CLINICAL BACTERIOLOGY MIDTERM

CLINICAL BACTERIOLOGY MIDTERM - Contains 33 defined species and 20 are species found
WEEK in man
WEEK 7:7: STAPHYLOCOCCUS
STAPHYLOCOCCUS AND
AND MICROCOCCUS
MICROCOCCUS
Two
Twogroups:
groups:
1. Coagulase-positive Staphylococci
a) S. aureus – golden-yellow colonies; pathogenic
2. Coagulase-negative Staphylococci (CoNS)
a) S. aureus – lemon yellow colonies; chromogenic
opportunistic pathogen
b) S. citreus – white pigment; chromogenic
opportunistic pathogen
c) S. albus – white pigment
d) S. hyicus – animal pathogen
e) S. intermedius – animal pathogen
f) S. epidermidis – predominant normal flora on the
skin; leading cause of Iantrogenic infection (most
common)
g) S. saprophyticus – opportunistic pathogen;
normal flora of skin; frequently cause UTI,
abortion/miscarriages (most common) (UTI in
adolescent girls and young women)
h) S. lugdunensis – opportunistic pathogen; normal
flora
i) S. haemolyticus – recovered in wounds, UTIs

GRAM
GRAMPOSITIVE
POSITIVECOCCI
COCCI
- Most gram-positive cocci are member of indigenous
microbiota
 Morphologically round in shape
 Violet/purple

Kingdom Monera Monera

Family Micrococcaceae Streptococcaceae
Genus Micrococcus Streptococcus
Staphylococcus Enterococcus
Stomatococcus Aerococcus
Planococcus Leuconcoccus
Gemella
Pediococcus

 Monera- made up of prokaryotes particularly

bacteria,

General
General Characteristics
Characteristics ofof Staphylococci
Staphylococci

- Common inhabitant of the skin and mucous

membranes, responsible for several suppurative
infections
- Gram-positive cocci, catalase-positive
- Spherical cells arranged in irregular clusters, appear
singly, in pairs
- Non-motile, non-spore forming
- Aerobic or facultatively anaerobe EXCEPT for S.
saccharolyticus (obligate anaerobe)
- Lack spores and flagella (non-motile), may have
capsules
STAPHYLOCOCCUS
STAPHYLOCOCCUSSPP.
SPP.  Colonies are medium sized (4 to 8 mm) and appear
cream-colored, white or rarely light gold, and
- STAPHLE (Greek: branches of grapes) “buttery-looking” (after incubation)
- Microscopy alone can’t differentiate staphylococci  Some species are β-hemolytic
from other gram- positive
  Rare strains of staphylococci are fastidious requiring
carbon dioxide, hemin, or menadione for growth.
o So-called small colony variants grow on
media containing blood, forming colonies
about 1/10 the size of wild-type strains after
at least 48 hours of incubation.
Staphylococcus
Staphylococcus
aureus
aureus
- Most clinically significant species in human
- Coagulase-positive
- It causes various cutaneous infections and purulent
abscesses. (most dangerous of all common)
o These cutaneous infections can progress to
deeper abscesses involving other organ
systems and produce bacteremia and
septicemia.
Coagulase-negative staphylococci
Coagulase-negative staphylococci (CoNS
(CoNS oror non-
non-
staphylococcus aureus)
staphylococcus aureus)
- Subdivided into two groups based on their novobiocin
susceptibility pattern:
1. Novobiocin susceptibility includes S. Epidermidis, S.
Capitis, S. Haemolyticus, S. Hominis subsp. Hominis, S.
Lugdunensis, S. Saccharolyticus, S. Warneri, and other
species
2. Novobiocin resistant group consists of such species as
S. Cohnii, S. Kloosii, S. Saprophyticus, and S. Xylosus
Other
Othercoagulase-producing
coagulase-producingStaphylococci
Staphylococci
- Often animal associated species and are less
frequently isolated
- Majority of clinical staphyloccocal isolates that are
identified by the true(?) coagulase test will be S.
aureus
 S. intermedius
 S. pseudintermedius
 S. hyicus
 S. delphini
 S. lutrae
 S. agnetis
 Some strains of S. schleiferi
o S. lugdunensis and S. schleiferi – mistaken as
coagulase positive staphylococci because of
the presence of clumping factor
VirulenceFactors
Virulence FactorsofofS.S.Aureus
Aureus
1) Cytolytic Toxins
- This extracellular proteins affects RBC and luekocytes
a. Hemolysin – (alpha, beta, delta, gamma)
o Pathogenic factor; destroys RBC
o Alpha – in addition to lysine erythrocyte can
damage platelets and macrophage
o Beta – aka. SPHINGOMYELINASE C acts as
sphingomyelin in plasma membrane and also
known as hot cold lysine
b. Panton-Valentine
Toxin/Leukocidin
o Rupture of WBCs
2) Enterotoxins - heat stable (100° C for 30 minutes);
cause various symptoms inc. diarrhea ,vomiting,
nausea (2-6 hr. after consuming contaminated food)
a. Enterotoxin A, B and D – cause of food poisoning
3) Toxic Shock Syndrome Toxin-1 (TSTT-1)
- Formerly known as Enterotoxin F and pyrogenic
exotoxin C
- Causes: Toxic Shock Syndrome (multiple organ dysf.)
- The enterotoxins and TSST-1 belong to a class of
polypeptides known as superantigens which are
potent superantigens which are potent activators of
T lymphocytes activators of T lymphocytes leading to
the release of cytokines such as interleukins and
tumor necrosis factor.
4) Epidermolytic Toxin (Exfoliative Toxin)
- Also known as ET or “exfoliatin”
- Responsible for the staphylococcal scalded skin
syndrome (SSSS)
- It is also has been implicated in bullous impetigo
o EXFOLIATIVE TOXIN –A – heat stable
o EXFOLIATIVE TOXIN –B - heat labile
5) Fibrolysin/Staphylokinase- responsible for lysin clot
o Activates plasminogen to form plasmin that
digests lysine clots
6) Protein A and Polysaccharide A –responsible for anti-
phagocytosis
o Has ability to bind to the fc portion of
Immunoglobulin, binding Ig can block
phagocytosis
7) Staphylocoagulase – localizes abscess; virulence
factor (important for virulence of S. aureus)
8) Hyaluronidase – degrades DNA components of tissue;
spreading factor (important for virulence of S. aureus)
9) Lipase – spreading factor (important for virulence of
S. aureus)
10) DNAse – spreading factor
- Other enzymes produced which are used to identify S.
aureus: Gelatinase, Thermonuclease, Penicillinase,
Catalase (table) (important for virulence of S. aureus)
Infections caused by Staphylococcus aureus
Infections caused by Staphylococcus aureus
- Determined by the virulence of strain, size of inoculum
and status of the host’s immune system
- Once the organism has crossed the initial barriers, it
activates the host acute inflammatory response which
leads to proliferation and activation PMN cells.
However, organisms are able to resist the action of
inflammatory cells by production of toxins and
enzymes establishing a focal lesion
A.A. Cutaneous
CutaneousInfections
Infections (Skin
(Skinand
andWound
WoundInfections)
Infections)
- Infections caused by S. aureus are suppurative
o The abscess is filled with pus and
surrounded by necrotic tissues and
damaged leukocytes
- Common skin infections caused by S. aureus:
1. Folliculitis – inflammation of hair follicle or oil gland;
infected area is raised and red
2. Furuncles (Boils) – extension of folliculitis; large,
raised and superficial abscess (pigsa)
3. Carbuncles – cluster of boils (fevers and chills)
4. Bullous impetigo – larger than streptococcal
nonbullous impetigo; surrounded by a small zone of
erythema (highly contagious infection and easily
spread by direct contact, fomites or auto inoculation)
 B.B. Deep
DeepInfections
Infections
- Include osteomyelitis, periostitis, tonsillitis,
pharyngitis, sinusitis, bronchopneumonia, empyema,
septicemia, meningitis, endocarditis, breast abscess,
renal abscess and abscesses in other organs.
C.C. Toxin-mediated
Toxin-mediatedDiseases
Diseases
1. Food Poisoning
- The enterotoxins do not cause any detectable odor
or change in the appearance or taste of the food
- Symptoms appear rapidly (approximately 2 to 8 hours
after ingestion of the food) and resolve within 24 to
48 hours
- Nausea, vomiting, abdominal pain and severe
cramping (common)
- Diarrhea and headaches (rare)
- No fever
- Salads, especially salads containing mayonnaise and
eggs, meat or meat products, poultry, egg products,
bakery products with grill fillings, sandwich fillings and
dairy products
2. Toxic Shock Syndrome (TSS)
- Fatal, multisystem disease characterized by sudden
onset of fever, chills, vomiting, diarrhea, muscle aches
and rash (predominantly on the trunk)
o Progress to hypotension and shock
- Most cases occur in menstruating women who use
tampons (2-5%)
- Caused by enterotoxin F, caused by release of toxins
from an overgrowth of S. aureus
3. Exfoliative Diseases
- Lesions are produced by the strains of S. aureus which
produce epidermolytic toxins
o Responsible for the ‘staphylococcal
staphylococcal scalded skin syndrome’
(SSSS), skin syndrome’ (SSSS), exfoliative skin
diseases in which the outer layer of
epidermis gets separated from the
underlying tissues (peeling skin overlarge
part of body)
o most common in summer and fall
o localizations contain purulent material or pus
and can progress to generalized form
characterized by a cutaneous erythema
followed by profuse spilling of epidermal
layer of the skin (from face, neck, axillary
region, and groin, trunk to extremeties)
o 2-4 days- duration
o Toxin is metabolized and excreted by kidneys
 Staphylococcal Scalded Skin Syndrome
- Bullous exfoliative dermatitis that occurs primarily in
newborns and previously healthy young children
(and adults with chronic renal failure and impaired
immune)
- Severe form: Ritter’s Disease
- Milder form: Pemphigus neonatorum and bullous
impetigo (localized form)
Laboratory identification of Staphylococcus and
Laboratory identification of Staphylococcus and Micrococcus
Micrococcus
ISOLATION
ISOLATIONAND
ANDIDENTIFICATION
IDENTIFICATION
- Rapid and direct identification of S. aureus is crucial
for proper management of patients with skin
infections, abscesses, septicemia/ bacteremia,
gastroenteritis, endocarditis, toxic shock syndrome
and certain food intoxications
- Grow easily on routine laboratory culture media –
sheep blood agar (SBA)
- Selective medium (for heavily contaminated
specimen)
o Mannitol Salt Agar (MSA), Columbia
Colistin–nalidixic Acid Agar (CNA), or
Phenylethyl Alcohol (PEA) agar
 High NaCl concentration (7.5%) in
MSA makes this medium selective
for Staphylococcus,
 Incorporation of mannitol and
phenol red distinguishes S. aureus
from most CoNS.
A.A. Microscopic
MicroscopicExamination
Examination
- Purulent exudates, joint fluids, aspirated secretions,
and other body fluids – cocci and PMN easily seen
when these sites are infected with staphylococci
- Culture should be done regardless of the results of the
microscopic examination – genus or species cannot
be appropriately identified (by microscope alone)
 Aspirate is the best sample, a single swab would be
less satisfactory for both culture and smear results
(must be 2 swabs)
- Spherical cocci; arranged characteristically in grape-
like clusters (large)
- May also be found singly, in pairs and in short chains
of three or four cells, especially when examined from
liquid culture.
o Long chains never occur
- Non spore forming, non-motile and usually non
capsulated with the exception of rare strains.
- Stain readily with aniline dyes and are uniformly gram-
positive
B.B. Cultural
Cultural Characteristics
Characteristics
1)1) Staphylococci:
Staphylococci:
o SBA (after 18-24 hours of incubation at 35° C
to 37° C) - round, smooth, white, creamy
colonies
o BAP - produce hemolytic zones around the
colonies (beta-hemolytic)
 Rarely exhibit pigment production
(yellow) with extended incubation
 Staphylococci completely lyse the
RBC surrounding it.
2)2) Staphylococcusaureus:
Staphylococcus aureus:
 Nutrientagar
Nutrient agar
- 1–3 mm in diameter 1 and have a smooth glistening
surface, an entire edge, a soft butyrous consistency
and an opaque, pigmented appearance
o Most strains produce golden-yellow (aureus)
pigment (staphyloxanthin) (when grown in
nutrient agar)
 o
White-colony strains of S. aureus are fully o Specimen: plasma
virulent o Detects bound coagulase (clumping factor)
  Blood
BloodAgar
Agar o Positive result: agglutination
o Colonies have the same appearances as on - A heavy suspension of suspected organism is prepared on a
nutrient agar, but may be surrounded by a glass slide in water or saline and mixed with drop of plasma,
zone of ß-hemolysis if agglutination occurs, the isolate can be identified as S.
aureus. 5% of S. aureus don’t produce clamping factor
o Hemolysis is more likely to be present if
2. Tube Method (confirmatory test)
sheep, human or rabbit blood is used.
o Specimen: 0.5 mL rabbit’s plasma
o Detects unbound coagulase
  Phenolphthalein
PhenolphthaleinPhosphate
PhosphateAgarAgar(PPA)
(PPA)
(staphylocoagulase)
o An indicator medium and assists in the
 Staphylocoagulase – is an
identification of S. aureus in mixed cultures
exocellular molecule that causes a
o Colonies is similar to those on nutrient agar
clot to form when bacterial cells are
 Become bright pink when culture
incubated with plasma
plate is inverted over ammonia for
o 35ºC incubation; initial time = 2 hours
a minute or so
o 4 hours incubation (if negative, continue
  Selective
Selective Salt
Salt Media:
Media:
incubation for 24 hours)
- S. aureus can tolerate high salt concentration that
o Positive result: coagulum/clot
can’t be tolerated by other Staphylococcus species
- Any negatives like coagulase test result
1. 7–10% of sodium chloride may be added to
- S. schleiferi
Nutrient Agar (Salt Agar) or Milk Agar (Salt Milk
Agar)
3)3) Oxidation-Fermentation
Oxidation-Fermentation(O/F)
(O/F)Reactions
Reactions
2. Mannitol Salt Agar with 1% mannitol
- Detect the oxidation or fermentation of
3. 7.5% NaCl
carbohydrates by bacteria
4. Nutrient Agar with Phenol Red
- Staphylococcus ferment glucose whereas micrococci
5. Ludlam’s Medium containing lithium chloride and
feel to produce acid under anaerobic conditions
tellurite
- Differentiate staphylococci from micrococci
6. Salt Cooked Meat Broth (10% NaCl)
- Medium used: O/F Glucose Medium
 S. epidermidis – small- to medium-sized, non-
- Two methods:
hemolytic, gray-to-white colonies; some weakly
1. Open Method
hemolytic
2. Closed Method – add mineral oil as a barrier for
 S. saprophyticus – slightly larger colonies, with about
oxygen
50% of the strains producing a yellow pigment
- Interpretation of results:
 S. haemolyticus – medium sized colonies, with o Closed = (+) yellow: Fermentative
moderate or weak hemolysis and variable pigment o Open = (+) yellow; Oxidative
production o If both (+) = Staphylococci
 S. lugdunensis – small to medium size colonies, often o If Closed is (-) and Open is (+) =
hemolytic Micrococcaceae
NOTE: Identification of Staphylococci on the basis of colony  Oxidative utilization of the carbohydrate – result in
morphology alone should not be done. acid production or yellow in open tube only
 Fermentative utilization- result in acid production or
(table) yellow in both open and closed tubes
 Acidic changes in overlaid tubes are considered to be
C.C. IdentificationMethods
Identification Methods
result of true fermentation
1)1) Catalase
CatalaseTest
Test
- demonstrate the presence of catalase - an enzyme  Acidic development in open tubes- due to oxidative
that catalyses the release of oxygen from hydrogen utilization of carbohydrate present
peroxide (H2O2) - Hugh and Leifson’s Medium
o To distinguish Staphylococci spp. And  Glucose, cylose, mannitol, lactose, sucrose and
Micrococci spp. (positive) from non-catalase maltose – added to the medium, serves as
producing bacteria such as streptococci fermentable carbohydrate
o Reagent: 3% H2O, 15% H2O2 for detection of
catalase in anaerobes 4)4) Modified
Modified Oxidase
Oxidase Test
Test
o Positive result: bubble formation or - Active Chemical component Tetra-methyl-para-
effervescence phenylene diamine dihydrochloride
- During this test, bacteria protect themselves from the little
effect of hydrogen peroxide which is accumulated as an end
product of aerobic carbohydrate metabolism

