Part II

 E. coli on Blood Agar (BA):

Antigenic Structures -Colonies are big, circular, gray, and moist
 Heat Stable Lipopolysaccharide (LPS) -Beta Hemolytic colonies are formed
is the major cell wall antigen.
 E. coli possess 4 antigens: H, O, K
and F.

 E. coli on MacConkey Agar (MAC):

H or Flagellar Antigen
-Colonies are circular, moist, smooth and of
- Heat and alcohol labile
entire margin
protein
-colonies appear flat and pink
- Present on the flagella
-they are pink (Lactose Fermenter)
- Genus specific
- Present as monophasic
 E. coli on Sorbitol MAC:
O or Somatic Antigen
-MacConkey Sorbitol Agar is only slightly
- Heat stable, resistant to selective, due to the low concentration of
boiling up to 2 hours. 30 bile salts
minutes
-Christal violet inhibits the growth of gram-
- Occur on the surface of the
positive bacteria, especially enterococci and
outer membrane
staphylococci. Sodium chloride maintains
- An integral part of the cell
osmotic equilibrium. Neutral red is an
wall
indicator. D-Sorbitol is the fermentable
K or Capsular Antigen carbohydrate. Differentiation of enteric
microorganism is achieved by the
- Heat labile
combination of sorbitol and the neutral red
- Acidic Polysaccharide antigen
indicator.
present in the enveloped
- Boiling removes the K -The growth of E. coli 0157:H7 on the
antigen MacConkey Agar with Sorbitol shows
- Inhibit phagocytosis colorless colonies and most of the fecal
flora ferment sorbitol and appear pink.
F or Fimbrial Antigen
Colorless or pink to red colonies are
- Heat labile proteins produce depending upon the ability of the
- Present in the fimbriae isolate to ferment the carbohydrate
sorbitol. (Greater sorbitol fermented the
greater the color pink to red)
Cultural Characteristics
-Sorbitol fermenters: pink to red colonies,
 E. coli on Nutrient Agar (NA): some surrounded by zones of precipitated
bile
- They appear large, circular, low convex,
grayish, white, moist, smooth, and opaque. -Sorbitol non-fermenters: colorless colonies
- They are of 2 forms: smooth (S) and Rough -It is used for isolation and detection of
(R) form. Enteropathogenic Escherichia coli 0157:H7
the primary serovar associated with
- Smooth forms are emulsifiable in saline
hemorrhagic colitis (HC) and hemolytic
-Due to repeated subculture, there is uremic syndrome (HUS)
smooth to rough variation (S-R variation).
 - it aids in the differentiation of E. coli
0157:H7 from other strains , especially
lactose fermenters E. coli since E. coli
0157:H7 DOES NOT FERMENT SORBITOL
( they will appear colorless)

 E. coli on Eosin Methylene Blue

Agar (EMB):
-Pink-purple colonies with a greenish
metallic sheen are formed

 E. coli on m-ENDO Agar:

-Colonies are green metallic sheen
-Metabolize lacrosse with the production of
aldehyde and acid

 E. coli on Liquid Media: (BROTH)

-They show homogenous (uniform) turbid
growth within 12-18 hours.
- R form agglutinate spontaneously, forming
sediment on the bottom of the test tubes
-after prolonged incubation (> 72 hours),
pellicle (like a covering a the top end in
STRAINS OF E. COLI
test-tube) are formed on the surface of the
liquid media  The effectiveness of E. coli as
pathogen is illustrated by the fact
-heavy deposits are formed which disperses
that :
on shaking
-the bacteria are the most common
Test Result Test Result gram-negative rods isolated from
patients with sepsis
Urease - Indole Production + -responsible for causing more than
Production 80% of all community-acquired UTIs
Catalase - Carbohydrate + as well as many hospital-acquired
test fermentation test infections
Motility + Methyl Red + -a prominent cause of gastroenteritis
Citrate - Voges Proskauer -
Utilization

TSI = A/A, G(+) , H2S (-)  The strains of E. coli that cause
gastroenteritis are subdivide. For the
IMVC= (+ + - -)
pathogenic enteric E. coli strains (6
pathotypes)

- Enterotoxigenic E. coli (ETEC)

