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

2.  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.  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.  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.  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.  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.  Organisms characterized as nonfermentative
GNB are first differentiated based upon the
combination of following three reactions-
1) Glucose oxidizer or glucose
inactive(asaccharolytic).
2) MacConkey agar growth present or absent.
3) 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.  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.

19.  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.

20.  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.

21.  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.

24.  Glucose utilisation: glucose oxidizer(TSI-
K/K, no gas, no H2S.)
 Oxidase: Positive
 Catalase:Positive
 Lactose: Negative
 Mannitol: Negative
 Sucrose: Negative
 42ºC growth: Positive
 Arginine dihydrolase: Positive
 Lysine decarboxylase: Negative

25.  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)

26.  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.

27.  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

28.  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.

30.  Pseudomonas aeruginosa is resistant to many
commonly used antibiotics like Penicillin,
Ampicillin, many 1st and 2nd generation
Cephalosporins.
 Sensitive to aminoglycosides,some 3rd
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.

31.  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)

32.  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.

33.  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.

34.  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.

35.  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.

36.  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.

37.  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.

38.  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.

39.  3rd 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.

40.  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.

41.  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.

42.  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.

43.  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.

44.  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.

46.  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.

47.  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 2nd and 3rd generation
cephalosporin.
 Combined rx with aminoglycoside+ticarcillin
or piperacillin;
sulbactum+cefoperazone;colistin for MDR sp.

48.  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.

49.  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

50.  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.

51.  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.

52.  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.

53.  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.

54.  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.

55.  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.

56.  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

57. 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.