2)2) Coagulase
Coagulase (Slide
(Slide oror Tube
Tube Method)
Method) - Results:
- Best single pathogenicity test for staphylococcus o (+) = blue to purple to black complex
- Basis of ability to produce coagulase enzyme (causes [Micrococci]
plasma to clot by converting fibrinogen to fibrin, o (-) = no color change [Staphylococcus]
produces 2 forms of coagulase bound in 3 coagulase)  Microdase test – differentiation of staphylococcus and
- 2 methods: micrococcus by detection of oxidase enzyme
1. Slide Method (screening test)
 5)5) Mannitol
MannitolFermentation
Fermentation o Catheterization
- If an organism can ferment mannitol, an acidic o Medical Implantation
byproduct is formed that will cause the final red in o Immunosuppressive therapy
agar to turn yellow - Most common cause of prosthetic valve endocarditis
- Staphylococcus spp. Fermentative (Glucose)
Staphylococcus
Staphylococcussaphrophyticus
saphrophyticus
- Medium: Mannitol Salt Agar
o (+) result: yellow [S. aureus] - UTIs in young women
o (-) result: pink [CONS] o Low numbers (<10,000 colony-forming
- Phenol red units/mL) in urine culture is considered as
- Non-pathogenic staphylococci- will not ferment significant
- Can be distinguished from S. epidermidis by its
6)6) DNAse
DNAseTest
Test resistance to novobiocin and by its failure to ferment
- Determine ability of an organism to hydrolyze DNA glucose anaerobically.
and utilize it as a source of carbon and energy for - adheres more effectively to the epithelial cells lining
growth the urogenital tract than other coagulase negative
- Medium: DNAse Medium staphylococci
- Incubate at 25ºC for several hours - Rarely found in other mucous membranes or skin
o (+) zone of inhibition surfaces.
o (+) = S. aureus
o (-) = S. epidermidis
 Pale green in color – because of DNA metal green
indicator complex
- 3-4 cm long (line) from rim to the center, Incubate the plate
at 37oC for 18-24 hr. After incubation, flood the plate with
one end hydrochloric acid, examine the plate within 5 min.
- Dark background

7)7) Gelatin
GelatinLiquefaction/Hydrolysis
Liquefaction/HydrolysisTest
Test
Staphylococcus lugdunensis
Staphylococcus lugdunensis
- Gelatin is a protein derived from connective tissues of
vertebrates that is collagen. Produced when collagen is - Cause both community-associated and hospital-
boiled in water acquired infections
- Gelatin hydrolysis detects the presence of gelatinases
- Can be more virulent and can clinically mimic S.
o Gelatinases- proteases secreted extracellularly by
aureus infections
bacteria that hydrolyze or digest gelatin
- 12% Gelatin Medium (Tube) - Infective endocarditis, septicemia, meningitis, skin
- Stabbing method and soft tissue infections, UTIs, septic shock
- Incubate at 35ºC for 16-18 hours o Frequently aggressive, requiring valve
o (+) Liquefaction replacement and infections have high
o Refrigeration (30 mins) mortality rate
o (+) = S. aureus - Can give positive clamping factor test result, it has
o (-) = S. epidermidis negative tube coagulase reaction.
 meCA - Contain the gene that encodes oxacillin
8)8) Novobiocin
NovobiocinSusceptibility
Susceptibility resistance
- Presumptive identification of staphylococcus is TREATMENT
TREATMENT
accomplished by testing for novobiocin using a 5
microgram novobiosintis (?)  Benzyl penicillin is the most effective antibiotic, if the
- Staphylococcus is resistant to novibiocin wherein strain is sensitive (highly adaptable, regenerates
most other CONs are susceptible rapidly)
o Penicillinase- bacteria produce, an enzyme
that degrades penicillin and can spread
throughout the bacterial population by
conjugation
 Cloxacillin, oxacillin, flucloxacillin are used - are
penicillinase resistant penicillin.
9)9) SugarFermentation
Sugar Fermentation
o Methicillin-resistant Staphylococcus aureus
- Ferments a range of sugars producing acid but no gas. (MRSA) are also resistant to other penicillin
- Sugar fermentation is of no diagnostic value except and cephalosporins.
for mannitol, which is usually fermented  Glycopeptides (vancomycin or teicoplanin) are the
anaerobically by S. aureus but not by other species. agents of choice in the treatment of systemic
(pic –chart) infection, but these agents are expensive and may be
Coagulase-negative toxic.
Coagulase-negativeStaphylococci
Staphylococci(CONS)
(CONS)
- For mild superficial lesions, topical applications of
Staphylococcus
Staphylococcusepidermidis
epidermidis drugs as bacitracin, chlorhexidine or mupirocin may
be sufficient.
- Invariably present on normal man skin.
- Infections are predominantly hospital acquired
- Predisposing factors:
 - The treatment of carriers is by local application of Vancomycin-Resistant
Vancomycin-ResistantStaphylococci
Staphylococci
antibiotics such as bacitracin and antiseptics
- 1996 the first vancomycin-intermediate
(chlorhexidine).
Staphylococcus aureus (VISA) strains were recovered
- In resistant cases, rifampicin along with another oral
in Japan.
antibiotic may be effective.
- Difficult to detect with automated antimicrobial
Other
OtherCoagulase-negative
Coagulase-negativeStaphylococci
Staphylococci susceptibility testing and disk diffusion procedure
- It has been suggested that clinical microbiology
 S. haemolyticus has been reported in wounds, laboratories use more than one method to detect
bacteremia, endocarditis, and UTIs. VISA.
- Other species include:
o S. lugdunensis Macrolide Resistance
Macrolide Resistance
o S. warneri
 Clindamycin, a macrolide, is frequently used in
o S. capitis
staphylococcal skin infections; additional testing
o S. simulans,
using a modified double disk diffusion test (D-zone
o S. schleiferi.
test) might be useful
Methicillin-resistant
Methicillin-resistantStaphylococcus
Staphylococcusaureus
aureus(MRSA)
(MRSA) o May be useful when discrepant macrolide
test result are obtained
Methicillin-resistant Staphylococcus aureus (MRSA)
- placing an erythromycin disk near a clindamycin disk -
- Group of gram-positive bacteria that are genetically if an isolate possesses inducible clindamycin
distinct from other strains of staphylococcus aureus resistance, the bacteria grow around the
- Responsible for several difficult to treat infections in erythromycin disk and in the area of the agar where
humans the two drugs overlap
- Resistant not merely to penicillin, but also to all other - a zone of inhibition is observed on the side of the
beta lactam antibiotics. clindamycin disk farther away from the erythromycin
- Isolates that are resistant have been traditionally disk, flattening the clindamycin zone, which looks like
termed methicillin-resistant staphylococci, with S. the letter “D”
aureus being called MRSA and S. epidermidis
Micrococci
Micrococci
referred to as MRSE.
- Any staphylococcus isolated is identified as being - resembles some members of the family microfoss
resistant to methicillin, this implies that it is also assay such as genus micrococci
resistant to nafcillin and oxacillin and to antibiotics, o Micrococci- genus of spherical bacteria
including the cephalosporins.
Introduction:
 Glycopeptides (vancomycin or teicoplanin) are the
agents of choice in the treatment of systemic - Catalase-positive, gram-positive, coagulase-negative
infection, but these agents are expensive and may be and usually oxidase-positive.
toxic. - They may occasionally colonize the skin or mucous
- The symptoms depends on where infected. Most membrane of humans, but they are rarely associated
often causes mild infections on the skin like sores, with infections.
boils or abscesses but can also cause some serious skin - Only two species, Micrococcus luteus and
infections or infect surgical wounds (blood stream, Micrococcus lylae, remain in the genus.
lungs or urinary tract)  M. luteus, have a tendency to produce a yellow
 Super bug- Spread of top strains of mrsa pigmented colony.
 Oxacillin is generally used for detection of methicillin
resistance.
o The use of an oxacillin-salt agar, such as the
oxacillin resistance screening agar can be
used as a screening test for MRSA in clinical
samples
o Latest CLSI M100 document recommends
cefoxitin be used to detect oxacillin
(methicillin) resistance.
o A high salt concentration (5.5% NACl) high
salt concentration (5.5% NACl) and
Micrococci: General Characteristics
polymyxin Bpolymyxin B make the medium
selective for staphylococci. - Appear as gram-positive cocci
 Gold standard for MRSA detection is the detection of in tetrads, rather than large
the meca gene by using detection of nucleic acid clusters.
probes or polymerase chain reaction (PCR) - Commonly isolated from
amplification. immunocompromised patients
o Cefoxitin is a better inducer of mecA
mediated resistance.
WEEK
WEEK8:8: Streptococcus, Enterococcus and
Streptococcus, Enterococcus andOther
OtherCatalase-
Catalase- - ß-hemolysis constitutes the principal marker for
Negative Gram-Positive Cocci
Negative Gram-Positive Cocci potentially pathogenic streptococci in cultures of
throat swabs or other clinical samples.
General Characteristics

- Streptococcus and Enterococcus spp. belong to the C)C) Gamma

Gamma(γ)(γ)ororNon-hemolytic
Non-hemolyticStreptococci
Streptococci
family Streptococcaceae - Produce no hemolysis on blood agar.
- Inhabit various sites, notably the upper respiratory - When RBC immediately surrounding the colony or
tract, and live harmlessly as commensals. (normal unaffected
flora in both man and animals)  Enterococcus faecalis - is an important organism of
- Both of the genera are catalase-negative (weak this group.
reaction),
Alpha Beta Gamma
o Catalase negative result differentiates
Color Green Clear Red
streptococci and enterococci from around
staphylococci colonies
- gram-positive cocci that are usually arranged in pairs Members S. S. equisimilis Some
or chains pneumoniae S. pyogenes Viridans
- The cells of Enterococci and some Streptococci S. agalactiae group
appear more elongated than spherical E. faealis
- The streptococcal cells are more likely to appear in
chains when grown in broth cultures
*S. pneumoniae, S. pyogenes, S. agalactiae, E. faealis – true
- Most members are aerotolerant anaerobes (but
pathogens
behave like facultative anaerobes)
o They grow in presence of oxygen but unable
to use oxygen for respiration
- Some species are capnophilic (need CO2)
 Poor growth on Nutrient Media such as Trypticase
Soy Agar
 More pronounced growth on media enriched with
blood or serum
 Colonies are usually small and transparent
 Most Streptococci, have a group or common C
carbohydrate (polysaccharide) which can be used to
classify an isolate serologically (Lancefield 2)2) LANCEFIELD
LANCEFIELDCLASSIFICATION
CLASSIFICATION
classification) Serological Properties (of streptococci)
 Rebecca Lancefield – developed in 1930s
Lancefield Grouping
CLASSIFICATION
CLASSIFICATION OFOF STREPTOCOCCUS
STREPTOCOCCUS SPP.
SPP.
- After first recognizing the antigen in b-hemolytic
streptococci
- It was developed by Rebecca Lancefield in the year
1930s
- Designated by letters
o Groups A, B, C, D, and G are most commonly
found associated with human infections.
- Basis of group specific carbohydrates (c) antigens in
the cell wall
1)1) SMITH
SMITH AND
AND BROWN
BROWN CLASSIFICATION
CLASSIFICATION  Group A- possess the same antigenic C carbohydrate
- Streptococci and similar organisms can produce  Group B – have the same C carbohydrate and so on.
numerous exotoxins that damage intact RBCs under
the Smith and Brown classifications of ff.
- Alpha, Beta and Gamma Hemolytic

A)A) Alpha-hemolytic
Alpha-hemolytic(α)(α) Streptococci
Streptococci
- Produces a zone of partial hemolysis with a greenish
discoloration around the colonies on blood agar
- Streptococci producing α-hemolysis are also known
as Viridans Streptococci.

B)B) Beta
Beta
(ß)(ß) Hemolytic
Hemolytic Streptococci
Streptococci
- Produces a complete hemolysis
o Sharply defined, clear, colorless zone of
hemolysis around the colony induced
bacterial hemolysins
o No red blood cell is visible on microscopic
examination in clear zone of complete
hemolysis.
 3)3) ACADEMIC
ACADEMIC OROR BERGY’S
BERGY’S CLASSIFICATION
CLASSIFICATION SpeA, SpeB, SpeC, and SpeF – 4 immunologically distinct types
- According to the temperature growth requirement found in streptococcus pyogenes

10ºC, 37ºC, 45ºC Toxin

Toxin

 Streptococcal pyrogenic exotoxin – formerly called

erythrogenic toxin; causes Scarlet Fever (red rash)
 Nephrogenic toxin– causes Acute Glomerulonephritis
 DNAse – spreading factors localized in the skin; S.
pyogenes DNases: A, B (most common), C, D
 Streptokinase – spreading factors localized in the skin
 Lipoteichoic acid and Protein F – adhesion molecules
o Lipoteichoic acid- affects protein on bacterial
surface in concert with M protein and fibronectin
binding protein
 Hyaluronidase – spreading factors localized in the skin
o Enzyme that solubilizes the ground substance of
mammalian CT, bacteria used this enzyme to
separate the tissue and spread infection (no
evidence)
 CAMP Factor – produced by S. agalactiae; heat stable
protein; enhances the beta-hemolysis of S. aureus

Laboratory
LaboratoryTests
Testsfor
forStreptococcus
Streptococcusspp.
spp.

A.A. Gram
GramStain
Stain
VIRULENCE FACTORS
Virulence Factors ofOF STREPTOCOCCI
Streptococci Spp. SPP. - Gram-positive cocci in pairs or chains
M Protein – S. Pyogenes – Genes emm
B.B. Cultural
CulturalCharacteristics
Characteristics
 M protein- Best defined virulence factor - Pinpoint colonies (smaller)
Streptococcus pyogenes encoded by genes emm
SBA
o Causes the streptococcal cell to resist
phagocytosis and plays a role in the adherence of  S. pyogenes – small, transparent and smooth; beta-
bacterial cell to mucosal cell
hemolytic
o Resistance to infection with streptococcus
pyogenes appears to be related to the type
 S. agalactiae – grayish white mucoid colonies; beta-
specific antibodies to the m protein hemolytic (small zone)
o Individual with antibodies against m5 is protected
C. Catalase Test
from infection by streptococcus pyogenes with m5 C. Catalase Test
protein but remains unprotected against infection - Negative (no effervescence)
with the roughly 100 remaining and protein - Differentiate if its staphylococci or streptococci
stereotypes.
o M1 stereotype – most common stereotype seen
D.D. Bacitracin
BacitracinSusceptibility
SusceptibilityTest
Test(Taxo
(TaxoA)A)
in pharyngitis
- Differentiates Group A Streptococci from other
Streptolysin S Streptolysin O Streptococci
Type of Surface of RBC Subsurface o Throat swab – used to inoculate SBA containing
hemolysin SMZ and a bacitracin disk placed directly onto the
Oxygen Stable Labile agar
Antigenecity Non-antigenic Antigenic  Positive: Group A Strep
 Negative: Other Beta-hemolytic Streptococci spp.

