Enterobacteriaceae Salmonella-Shigella agar

Dr. Natividad 8/25/2015 Species differentiation is done by:

o Biochemical tests a battery of tests
I. Members of the Enterobacteriaceae (encountered in infection): Triple Sugar Iron (TSI)
Indole, Methyl Red, Voges-Proskauer, Citrate Tests (IMViC)
Urease
Sugar fermentation
All members of this family ferment glucose
Enzyme tests
Again, all members of this family are oxidase negative
Tests for other enzymes are available
o Serologic typing using specific antisera
Antigen O somatic polysaccharide (LPS)
Antigen H Flagellar antigen
Antigen K Envelope or capsular Antigen
Serologic tests are useful for Salmonella, Shigella and E. coli
Virulence factors
*Take note: o Endotoxin recall that this is an exclusive characteristic of Gram negative
All coliforms are Lactose fermenters o Capsule
Non-Lactose fermenters are further divided into Intestinal pathogens and K1 Ag- E. coli
Opportunistic/commensal pathogens Vi Ag S. typhi
o Exotoxins
II. General Characteristics of Enterobacteriaceae members: Enterotoxins
Gram negative bacilli Shiga toxin
Facultative anaerobes o Invasiveness
Oxidase negative o Type III secretion system
Most are motile with peritrichous flagella Proteins that are involved in the secretion of virulence factors
o Exceptions are the non-motile members: coded by a cluster of genes found in pathogenicity islands
Klebsiella Genes are clustered in one area of the chromosome
Shigella
Yersinia III. Clinical Divisions of Enterobacteriaceae
Cultivation A. Opportunistic group
o They are not fastidious. They grow very well in ordinary media, like Blood E. coli
Agar Plate (BAP), Chocolate Agar Plate (CAP) and Nutrient Agar Plate (NAP)
Proteus
o If we are to isolate them from clinical specimen (primary isolation) such as
Serratia
stools, we need to use differential and selective media to enhance their growth
and inhibit others Cedecea
Differential media: Klebsiella
Eosin Methylene Blue Agar (EMB) Providencia
Mac Conkey Agar Citrobacter
o Lactose fermenters grow as pink colonies Edwardsiella
o Non-Lactose fermenters grow as colorless colonies Enterobacter
o Media inhibits the growth of gram positive but allow the Morganella
growth of other gram negative (MCA is also selective Kluyvera
because of this)
Desoxycholate Citrate Agar Members of this group are commensals of the large intestines
Selective media: Disease is produced only when there are alterations in the defenses of the
Hektoen Enteric Agar host
Xylose-Lysine Desoxycholate Agar (XLD)

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 Infections are found on any tissue of the body outside the
intestines/extraintestinally (since they are intestinal commensals)
May be community-acquired or nosocomial infections
May be iatrogenic infections introduced by health care providers (e.g.
insertion of catheter, nonsterile phlebotomy)
Usual type of infections produced:
UTI
Pneumonia
Septicemia Donovan bodies indicates donovanosis
Wound infection Patients dont exhibit buboes like lymphogranuloma venereum (LGV)
Meningitis caused by Chlamydia trachomatis
Various GIT disorders except diarrhea

Klebsiella-Enterobacter-Serratia group
Grouped as such because they have the genetic and biochemical similarity
a. Klebsiella pneumoniae
Also called Friedlanders bacillus
Encapsulated, non-motile, urease positive
Capsule is their most important virulence factor
Colonies are mucoid f. Enterobacter (formerly Aerobacter)
Causes lobar pneumonia Includes E. cloacae, E. aerogenes, E. sakazaki
Characterized by extensive necrotizing consolidation of the Motile, no urease
lungs Any tissue may be infected most frequently associated with UTI
Currant jelly sputum (Blackish red) Methyl Voges-
Predisposing factors include alcoholics, and pulmonary Organism Indole Red Proskauer Citrate Urease Capsule Motility
K. non-
immunocompromised patients pneumoniae - - + + + + motile
Can also cause infection in other tissues (UTI, bacteremia)
Enterobacter - - + + - - motile
b. K. oxytoca produces same infection as K. pneumoniae Urease test is usually done to differentiate between K. pneumoniae and
Can be differentiated by Indole test (K. pneumoniae is Indole negative Enterobacter
while K. oxytoca is Indole positive)

c. K. ozaenae causes chronic atrophic rhinitis g. Serratia

Produces DNAse, lipase, gelatinase
d. K. rhinoscleromatis granulomatous disease of the nose o Other members of Enterobacteriaceae can produce only one of the
three enzymes. Serratia can produce all three.
e. K. granulomatis previously Calymmatobacterium granulomatis Major cause of nosocomial infections
Causes granuloma inguinale (donovanosis) chronic genital ulcerative Most common spp. is Serratia marcescens
disease Produces a characteristic red pigment called prodigiosin
Does not grow in artificial culture/agar causing red-colored colonies
Diagnosis is made by staining a smear from the specimen
Presence of 1-25 bacteria per mononuclear cell using Giemsa or Wright
stain for blood cells

