxidatively metabolise glucose through Entner-Doudoroff pathway by forming 6-phosphogluconate before forming pyruvate and then transfer H+ into Krebs cycle. They are called Non- fermenters .

8. The acids produced in ED pathway and Kreb ’ cycle are extremely weak compared with the mixed acids produced in fermentation. As the end product of oxidative metabolism is water, no gas formation occurs here. So the nonfermentative gram-negative bacilli are a group of aerobic,non-sporeforming bacilli that either do not use carbohydrate as a source of energy( eg . Moraxella ) or degrade them through pathways other than fermentation.

9. In Microbiology lab. nonfermenters are used to mean all aerobic gram-negative rods that show abundant growth within 24hrs on the surface of KIA or TSI media, but neither grow in nor acidify the butt of media

10. Clinically important non- fermenters Achromobacter Acidovorax Acinetobacter Agrobacterium Alcaligenes Bordetella Brevundimonas Burkholderia Stenotrophomonas Chryseobacterium Chryseomonas Comamonas Flavimonas Flavobacterium Methylobacterium Moraxella Weeksella Ochrobactrum Oligella Pseudomonas Psychrobacter Roseomonas Shewanella Sphingobacterium Delftia Ralstonia

11. General information They are predominantly opportunistic. Pathogenecity of the organism is usually related to an altered or already debilitated host. Nomenclature of these organisms changes rapidly due to defining of new genera with the use of molecular techniques.

12. Characteristics of non- fermenters They will grow on routine isolation media like blood agar, chocolate agar plate. Growth on MacConkey agar is variable and this property is used for identification. Optimum temperature of incubation ranges from 22-35ºC. Most of them require an incubation time of at least 24 hrs, sometimes 48-72 hrs.

13. NonferMenters are differentiated from enterobacteriaceae based on: Most nonfermenters are oxidase + ve , but enterobacteriaceae are oxidase − ve . Not all nonfermenters grow on MacConkey agar. Nonfermenters that grow on MacConkey agar are lactose negative. Acid produced by nonfermenters are so weak that normal culture broths designed to detect acid produced by fermenters are not suitable for them.It can be easily identified in Hugh- Leifson OF media.

14. Some nonfermenters that are unable to utilize carbohydrates as energy sources are termed nonsaccharolytic or asaccholytic , eg-Moraxella , Alcaligenes etc. Nonfermenters can rapidly develop resistance to antimicrobial agents used in treating infection. A number of pigments are produced by nonfermenters , which are helpful in species identification,eg - carotenoids , violacein , phenazines , fluorescein , pyocyanine etc.

15. LABORATORY IDENTIFICATION OF NONFERMENTERS: Organisms characterized as nonfermentative GNB are first differentiated based upon the combination of following three reactions- Glucose oxidizer or glucose inactive( asaccharolytic ). MacConkey agar growth present or absent. Oxidase+ve or oxidase−ve . Once placed on one of the above groupings, the organisms are identified using a specific

16. set of differential media(can be conventional or commercial systems like API 20NE, Vitek 1 and 2 system, Phoenix-Becton Dickinson etc.) or by species-specific rRNA based PCR assays. Speciation of these organisms can be difficult.Reliability of commercial systems with some of these organisms is variable.

17. GLUCOSE OXIDIZER,MAC+VE,OX+VE GENUS-PSEUDOMONAS Pseudomonas is the most commonly isolated nonfermenting GNB. Distribution is world wide and associated with water and moist environment. Obligate aerobe,slender GNB,1.5-3x0.5µm. Motile by polar monotrichus or multitrichus (tufts of) flagella. Cytochrome oxidase positive.

18. Utilize carbohydrates oxidatively . Grow on MacConkey agar producing non-lactose fermenting colonies. Most commonly isolated species in the genus is Pseudomonas aeruginosa . One of the leading causes of hospital acquired infection. Not usually part of normal flora of healthy individuals.