 M protein – part of cell wall; antiphagocytic E.E. CAMP

CAMPTest
Test(Christie
(ChristieAtkins
AtkinsMunch
MunchPetersen
PetersenTest)
Test)
 Capsule – principal virulence factor of Group B - Test for Group B Streptococci (1994)
Streptococci; antiphagocytic - Differentiate Streptococcus agalactiae from other
o Hyaluronic capsule is weakly immunogenic, Strep.
prevents oxidized phagocytosis by neutrophils or
 Known organism (S. aureus)
macrophages
 Unknown organism [Beta-hemolytic, Catalase (-),
o Allows bacterium to mass its antigen and remain
unrecognized by its hosts pinpoint, Bacitracin Resistant]
 Hemolysin o Group B Strep (strict, perpendicular to a
o Streptolysin O (oxygen labile) – highly streak of Strep. aureus on SBA)
immunogenic; measured using ASO o S. agalactiae
(antistreptolysin O) test  Positive: arrowhead hemolysis [Group B Strep] or
 Responsible on SBA plate incubated bowtie appearance [S. agalactiae]
anaerobically, active only on  Group B Strep – produces a diffusible extracellular
reduced form hemolytic heat stable protein (CAMP factor) –
o Streptolysin S (oxygen stable) – enhance lysis of RBC
nonimmunogenic;
 Lyses leukocytes
F.F. PYR
PYR Hydrolysis
Hydrolysis Test
Test - Their resistance allows them to selectively grow out from
 Positive: Cherry Red/Pink contaminating bacteria that are inhabited or inhibited by
- Test for Group A and Group D Streptococci this antibiotic
 Substrate: L-pyrrolidonyl-beta-napthylamide
L. L. Serological
Serological Tests
Tests
 Reagent: p-dimethyl-aminocinnamaldehyde
- Detect carbohydrate component of the cell wall of
 Enzyme: pyrroglutamylamino peptidase or
Streptococci
pyrrolidomylaryl amidase
 Name of test: Streptex
- Identification of group A b-hemolytic streptococci-
o Streptex- a rapid latex test system for
Streptococcus pyogenes
qualitative detection and identification of the
- Differentiation of enterococcus species, PYR-positive
Lancefield group of Streptococci
from group D streptococci
 Principle: Antigen(from colonies)-Antibody(from
 Streptococci pyogenes – only species of
Reagent) Reaction
Streptococcus that is PYR positive, susceptible to
- Tube
bacitracin and hydrolyzes PYR
o Add diluent (PBS/NSS) [0.5mL]
o Transfer bacteria
G.G. Hippurate
Hippurate Test
Test
o Transfer to test card labelled A-G
- Test for Group B Streptococci
o Add antibody
 Medium: Broth with hippurate
 Positive: agglutination/clumping [Group A Strep]
 Reagent Indicator: Ninhydrin
 Negative: no agglutination [S. agalactiae]
 Hippurate -- Benzoic Acid + Glycine
o Detect the ability of bacteria to hydrolyze
substrate hippurate into Glycine and benzoic acid
- Add indicator after incubation
 Positive: Purple (S. agalactiae)
 Hippuricase – constitutive enzyme that hydrolyzes the
substrate hippurate to produce glycine

Clinically Significant
Clinically Significant Streptococci and
and Streptococcus-like
Streptococcus-like
H.H. Bile
Bile Esculin
Esculin Test
Test
organisms
organisms
- Group D (Enterococci/Non-Enterococci)
- Differentiate Group D from Beta-hemolytic Streptococcus
Streptococcuspyogenes
pyogenes
- Bile tolerant and can hydrolyze esculin to esculetin with the
help of the enzymes Esculinase from non-group D variants - A Group A streptococci under Lancefield classification
 Enzyme: Esculinase
Clinical
ClinicalInfections
InfectionsofofS.S.pyogenes
 Positive: blackening of medium [Group D] pyogenes
 ESCULIN – ESCULETIN GAS (Group A Streptococcal) Infection

I. I. Salt
Salt Tolerance
Tolerance Test
Test (6.5%
(6.5% NaCl)
NaCl) A)A) Bacterial
BacterialPharyngitis
Pharyngitis

- Further test from Bile Esculin Test  “Strep throat” – most often seen in children between
- To identify Enterococci (group D streptococcus) 5 and 15 years of age
- Used to characterize several bacteria including - Spread by droplets and close contact
Viridans Streptococci - 1-4 days incubation period
 Negative: Clear/Transparent [Non-enterococci] o Sore throat
 Positive: Turbid [Enterococci] o Malaise
- Broth medium o Fever
o Headache
J. J. Leucine
Leucine Aminopeptidase
Aminopeptidase Test
Test o Nausea, vomiting and abdominal pain
- Peptidase that hydrolyzes peptide bonds adjacent to a (unusual)
free amino group o Tonsils and pharynx are inflamed
 Substrate: Leucine-β-naphthylamide –- (hydrolyze o Cervical lymph nodes are swollen and tender
- Disease varies in intensity and not unusual to isolate a
into β-naphthylamine
nearly pure culture of streptococcus pyogenes from the
 Reagent: paradimethylaminocin namaldehyde throat of a child with fever and complete for only of a mild
reagent sore throat
 Positive color: Red color  Group C and G – capable to produce significant acute
 Positive: Viridans Streptococci Pharyngitis but are less commonly seen
 3-4 days – symptoms subside
K.K. SXT
SXT Susceptibility
Susceptibility Test
Test
- Sulfametoxazole in conjuction with bacitracin is used B)B) Pyodermal
Pyodermal Infections
Infections
for identification of b-hemolytic strep. on blood agar 1. Impetigo - a localized skin disease, begins as small
 R – Group A & B Strep vesicles that progress to weeping lesions; inoculation
 S – Other Strep spp. of organisms through minor abrasions or insect bites
- Susceptibility of sulfametoxazole is used for the primary o Seen in children 2-5 years old
recovery of Group A and B from specimen with mixed 2. Cellulitis – followed by deeper invasion of
culture.
streptococci; life-threatening; with bacteremia or
sepsis
 o Patients with peripheral vascular disease or
diabetes – leads to gangrene
3. Erysipelas - is a rare infection of the skin and
subcutaneous tissues observed frequently in elderly
patients
o Lesion characteristics: acute spreading,
intensely erythematous with plainly
Clinical
demarcated but irregular edge
4. Scarlet Fever – cause by streptococcal pyrogenic
exotoxin
o Diffuse red rash on upper chest and spreads
to the trunk and extremities
o Strains of Streptococcus pyogenes infected with
temperate bacteriophage T12 produce pyogenic
exotoxins
o Appears 1-2 days after bacterial infection and
disappears over the next 5-7 days
C)C) Necrotizing
Necrotizing Fasciitis
Fasciitis
- “Flesh-eating disease or syndrome”, “Suppurative
fasciitis”, “Hospital gangrene”, “Necrotizing
erysipelas”
- An invasive infection characterized by rapidly
progressing inflammation and necrosis of the skin,
subcutaneous fat, and fascia
- May be prevented if early intervention is instituted.
Mortality rate may reach greater than 70% if left untreated
D)D) Streptococcal
Streptococcal Toxic
Toxic Shock
Shock Syndrome
Syndrome
- A condition in which the entire organ system
collapses, leading to death
- Portal of infection is unknown, although minor
injuries or surgeries have been implicated
- Caused by a type of streptococcal pyrogenic exotoxin
– SpeA
o Has a major role in the pathogenesis
o Functions as superantigen leading to
overstimulation of the immune response
- Initial streptococcal infection is severe (e.g.,
pharyngitis, peritonitis, cellulitis, wound infections)
o Then it develops into staphylococcal TSS-like
symptoms
- Patients are often bacteremic and have necrotizing
fasciitis
 Streptolysin O and various cell wall antigens can also
contribute to toxic shock
 Young children with chicken pox or varicella and
elderly adults – greater risk
E)E) Poststreptococcal
Poststreptococcal Sequelae
Sequelae
- Two serious complications or sequelae of GAS disease:
1. Rheumatic Fever
o Follows after S. pyogenes pharyngitis
o Characterized by fever and inflammation of
the heart, joints, blood vessels and
subcutaneous tissues
o Most serious result: chronic, progressive
damage to the heart valves
o Usually begin 1 month after infection
2. Acute Glomerulonephritis
o Follows after cutaneous or pharyngeal
infection
o More common in children than in adults
o Immunologically mediated for acute
glomerulonephritis
 GAS are susceptible to penicillin (drug of choice for
treatment)
 Erythromycin- if allergic to penicillin
- Patients with history of rheumatic fever, prophylactic doses
of penicillin are given to recurrent infections that may cause
additional damage to the heart
ClinicalInfections
InfectionsofofOther
OtherStreptococcus
Streptococcusspp.
spp.
1)1) Streptococcus
Streptococcus agalactiae
agalactiae
- Only species that expresses group b antigen
 Todd-Hewith Broth
- Significant cause of invasive disease in newborn
(1970)
o Most infections of infants occur in the first 3
days after birth, usually within 24 hours
- Two clinical syndromes are associated with neonatal
GBS disease: early-onset infection (<7 days old) and
late-onset infection (at least 7 days old to about 3
months old)
o Early-onset infection – pneumonia and
sepsis
 80% in newborns and caused by vertical
transmission from mother
o Late-onset infection – meningitis and sepsis
 Organisms is rarely found in the
mother’s vagina before birth. Mortality
rate is less
- Commonly associated with obstetric complications,
prolonged rupture of membranes, and premature
birth
- It is recommended that all pregnant women be
screened for GBS at 35 to 37 weeks’ gestation
(presence of GBS in the vagina of mother)
- Collecting vaginal and rectal material with swabs,
samples should be inoculated into selective broth
- In adults, the infection affects two types of patients:
o Young, previously healthy woman who
become ill after childbirth or abortion
 Endometritis and wound infections
o Elderly person with a serious underlying
disease or immunodeficiency
 Drug of choice: Penicillin (for treating GBS infection)
o Some clinicians recommend a combination
of ampicillin and aminoglycosides
o Although GBS are less susceptible to penicillin
than GAS, the clinical response to antimicrobial
therapy is often poor despite heavy doses given
2)2) Group
GroupCC and
and GG Streptococci
Streptococci
 S. equi subsp. Zooepidemicus – animal pathogen and
associated with glomerulonephritis and fever
- Subdivisions:
1. Large-colony forms
o Classified with the pyogenic streptococci
o Beta-hemolytic isolates – belong to the
subspecies S. dysagalactiae subsp.
equisimilis (also exhibited group A and L
antigens)
 Have involved several body sites and are
thought to be uncommon in domestic
animals
 Includes upper respiratory tract
infections, skin infection, soft tissue
infections and invasive infections such
as necrotizing fasciitis
 2. Small-colony forms Laboratory Diagnosis
Laboratory Diagnosis
o Beta-hemolytic isolates – belong to the S.
anginosus group (Under viridans group)  Gram Stain
 With groups C and G antigens o Gram-positive cocci in pairs (diplococci)
o Ends of the cells are slightly pointed (oval or
3)3) Streptococcus
Streptococcus pneumoniae
pneumoniae lancet shape)
- Also known as pneumococcus or diplococcus  Culture
- Member of S. mitis group (but much more virulent) o BHIA, TSA with 5% sheep RBCs or Chocolate
- No Lancefield Classification Agar are necessary for good growth
 C substance - Antigen present in the cell wall o SBA – large zone of alpha-hemolysis
 C-reactive protein (CRP) reacts with C substance to  Cultural Characteristic
form a precipitate in human serum (a chemical o Young cultures – round, glistening, wet,
reaction not antigen-antibody combination) mucoid, dome-shaped appearance
o It increases during inflammation and o Old cultures – coin with a raised rim
infection appearance
- Gram-positive cocci in pairs - The colonies may closely resemble colonies of the
o As culture ages, the gram stain reaction becomes viridans streptococci
variable and the gram-negative cells are seen - Can autolysis which makes it difficult to keep isolates alive.
- Alpha-hemolytic It require frequent subculturing every 1-2 days to ensure
viability
- Dome-shaped colonies
 Optochin Test or Taxo P
Clinical Infections
Clinical Infections o Chemical composed of ethylhydrocupreine
hydrochloride
 Lobar Pneumonia o S = Pneumococci
- It is an important human pathogen that causes o R = Viridans Streptococci
pneumonia, sinusitis, otitis media, bacteremia, and  Bile Solubility Test
meningitis o For S. pneumonia only
- Most frequently isolate in children younger than 3 o Determines the lysis of S. pneumoniae in the
years old with recurrent otitis media presence of bile salts
- No. 1 cause of bacterial pneumonia – prevalent in o Can only be done with a-hemolytic organisms
elderly persons and in patients with underlying
 Inulin Fermentation
conditions
o Only S. pneumoniae can ferment inulin
- Not usually a primary infection but rather a result of
(carbohydrate)
disturbance of the normal defense barriers
o Indicator: Phenol Red
- Predisposing factors:
o (+) = Yellow [S. pneumoniae ]
o Alcoholism
o (–) = Red/Pink [Viridans Group]
o Anesthesia
 Capsular Swelling Reaction or Nueffeld Quellung
o Malnutrition
Reaction
o Viral infections of the upper respiratory tract
o Methylene Blue + Antitoxin + Bacteria
- Causative agent of Lobar Pneumonia or
o Positive: Obvious cell wall, opaque and
Pneumococcal Pneumonia
enlarged [S. pneumoniae ]
o Lobar distribution of the infection
o Negative: No capsule [All other Alpha
o Sudden onset with chills, dyspnea and cough
hemolytic Streptococci]
o Transmitted via aspiration of respiratory - Antibodies bind to the bacterial capsule of S. pneumoniae
secretions
 Mouse Virulence Test
- Begins with aspiration of respiratory secretions which often
o Positive: Death of Test Animal
contain pneumococci, the infective organisms in the alveoli
stimulates an outpouring fluid which serves to facilitate the o Fred Neufeld – identified several strains of
spread of organism to adjust an alveoli, this accounts for the bacterium Strep. pneumoniae
lobar distribution of the infection, hence the name, lobar  Francis Skin Test
pneumonia o Test for previous infection
- Pneumonia may be complicated by a pleural effusion o Erythrogenic test
that is usually sterile (empyema) o Patient must not have rashes
- Also causes bacterial meningitis in all age groups – o 2-3 days
usually follows after otitis media or pneumonia o Positive: induration or Wheale formation
o Direct smears of CSF – reveal leukocytes and
numerous gram-positive cocci in pairs Treatment
Treatment
 Secondary atypical hemolytic uremic syndrome  Drug of choice: Penicillin
caused by S. pneumoniae has been reported in  Penicillin resistance – Erythromycin or
children Chloramphenicol
 Vaccines are available – heptavalent pneumococcal
conjugate vaccine (PCV7) Viridans
Viridans Streptococci
Streptococci
o Part of routine pediatric immunization
- Normal flora of the upper respiratory tract, female
schedule
genital tract and gastrointestinal tract
o Recommended for: asplenic individuals,
 Viridans – means “green” (a-hemolysis)
elderly patients with cardiac or pulmonary
- Fastidious, some strain requires CO2 for growth
disease
Clinical Infections
Clinical Infections - Both groups are nonhemolytic
- Both groups are positive for Bile Esculin Test
- Are opportunistic pathogens but can, cause disease.
- Tests that differentiate the two groups:
(orophayngeal commensals)
o PYR Test
- The most common cause of subacute bacterial
• Positive: Enterococci
endocarditis – a condition associated with a transient
•Negative: Nonenterococci (Group D
bacteremia (children and with hematological
Streptococci)
malignancies)
o 6.5% NaCl Test
o Symptoms may be present for weeks or months
• Positive: Enterococci (growth)
and individuals whose heart valves have been
damaged by rheumatic fever • Negative: Non-enterococci (no growth)
 Oral infections such as gingivitis and dental caries Penicillin
PenicillinResistance
Resistance
(cavities)
- They have also been implicated in meningitis,  Resistant – Enterococci
abscesses, osteomyelitis, and empyema  Susceptible – Group D Streptococci (Nonenterococci)