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 Proteeae Group o Helps in synthesis of vitamins
Three (3) Genera: o Is part of 1st line of defense in suppressing invading pathogens
o Proteus P. mirabilis (most common species), P. vulgaris o Produce bactericidal substances that kill small populations of
o Morganella M. morgani pathogens in GIT (e.g. bacteriocin)
o Providencia P. rettgeri, P. stuarti, P. alkalifaciens index for fecal pollution of water
Produces Phenylalanine deaminase (PAD) o presence of E. coli in drinking water does not automatically mean it
Three genera mentioned above are Urease positive can induce disease but it may suggest presence of fecal matter in
o Urease is an enzyme used for the hydrolysis of urea into ammonia the water
and CO2. Specialized Virulence factors
Majority cause UTI o Adhesins
o Ammonia that is produced induces alkalinity of urine Colonization factor antigens CFA/I, CFA/II, and CFA/III
o Alkaline pH of urine Aggregative adherence fimbriae AAF/I and AAF/II
as seen in patients with UTI Bundle-forming pili (Bfp)
causes precipitation of calcium and magnesium salts Intimin
formation of urinary calculi P pili
causes damage to the renal epithelial cells Ipa protein
o P. mirabilis 2nd most common cause of UTI (Recall that when we Dr fimbriae
talk of Gram negative organisms, 1st common cause of UTI is E. o Exotoxins
coli) Heat-labile toxins LT1 and LT2
Proteus vulgaris and Proteus mirabilis exhibit swarming growth on blood agar Activates adenyl cyclase
Heat-stable toxins STa and STb
Activates guanylate cyclase
Shiga toxins Stx1 and Stx2
Hemolysin H1yA
Extraintestinal infections:
o most common cause of UTI in man
o Uncomplicated cystitis caused by uropathogenic strains that
produces hemolysin which is an exotoxin
Proteus - Antigenically cross-reactive with rickettsiae o Complicated (presents as pyelonephritis) non-uropathogenic strains
o Weil-Felix test (obsolete but inulit ni Doc because lumabas daw ito with P fimbriae virulence factor
sa 2012 boards) o Neonatal meningitis
Proteus Antigens OX2, OX19, OXK are used for the Acquired from birth canal
diagnosis of Rickettsiae because Rickettsiae is very Due to capsular K1 antigen
dangerous to handle 3 pathogens are important in neonatal meningitis
Streptococcus agalactiae (Group B Strep)
Citrobacter Group Listeria monocytogenes
Formerly Bethesda-Ballerup group Escherichia coli
Majority of the isolates are from the urinary tract, neonatal infections o Nosocomial infections
Pneumonia
Other organisms have been isolated from a variety of opportunistic infections Wound infection
Septicemia/Bacteremia E. coli is the most common
B. True Intestinal Pathogens cause of Gram negative sepsis
a. Escherichia coli Endotoxic shock
may be nonpathogenic, opportunistic or true intestinal pathogen
most abundant of the aerobic flora of the colon (but not the most abundant
flora! Pinakamarami lang siya among aerobes in GIT)
o 90% anaerobes
o 10% aerobes
Beneficial in the GIT because it:

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 Five (5) groups
1. EnteroPathogenic E. coli (EPEC) 3. EnteroHemorrhagic E. coli (EHEC)

Shiga toxin
(STx) is phage-
coded

A/E Attachement
effacement
Subunit A
toxic; binds to
60S ribosomal
subunit

Subunit B
binds to
receptors

4. EnteroInvasive E. coli (EIEC)

2. EnteroToxigenic E. coli (ETEC)
Ulceration of the
intestinal mucosa
Heat-labile toxin
(LT) activates
Adenyl C yclase

Heat-stable toxin
(ST) activates
Guanylyl Cyclase

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 5. EnteroAggregative E. coli (EAEC) STx (Shiga toxin)
Phage-coded
Subunit A Toxic, binds to 60S ribosomal subunit
o Induces depuration of specific adenine residue in ribosomal RNA
o Leads to cessation of protein synthesis and cell death
Adheres to small o Altered surface of the intoxicated endothelial cell activation of
and large bowel
epithelia in a
coagualation cascade formation of microthrombi cause distal
thick biofilm and ischemic necrosis, platelet consumption and red cell fragmentation =
elaborates hallmarks of Hemolytic-Uremic Syndrome (HUS)
secretory Subunit B binds to receptors
enterotoxins and
cytotoxins
STEC (Shiga toxin-producing E. coli)
Causes Hemolytic-Uremic Syndrome (HUS)
o Microangiopathic hemolytic anemia
o Results from Shiga toxin-induced damage to endothelial cells
o Kidney is particularly susceptible
o Ischemic necrosis of the bowel, brain, eye, or virtually any organ can
occur