 - Enteropathogenic E. coli (EPEC) -the disease is usually nosocomial and
- Enterohemorrhagic E. coli (EHEC) causes watery diarrhea with mucus bot no
- Enteroaggregative E. coli (EAEC) blood
- Enteroinvasive E. coli (EIEC)
- Diffusely Adhering E. coli (DAEC)
 Enteroaggregative E. coli (EAEC)
-produces stable and labile toxins and
 Enterotoxigenic E. coli (ETEC)
colonization factors that lead to a watery,
-commonly known as “Traveler’s Diarrhea”, mucoid diarrhea without red cells or white
“Weanling Diarrhea”, “Montezuma’s cells in the stool
revenge”, “Turista”, “New Delhi fever”,
-characterized by their autoagglutination in
upon ingestion of contaminated food or
a “stacked-brick” arrangement over the
water
epithelium of the small intestine and, in
-Produces heat-labile toxin (LT) and/ or some cause, the colon
heat-stable toxin (ST) which leads indirectly
-Most strains are “O” untypeable but “H”
to fluid loss. (by diarrhea)
typeable
-Toxigenic but NON-INVASIVE
-manifestation of the disease include acute
-Pathogenesis involves attachment to the and or chronic diarrhea
intestinal mucosa mediated by fimbrial
protein called CFA (COLONIZATION FACTOR
ANTIGEN) and elaboration of heat-stable  Enteroaggregative E. coli (EAEC)
and heat- labile enterotoxins.
-also known as Shig toxin-producing E. coli
-the illness is characterized by nausea, (STEC) or Vero-cytotoxic E. coli (VTEC).
diarrhea without blood, pus or mucus, mild Presence of Shiga toxin 1 (Stx1) or 2 (Stx2)
vomiting, chills and headache.
-pathogenicity is cause by verotoxin
production, which is a cytotoxin resembling
toxins produced by Shigella spp. Most
 Enteropathogenic E. coli (EPEC)
common nut severe serotype is
-Known for causing nursery outbreaks of EHEC: 0157:57
diarrhea (watery diarrhea)/infantile
-consumption of undercooked ground beef
diarrhea and occasionally causes sporadic
or meat water, unpasteurized milkor fruit
diarrhea in adults through fecal-oral
juices (e.g. cider made from apples
transmission.
contaminated with feces from cattle),
-NON-toxigenic and NON-invasive uncooked vegetables.
-Mechanism of Diarrhea involves the -cause HUS (Hemolytic Uremic Syndrome) ,
adhesion to intestinal mucosa, which form characterized by acute renal failure,
cup-like projections called pedestals thrombocytopenia, and microangiopathic
hemolytic anemia.
-cause Hemorrhagic colitis with severe
-A/E lesions (attacking and effacing lesions)
abdominal pain, lower gastrointestinal
are produced on the intestinal epithelium
hemorrhage, and bloody diarrhea.
which leads to disruption of brush border
epithelium causing increased secretions and
watery diarrhea
 Enteroinvasive E. coli (EIEC)
-Invades the intestinal epithelium causing a - found in the digestive and
Shigella-like infection. This process of respiratory systems of humans and
epithelial cell destruction with inflammatory
animals
infiltration can progress to colonic
ulceration. -can cause opportunistic infections
-causes dysentery (Shigellosis-like disease) - produce a capsule that protect the
usually in children characterized by fever,
bacteria from phagocytosis
cramps and bloody stools that contains
RBCs, neutrophils, and mucus -K. pneumonia is the most common
-this invasive E.coli is identified by the isolated pathogenic species
Sereny Test, which is characterized by the
-causes pneumonia
development of Keratoconjunctivitis in
guinea pigs - may be involved in
bacteremia, meningitis , wound
infections , UTIs
 Diffusely Adhering E. coli (DAEC)
-Recently , DAEC strains have been
recognized as the sixth class of
diarrheagenic E.coli and appear as
heterogenous group
-Adhere to the epithelial cells in diffuse
pattern and also causes a diarrheal
syndrome with a watery stool withour RBC
or White Blood Cells Present
-It expresses diffuse adherence fimbria
which contribute to the pathogenesis
-These strains are age dependently
involved in diarrhea in children , are
apparently not involved in diarrhea in
adults, and can also be asymptomatic
intestinal microbiota strains in children and
adult