Enterococcus
Enterococcus
- More than 30 species are classified
 Strep. anginosus group – can possess Lancefield group A, C
and F, G or N antigen and may not be groupable. Can cross-
react with other grouping antisera
 Srep. bovis group – possess group Bantigen. No longer a
valid species name (just same as equinos)
1) S. anginosus group – normal flora of oral cavity and
gastrointestinal tract
- Associated with abscess formation in the oropharynx,
brain and peritoneal cavity
- S. constellatus subsp. pharyngis – pharyngitis
2) S. mitis group – normal flora of oral cavity,
gastrointestinal tract and female genital tract; also
transient normal flora of the skin.
- Most common isolates associated with bacterial
endocarditis in native valves and less frequently, in
prosthetic valve infections
 S. bovis group – often encountered in blood cultures
of patients with bacteremia, septicemia and
endocarditis (can be isolated from blood of
asymptomatic individuals)
o Gastrointestinal carcinoma - Presence of S.
gallolyticus subsp. gallolyticus in blood
cultures
1. Strep. salivarius – linked to bacteremia, endocarditis
and meningitis
2. Strep. vestibulais – not associates with the disease
- Consist of gram-positive cocci
- Previously classified as Group D Streptococci
- Natural inhabitants of the intestinal tracts of humans
and animals
- Common species: E. faecalis, E. faecium
- All species produce the cell wall–associated group D
antigen in the Lancefield classification system
- Most enterococci are nonhemolytic or α-hemolytic,
although some strains show β-hemolysis
- Sometimes exhibit a pseudocatalase reaction – weak
bubbling in catalase test
- Ability to grow under extreme conditions: presence
of bile, 6.5% NaCl or alkaline pH, 45oC
- Can hydrolyze PYR – differentiates them from Group
D Streptococci
- Frequent cause of nosocomial infection – UTI (most
common) followed by bacteremia (catheterization)
- Prolonged hospitalization is a risk factor for acquiring
enterococcal bacteremia
 Bacteremia: receiving hemodialysis,
immunocompromised patients with a serious
underlying disease, prior surgical procedure
 Endocarditis: elderly patients with prosthetic valves
or valvular heart disease
- account for about 5-10% of infections in patients with
bacterial endocarditis

3) S. mutans group – most commonly isolated among

the viridans streptococci
o Usually isolated from the oral cavity
o S. mutans – primary contributor to dental
caries
o Most common member of the mutans group
associated with bacteremia

Group
GroupDD
Streptococci
Streptococci
- Subdivided into: Chart
o Enterococci – placed into a new genus enterococcus
o Non-enterococci –remained in group D
WEEK
WEEK9:9:
NEISSERIA AND
NEISSERIA MORAXELLA
AND MORAXELLA
General Characteristics – Neisseria spp.
- 25 species about 12 species biovarscan be solated
from humans
- Most Neisseria spp. are aerobic, nonmotile, non–
spore-forming, gram-negative diplococcic (Adjacent
sides, flattened together resembling a coffee bean)
o Except Neisseria elongata, Neisseria
weaveri, and Neisseria bacilliformis –
rod-shaped
- Oxidase positive
- Catalase positive (except N. elongata and N.
bacilliformis)
- Many Neisseria spp. are capnophilic (need CO2) and
Neisseria gonorrhoeae – Epidemiology
have optimal growth in a humid atmosphere
- They can grow anaerobically if alternative electron
acceptors (e.g., nitrites) are available
 Natural habitat - mucous membranes of the
respiratory and urogenital tracts
Important species of the genus Neisseria are:
- Fastidious requiring enriched media for optimal
recovery
- Sensitive to unfavorable environmental conditions
- Iron – required for growth
1. N. meningitidis, -Meningococci (may be found as
commensal, inhabitant of the upper respiratory tract
of carriers, but can also become an invasive pathogen)
2. N. gonorrhoeae, - Gonococci (not considered to be
part of the normal biota & always pathogenic)
3. N. flavescens,
4. N. subflava,
5. N. sicca,
6. N. mucosa,
7. N. lactamica
8. N. polysacchareae
 N. gonorrhoeae and N. menin-gitidis are the primary
human pathogens of the genus.
Neisseria
Neisseriagonorrhoeae-
gonorrhoeae-Gonococcus
Gonococcus
 Humans are the only natural host
 Agent for gonorrhea “flow of seed”(
o Flow of seed – 2nd century when the urethral
discharge was mistaken as semen
Neisseria
o Gonorrhea- STD that can infect both men
and women
o Gonorrhea and Syphilis – were confused
because they were often present together in
the same individual
- Also known as “clap” from the French word clapoir
meaning “brothel”
- An acute pyogenic infection of non-ciliated columnar
and transitional epithelium
- Infection can be established at any site where these
cells are found – urethra, endocervix, anal canal,
pharynx, and conjunctiva
- Most commonly transmitted by sexual contact
Neisseria gonorrhoeae – Clinical Infections
Clinical Infections
 Primary reservoir – asymptomatic carrier
 Incubation period – 2-7 days
 Most common clinical presentation is acute urethritis
in men
o Purulent discharge and dysuria (painful
urination) – common manifestation
o Complications – prostatitis and epididymitis
 Most common site of infection in women –
endocervix
o Dysuria, cervical discharge, lower abdominal
pain
 50% of cases in women – asymptomatic
o Complications – pelvic inflammatory
disease – which may cause sterility, ectopic
pregnancy, or perihepatitis (Fitz-Hugh-Curtis
Syndrome)
o Only 3% - 5% of cases are asymptomatic in
men
Epidemiology
 1% cases – Blood-borne dissemination
o Resulting in purulent arthritis and
septicemia (rare)
o Fever and rash can also be present
 It is inhibited by SPS (sodium polyanethol sulfonate) –
anticoagulant in blood culture media
o Gelatin is added to neutralize the effects of
SPS
o Neisseria gonorrhoeae- might not be
recovered from blood cultures
 Other conditions associated – rectal and
oropharyngeal infections
o Common in men who have sex with men but
can also occur in women
o Mostly asymptomatic
o Symptomatic oropharyngeal infection –
pharyngitis
o Symptomatic rectal gonorrhoeae –
discharge, rectal pain or bloody stools
 A non-venereal infection is ophthalmia neonatorum
in the newborn (gonococcal eye infection) –
conjunctivitis of newborn
o The eyes are coated with gonococci as the
baby passes down the birth canal
o Can result in blindness if not treated
immediately
o During vaginal delivery through an infected
birth canal
Laboratory
Diagnosis – Laboratory Diagnosis
gonorrhoeae
A.A. Specimen
SpecimenCollection
Collectionand
andTransport
Transport
- Specimens are collected from genitals or other sites,
such as rectum, pharynx, and joint fluid
- Specimen of choice for genital infections:
o Men – urethra (purulent discharge)
 When no apparent discharge is present,
the swab is inserted up to 2 cm into the
anterior urethra and slowly rotated
 4-5 cm – swab for rectal culture into the
anal canal
o Women – endocervix
- It is inhibited by cotton and calcium alginate swab
o Preferred – dacron or rayon swab
 Disinfectant should be avoided in
preparing patient for the collection
 Transport media – Amies medium with charcoal
(within 6 hours)
o N.gonorrhoeae – susceptible to drying and
temperature changes, direct plating of the
 specimen to gonococcal selected media
gives optimal results
o When direct plating is not possible,
inoculated swabs should be placed in a
transport system
B.B. Direct
Direct Microscopic
Microscopic Examination
Examination
- Gram-negative intracellular diplococci
o Correlates at 89% with culture
- The gonococci are in pairs with adjacent sides
flattened, giving them a kidney shape
- Gram stain with more than five polymorphonuclear
neutrophils per field but no bacteria – nongonococcal
urethritis with organisms such as Chlamydia
trachomatis or Ureaplasma urealyticum
- Not recommended for pharyngeal specimens
because commensals Neisseria species can be present
- Because women have vaginal commensal microbiota
that resembles camicoxide – direct gram stain
correlates only 15-70% of cases with culture
- May be helpful in asymptomatic women with
discharge, but culture is necessary for confirmation
C.C. Culture
Culture
- Does not grow on SBA
 Medium of choice – CAP (Chocolate Agar Plate)
 Selective medium:
1. Thayer-Martin
2. Modified Thayer-Martin
3. Martin-Lewis
4. New York City
5. GC-LECT
- Other bacteria can grow on selective gonococcal
media, these isolates can be differentiated from the
N. gonorrhoeae by the oxide and catalase test
o Inhibitory agents – vancomycin and colistin
– inhibit gram-positive bacteria, gram-
negative bacteria and fungi; trimethoprim –
inhibit Proteus spp.
- All specimens received in the laboratory for recovery
of Neisseria spp. should be held at room temperature
and plated as soon as possible
- Neisseria spp. are susceptible to cold – media should
be warmed to room temperature before inoculation
D.D. Incubation
Incubation
- Inoculated plates should be incubated at 35° C in a 3%
to 5% CO2 atmosphere
- Incubation is accomplished by use of a CO2 incubator,
CO2 generating pouch, or a candle extinction jar
(white wax candles only)
o Scented or colored candles – may be
inhibitory to the gonococci (do not use)
 Sufficient humidity – provided by the moisture
evaporating from the media in a closed jar
E.E. Laboratory
LaboratoryIdentification
Identification
 CAP or Selective Agar colony morphology – small,
gray to tan, translucent, and raised after 24-48 hours
of incubation
 Oxidase Test – positive (purple color)
 Carbohydrate Utilization – traditional method for the
identification of Neisseria spp.
o Medium used – CTA (Cystine Trypticase
Agar) –containing 1% the individual
carbohydrate and phenol red (pH indicator)
o If the organism uses the particular
carbohydrate – acid production (yellow
color) – produced in 24-72 hours of incubation
o N. gonorrhoeae is glucose fermenter only
 Genetic Probes – probes specific for the nucleic acids
of N. gonorr-hoeae have been developed for the direct
detection of bacteria in clinical specimens
Treatment
Neisseria gonorrhoeae – Treatment
 Penicillin is no longer the antibiotic of choice for
treatment of gonorrhea since the development and
widespread use of penicillin, gonococcal resistance to
penicillin has gradually risen
 Cephalosporin – recommended
Neisseria
Neisseriameningitides
meningitides
- Also found only in humans, can be found as
commensal and as an invasive pathogen
- Meningococcus, Diplococcus Intracellularis
Meningitidis
Neisseria meningitidis – Epidemiology
Epidemiology
 It is an important etiologic agent of (endemic and
epidemic) meningitis and meningococcemia
o Rarely pneumonia, purulent arthritis, or
endophthalmitis
o N. meningitidis- has also been recovered
from urogenital and rectal sites as a result of
oral genital contact
 Can be found on the mucosal surfaces of the
nasopharynx and oropharynx (30%)
 Mode of transmission – Droplet transmission
- Only a few new colonized hosts developed
meningococcal disease with highest incidence being
found in infants and adolescents
Neisseria meningitidis – Clinical Infections
Clinical Infections
- Incubation period – 1-10 days
- The bacteria adhere to the nasopharyngeal mucosa
leading to colonization
o Can gain access to the bloodstream and CNS
  Resulting in meningitis, sepsis or Neisseria
Treatmentmeningitidis – Treatment
both
Drug of choice:
– two main diseases can occur: fulminant
meningococcemia or meningitis  Meningitis – penicillin
 Meningococcemia – third generation cephalosporins
1)1) Meningococcemia
Meningococcemia
 Close contact with patient positive for meningococcal
- A sepsis, may occur with or without meningitis disease – chemoprophylaxis with rifampin or
- Carries 25% mortality rate even if teated ciprofloxacin
 Purpura (hemorrhaging of blood into the skin and o Resistance to ciprofloxacin – azithromycin
mucous membranes producing bruises) with petechial o Chemoprophylaxis – not recommended for
skin rash (pinpoint red spot caused by hemorrhage) asymptomatic carriers
 Tachycardia (develop in bacteremia)
 Hypotension (develop in bacteremia) Moraxella
Moraxellacatarrhalis
catarrhalis
 Thrombosis
- Resembles the morphology of Neisseria, isolated only
- In some cases, the disease becomes fulminant and
from humans and commensal of upper respiratory t
spreads rapidly, causing:
- Opportunistic pathogen
o Disseminated intravascular coagulation,
- Third most common cause of acute otitis media
o Septic shock
- and sinusitis in children
o Hemorrhage in the adrenal glands
- Cause of upper respiratory tract infection
(Waterhouse-Friderichsen syndrome)
o Can cause lower respiratory infections,
- Death may occur in 12-48 hrs. onset
especially in adults with chronic obstructive
pulmonary disease
2)2) Meningitis
Meningitis
- Frontal headache Laboratory
Moraxella Diagnosis– Laboratory Diagnosis
catarrhalis
- Stiff neck (nuchal rigidity)
A.A. Specimen
SpecimenCollection
Collectionand
andIdentification
Identification
- Confusion
- Middle ear effusion, nasopharynx, sinus aspirates,
- Photophobia
sputum aspirates, or bronchial aspirates (collected)
 10-20% - neurologic complications or seizures
 SBA and CAP colony morphology - smooth, opaque,
(sequelae or complication)
gray-to-white colonies “hockey puck” – colony
 10-15% - fatality rate
remains intact when pushed across the plate with a
Laboratory
Neisseria Diagnosis– Laboratory Diagnosis
meningitidis loop
o Older colonies – “wagon-wheel” appearance
A.A. Specimen
Specimen Collection
Collection and
and Transport
Transport - Most strains can tolerate lower temperature and
- CSF, blood, nasopharyngeal swabs and aspirates, joint grow well at 28° C
fluids, and less commonly sputum and urogenital sites - Inhibited by colistin on gonococcal selective agars
- Also inhibited by SPS
 Non-capsulated and non-motile
B.B. Direct
Direct Microscopic
Microscopic Examination
Examination
 Oxidase positive
- Appear as intracellular and extracellular gram-
 Catalase positive
negative diplococci (with adjacent sides flattened)
 DNAse positive
- Such as CSF
 Butyrate esterase positive – using
tributyrin (substrate)
C.C. Culture
Culture and
and Incubation
Incubation
 Asaccharolytic – unable to metabolize carbohydrates
- Can grow on SBA and CAP
in the presence or absence of oxygen & must rely on
- Same atmospheric conditions with N. gonorrhoeae
other carbon sources for energy
- Specimens from mucosal surfaces that contain
normal microbiota should also be inoculated to a Commensal
CommensalNeisseria
Neisseriaspecies
species
selective medium as described under the culture of
N.gonorrhoeae - Inhabitants of the upper respiratory tract referred to
as commensals, saprophytes or non-pathogens
D.D. Laboratory
Laboratory Identification
Identification - Occasionally isolated from the genital tract
 CAP and SBA colony morphology – medium-sized, - Incidence of infections caused by the commensal
gray, and convex, and encapsulated strains are Neisseria spp. Is extremely low - main reason for the
mucoid strains are mucoid clinical microbiologists to be familiar with these
o Grow within 18-24 hours organisms to accurately separate them from the
 Blood agar underneath – colonies tends to have a pathogenic species.
1. N. cinerea – misidentified as N. gonorrhoeae
green tinge
- Oxidase positive - Morphology is also similar to one of strain of the N.
- Catalase positive gonorrhoeae on chocolate agar
- Glucose and maltose fermenter o Non-glucose fermenter
o Grows on SBA
 Gamma-glutamyl aminopeptidase positive (negative
o Susceptible to colistin (can differentiate)
for N. gonorrhoeae, N. lactamica, and M. catarrhalis)
 N. gonorrhoeae – resistant to
colistin
2. N. flavescens –yellow-pigmented Neisseria species
that is asaccharolytic
3. N. mucosa – large, very mucoid colonies (often adhere
to the agar)
4. N. lactamica – glucose, maltose and lactose fermenter
o Misidentified as N. meningitidis (glucose
and maltose fermenter only)
o Slightly smaller
5. N. polysaccharea – produces large amounts of
extracellular polysaccharide when grown in media
containing 1% or 5% sucrose
6. N. sicca – dry, wrinkled, adherent, and breadcrumb-
like colonies (sicca – Latin= “dry”)
7. N. subflava – “less yellow”
8. N. elongata, N. weaveri, N. bacilliformis – rod-shaped
 WEEK
WEEK 10:
10: ENTEROBACTERIACEAE
ENTEROBACTERIACEAE Classification
Classification