19. Colonization occurs in the GI tract, throat, nasal mucosa, axilla and perinium . Produces pigments like pyocianin (blue green) pyoverdin (yellow-green or yellow-brown, fluorescent), pyorubin (red), pyomelanin . Cetrimide agar can be used to detect pyocyanin and pyoverdin production and selectively isolate the organism.

20. Lab. identification Colony morphology: BAP- spreading, flat, irregular edge, gray-green, with a metallic sheen, possibly mucoid , beta-hemolytic, and a grape like or corn taco-like odor. MAC- clear, nonlactose fermenting colony. Gram Stain morphology: thin gram − ve rod. Pigment production: Pyocyanin , Pyoverdin , Pyomelanin , Pyorubin .

21. Glucose utilisation : glucose oxidizer(TSI-K/K, no gas, no H 2S.) Oxidase : Positive Catalase: Positive Lactose: Negative Mannitol : Negative Sucrose: Negative 42ºC growth: Positive Arginine dihydrolase : Positive Lysine decarboxylase : Negative

22. Ornithine decarboxylase : Negative Gelatine hydrolysis: Variable Nitrate: Positive Citrate: Positive Urease : Variable Acetamide : Positive Polymyxin -B: Sensitive VIRULENCE FACTORS: Pilli(attach to cell surface) Lipopolysaccharide ( endotoxin )

23. Alginate(capsular polysaccharide inhibit phagocytosis and form biofilms ) Exotoxin A: inhibit protein synthesis. Other extracellular enzymes and toxins like proteases, elastase , neuraminidase, phospholypase -C, leucocidin , enterotoxin . Pyocyanin : suppress other bacteria, disrupt respiraory cilliary activity.

24. Clinical significance: Type of patients infected by Pseudomonas: Leukocytopenic pt. Immunosuppressed pt. Extensive burn pt. Cystic fibrosis and Diabetic pt. Presence of indwelling foreign devices. IV drug abuser. Antibiotic therapy. Immature immunologic system

25. Types of infections associated with pseudomonas: Infected burn wounds. Nosocomial infections like pneumonia. Chronic pulmonary disease in Cystic fibrosis. Septicemia. Swimmer’s ear or otitis externa . Folliculitis (swimming pool associated) Corneal ulcer/ keratitis , Urinary tract infection, Osteomylitis , Ecthyma gangrenosum .

26. Antibiotic therapy Pseudomonas aeruginosa is resistant to many commonly used antibiotics like Penicillin, Ampicillin , many 1 st and 2 nd generation Cephalosporins . Sensitive to aminoglycosides,some 3 rd generation cephalosporins , antipseudomonal penicillins ( piperacillin,ticarcillin ) and quinolonnes , carbapenems like imipenem , meropenem and aztreonam . Aminoglycosides : affected by conc. of Ca, Mg ion of the medium.

27. CHRISEOBACTERIUM MENINGOSEPTICUM: The organisms are found in soil, water and hospital environment and on plants and foodstuffs. Not part of normal human flora but can cause nosocomial infection. LABORATORY IDENTIFICATION: Colony morphology:BAP - smooth fairly large colonies, may have a pale yellow pigment. MAC- growth (ambient air)

28. Gram morphology: thin GNB, sometimes with swollen ends, may be filamentous. Motility: Negative. Oxidase : Positive. Glucose: Oxidizer. Indole : Positive(may be weak,use Ehrlich’ method). Nitrate reduction: Negative . Esculine : Positive. ONPG: Positive. Polymyxin : Resistant.

29. CLINICAL SIGNIFICANCE: Neonatal meningitis and sepsis. Highly pathogenic for premature infants. High mortality rate and chance of nursery epidemic. Bacterimia associated with implanted catheters. Other opportunistic infections.

30. Antibiotic therapy: MIC determination is recommended for clinically significant isolates. Disc diffusion test is unreliable. Susceptible to penicillin(usually), vancomycin (which is unusual for a gram negative organism), co- trimoxazole , fluoroquinolones , piperacillin / tazobactum .