 KLEBSIELLA
- All are encapsulated, the capsule is
responsible for the mucoid colonies
- all are NONMOTILE
-all are lactose fermenters except K.
rhinsc;erpmatis and K. ozaenae
 1. Klebsiella pneumoniae  ENTEROBACTER
- “Friedlander’s bacillus” - gram negative rods most species are
Lactose fermenter
-Normal florain the URT (Upper
Respiratory Tract ) and GIT -often confused with K. pneumoniae
(Gastrointestinal Tract) because most members are urease
(+)
-Encapsulated and appears as mucoid
colonies that tend to string - Differentiated from K. pneumoniae
in the following :
-nosocomial pneumonia
- MOTILITY = positive
-Pneumonia is necrotic and
- OD = positive
hemorrhagic; currant jelly-like
- TSIA = A/A G H2S (-)
sputum; lung abscess
- IMVC = (- - + +)
-Meningitis, surgical wound
-E. sakazakii – produce yellow
infections, UTI , bacteremia
pigment
- E. aerogenes – free living bacteria ;
Klebsiella pneumoniae in causes UTI and sepsis
EMB/MacConkey/XLD
-Large, Pink, moist mucoid colony
- TSIA = A/A G H2S (-)
-IMVC = (- - + +)
-Neufeld quellung (+)

2. Klebsiella oxytoca -indole (+),

resembles K. pneumonia in
disease spectrum.