General
GeneralCharacteristics
Characteristics Based
BasedononLactose
LactoseFermentation
Fermentation

 ENTERICS – contains numerous diverse organisms - Several selective and differential media used to
 Gram-negative bacilli – large heterogeneous group isolate distinguishes between LF & LNF
 Facultative anaerobes - The most important media are:
 All species ferment glucose with the production of o MacConkey agar – most important media
acid or acid and gas used
 All are motile at 35oC with peritrichous flagella  Lactose fermented-
(except for Klebsiella, Shigella and Yersinia) pink colony
 Catalase positive (except for Shigella dysenteriae  Non- lactose-
type 1 which is catalase-negative) Colorless colony
 Oxidase-negative (except for Plesiomonas) o Eosin Methylene Blue (EMB) agar
 Non encapsulated (except for Klebsiella and o Salmonella Shigella (SS) agar
Enterobacter) o Triple Sugar Iron (TSI) agar
 Non-spore forming
A) Lactose Fermenter
1. Escherichia coli
 Reduce nitrate to nitrites (except for Photorhabdus
2. Klebsiella
and Xenorhabdus)
3. Enterobacter
 Most are commensal of the GI tract (except for
4. Serratia (except S. fonticola) – SLOW or LATE
Plesiomonas, Salmonella, Shigella and Yersinia)
5. Citrobacter – SLOW or LATE
 Serratia and Yersinia may grow at 1oC to 5oC
 Coliform bacilli – Escherichia, Klebsiella, Enterobacter
 They do not produce cytochrome oxidase except for
and Citrobacter
Plesiomonas
 Common habitat: Intestinal tract of humans and
B) Non-Lactose Fermenter
animals
1. Proteus
Microscopic
Microscopicand
andColony
ColonyMorphology
Morphology 2. Morganella
3. Providencia
 Gram-stain 4. Hafnia
- Gram-negative coccobacilli or straight-rods 5. Edwardsiella
 SBA or CAP (Chocolate Agar Plate) – no value for initial 6. Salmonella
identification 7. Shigella (except S. sonnei –
o Except for Klebsiella and sometimes “Late” LF)
Enterobacter – large and very mucoid 8. Yersinia
colonies
o Some isolates of E. coli – Beta-hemolytic Based
Basedononthe
theClinical
ClinicalInfection
Infection
(produces clear zones around colonies)
1) Opportunistic pathogens
 Differential and Selective Media - The opportunistic pathogens are often a part of the
o EMB (Eosin-Methylene Blue) usual intestinal microbiota of both humans and
o MAC animals. However, outside their normal body sites,
o HEA (Hektoen Enteric Agar), XLD (Xylose- these organisms can produce serious extra-intestinal
Lysine Desoxycholate) – highly selective opportunistic infections
 (H2S) Hydrogen Sulfide Production
can be seen 2) Primary pathogens
 Contains sodium thiosulfate and - Plesiomonas, Salmonella, Shigella and Yersinia
ferric ammonium citrate – - Not present as commensal biota in the GI tract of
produces blackening of (H2S)- humans. Produces infections from ingestions of
producing colonies contaminated food or water, or other sources.
 Have been used to differentiate and
characterize the genera. Definitive Virulence
Virulenceand
andAntigenic
AntigenicFactors
Factors
identification depends mainly on
- Affected by many factors such as the ability to adhere,
the biochemical reactions and
colonize, produce toxins and invade the tissue.
serological antigenic structures
- Has antigens that can be used in the identification
- Contains 1 or more Carbohydrate such as lactose and
 O antigen (somatic antigen) – this is a heat-stable
sucrose which show the ability of the species to
antigen located on the cell wall.
ferment specific Carbohydrate
 H antigen (flagellar antigen) – this is a heat-labile
 Fermentation – indicated by color change on the
antigen found on the surface of flagella, structures
medium – results from decrease in pH detected by pH
responsible for motility.
indicator
 K antigen (capsular antigen) – this this is a heat-labile
 Non- fermenting- Lack of color change and colonies
polysaccharide found only in certain encapsulated
retain original color of medium
species
o K1 antigen – E. coli
 o Vi antigen – Salmonella enterica subsp. - Test the ability of the bacteria to produce the enzyme
enterica serotype Typhi. tryptophanase and deaminate
tryptophan to indole, pyruvic acid, and
Media
Media andand Tests
Tests used
used for
for the Identification
the Identification ofof
ammonia
Enterobacteriaceae
Enterobacteriaceae - Two reagents can be used:
 Colony morphology, Gram-staining, Biochemical test 1. Ehrlich’s reagent – more sensitive
 Biochemical Testing – in test tube, more accurate but 2. Kovac’s reagent
requires a lot of time to inoculate and need a lot of space in o Both contains PDAB
incubator (paradimethylaminobenzaldehyde)
TripleSugar
SugarIron
IronAgar
Agar(TSI)
(TSI)  Positive reaction – pink/red
Triple
o An alcoholic layer concentrates the red color
- Used for presumptive identification of Gram-negative as a red ring at the top
enteric bacteria particularly in screening fo intestinal  Tryptophan
pathogens
- Ability to ferment glucose, lactose, and sucrose and 2)2) Methyl
MethylRedRedTest
Test
to produce hydrogen sulfide - Detects the end products of glucose fermentation and
 Composition: 1% lactose, 1% sucrose, 0.1% glucose mixed acid producers
 Ferrous sulfate and sodium thiosulfate are added – - If glucose is metabolized by the
to detect the production of hydrogen sulfide H2S mixed acid fermentation
(black precipitate in the medium) pathway, stable acid end
 pH indicator: Phenol red products are produced, which
 Original color of medium: red results in a low pH
 Kligler’s iron agar (KIA) is a similar medium, but only  Negative reaction – remain yellow after addition of
incorporates the carbohydrates glucose and lactose. MR pH indicator
o H2S production can also be detected with  Positive reaction – red color after addition of MR pH
this formulation indicator
 Slant – aerobic – both  MRVP MEDIUM – used for 2 tests
 Butt- anaerobic - This bacteria initially metabolize the glucose to pyruvic acid
- An agar slant of a special medium with a multiple which is further metabolized through the next acid
fermentation pathway to produce the stable acid
sugars
- Acid soap reduced- decreases the pH to 4.4 or below and
Reactions on TSI agar or KIA indicated by color change from yellow to methyl red (acidic
pH)
- Read within 18-24 hour incubation period
1. No fermentation – Alkaline slant/alkaline butt 3)3) Voges-ProskauerTest
Voges-Proskauer Test
(ALK/ALK or K/K) or alkaline slant/no change (ALK/no - Detects 2,3-Butanediol
change or K/NC) –Red/Red (red- buffered at 7.4) - It measure the production of acetoin
o Due to absence of carbohydrate after the addition of α-naphthol
fermentation (catalyst or color intensifier) followed
2. Glucose fermentation only, no lactose (or sucrose in by 40% KOH or NaOH
TSI) fermentation – Alkaline slant/acid butt or K/A – - Acetoin then oxidized into diacetyl -
Red/Yellow (yellow- below 6.8) 2,3-Butanediol
3. Lactose (or sucrose or both) fermentation –  Positive reaction – red complex
Acid/acid, Yellow/Yellow - pH becomes relatively neutral
4. H2S production – blackening of the medium - bacteria positive (either voges and methyl red test)
5. Gas production (aerogenic) or no gas production but not both
(nonaerogenic) – formation of bubbles or splitting of
the medium from the bottom of the tube 4)4) Citrate
CitrateUtilization
UtilizationTest
Test
- It determines whether an organism can use sodium
citrate as a sole carbon source
 Medium used – Simmons’ Citrate Medium (SCA) –
routinely used
 Ammonium salts are the nitrogen source in the
medium and utilization of these
salts results in the release of
ammonia, causing a pH change pH
IMViC indicator – bromthymol blue
IMViCReactions
Reactions
 Positive reaction – blue
1. Indole Test o From green to blue
2. Methyl Red Test - Bacteria able to use citrate will use
3. Voges-Proskauer Test the Ammonium salts releasing ammonia.
4. Citrate Utilization Test
Nitrate
NitrateReduction
ReductionTest
Test
1)1) Indole
IndoleTest
Test
- Determines whether an organism has the ability to
- Degradation products of tryptophan reduce nitrate to nitrite
 - Inoculated to nutrient broth containing a nitrogen Hydrogen
HydrogenSulfide
SulfideProduction
Production(H2S)
(H2S)
source, after 24 hrs. of incubation..
- Some reduce sulfur containing compounds to
- Detected by the addition of N,N-Dimethyl-α-
hydrogen sulfide during metabolism
naphthylamine and sulfanilic acid
- Utilizes the sodium thiosulfate sulfur source to form
 Positive reaction – red color (diazo red dye) presence
H2S, a colorless gas –H2S combines with the
of nitrite
indicator, ferrous sulfate producing black color
(positive reaction)
 Metal salts – used to indicate H2s formation
- Media demonstrate the production of H2S
o Sulfide-indole-motility agar
 More sensitive in the detection of
Oxidase
OxidaseTest
Test H2s because of its semi-solid nature,
its lacking of interfering
- Identify microorganisms with enzyme Cytochrome C
carbohydrates and the use of
Oxidase that is important in the ETC
peptonite ion as an indicator
- Determines the presence of the cytochrome oxidase
 Contains Ferrous sulfate – serves as
system that oxidizes reduced cytochrome with
an indicator for the production of
molecular oxygen
hydrogen sulfide
- Differentiates Enterobacteriaceae (oxidase-negative)
o Motility-indole-ornithine agar
from Pseudomonads (oxidase-positive)
o Hektoen enteric agar
 Reagent – tetramethyl-p-phenylenediamine
o Salmonella-Shigella agar
dihydrochloride added to filter paper and a wooden
o Triple sugar iron agar
applicator stick is used to wrap a colony onto the
o Kligler Iron agar
moist and filter paper
o Lysine iron agar
 Positive reaction – purple or lavender color (within
 FERROUS SULFIDE –production of hydrogen sulfide; a
10-15 sec.)
black precipitate is produced as a result of Ferrous
sulfate reacting with hydrogen sulfide gas

MotilityMotility
Test Test
- 0.4% agar
- Single stab in the medium
Urease
UreaseTest
Test  Routine medium used – SIM
(Sulfide-Indole-Motility) Agar
- Identify bacteria capable of hydrolyzing urea using
 Positive reaction – movement away
enzyme urease
the stab line or hazy appearance
 Proteus – differentiate from other enteric bacteria
throughout the medium (indicates a motile organism)
- Determines whether a microorganism can hydrolyze
- Some are motile only at room temperature
urea (by the action of urease enzyme), releasing a
- 2 motility tubes be inoculated (1- incubated at room
sufficient amount of ammonia to produce a color
temperature, 2 –incubated at 35oC)
change by a pH indicator
o Ammonia then reacts to Amino Amino
Acid Utilization
Acid Utilization
solution to form
 Decarboxylase and Dihydrolase Test
Ammonium carbonate,
increases the pH  Deamine Test – PAD Test (Phenylalanine Deaminase
Test)
 Preferred medium - Christensen’s
Urea Agar
A) Decarboxylase and Dihydrolase Test
 pH indicator – phenol red
- Decarboxylase tests determine whether the bacterial
 Positive reaction – bright pink color
species possess enzymes capable of decarboxylating
GelatinGelatin
Hydrolysis Test Test (removing the carboxyl group, COOH) specific amino
Hydrolysis
acids in the test medium – ability to use amino acids
- Also known as Gelatin Liquefaction test
as the energy and carbon sources
- Detects the presence of gelatinase that are proteases
 Two amino acids commonly used – lysine and
secreted extracellularly by some bacteria that
ornithine
hydrolyze or digest gelatin
 Products of decarboxylation – amine or diamine
- Bacteria that produce gelatinases that break down
molecules and CO2Agmatine (with resulting alkalinity)
gelatin into amino acids.
- Liquefaction of the gelatin is a positive test