31. Glucose oxidizer, mac+ve , ox- variable,BURKHOLDERIA CEPACIA Natural distribution is in water sources, detergents, disinfectants.Clusters of 9 closely related genomic species. Isolated from pts. of CF and CGD. LAB. IDENTIFICATION: Colony morphology in BAP: smooth, slightly raised, may be mucoid , non-pigmented, strong earthy odor. MAC: punctate and tenacious, may become dark pink red after 4-7 days.

32. Selective media for isolation is OFPBL media containing polymyxin B,bacitracin and lactose. Form yellow colony. PC agar: polymyxin B, crystal violate, ticarcillin . Form pink colony due to lactose utilization. Gram negative rod. Oxidase : Positive. Growth at 42ºC: Variable. Glucose: Oxidizer.

33. Arginine dihydrolase : Negative. Lysine decarboxylase : Positive. Ornithine decarboxylase : Variable. Polymyxin B: Resistant. CLINICAL SIGNIFICANCE: Organisms can be isolated from anesthetics, nebulizer solutions, IV fiuids and disinfectant like povidone -iodine, quaternary ammonium compounds and chlorhexidine .

34. Opportunistic pathogen primarily related to nosocomial infections and cystic fibrosis. ANTIBIOTIC THERAPY: Resistant to aminoglycosides . Sensitive to co- trimoxazole (drug of choice). CLSI: if susceptibility done with disc diffusion only report ceftazidime , meropenem , minocycline and co- trimoxazole .

35. Glucose oxidizer/ inert,mac+ve,ox−ve , stenotrophomonas maltophila 3 rd most commonly found nonfermenter in clinical specimen. Ubiquitous in nature.Can be recovered from any clinical site. Important nosocomial pathogen. Not considered part of normal human flora. Can quickly colonize respiratory tract of hospitalized pts. Have considerable genetic diversity.

36. Lab.identification : BAP: colonies are pale yellow to lavender green(good growth at 24 hrs). Gram negative rods. Oxidase : Negative Glucose: Oxidizer(weak). Growth at 42ºC: Negative. Maltose: Strong oxidizer. Arginine dihydrolase : Negative. Lysine decarboxylase : Positive. Ornithine decarboxylase : Negative.

37. DNase : Positive . Polymyxin B: Sensitive. CLINICAL SIGNIFICANCE: Usually cause pneumonia, wound infection, urinary tract infection, bacterimia in compromised host. ANTIBIOTIC THERAPY: Inherently resistant to commonly used anti- pseudomonal drugs.

38. Inherently susceptible to co- trimoxazole . CLSI recommends broth dilution. Common antimicrobials include ticarcillin-clavulanate , levofloxacin and tetracycline. If disc diffusion performed, only report minocycline , levofloxacin and co- trimoxazole .

39. Glucose oxidizer/ asaccharolytic,mac+ve,ox−ve . acinetobacter After the genus Pseudomonas,it is the most frequently isolated nonfermenter . Ubiquitous in soil, water and sewage.Can be part of normal skin flora. Organism can survive on moist and dry surface. Strict aerobe. In hospital environment isolated from ventilators, humidifiers and catheters.

40. Lab. identification Colony morphology on BAP is translucent to opaque, non-pigmented, convex. On MAC colonies are colorless to slightly pink Gram stain morphology is plump gram − ve coccobacilli , often appear to be diplococci , can be confused with Neiserria sp. Oxidase : Negative. Motility: Nonmotile .

41. Nitrate reduction: Negative. Glucose: Oxidizer or asaccharolytic . A.baumannii is saccharolytic glucose oxidizer. Definitive identification: rapid production of acid from lactose. A.lwoffi , A.johnsonii , A.radioresistens are asaccharolytic . Penicillin: Resistant. CLINICAL SIGNIFICANCE: Generally considered non-pathogenic in healthy individuals.