3. Klebsiella ozaenae – ccauses

ozaena , a fetid chronic atropic
disease of the nose

4. Klebsiella rhinoscleromatis –
causes rhinoscleroma
(granulomatous destruction of
nasal passages and pharynx)
  HAFNIA
- Gram negative straight rods,
peritrichous flagella
-non- encapsulated, non spore
forming
-Non-Lactose Fermenters, may
resemble Salmonella
(TWO TABLE KASALI SA QUIZ) -most strains are translucent or
colorless; rare strains may
produce red or pink colonies
on media containing sucrose
-facultative anaerobic
 Serratia marcescens
-glucose is fermented with the
-Gram negative , motile , non spore- production of acid and gas
forming bacilli -chemoorganotrophic; utilizing
-routinely isolated from bloodstream both fermentative and
and wound sites using blood agar respiratory type metabolisms.
culture or from respiratory and - utilizes citrate, acetate and
urinary sites using selective culture malonate as sole source of
methods. carbon
-possible causative agent of
-opportunistic pathogens intestinal disorder
-produce RED PIGMENT called
PRODIGIOSIN RECOMMENDED MEDIA
1. For culture :
- 3rd leading cause of nosocomial
infections -Tryptic Soy Agar , or Blood Agar 5%
2. For Selective isolation :
BIOCHEMICAL REACTIONS
-EMB, MacConkey Agar , Hektoen
- DNASE = positive Enteric Agar , SS or XLD agar.
- GELATINASE = POSITIVE
3. For maintenance:
- LIPASE = POSITIVE
- ONPG = POSITIVE -CTA at room temperature for up to 1
- TSIA = K/A G H2S (-) year
- IMVC = ( - - + +) -Lyophilization is required for long-
term storage
  PROTEUS -Proteus mirabilis is the source of OX-
K
-Gram negative , motile , pleomorphic
, piliated rods
-swarming growth with bluish-gray
confluent surface growth
-burnt gun powder/ burnt chocolate
odor
CULTUE
-Glucose fermenter but NON Lactose
Fermeter, non spore-forming , - Swarming growth in BAP
facultative anaerobes - No swarming in MAC and form
,smooth, pale or colorless
-urease (+) , oxidase (-) , reduces
(NON LACTOSE FERMENTER)
nitrate to nitrite
colonies
-PAD- phenylalanine decarboxylase - Trimethoprim kill proteus
(+)
-Ornithine decarboxylase
DIENE’S PHENOMENON
-P. vulgaris = negative
-Proteus mirabilis is well known for
- P. mirabilis = positive its ability to differentiate into
hyperflagellated, motile and
- the bacilli possess thermostable , ‘O’
elongated swarmer cells that rapidly
(somatic) and thermostable ‘H’
spread over a surface.
(flagellar) antigens, based upon which
several serotypes have been -when two different strains of P.
recognized mirabilis swarm on the same agar
plate, a visible demarcation line with
-certain strains of Proteus vulgaris
lower cell density forms at the
(OX-19, OX-2 and OX-K) produce O
intersection, and this line is known as
antigens that are shared by some
Dienes line (after Louis Dienes, who
rickettsiae.
described the phenomenon in 1946)
- these Proteus strains are used in an
- BUT when two identical isolates
agglutination test ( the Weil-Felix
meet, the swarming edges merge
test) for serum antibodies produced
without formation of Dienes line
against rickettsiae of the typhys and
spotted fever groups.
-the 2 types of P. vulgaris , namely
type OX-2 and OX-19 are used as
antigens in the Weil-Felix test ( a
hetrophile antibody test for
rickettsial infection)
  MORGANELLA  PROVIDENCIA
-Gram negative straight rods, motile -Gram negative straight rods,
motile
-non-capsulated, non spore-forming
-non-encapsulated, non spore-
-some Morganella strains appear
forming
hemolytic when cultured on Blood
Agar, while others produce a reddish- -relatively large, dull gray
brown pigmentation. colonies; non-swarming
-facultatively anaerobic. -appear colorless on EMB, HEA,
Chemoorganotrophic, having both and SSA
fermentative and respiratory type
-strains of P. stuartii and P.rettgeri
metabolism.
may produce wound and burn
-Acid production from mannose, but infections
gas is not produced.
- the rise in medical importance of
-nosocomial UTI, bacteremia, these organism is associated with
respiratory tract infection and wound its tendency to cause nosocomial
infections infections and with their marked
resistance to numerous antibiotics
BIOCHEMICAL REACTION
-this species is also associated
TEST REACTION
with diarrhea, urinary tract
OXIDASE NEGATIVE
CATALASE POSITIVE infections, wound and burn
UREASE POSITIVE infections, bacteremia, and
INDOLE POSITIVE poultry disease
VOGES- POSITIVE TEST REACTION
PROSKAUER Catalase Positive
SIMMONS- NEGATIVE Oxidase Negative
CITRATE H2S Negative
METHYL RED POSITIVE Urea is not
H2S NEGATIVE hydrolyzed,
ORNITHINE- POSITIVE except P.
DECARBOXULASE rettgeri (P.
PRODUCE ACID stuartii may be
FROM urease
MANNOSE positive ,
IMVIC REACTION ++-- approximately
15%)
Lysine- and Negative
Ornithine-
Decarboxylase
Arginine - Negative
Dihydrolase
 Indole Positive media, wounds, abscesses, the
Methyl Red Positive throat and autopsies.
Voges Negative BIOCHEMICAL REACTIONS
Proskauer  Citrate can be utilized as
Citrate positive the sole carbon source.
ONPG negative  Catalase- Positive
IMVC ++-+  Oxidase – Negative
LIA R/A  Nitrate reduced to
TSIA K/A H2S (-) nitrite.
 Lysine-Decarboxylase-
 CITROBACTER negative
- Gram negative straight rods in  Methyl Red- positive
singles/pair, motile  Voges-Proskauer –
- Non – encapsulated non-spore negative
forming.
- At 24 hours, colonies on
nutrient agar are generally 2-4
millimeters in diameter,
smooth, low, convex, and
moist.
- They are usually appearing  SALMONELLA
translucent or opaque and gray - Gram negative bacilli ,short
with a shiny surface and an straight rods, encapsulated (Vi
entire edge. antigen) , Non spore-former ,
- Mucoid or rough strains may motile
occur occasionally. - All are motile except S.
- -colonies which slowly ferment pullorum and S. gallinarum
lactose can resemble - All produce gas (aerogenic)
Salmonella colonies on enteric except S. typhi and S.