 Agmatine – is metabolized further to putrescine and

also urea
 B) Decarboxylase and Dihydrolase Test  Oxidative deamination of lysine occurs – a compound
- Media include Moeller decarboxylase base medium, is formed that in the presence of
motility-indole-ornithine (MIO), or lysine iron agar ferric ammonium citrate forms a
(LIA) burgundy color on the slant
 Positive reaction – purple  Alkaline – K
o Caused by an alkaline pH shift is returned to  Acid – A
the original color of the uninoculated  Red Production (positive for
medium lysine deamination)- R
 Negative reaction – yellow
Opportunistic
Opportunistic Members
Members of of
thethe Family
Family Enterobacteriaceae
Enterobacteriaceae
o Yellow- Initially indicates glucose
fermentation & results in acid pH that is Escherichia coli (Colon
1) Escherichia coli Bacillus)
(Colon Bacillus)
lowered enough that can activate - The most significant species in the genus Escherichia
decarboxylase enzymes (considered harmless but now recognized as an
important human pathogen)
Phenylalanine Deaminase
Phenylalanine Test (PAD
Deaminase TestTest)
(PAD Test) - Associated with UTIs, diarrheal disease and CNS
- Amino acids can be metabolized by deaminases that infections
remove an amine group (NH group) to grow o Most common cause of UTIs in humans
- Determines whether an organism possesses the - Primary marker of fecal contamination in water
enzyme that deaminates phenylalanine to quality testing
phenylpyruvic acid - Most strains are motile and generally possess
- The surface of the slant is inoculated with a bacterial adhesive fimbriae and sex pili
colony after incubation, an addition of…  O, H, K antigen – serotyping useful in
 Reagent – 10% ferric chloride identification of strains
 Positive reaction – green color - Cultural Characteristics:
(phenyl pyruvic acid is present) o MAC – lactose-positive (pink)
- Proteus, Morganella, and colonies
Providencia species are o EMB – greenish metallic
phenylalanine deaminase-positive sheen
- Fermentation of glucose, lactose, trehalose, and
Lysine Iron Agar
Lysine Slant
Iron Agar Slant xylose
- Inoculated in the same manner as the PSI agar slant. - IMViC + + - - (Production of indole from tryptophan,
Most useful in conjunction with TSI and screening two methyl red positive, Voges-Proskauer & citrate
specimens for the presence of enteric pathogens negative)
- It is used primarily to determine whether the bacteria - Does not produce H2S, DNase, urease, or
decarboxylate or deaminate lysine phenylalanine deaminase
- Useful for differentiating Salmonella spp.
(lysine-positive) from Citrobacter spp. Pathogenic
PathogenicStrains
Strains
(lysine-negative)
1. Uropathogenic E. coli
- Also useful in differentiating Proteus,
2. Enterovirulent E. coli or Diarrheogenic E. coli
Morganella, and Providencia spp. from most
other members of Enterobacteriaceae Categories: also referred as enterovirulent E. coli
 Butt-slant medium
o aerobic process in slant) o Enterotoxigenic E. coli (ETEC)
o Lysine decarboxylation - anaerobic process o Enteroinvasive E. coli (EIEC)
in butt o Enteropathogenic E. coli (EPEC)
o Enterohemorrhagic E. coli (EHEC)
 Contains amino acid lysine, glucose, ferric ammonium
o Enteroadherent
citrate and sodium thiosulfate
 Diffusely adherent E. coli (DAEC)
 pH indicator- bromcresol purple
 Enteroaggregative E. coli (EAEC)
Lysine Iron Agar – Interpretation of Results
Uropathogenic
UropathogenicE.E.coli
coli(UPEC)
(UPEC)
 Lysine Decarboxylation (detected in butt):
- Strain of E. coli that is considered as the most common
o Positive Test: Purple slant/purple butt
cause of UTIs in humans
(alkaline) – K/K
 Pili – primary virulence factor associated with the
o Negative Test: Purple slant/yellow butt
ability of E.coli to cause UTIs
(acid), fermentation of glucose only – K/A or
o Other factors include cytolysins and
R/A
aerobactins
- If the organism produces Lysine decarboxylase,
o Usually originate in large intestine and can either
cadaverine is formed – it neutralizes the organic acid
exist as the predominant E. coli population or as a
formed by glucose fermentation and the butt of the small part of E. coli strains in the large intestine
medium reverts to the alkaline state which is color o Strains that cause UTIs produce factors that allows
purple them to attach to urinary epithelial mucosa
 Lysine Deamination (detected on slant):
o Positive Test: Red slant – R/A A)A) Enterotoxigenic
EnterotoxigenicE.E.coli
coli
o Negative Test: Slant remains purple – K/K or - Associated with diarrhea of adults and especially
K/A Alkaline – K children in tropical and subtropical climates
- It is the most common cause of diarrheal disease  Primary screening test – Stool culture using MAC
- Also referred to as “traveler’s diarrhea” containing sorbitol (SMAC)
- Consumption of contaminated food or water o Non-sorbitol fermenter – E. coli O157:H7
- poor hygiene educe availability of potable water and (colorless)
inadequate sanitation – contributing factors in spread o Sorbitol fermenter – other E. coli strains
and transmission of disease (pink colonies)
 High risk – “Achlorydia” – deficiency of hydrochloric  Additional test – 4-methylumbelliferyl-β-D-
acid within the stomach glucuronide (MUG) assay
o Inhibitor – stomach acidity - A biochemical test used to screen isolates for E.coli
- Colonization of ETEC on the small intestine is O157:H7
mediated by fimbriae o E. coli O157:H7 rarely produces the enzyme
o Permit ETEC to bind to specific receptors on β-glucuronidase (92% others produce it)
the intestinal microvilli o Other strains – producer
- Produce a heat-stable toxin (ST) or a heat-labile toxin o (+) Flourescence
(LT) or both
- Infection is usually mild, self-limiting E)E) Enteroadherent
EnteroadherentE.E.coli
coli
- Characterized by watery diarrhea, abdominal - Associated with two kinds of human disease: diarrheal
cramps, nausea (rare),usually no vomiting or fever syndromes and UTIs
 106 to 1010 organisms  Two types: DAEC and EADC
1. DAEC – UTIs and diarrheal disease (diffusely
B)B) Enteropathogenic
Enteropathogenic E.E. coli
coli adherent E. coli)
- Causes “infantile diarrhea” o Cystitis in children and acute pyelonephritis
o Severe diarrhea (<1 year old) in pregnant women
o Outbreak occurs in hospital nurseries and o Chronic or recurring UTIs
daycare centers 2. EAEC – causes diarrhea by adhering to the
o Rarely seen in adults surface of the intestinal mucosa (entero-
- Characterized by low-grade fever, malaise, vomiting aggregative E. coli)
and diarrhea o Produces watery diarrhea, vomiting,
- Stool contains large amount of mucus, no blood dehydration and occasionally abdominal
present pain mostly in children
o WBCs and RBCs are absent from the stool
C)C) Enteroinvasive
Enteroinvasive
E.E. coli
coli o Cause of diarrhea in HIV positive
- Produce dysentery with direct penetration, invasion,
and destruction of the intestinal mucosa F)F) E.E.Coli
Coli– –Extraintestinal
Extraintestinalinfections
infections
o Very similar to the diarrheal illness produced - G) One of the most common cause of septicemia and
by Shigella spp.(motile and LF) meningitis (40%) in neonates
o Differentiated by motility test and lactose - Newborn usually acquires the infection in the birth
fermentation canal before or during delivery
o May be nonmotile, NLF  K1 capsular antigen – virulence factor associated with
- Characterized by fever, sever abdominal cramps, neonatal meningeal infections
malaise and watery diarrhea - Clinically significant isolate in blood cultures from
o Occur in adults and children. Direct adults (bacteremia)
transmission Enteroinvasive E. coli from o From urogenital tract infection or from GI
person-to-person via the fecal order route source
- Rare compared to the 2 strains OtherEscherichia
Escherichiaspecies
species
Other
D)D) Enterohemorrhagic
EnterohemorrhagicE.E.coli
coli
 H) E. hermannii – yellow-pigmented organism; isolated
- O157: H7 strain of E. coli (1982) I) from CSF, wounds and blood
- Associated with hemorrhagic diarrhea and HUS o Foodstuffs such as raw milk and beef
(hemolytic uremic syndrome)
 E. vulneris – yellow-pigmented colonies (more than
o HUS – characterized by low platelet count, half of strains); isolated from humans with infected
hemolytic anemia and kidney failure wounds
- Fatal especially in young children and elderly adults in
 E. albertii – associated with diarrheal disease in
nursing homes
children (newest specie added)
- Watery diarrhea --Bloody diarrhea with abdominal
cramps, low grade fever or an absence of fever
- Stool contains no leukocytes
o Distinguishing characteristic from Shigella
spp. and EIEC infections
- Processed meats (undercooked hamburgers),
unpasteurized dairy products, apple cider, bean
sprouts and spinach
Klebsiella
Klebsiella
 Produces two cytotoxins (Shiga-like toxins) –
verotoxin I (Stx1) and verotoxin II (Stx2) - Found in the intestinal tract of humans and animals
or free-living in soil, water, and on plants
 - Associated with various opportunistic and hospital- 2)2) K.K.oxytoca
oxytoca
acquired infections, particularly pneumonia, wound - Produces infections similar to those caused by K.
infections, and UTIs pneumoniae
- Linked to antimicrobial-associated hemorrhagic
Common
CommonCharacteristics
Characteristics colitis
- LF on MAC - Identical to K. pneumoniae except for its production
- Most grow on SCA and in Potassium Cyanide Broth of indole, and there are reports of ornithine-positive
(KCN) isolates as well
- H2S negative
- A few hydrolyze urea slowly 3)3) K.K.pneumoniae
pneumoniaesubsp.
subsp.Ozaenae
Ozaenae
- All are MR- and VP+ - Isolated from nasal secretions and cerebral abscesses
- With a few , no indole is produced from thryptophan - Causes atrophic rhinitis – a tissue-destructive disease
- Variable motility –it distinguishes Klebsiella species restricted to the nose (“foul-smelling” atrophic
from most other members of the family rhinitis) – affects the interior of nose
enteobacteriaceae - Condition occurs when the tissue that lines the nose
(mucosa) and bone underneath shrink down
Species:
Species:
1. K. pneumoniae subsp. Pneumonia
4)4) K.K.pneumoniae
pneumoniaesubsp.
subsp.Rhicoscleromatis
Rhicoscleromatis
2. K. oxytoca, - Causes rhinoscleroma – an infection of the nasal
3. K. pneumoniae subsp. ozaenae cavity that manifests as an intense swelling and
4. K. pneumoniae subsp. rhinoscleromatis, malformation of the entire face and neck
5. K. ornitholytica,
6. K. planticola, 5)5) Enterobacter,
Enterobacter,Cronobacter
Cronobacterand
andPantoea
Pantoea
7. K. terrigena. - 6) Genus Enterobacter is composed of at least 12 species
- Clinically significant species:
1)1) K.K. pneumoniae
pneumoniae (Friedlander’s
(Friedlander’s bacillus)
bacillus) o Enterobacter cloacae
- Most commonly isolated specie o Enterobacter aerogenes
- Posses a large polysaccharide capsule o Enterobacter gergoviae
o Responsible for the moist, mucoid colonies o Enterobacter hormaechei
characteristic of K. pneumoniae on MAC - Motile
o Colonization of gram-negative bacilli in the - MAC – colony morphology resembles Klebsiella
respiratory tracts of hospitalized patients by  E. cloacae and E. aerogenes – two most common
K. pneumoniae increases with the length of isolates
hospital stay o Isolated from wounds, urine, blood and CSF
- Frequent cause of lower respiratory tract infection  Pantoea (Enterobacter) agglomerans – nationwide
among hospitalized patients and outbreak of septicemia resulting from contaminated
immunocompromised hosts such as newborns, IV fluids
elderly patients and seriously ill patients of respirators
 E. agglomerans complex – lysine -, ornithine -, and
o Resistant to multiple antimicrobial agents in
arginine - or “triple decarboxylases-negative”
newborn nurseries
 E. gergoviae -- found in respiratory samples and is
 Plasmid transfer of antimicrobial resistance –most
rarely isolated from blood cultures
severe with K.pneumoniae because of presence of
 Cronobacter (Enterobacter) sakazakii – produces a
Carbapenemase
yellow pigment and has been documented as a
- Causes community – acquired pneumonia – cough up
pathogen in neonates causing meningitis and
“currant-jelly like” sputum
bacteremia, often coming from powdered infant
o Blood tinged or rust-colored – sputum
formula
produced with Streptococcus pneumoniae
 E. hormaechei – isolated from human sources such as
- Also causes wound infections, UTIs, liver abscesses
blood, wounds, and sputum.
and bacteremia
 E. asburiae – is similar biochemically to E. cloacae and
has been isolated from blood, urine, feces, sputum,
and wounds.
 E. cancerogenus (formerly E. taylorae) – associated
with osteomyelitis after traumatic wounds.

Enterobacter
Enterobacter

 Virulence factor – polyssacharride capsule - Ornithine decarboxylase (ODC) positive