Laboratory Diagnosis
Non-GIT infections
Use differential media for Lactose fermenters
Biochemical tests
E. coli that causes diarrhea acquire virulence via plasmid mediation except GIT pathogens
EHEC which uses a bacteriophage Identifaction done in reference laboratories except:
o EHEC in culture use Sorbitol containing Mac
Conkey Agar (S-MAC)
Non-sorbitol fermenting EHEC
Confirm it by serotyping
O157:H7 most
important and most
common
Immunoassay for toxin
production
All strains of E. coli except for EHEC produces pink
colonies because of their ability to ferment sorbitol

b. Shigella
Primarily a human pathogen and does not infect animals
Infection is usually confinedin the GIT and no bacteremia seen,
therefore it cannot be diagnosed using blood specimen
Shigella spp. are categorized based on their O antigen. Note that the
species doesnt have and H antigen
Four species that can cause bacillary dysentery
o S. dysenteriae (shiga bacillus) - Group A
Causes the most severe disease of all the species
o S. flexneri - Group B
Most common in developing countries
o S. boydii - Group C

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 o S. sonnei - Group D Ciprofloxacin, Ampicillin, Doxycycline, Trimethoprim-
Most common in industrial world Sulfamethoxazole
Source of transmission is by food handlers o Susceptibility testing is required before
Infection of Shigella spp. is also called Shigellosis testing
Very low infective dose: o Because Shigella harbors the R plasmid
Only 100 bacilli is needed to cause disease which gives multidrug resistance via
Compared to conjugation
o Shigellosis 102
o S. Typhi (Typhoid Fever) 103 c. Salmonella
o Other Salmonella 105 Single species: Salmonella enterica
o Vibrio cholera - 108 o Other previous species are now called serotypes
Transmitted by four (4) Fs: Salmonella enterica serotype Typhi
Fingers How to write it: Salmonella Typhi
Flies Transmission is via fecal-oral route
Food o Important sources of most serotypes are animals
Feces o Typhi, Paratyphi, and Cholerasuis human source only
Three (3) clinical entities
No prolonged carrier state
1. Enterocolitis Salmonellosis; most common manifestation
Prominent signs and symptoms:
Most common serotype: Typhimurium
Painful passage of bloody, mucoid stools (Tenesmus)
Incubation period: 8-48 hours
Cramping (dysentery)
Nausea, headache, vomiting and profuse diarrhea
Fever Often self-limited
Convulsions Antibiotics are not recommended because they prolong
clinical signs and symptoms by induction of antimicrobial
Pathogenicity therapy
i. Invasiveness Infection limited to the gut
Ulceration of the terminal ileum and colon, with blood ID: 10
and mucus in stool 2. Septicemia
Rarely penetrate through wall or the blood stream Occurs in one of 2 settings:
Shigella only invades the lining. It doesnt penetrate the Chronic disease such as sickle cell anemia
wall A cancer patient with enterocolitis
ii. Shiga toxin
Mainly secreted by S. dysenteriae Type 1 Ingestion Intestine Early blood stream invasion Seeding in many
Subunit B for binding to intestinal epithelium organs: Osteomyelitis, pneumonia, meningitis
Subunit A1 inhibits protein synthesis by binding with
60S ribosomal subunit Intestinal symptoms usually absent because Salmonella
Enterotoxic, cytotoxic, neurotoxic immediately leaves the intestine and proceeds to the
Other species produce shiga-like toxin blood stream
3. Enteric fever: Typhoid and Paratyphoid Fever
Laboratory Diagnosis Vi Ag antiphagocytic, capsular antigen
Rectal swab of an ulcer best specimen Severe life threatening
Biochemical test results: Incubation period: 10-14 days
o Non-lactose fermenting Facultative intracellular parasite
o TSI: K/A (alkaline slant & acid butt), no gas, no H2S Fever, malaise, headache, constipation, bradycardia,
Confirmation by serotyping by slide agglutination myalgia, hepatomegaly, splenomegaly, Rose spots in
abdomen and chest (rare)
Treatment Chronic carrier state : in gallbladder
Usually self-limited Typhoid Mary (read on her case)
Replacement of fluids and electrolytes

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 If patient is not treated, cholecystitis, intestinal
perforation will develop

Intestine Lymphoid tissues Bacteremia (Salmonella in blood, no

diarrhea yet) Various organs + intestines (after 2 weeks, diarrhea is
present)

Laboratory Diagnosis
Enteric fever
Culture from blood or bone marrow specimen best specimen
1st and 2nd week blood specimen is collected since
bacteria is not yet in the intestine
after 2nd week stool or urine specimen is collected
since the bacteria is already in the intestines
S. Typhi non-lactose fermenter; TSI K/A no gas with
small amount of H2S
Widal test obsolete (uses O and H antigen)
Typhi Dot specific IgG and IgM

Treatment
Enteric fever: Typhoid and Paratyphoid fever
o Drug of choice: Ceftriaxone or Ciprofloxacin
Prevention vaccine (50-80% protection)
o Intramuscular contains Vi polysaccharide capsule
o Oral live attenuated S. Typhi; produces IgA in
intestine

d. Yersinia enterocolitica
Cause Yersiniosis
Zoonotic from animals
Majority of human infections come from Northern Europe (cold countries)
because they grow better in 22O C
Not diagnosed in the Philippines; often missed because incubators are of
different temperature (37OC)
Mode of transmission:
o Feces of animals - most common
o Person to person
Manifestations:
o Bloody diarrhea
o Mimics appendicitis

Laboratory Diagnosis
Positive culture of the organism
Requires cold enrichment (-4OC)

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