media gallinarum
- Citrobacter spp. Often occur in - Most strains produce H2S
water sewage, soil and food - Most species invade intestinal
and may be present in clinical mucosa; S. typhi and S.
specimens where they can be choleraesuis invade the
opportunistic pathogens. bloodsream
- Citrobacter is found in clinical - found in the intestine and feces
bacteriology as an of most birds, reptiles, and
opportunistic pathogen, not mammals.
only in stools but also in urine, - some strains produce and
sputum and specimens from enterotoxin, fecal-oral is the
bacteremia, meningitis, otitis mode of transmission.
 - poultry and eggs are
particularly common source of
Salmonella in Xylose Lysine
Salmonella.
Deoxycholate
- 2 important pathogens
o S. typhimurium – causes Salmonella typhi
salmonellosis -Red Colonies
o S. typhi – cause typhoid
fever -Black Centers
- Salmonellois requires 10^5 – Salmonella choleraesuis
10^8 organism
-Red colonies
- Typhoid characterized by
spiked fever, enlarge liver,
spleen, rose spots on abdomen
BIOCHEMICAL REACTION
or chest , hyperplasia of Peyer’s
patches
- Those with sickle cell disease -Bismuth Sufite agar = black colonies
are susceptible to salmonellosis
-TSIA- K/A G H2S (+)
IMVIC
S. typhi = - + - -
Others = - + - +
Test Reaction
Non-Lactose
fermenter and
Non Sucrose
fermenter
Bismuth Sufite
agar = black
colonies
TSIA K/A G H2S (+)
IMVIC S. typhi = - + - -
-\ Others = -
+-+
-Salmonella – Shigella Agar
-Salmonella will not ferment lactose, Oxidase Negative
but produce hydrogen sulfide (H2S) Catalase Positive
gas. The resulting bacterial colonies
will appear colorless with black
centers.
IMMUNOLOGICAL TEST 3 CATEGORIES OF INFECTION
1. Widal Test- Screening Test 1. ENTERIC FEVER (TYPHOID
FEVER)
-usually positive on the second and
- Salmonella typhi ( Elbert’s
third week of infection
Bacillus)
-high or rising titer O suggest an - Salmonella paratyphi A
active infection ( Paratyphoid bacilli)
-High or rising titer H suggest past - Salmonella paratyphi B
immunization (Salmonella schottmuelleri)
- Salmonella paratyphi C
-High or rising titer Vi occurs in some (Salmonella hirschfeldii)
carriers
2. Slide Agglutination Test 2. Bacteremia with focal lesion
- Salmonella choleraesuis
- O or somatic antigen – basis for sub-
grouping; uses S. typhi 0 90
3. Enterocolitis / Gastroenteritis
- H or flagellar antigen – basis for - Salmonella typhimurium
typing ; uses S. typi H 901 - Salmonella enteritidis serotype
gallinarum (Gartner’s Bacillus)
-Kaufmann-White Scheme types the
- Salmonella pullorum
Salmonellae into more than 2,000
serovars.
3. Phage typing
Salmonella typhi
- using bacteriophages; most specific
-humans are the only host
-note : in typhoid fever, women are
-causes typhoid fever
more likely to become carriers than
males; persons who contact typhoid -infection occur via ingestion of food
fever later in life are more likely to or water contaminated with sewage
become carriers than those who containing bacteria from carriers
contact it early
-Bacteria can pass through the
-first female who had typhoid (cook) intestines into the bloodstream and
– Typhoid Mary (In new York- Spread into the liver, spleen, bone marrow,
Salmonella) – Mary Mallon (Real and gall bladder
name)
-Bacteria from the gall bladder can
4. Typhidot reinfect the intestine, producing
gastroenteritis and recurrence of
-Test for salmonella Ab
bacteremia
-IgM – new infection
-S. typhi is anaerogenic
-IgG – old infection
-Blood – POSITIVE on 1st week of
infection
-Stool and Urine – POSITIVE on 2nd
- A parasite of the digestive tract
and 3rd week of infection
of humans
- Produce a diarrhea- inducing
enterotoxin
 Shigella
- Cause a severe form of
-resembles E. coli but are non-lactose bacillary dysentery called
fermenter and non-motile Shigellosis which can be
-fecal-oral is the mode of disseminated to other site of
transmission the body
- Shigella should be suspected
when a Non lactose fermenter
which is nonmotile and H2S
negative is isolated from the
stool
- Shigellosis involves watery
diarrhea lasting up to 3 days
and followed by less frequent
bowel movement and then the
dysenteric phase characterized
by frequent stools with
presence of RBC, WBC and
Mucus
 -Shigella do not ferment lactose or
produce hydrogen sulfide gas , so the
resulting colonies will be colorless
SHIGELLA ACCORDING TO
SEROGROUPING
 GROUP A- S. dysenteriae –
Shiga’s bacillus
-causative agent of the
most severe type of
dysentery (Japanese
epidemic) , non-mannitol  YERSINIA
fermenter - the name Yersinia is given after
the French bacteriologist
 GROUP B – S. flexneri -Strong’s Alexander Yersinia who
bacillus discovered the organism
-commonly isolated in causing plague in 1984
developing countries , - at present 11 species of
causative agent of the Yersinia are known and only 3
Philippine dysentery species ( Y. pestis , Y.
epidemic enterocolitica and Y.
pseudotuberculosis ) are
 GROUP C – S. boydii – Boyd’s pathogenic to humans
bacillus - other species can occasionally
 GROUP D- S. sonnei – Sonno- cause opportunistic infections
Duval bacillus to humans
-most isolated in - Yersinia Pestis (formerly
industrialized nations Pasteurella pestis ) cause
- The LPS (Lipopolysaccharide) in its plague (Black Death)
cell wall is responsible to their - Causes plague – primarily in
resistance to gastric acidity. rodents, transmitted to
humans by bite of rat fleas 3
-Shiga toxin interferes with protein
types:
synthesis and is neurotoxic and
1. Bubonic (80-85% mortality)
enterotoxic
– is hemorrhagic and
-Shigellosis is treated with fluid and characterized by
electrolyte replacement (eat banana) Schwartzmann
phenomenon where there
- Produces reddish colonies in XLD
is presence of black spots
-TSIA = K/A H2S (-) over the body.
-IMVC = ( V + - - ) 2. Septicemic (97-99%
mortality)
-salmonella-Shigella Agar
 3. Pneumonic (>90 %
mortality)