 Differential test – string test
o Viscose strings of greater than 5 mm in length
when a loop is used to stretch a colony on an
agar plate
 Neufeld-Quelleng test: Positive
 Growth in potassium cyanide (KCN): positive
o IMViC: - - + +
o TSI: A/A, gas+, H2S-
- Lysine decarboxylase (LDC) is positive by most species
(except E. gergoviae and E. cloacae)
- Growth on KCN agar
- Sorbitol fermentation +
- IMVic - - + +
- TSI: A/A (except E. agglomerans K/A), gas+ (except E.
- agglomerans neg), H2S- (table)
Serratia
Serratia - Can produce “swarming” colonies on nonselective
media, such as SBA
- opportunistic pathogens associated with outbreaks in
 “Burnt chocolate” odor
healthcare settings
- Both species are:
Species: o Urease positive
o H2S producer
1. S. marcescens (most clinically significant) 1. P. mirabilis – Indole neg; Ornithine pos
2. S. liquefaciens,
2. P. vulgaris – Indole pos; Ornithine neg
3. S. Rubidaea
o Sucrose fermenter; A/A in TSI
4. S. Odorifera
5. S. Plymuthica Morganella
Morganella
6. S. ficaria,
7. S. Entomophila - Only one specie – M. morganii
8. S. fonticola o Two subspecies: M. morganii subsp.
- Late LF on MAC except for S. fonticola morganii and M. morganii subsp. sibonii
 S. marcescens, S. rubidaea, and S. plymuthica – - Causes UTI and neonatal sepsis
produces “prodigiosin”, pink-red pigment - Motile but does not swarm
 S. odorifera – musty and pungent odor or “rotten  Same biochemical ID as P. vulgaris (except that P.
potato –like” odor vugaris is citrate +)
 S. liquifaciens – ferments arabinose growth in KCN  NLF on MAC
media  PAD test +
 S. marcescens – most significant  IMViC + + - -
o Causes outbreak in nurseries, burn units and  LIA rxn: R/A
cardiac surgery units  TSI rxn: K/A, gas +, H2S –
o Found frequently in hospital-acquired  Urease, KCN, ornithine decarboxylase positive
infections of the urinary or respiratory tract
 Motile but does not swarm
and bacteremic outbreaks
o Contamination of antiseptic solution used Providencia
Providencia
for the joint infections has resulted in the
- Five species:
epidemic of septic arthritis
1. P. alcalifaciens – is most commonly found in the feces
o Urease, gelatinase positive, arabinose
of children with diarrhea
fermentation negative
2. P. stuartii – outbreaks in burn units; isolated from
- General characteristics:
urine cuture (resistant to antimicrobial)
o IMViC - - + +
3. P. rettgeri – documented pathogen of urinary tract;
o TSI rxn: K/A, gas +, H2S –
also diarrheal disease among travelers. Has caused
occasional outbreaks in healthcare settings (resistant
to antimicrobial)
4. P. rustigianii – formerly identified as a strain of P.
alcalifaciens (rarely isolated and its pathogenicity also
remains unproven)
5. P. heimbachae
Hafnia
Hafnia Genus proteus providencia and morganella is distinguished
from other members of enterobacterius by the virtue of the
- Composed of only one specie: H. alvei
ability to deaminate the amino acid phenylalanine
- Gastroenteritis
- Major characteristic: delayed positive citrate reaction Edwardsiella
Edwardsiella
Proteus
Proteus - Three species:
1. E. tarda – only recognized human pathogen
 P. vulgaris and P. mirabilis – widely recognized
o Causes bacteremia and wound infections
human pathogens (has at least 4 species)
2. E. hoshinae
o P. mirabilis – most common clinical isolate
3. E. ictaluri
- Ascend the urinary tract causing infections in both
 NLF in MAC, urease test –
lower and upper urinary tract, can infect…
 Positive for lysine decarboxylase (LDC)
 Proximal kidney tubules – causing Acute
glomerulonephritis  IMViC + + - -
- Disseminated in the environment, are normal  TSI rxn: K/A, gas +, H2S +
intestinal microbiota, and are recognized as Citrobacter
Citrobacter
opportunistic pathogens
- Distinguished from the other members of the - Consist of 11 species that all have been isolated from
Enterobacteriaceae by virtue of the ability to clinical specimens
deaminate the amino acid phenylalanine o Most often isolated are C. freundii, C. koseri,
 NLF and C. braakii
 Urease activity of P. mirabilis can lead to struvite - Produces colonies on MAC that resemble E. coli
kidney stones (calculi) o Citrobacter – Late LF (ferment lactose slowly)
 - Considered as inhabitants of the GI tract and are
associated with hospital-acquired infections – most
frequently UTIs
 C. freundii has been associated with infectious
diseases acquired in hospital settings: UTIs,
pneumonias and intraabdominal abscesses
o Also endocarditis in intravenous drug
abusers
o Because it is H2S positive and 50% of the
strains fail to ferment lactose – the colony
morphology on selective media can be
mistaken for Salmonella when isolated from
stool cultures
 Differentiated by urea hydrolysis
and lysine decarboxylase (test)
 70% C. freundii urease pos;
Decarboxylate lysine neg
- Most hydrolyzes urea but all failed to decarboxylate
lysine whereas salmonella fails to hydrolyze urea and
mostly isolates the carboxylate lysine
 Urease +
 IMViC:
o C. freundii - + - +
o C. koseri + + - +
 TSI rxn:
o C. freundii A/A or K/A, gas +, H2S +
o C. koseri K/A, gas +, H2S –
Plesiomonas
Plesiomonas
 Vibrionaceae – formerly a member however studies
have shown that it is closely related to the family
enterobacteriaceae
- Only one specie – P. shigelloides
o It is found in soil and aquatic environments
(fresh and estuarine waters of tropical and
subtropical climates)
o Does not tolerate increased NaCl
o Minimum growth temperature of 8° C
- Widely-distributed among warm and cold-blooded
animals
o Dogs, cats, vultures, snakes, lizards, fish,
newts, and shellfish
- Similar colonies with E. coli on ordinary enteric media
- Also a potential cause of enteric disease
- Most common mode of transmission – ingestion of
contaminated water or food (uncooked or
undercooked seafood such as oysters, clams, or
shrimp)
 Three major clinical types of gastroenteritis
1. The more common watery or secretory diarrhea
2. A subacute or chronic disease that lasts from 14
days to 2 to 3 months
3. A more invasive, dysenteric form that resembles
colitis
 25% to 40% of all patients present with fever,
vomiting, or both, and the single most common
clinical symptom for all such patients is abdominal
pain
 Self-limiting, but antimicrobial therapy is indicated in
severe and prolonged cases
o Infected with HIV and are associated with
inflammatory bowel disease
 Shown a general resistance to the penicillin class of
antibiotics
- Straight gram-negative bacilli that occurs singly in
pairs or in short chains or filamentous forms, non-
spore-forming, non-capsulated and motile by
monotrichous or 2-5 lopotrichous flagella
- Vibriostatic test 0/129: Sensitive
- NLF on MAC (but some strain will not grow)
- Non- hemolytic, smooth and opaque in BAP
- Inositol-brilliant green-bile salt agar : white or green
to pink color – enhances the recovery from specimens
- Growth in HEA and CIN, no growth in TCBS and media
with NaCl
 TCBS – THIOSULFATE-CITRATE-BILE
– SALT-SUCROSE AGAR - not grow
 CIN – CEFSULODIN-IRGASAN-
NOVOBIOCIN AGAR - grow
 Yersinia spp.
- Oxidase positive
- Decarboxylase test: positive to trio decarboxylase test
- Inositol fermentation: positive
- IMViC + + - -
- TSI rxn: K/A, gas -, H2S –
- Antigenic structure: O and H antigens
Primary
PrimaryIntestinal
Intestinal Pathogens ofthe
theFamily
FamilyEnterobactericeae
Enterobactericeae
Pathogens of
- Salmonella, Shigella and Yersinia
Introduction
 Salmonella and Shigella organisms produce GI
illnesses in humans --- not considered normal biota
of the human intestinal tract – inhabit the GI tracts of
animals
 Humans acquire the infection by ingesting the
organisms in contaminated animal food products or
insufficiently cooked poultry, milk, eggs, and dairy
products
 Some Salmonella infections are transmitted by
human carriers (no animal reservoir)
 Shigella dysentery usually indicates improper
sanitary conditions and poor personal hygiene
 Yersinia infections include GI disease, mediastinal
lymphadenitis, fulminant septicemia, and pneumonia
Serotype
Serotypeororserovar
serovar
- Are groups within a single species of microorganisms
which share distinctive surface structures
o Type and number of cell-surface antigens
o Share similar antigen (antigenically-similar)
 Strain- low level taxonomic rank standing below the
species, represents diversity within the species
 Serotype – subgroup of species which are grouped
according to their antigenic properties. Subgroup of
same species of microorganisms which shares similar
antigens and the antibodies that are directed against
those antigens are the same
Salmonella
Salmonella
- Found in cold-blooded animals as well as in rodents
and birds (natural host)
- Most pathogenic Enterobacteriaceae
- Causes enteric fever (typhoid fever) and acute
gastroenteritis (food poisoning)
  MAC – produces clear, colorless,
non-lactose fermenting colonies
 HEA and XLD – colonies with black
centers are seen (due to production
of hydrogen sulfide)
- Gram-negative, facultatively and
aerobic bacilli
Salmonella – Biochemical
Biochemical Features Features
- NLF
- IMVIC - + - -
- Phenyalanine and Urease Neg
- H2S Producer (except S. paratyphi A)
- Do not grow in KCN medium
Salmonella
Salmonella
- S. enteritidis, S. cholerasuis and S. typhi
 S. enterica – type specie of the genus
o Subspecies:
o S. enterica subsp. enterica (subspecies I)
o S. enterica subsp. salamae (subspecies II)
o S. enterica subsp. arizonae (subspecies IIIa)
o S. enterica subsp. diarizonae (subspecies IIIb)
o S. enterica subsp. houtenae (subspecies IV)
o S. enterica subsp. Indica (subspecies VI)
- Nearly all former salmonella species have been places
as stereotypes below the level of salmonella enterica
subsp. Enterica
o Ex: S. enterica subsp. enterica serotype Typhi
or Salmonella Typhi
 S. bongori is a rarely isolated species – named after
the town of Bongor in Chad, Africa
1) Antigenic Structures
 Somatic O antigen (heat-stable) and flagellar H (heat-
labile) antigen – primary; used for serologic grouping
 Capsular K antigen (Vi antigen) – few strains; prevents
phagocytosis – important in identifying Salmonella
Serotype Typhi
2) Virulence Factors
 Fimbriae – role in adherence in initiating intestinal
infection
 Enterotoxin – causes gastroenteritis
Clinical
ClinicalInfections
Infections
1. Acute gastroenteritis or food poisoning – vomiting
and diarrhea
2. Typhoid fever – most severe form of enteric fever
o Caused by Salmonella serotype Typhi
3. Enteric fevers
o Caused by other Salmonella serotypes (e.g.,
Salmonella Paratyphi and Choleraesuis)
4. Nontyphoidal bacteremia
5. Carrier state following Salmonella infection
 Salmonellosis
- Humans acquire the infection by ingesting the
organisms in food, water, and milk contaminated with
human or animal excreta
 Salmonella serotypes Typhi and Paratyphi – no
known animal reservoirs; infections only occur in
humans
A)A) Salmonella
Salmonella– –Gastroenteritis
Gastroenteritis
- One of the most common forms of “food poisoning”
- GI infection caused by salmonellae results from the
ingestion of the organisms through contaminated
food
- Source of infection – poultry, milk, eggs and egg
products as well as to handling pets
o Common – insufficiently cooked eggs and
domestic fowl, such as chicken, turkey and
duck (1 mill bacteria higher than the dose
required for shigelloses)
o Cooking utensils, such as knives, pans, and
cutting boards used in preparing the
contaminated meat, can spread the bacteria
to other food
- Symptoms may appear 8 to 36 hours after ingestion
o Nausea, vomiting, fever, and chills,
accompanied by watery diarrhea and
abdominal pain
- Most cases of Salmonella gastroenteritis are self-
limiting
 Susceptible to infection – patients with sickle cell
disease and other hemolytic disorders, ulcerative
colitis and malignancy
o Severe in very young children, elderly adults
and patients with other underlying diseases
 Antimicrobials of choice include chloramphenicol,
ampicillin, and trimethoprim-sulfamethoxazole
B)B) Salmonella
Salmonella– –Typhoid
TyphoidFever
Fever
- Enteric fever caused by Salmonella Typhi is known as
typhoid fever
- A febrile disease that results from the ingestion of
food contaminated with the organisms originating
from infected individuals or carriers
- Clinical features:
o Prolonged fever
o Bacteremia
o Involvement of the reticuloendothelial
system, particularly the liver, spleen,
intestines, and mesentery
o Dissemination to multiple organs
- No known animal reservoir
 Paratyphoid fevers – caused by Salmonella serotypes
Paratyphi A, B, and C and Salmonella serotype
Choleraesuis – less severe
o Similar to typhoid fevers but are less severe
and lower fatality rate
- Occur more in tropical and subtropical areas where
international travelers are more likely to acquire the
infection
- Improper disposal of sewage, poor sanitation and lack
of modern potable water systems have caused
outbreaks of typhoid fever (food handlers- carriers)
- Typhoid fever develops approximately 9 to 14 days
after ingestion of the organism
- The onset of symptoms depends on the number of
organisms ingested – the larger the inoculum, the
shorter the incubation period
 First week of disease – fever accompanied by malaise,
anorexia, lethargy, myalgia, and a continuous dull
frontal headache
 Second and third week of disease – sustained fever
with prolonged bacteremia
o 2nd week – “Rose spots” (blanching, rose-
colored papules around the umbilical region)
appear
 - Organisms invade the gall bladder and Peyer’s patches
of the vowel, also reaches the intestinal tract via
Biliary tract
C)C) Salmonella
Salmonella – Bacteremia
– Bacteremia
- The serotypes most commonly associated with
bacteremia are Typhimurium, Paratyphi, and
Choleraesuis (nontyphoidal Salmonella)
- Observed in two different groups:
(1) Young children, who experience fever and
gastroenteritis with brief episodes of bacteremia
(2) Adults, who experience transient bacteremia during
episodes of gastroenteritis or develop symptoms of
septicemia without gastroenteritis (adults with
underlying diseases such as malignancies, and liver
diseases)
- Risk of metastatic complications could be more severe
than the bacteremia itself
D)D) Salmonella
Salmonella– –Carrier
CarrierState
State
- Who recover from infection may harbor the
organisms in the gallbladder, excreted in feces
 Antimicrobial therapy if gallbladder infection is not
evident
 Cholecystectomy has been the only solution to the
chronic state of enteric carriers
Shigella
Shigella
- Named after the Japanese microbiologist Kiyoshi
Shiga who first isolated the organism in 1896
- Closely related with Escherichia
- Not a member of the normal GI microbiota – all spp.
can cause bacillary dysentery
- Species: S. dysenteriae, S. flexneri, S. boydi, S. sonnei
o S. dysenteriae – most virulent
o S. flexneri – one of the causes of gay bowel
syndrome
 Characteristics:
o NLF (except S. sonnei)
o Non-motile
o Produce gas from glucose (except S. flexneri)
o Urease neg
o Does not produce H2S
o They do not decarboxylate lysine
o Fragile organisms- susceptible to various
effects of physical and chemical agents such
as disinfectants and high concentrations of
acids in bile)
 Serogroups: A (S. dysenteriae), B (S. flexneri ), C (S.
boydi), D (S. sonnei)
 Antigenic structure: Somatic O
 Specimen: stool or rectal swab
 Sensitive to pH change, must be process immediately
in lab
Shigella – Clinical Infections and Identification
Shigella – Clinical Infections and Identification
 S. dysenteriae – causes the enteric disease bacillary
Dysentery
o Characterized by the presence of blood,
mucus, and pus in the stool
o IMViC v + - -
o TSI rxn: K/A, gas -, H2S –
 S. sonnei – unique in its ability to decarboxylate
ornithine
o Produces a “delayed” positive fermentation
of lactose with the formation of pink
colonies on MAC agar only after 48 hours of
incubation
o ONPG-positive
o Self-limiting infection – characterized by
fever, watery diarrhea without blood
- Humans are the only known reservoir
- Shigellosis is highly communicable
o Low infective dose is required to produce
the disease (<100 bacilli are needed to
initiate the disease)
 Mode of transmission:
1. Direct person-to-person contact
o Via fecal oral route
o Anal-oral sexual activity
o Anal intercourse (men-to-men) – S. flexneri
2. Transmitted by flies, fingers and food or water
contaminated by infected persons
- Young children in day care centers particularly infants
younger than 1 year – most susceptible
Yersinia
Yersinia
- 14 named species, most are considered
environmental species
- Three species (human pathogen):
 Y. pestis – causative agent of plague, a disease
primarily of rodents transmitted to humans by fleas
 Y. pseudotuberculosis and Y. enterocolitica – caused
sporadic cases of mesenteric lymphadenitis in
humans, especially in children, and generalized
septicemic infections in immunocompromised hosts
 Y. enterocolitica – produces an infection that mimics
appendicitis (cause of diarrhea in community
outbreaks)
1)1) Yersinia
Yersiniapestis
pestis
- Three forms of plague in humans:
1. Bubonic or glandular form – most common;
results from the bite of an infected flea (appear 2-
5 days after infection)
o High fever with painful regional lymph nodes
known as buboes (swollen lymph nodes)
2. Septicemic form – bacteria spread to the
bloodstream
3. Pneumonic form – occurs secondary to bubonic
or septicemic plague when organisms proliferate
in the bloodstream and respiratory tract
o Can be a primary infection when the bacteria
is inhaled
o High fatality rate – 100%- untreated
- Gram-negative, short, plump bacillus 3)3) Yersiniapseudotuberculosis
Yersinia pseudotuberculosis
- Class A bioterrorism agent - Pathogen of rodents, particularly guinea pigs
 Methylene blue or wayson stain – shows intense - Reservoir are farm and domestic animals , usually
staining at each end of the bacillus – referred to as the birds
bipolar staining “safety pin” appearance - Causes a disease characterized by caseous swellings
- Preferred growth temperature – 25° C to 30° C called pseudotubercles – fatal in animals
- NLF in MAC, pinpoint at 24hrs in BAP - Human infections are rare
- “ Stalactite-shaped” pattern in broth – when o Close contact with infected animals or their
unshaken fecal material or ingestion of contaminated
- IMViC - + - - drink and food stuff
- TSI rxn K/A. gas -, H2S – o They spread to the mesenteric lymph nodes
producing a generalized infection that is
2)2) Yersiniaenterocolitica
Yersinia enterocolitica usually self- limiting
- Most commonly isolated specie  To differentiate from Y. pestis – motile at 18° C to 22°
- Can be acquired from contact with household pets C, urease positive and rhamnose fermentation
(organisms are found- domestic swine, cats and dogs) - MAC is NLF
- Causes enterocolitis or waterborne gastroenteritis - TSI rxn: K/A, gas -, H2S –
o Stools may contain blood
o Mild and self-limiting
- Associated with the transfusion of contaminated
packed red blood cells (sepsis) – survives in cold
temperature
o Food refrigeration becomes an ineffective
preventive measure
 Related infections – appendicitis-like syndrome,
arthritis, and erythema nodosum
o Appendicitis-like syndrome – severe
abdominal pain that is concentrated in the
right lower quadrant and fever
o Arthritis – common extraintestinal form of Y.
enterocolitica infection
 Following a gastrointestinal episode
or Appendicitis-like syndrome
o Erythema nodosum – inflammatory reaction
characterized by tender, red nodules that
may be accompanied by itching and burning
 Anterior portion of the leg, arms,
more common in female patients
- Incidence of generalized infection is higher among
adults with underlying diseases such as liver cirrhosis,
diabetes and AIDS, leukemia, aplastic anemia