42. Associated with opportunistic/ nosocomial infection of respiratory tract, urinary tract, wound and blood. ANTIBIOTIC SUSCEPTIBILITY: Resistant to a variety of antibiotic like penicillin, ampicillin , cephalothin ; variable susceptibility to 2 nd and 3 rd generation cephalosporin. Combined rx with aminoglycoside+ticarcillin or piperacillin ; sulbactum+cefoperazone;colistin for MDR sp.

43. Glucose asaccharolytic,mac+ve,ox+VE,ALCALIGENES /ACHROMOBACTER Occur in water, soil, moist area of hospital, respirators, hemodialysis system, iv soln. May be found on skin and in GI tract. LAB. DIAGNOSIS: Colony morphology of Alcaligenes fecalis is flat, thin, spreading and rough with an irregular edge, may have a fruity odor. Gram − ve coccobacilli .

44. Oxidase : Positive. Grow on MacConkey agar. Glucose: weak oxidizer or asaccharolytic . Motile by peritrichous flagella. Reduce Nitrite(not Nitrate). Achromobacter xylosoxidans is saccharolytic , Glucose: weak oxidizer. Xylose : strong oxidizer. CLINICAL SIGNIFICANCE: Alcaligenes fecalis is opportunistic pathogen

45. Isolated from blood, sputum, urine. Achromobacter xylosoxidans causes nosocomial septicemia and pulmonary complications in Cystic Fibrosis pts. and intubated children. ANTIBIOTIC THERAPY: Susceptibility pattern varies.

46. Glucose asacchrolytic,mac variable,ox+ve,moraxella . Normal flora of skin and mucous membrane. Non motile gram negative plump coccobacilli or diplobacilli.May resist decolorization . On BAP tiny pinpoint colonies at 24 hrs., may pit the agar. Most common sp. Moraxella catarrhalis is very similar to Neisseria sp. Oxidase : Positive.

47. Catalase : Positive. Glucose: non oxidizer( asaccharolytic ) Indole : negative. Penicillin: highly susceptible . CLINICAL SIGNIFICANCE: Causes nosocomial and opportunistic infection like respiratory tract infection and conjunctivitis. Production of β - lactamases has been reported mainly in M.catarrhalis .

48. Apart from these organisms some other medically important nonfermenters , though they are very rarely isolated from clinical specimens are Burkholderia mallei and pseudomallei . B.mallei is an obligate parasite of animals like horses, mules and donkeys causing a RTI glanders . In rare instances,it can be transmitted to humans through an abrasion of the skin.

49. It is gram-negative coccobacillus,only nonmotile sp. in the genus, oxidase − ve . B.pseudomallei causes melioidosis , a glander like disease in animals and humans. The organism has a specific ecological niche, existing in soil and stagnated water in parts of south-east asia like- Thailand,Vietnam,China,Taiwan and part of north Australia.

50. Infection is acquired by direct contact through break in the skin or by inhalation of contaminated dust. ACUTE DISEASE: presenting as septicemia with metastatic lesion. SUBACUTE DISEASE: presenting as TB like pneumonia with cellulitis and lymphangitis . CHRONIC DISEASE: presenting as localized cellulitis .

51. The unusual antibiotic profile( gentamycin , colistin resistance and amoxiclav susceptible) in an oxidase+ve,gram−ve,motile,arginine+ve rod is useful in confirming the diagnosis. Another important nonfermenter is genus Bordetella . Three most common human species are B.pertusis , B.parapertusis and B. bronchiseptica . B.bronchiseptica is motile by peritrichous

52. flagella and grow readily on ordinary media.It is an infrequent isolate in clinical lab which rapidly convert Christensen’s urea agar positive. B.pertusis and parapertusis both cause whooping cough, nonmotile and have special growth requirement.They are grouped as fastidious gram− ve rods.

53. thank you