GENERAL CHARACTERISTICS
Yersinia pestis
- Gram negative pleomorphic
- Oxidase-negative.
coccobacilli in singles , pairs ,
- Catalase-positive.
chains. Non motile, non spore
- GF w/o gas, NLF
forming , and non -acid fast.
- When stained with Giemsa ,
Wayson’s or methylene blue, it
shows bipolar staining with two
\
ends densely stained and the
central area clear giving a Yersinia enterocolitica
characteristic “ safety pin - ram-neg coccobacilli, short, bipolar
appearance” staining, NLF
- Capsules are present but my be
seen in cultures grown at 37 -facultative anaerobe, slow growers
degree Celsius (250C), motile at 250C NOT at 370C
- Yersinia pestis are found in
- transmitted by consuming or
many wild animals like dog,
squirrels, rabbits, mice etc. handling contaminated food, most
CULTURAL CHARACTERISTIC commonly raw or undercooked pork
- recommended medium is CIN products; milk or milk products that
(CefsuIodin-Irgasan-NovobIocin)
were not pasteurized, inadequately
-grows fairly rapid on BAP producing
pasteurized, or contaminated after
small , non hemolytic round ,
transparent , glistering colorless pasteurization; or untreated water.
colonies with undulate margin, but - can invade the intestinal mucosa
grows slowly in NA
leading to systemic diseases
-old colonies are like shiny
‘hammered copper surface’ , large, - produce enterotoxin similar to E. coli
mucoid , opaque with yellowish -can survive within macrophages and
centers and whitish edges PMNS
-XLD -reddish pinpoint colonies -causes gastroenteritis , septicemia,
-in broth culture overlaid with sterile exudative pharyngitis and arthritis
oil, old cultures shows a characteristic -Cultured on CIN (Cefsulodin Irgasan
“stalactite streamers” type of growth Novobiocin)- the bacteria produces
then settle to the bottom (“cotton “bull’s eye colonies”
fluff”)
-associated with erythema nodosum-
red nodules
CULTURAL CHARACTERISTICS
MacConkey Agar
-Lactose negative colonies
-flat , colorless , or pale pink
- 1-2 mm diameter
YSA (CIN) Agar
-deep red center
-surrounded by clear , colorless
zone
-1-2 mm diameter

LAIA Slant
- Y. enterolitica = ( K / A - -)
- Salmonella = ( k / k + - )
Christensen’s Urea Agar
-Yersinia enterocolitica = pink
color ( urease positive)
- E. coli = no color (urease
negative

Bile Esculin Agar

-Yersinia enterocolitica ( except
biotype 1A) are esculin negative
( absence of black color)
-Ent. Faecalis = esculin positive (black
color)