- Acquired thru ingestion of undercooked food (pork,

pork intestines and vacuum packed meat), dairy
products (chocolate milk) and handling of pets
- Gram-negative coccobacilli with bipolar staining
- SBA or MAC
- Optimal growth temperature – 25° C to 30° C
- Motile at 25° C but not at 35° C
o CIN (Cefsulodin-irgasan-novobiocin) agar –
selective medium – “with bull’s-eye
appearance or dark red or burgundy center
transparent borders” at 48 hrs
o Produces pink to red-centered colonies
surrounded by a transparent border giving
the appearance of a bull’s eye
 Inhibitory agents – cefsulodin, irgasan, novobiocin,
bile salts, and crystal violet
o Inhibits normal colon microbiota
- MAC is NLF
- IMViC v + - -
- TSI rxn: K/A, gas -,
- H2S –
WEEK
WEEK11:
11:Vibrio,
Vibrio, Aeromonas, Campylobacter
Aeromonas, Campylobacter and
and Helicobacter
Helicobacter  Vibriostatic 0/129 susceptible
o Exhibiting a zone of inhibition to 150
- Important because some of them (vibrio species) have been
microgram vibriostat disc on Weller Hinton
associated with the large epidemics and pandemics
- Campylobacter species infections may play a role in GBS on Trypticase Soy Agar
- Helicobacter pylori can cause ulcers and has been linked to  Do not grow on MAC
gastric carcinoma  Routine media used: TCBS
- Most species exhibit a positive string test including V.
Vibrio
Vibrio
cholerae
- Resides in family Vibrionaceae o Observed as a mucoid “stringing” reaction
- Encompasses more than 110 validly published after emulsification of colonies in 0.5%
species, today only 10 of these species have been sodium desoxycholate
found in human clinical specimen - All species are halophilic or “salt-loving” (except for
- Commonly found in a wide variety of aquatic V. cholerae and V. mimicus)
environments, including fresh water, brackish or o Require the addition of sodium for growth
estuarine water, and marine or salt water
o Risk of infection can be reduced by avoidance of
eating raw/ undercooked shellfish particularly in
warmer summer months
o Pertinent to immunocompromised or have
serious underlying disease
o Those who are immunosupressed should avoid
exposure of wounds to fresh, estuarine and
marine water sources
- Most commonly isolated species:
 V. cholerae – cause a devastating diarrheal disease:
Cholera (has been documented since 1817)
 V. parahaemolyticus
 V. vulnificus
 V. alginolyticus

Vibrio – Clinical Manifestations - Can be differentiated from the similar genera

Vibrio – Clinical Manifestations
Aeromonas and Plesiomonas by key biochemical and
- Factors to consider when identifying Vibrio infection: growth requirement characteristic
1. Recent consumption of raw seafood (especially
oysters) Vibrio – Antigenic
Vibrio Structure
– Antigenic Structure
2. Recent immigration or foreign travel
- Three major subgroups of V. cholerae
3. Gastroenteritis with cholera-like or rice-water stools
 V. cholerae O1, V. cholerae O139, and V. cholerae
4. Accidental trauma incurred during contact with fresh,
non-O1
estuarine, or marine water or associated products
- All of which share a common flagellar (H) antigen and
(e.g., shellfish, oyster or clam shells, fishhooks)
somatic (O) antigen
- Based on the composition of O antigen, V. cholerae
O1 organisms are divided into the following serotypes:
1. Ogawa – India
2. Inaba – Philippine
3. Hikojima – Japan

Vibrio
Vibriocholera
cholera
 V. cholerae O1 – causative agent of cholera (aka
Asiatic cholera or epidemic cholera)
- Most epidemics occurring in developing countries in
Vibrio – Microscopic
Vibrio Morphology
– Microscopic Morphology which it is endemic in particular, cholera is prevalent
in the Bengal region of India and Bangladesh
 Asporogenous
 Curved (comma) or pleomorphic gram- CHOLERA
CHOLERA
negative rods
 In broth – possess polar, sheathed flagella - Spread through contaminated water
 In solid media – peritrichous, unsheathed o Improperly preserved and handled foods
flagella (fish and seafood, milk, ice cream, and
unpreserved meat) have been responsible
Vibrio – General
Vibrio Characteristics
– General Characteristics for the outbreaks
- The disease manifests in acute cases as a severe
 Facultative anaerobe gastroenteritis accompanied by vomiting and
 Catalase negative (except for V. metschnikovii) followed by diarrhea
 Oxidase positive (except for V. metschnikovii) - Rice watery stool (contain mucus flecks) that can
 Reduce nitrate to nitrite (except for V. metschnikovii) rapidly lead to dehydration and death
 Glucose Fermenter o Responsible: Cholera toxin, or choleragen
 o Once ingested, the bacteria colonize the o Differentiates sucrose-fermenting (yellow
small intestine in which they multiply and colonies) species from nonsucrose-
produce this toxin. fermenting (green colonies) species
- Treatment and management: intravenous or oral
fluids to replace fluids lost from the severe diarrhea
o Can be shorten by antibiotics such as
azithromycin or ciprofloxacin

TWO TYPES OF CHOLERA:

 Classical Type of Cholera

 El Tor Type of Cholera (more serious, fatal if not given Non-sucrose fermenter
an immediate treatment) 1. V. mimicus,
Differentiated by: 2. V. parahaemolyticus,
3. P. damsela, and
o Voges Proskauer Test – Pos: El Tor 4. most V. vulnificus strains and
o Polymixin B Susceptibility Test – Susceptible: 5. some V. vulnificus
El Tor
o Serological Test using Chicken RBC as Sucrose fermenter:
Antigen – Agglutinate chicken RBCs – El Tor 1. V. cholerae,
Other
Other Vibrio
Vibrio spp.
spp. 2. V. alginolyticus,
3. V. fluvialis,
1) Vibrio parahaemolyticus 4. V. furnissii,
- Summer Diarrhea 5. V. cincinnatiensis,
- Second most common Vibrio species implicated in 6. V. metschnikovii
gastroenteritis
- Most cases of gastroenteritis can be traced to recent  Enrichment procedure – alkaline peptone water with
consumption of raw, improperly cooked, or 1% NaCl can be inoculated (at least 20 mL) and
recontaminated seafood, particularly oysters incubated for 5 to 8 hours at 35° C before subculturing
- Self-limited disease to TCBS
o Watery diarrhea, moderate cramps or  All Vibrio spp. are inositol fermenter (except V.
vomiting and little if any, fever cincinnatiensis and some strains of V. metschnikovii)
o 24 – 48 hrs. - Symptoms begin after ingestion  Vibriostatic agent O/129 susceptible
 Kanagawa-toxin Positive – Produces a heat-stable
hemolysin that is able to lyse human erythrocytes in a Aeromonas
Aeromonas
special, high salt mannitol medium – Wagatsuma Agar
- Prefer aquatic habitat usually freshwater
o Possible association between hemolysin
production and virulence known as MAJOR
MajorCLINICAL SPECIES:
Clinical Species:
Kanagawa phenomenon.
- First recognized as a pathogen in Japan in 1950 when  A. hydrophila
it was the cause of a large food poisoning outbreak  A. sobria
 A. caviae – most commonly isolated specie
2) Vibrio alginolyticus
- Least pathogenic; most frequently isolated GENERAL
General CHARACTERISTICS
Characteristics

3) Vibrio vulnificus - Ubiquitous

- Two categories of infection: primary septicemia and
wound infections
Laboratory Diagnosis
Laboratory ofof
Diagnosis Vibrio spp.
Vibrio spp.
 SBA and CAP colonies – medium to large colonies that
appear smooth, opaque, and iridescent with a
greenish hue
o SBA - α- or β-hemolysis
 MAC – NLF colonies (except for V. vulnificus)
o May be overlooked and incorrectly
considered to be the members of family
Enterobacteriaceae(E.coli)
 Oxidase positive – imperative in testing
 MAC and CIN agar can give false-positive oxidase
reactions
 Transport media – Carry-Blair Media
o Not recommended – Buffered Glycerol Saline
 Recommended selective medium – TCBS (thiosulfate
citrate bile salt sucrose agar)
- Oxidase positive
- Glucose fermenter
- Gram-negative rods
- Motile with single tuft of flagella
- Widely distributed in freshwater, estuarine, and
marine environments worldwide
- Frequently isolated from retail-produced sources and
animal meat products
- Previously resided in the family Vibrionaciae however,
phylogenetic evidence from molecular studies has
resulted in the proposal of a separate family
CLINICAL MANIFESTATIONS
Clinical Manifestations
- Recognized as enteric pathogens but not as the same
manner of other common enteric pathogens
- Not only from aquatic infections but also associated
with untreated groundwater or consumption seafood
particularly raw oysters or clams / from fresh produce
and dairy products
  They may cause diarrheal disease as well as other
miscellaneous infections
 Diarrhea diarrheal infection is usually self-limiting
o But in pediatric, geriatric and
immunocompromised population
supportive therapy and antimicrobials are
often indicated.
 A. caviae – most frequently associated with
gastrointestinal infections
 Extraintestinal infections – septicemia, meningitis
and wound infections
 Most common presentation of wound infection –
cellulitis
o Wound infections invariably involves a
recent aquatic exposure (boating/ fishing
accident)
Laboratory Diagnosis
LABORATORY DIAGNOSIS
- Grow readily on most media used for routine and
stool cultures after 24 hr. incubation at 35oC
 Colony appearance – large, round, raised, opaque
colonies with an entire edge and a smooth, often
mucoid surface
o extremely strong odor is present, and
pigmentation ranges from translucent and
white to buff-colored
 Major clinical species hemolytic pattern – strong β-
hemolysis
 CIN colony appearance – pink-centered colonies
(mannitol fermenter) with an even clear apron-
resembling Yersinia enterocolitica
 Inositol negative – (-) Aeromonas,(+) Pleisomonas
 Oxidase positive –easily separate the (+) Aeromonas
from (-) Yersinia,
o A positive oxidase distinguishes Aeromonas
from the family Enterobacteriaceae except
P. shigelloides
 Glucose fermenter with or without the production of
gas (distinguishes Aeromonas from oxidase + non-
fermenting Pseudomonas isolates)
o A. caviae – Lactose fermenter
 String test negative – (+) Vibrio
 Vibriostatic O/129 resistant – Vibrios are susceptible
 Can grow in a media not containing salt (same with
Pleisomonas)
Campylobacter
Campylobacterand
andHelicobacter
Helicobacter
Campylobacter
Campylobacter
- Formally classified with a Vibrio because of their +
oxidase and characteristic microscopic morphology
but DNA homology studies have shown that they do
not belong with Vibrios
- Most are asaccharolytic
Most clinically relevant species:
1. jejuni subsp. Jejuni
2. C. coli – have similar clinical manifestations with C.
jejuni
3. C. fetus subsp. Fetus – isolated most frequently from
blood cultures and rarely associated with
gastrointestinal illness (immunocompromised and
elderly patients)
 C. jejuni – most common cause of bacterial
gastroenteritis worldwide
- Self-limited – results in 2-6 days
- Mode of transmission:
o Direct contact with animals and handling
infected pets, such as dogs, cats, and birds
o Indirectly by the consumption of
contaminated water and dairy products and
improperly cooked poultry
o Person-person transmission and can be also
sexually transmitted
- Present with a diarrheal disease that begins with mild
abdominal pain within 2-10 days after ingestion of the
organism
o cramps and diarrhea often follow the initial
signs
o may experience fever and chills and rarely
nausea and vomiting
- untreated patients can remain carriers for several
months
 C. fetus- is the causative agent of bacteremia
 GBS – Guillain-Barré Syndrome
o Campylobacter infection plays a role in GBS,
an autoimmune disorder characterized by
acute paralysis caused by damage to the
peripheral nervous system
Helicobacter
Helicobacter
- Important specie: Helicobacter pylori – major cause
of type B gastritis or peptic ulcer
o It colonizes the stomach for a long time and
can cause a low-grade inflammatory process
o Peptic ulcer – a chronic condition formerly
associated primarily with stress and chemical
irritants
- Classified as carcinogen
- Important risk factor for gastric carcinoma – long
term H. pylori infection (resulting in chronic gastritis)
Laboratory
LaboratoryDiagnosis
DiagnosisofofCampylobacter
Campylobacterand
andHelicobacter
Helicobacter
A)A) SPECIMEN
SPECIMENCOLLECTION
COLLECTIONANDANDTRANSPORT
TRANSPORT
 Specimen for Campylobacter causing gastroenteritis
(C. jejuni):
o Stool
o Rectal swab (less preferred)
 Delay in processing – transport medium used is Cary-
Blair
o Buffered-glycerol saline – avoided (toxic to
enteric Campylobacters)
 Specimen of choice of C. fetus – blood with
incubation at 35◦C to 37◦C
  Specimen for Helicobacter – gastric biopsy material  Helicobacter spp. – morphologically similar with
o Transport medium – Stuart medium (to Campylobacter
maintain the viability of H.pylori if a delay in o Curved or U-shaped
processing is anticipated) o Motile with multiple flagella at one pole
o Tissue samples may also be placed in
Cysteine-brucella Broth with 20% glycerol
and frozen at −70° C

B)B) CULTURE
CULTURE MEDIA
MEDIA
- Commonly used selective media for C. jejuni – Campy-
BAP (blood agar plate)
o Other media used: Butzler medium and E)E) COLONY
COLONY MORPHOLOGY
MORPHOLOGY
Skirrow’s medium  C. fetus subsp. fetus – smooth, convex, translucent
- Commonly used media for H. pylori – CAP or Brucella colonies
agar with 5% Horse red blood cells (nonselective  C. jejuni and other enteric Campylobacters – moist,
medium) runny looking, and spreading
o Selective media: Skirrow’s agar o Non-hemolytic
- Important that the inoculated medium be fresh and o Some are round and raised and others may
moist and must be incubated in a microaerophilic be flat
environment with an increased humidity.
DEFINITIVE IDENTIFICATION

 Both genus – Oxidase positive

 Urease test: Pos – H. pylori; Neg – C. jejuni
 Hippurate hydrolysis test positive – C. jejuni

C)C) INCUBATION
INCUBATION

Incubation temperature:

 Campylobacter jejuni – 42° C (growth of colon

microbiota is inhibited at this higher temperature)
 H. pylori – 37° C

Both Campylobacter spp. and Helicobacter spp. require a

microaerophilic and capnophilic environment

 Campylobacter – 5% O2, 10% CO2

 Helicobacter – 5% to 10% O2 and 5% to 12% CO2

D)D) MICROSCOPIC
MICROSCOPICMORPHOLOGY
MORPHOLOGY
 Campylobacter spp. – (campylo means) curved non–
spore-forming, gram-negative rods
o May appear as long spirals or ‘S’ or seagull-
wing shapes
o Stain poorly on gram-stained smears
o Carbol fuchsin is recommended as a counter
stain
 If Saffranin is used, counterstain
should be extended to 2-3 min.
o “Darting” motility (corkscrew movement)
on hanging drop preparations or when
visualized under phase contrast microscopy
o Single polar flagellum
o Brucella or Tryptic Soy broth - to observe the
typical motility
o Water and Saline – inhibit the motility