ENTEROBACTERIACEAE (PART 1)

Investigation of an Unknown Isolate

 In the isolation of a specific unknown organism from clinical specimen, the causative agent that is causing the infection has to be known
and the investigation involves 3 basic steps:
1. Isolate the organism from the clinical specimen
o When the specimen is received in microbiology laboratory, the first thing that is to be done is microscopic examination.
 In order for the Microbiology lab to know what specific culture medium/media to be used in the isolation of the organism of
interest, the characteristic of the unknown organism in terms of gram staining reaction has to be known first.
o 1st step: make a bacterial smear, stain with Gram staining, and evaluate its appearance under the microscope so that whatever
findings that the microbiology lab has discovered in the microscopic evaluation, the next step comes in
o 2nd step: Identification of the correct culture media to use so that proper isolation of the unknown organism can be obtained.
 The culture media needs to be inoculated properly and the technique that needs to be used in the inoculation of the culture medium
is the four quadrant streak plate method.
 Four quadrant streak plate method is the semi-qualitative/qualitative method of isolating pure colonies to make sure that pure
culture is dealt with.
 Pure colonies are the colonies of the organism of interest without any contaminants/contamination
 Colonies in the 4th quadrant are always used first
 If colonies in the 4th quadrant do not show any colonies/apparent growth, proceed to the 3rd quadrant.
 If 3rd quadrant does not show any growth, proceed to the 2nd quadrant
 Never use the colonies in the 1st quadrant because getting a specific volume of the specimen to be investigated, when starting
to streak in the culture medium with 1st quadrant, it contains organisms of interest plus the contaminants present in the clinical
specimen.
 When 4th, 3rd, and 2nd quadrants do not show growth, if the sample is still credible, the inoculation has to be redone.
 If the specimen is considered sterile, the wire loop is not heated in between quadrants anymore to elicit a higher chance of
isolating and growing the organism of interest in that specimen. Example:
 Example: CSF (collected from lumbar puncture)
 Sterile specimen- a specimen wherein the only organism present in it is the organism of interest or the organism really causing
the infection.
 Heating the wire loops between quadrants will kill the organism of interest and when proceeding to the next quadrants, there
is lesser chances to isolate the organism of interest.
 If the specimen is not sterile/contaminated, four quadrant streaking should involve heating the wire loop in between the
quadrants.
 Example: Urine, Sputum (collected from alveoli)
 Heating the wire loops between quadrants will increase the chance of killing the contaminants in the non-sterile sample and
also increasing the chance of isolating the organism of interest since in a non-sterile sample, the contaminants are dominating.
 Serial dilution pour plate method is the quantitative method of counting colonies; uses Quebec colony counter to count colonies
and a specific formula is used to get the CFU/mL
 Not used because it can be a mixture of the contaminants and the organism of interest.
o 3rd step: Biochemical reactions will only come into play once the colonies are already apparent in the culture media.
 Colonies should be used to inoculate the different culture media involved in the biochemical tests/reactions
 The characteristic colonies needed to be used for the biochemical tests should also be known
2. Inoculate the culture media using four quadrant streak plate method
3. Incubate the culture media at 37°C for 24-48 hours
4. Colonies now are apparent in the culture media

Enterobacteriaceae
 Is a wide family of organisms composed of different genera/genuses under the family and became a very important organism of interest
in bacteriology laboratory because it causes wide variety of infections to humans
 Causes common infections to humans that is why isolation and identification of these organisms in microbiology/bacteriology
laboratory has to be understood correctly so that proper diagnosis can be achieved or obtained at the end of the investigation.
 Escherichia  Enterobacter  Proteus
 Shigella  Erwinia  Providencia
 Salmonella  Serratia  Morganella
 Citrobacter  Hafnia  Yersinia
 Klebsiella  Edwardsiella
 The collective term used to describe the Enterobacteriaceae organisms is enterics/enteric organisms.
 Divided into two types of organisms depending on the clinically relevant members/species (on what type of infection cause to humans)
o Opportunistic Pathogens
 Coliform organisms are gram-negative bacilli/gram-negative organisms which are non-spore forming (vegetative organisms)
and it has the ability to ferment lactose to acid with hydrogen gas
 Escherichia  Citrobacter  Klebsiella
 Enterobacter  Edwardsiella  Morganella
Erwinia  Proteus (only group who does
Serratia not ferment lactose)
Hafnia  Providencia
They can cause infection; are normal flora in the gastrointestinal tract of any human. These opportunistic pathogens do not cause
infections in the GI tract because it is their habitat.
 They will only cause infection outside of GI tract like pneumonia (infection in the lungs), Meningitis (CNS), UTI (Kidneys),
Septicemia (Blood).
 It is hard for these organisms to go out of the GI tract because the risk of these organisms going out of the GI tract is high. The
immune system of any human host can fight off any infection that is outside of their comfort zone
o Overt/True Pathogens
 These group of pathogens/enteric organisms causes infections in the GI tract. The presence of these organisms in the GI tract of any
human host always means infection.
 Shigella  Salmonella  Yersinia
 Mode of Transmission: fecal-oral route
 Two types of enteric organisms based on the ability of the enteric organisms to ferment the sugar lactose
o Lactose Fermenters
 Enteric organisms that ferments lactose to acid with hydrogen gas; usually the opportunistic pathogens (except for Proteus)
 Always produces colored colonies in the culture media
o Non-lactose Fermenters
 Enteric organisms not fermenting lactose; usually the true pathogens
 Always produces colorless colonies in the culture media

Biochemical and Morphological Characteristics of Enteric Organisms

 Gram-negative coccobacilli, facultative anaerobe organisms, oxidase negative (do not possess indophenol oxidase) and catalase
positive (produce enzyme catalase) organisms.
 Can be motile and non-motile. Principle: other genera of the family have flagella and the others do not. Example: Escherichia,
Salmonella, Proteus are motile and has Peritrichous flagella). Klebsiella has capsule, Escherichia also have a slime layer.
 Do not possess spores. (Vegetative cells)
 All of them ferment glucose to acids or Ferment glucose→ acid with gas
 Reduce nitrates→ nitrites

Different Culture Media for Enteric Organisms

 Criteria: level of its selectivity
1. Differential, Slightly/Mildly Selective Media
 Clear appearance: undergone autoclave so that all other content are eliminated
A. Eosin-Methylene Blue Agar (EMB)
o Slight selective for isolation gram (-) intestinal bacteria; promotes the growth of gram (-) organisms by
inhibiting the growth of gram (+) organisms. Its level of selectivity is mild because all gram (-) organism
can grow in the culture medium.
 Non-enteric gram (-) organisms can grow in this culture medium
o Sugars: Lactose and Sucrose
o pH indicators: Eosin Y, Methylene blue (aniline dyes); There are pH indicators because there is SUGAR in the medium, pH
sensitive dyes.
o Inhibitors: Eosin Y (orange in color), Methylene blue (blue in color) AKA “aniline dyes”; responsible for the selectivity of
the culture medium
o Differentiates Lactose Fermenters from Non-lactose Fermenters
 If an organism is growing in the EMB agar and that organism is a lactose fermenter, then the pH indicators will indicate that
the organism have fermented the lactose by giving off colored colony appearance.
 Since the organisms growing in EMB is not fermenting lactose, then the pH indicators were not challenged to produce their
colors→ COLORLESS
o The lactose content is responsible for the differential property of the EMB agar because it is incorporated in the EMB agar and
that would immediately mean it can differentiate lactose fermenters from non-lactose fermenters
o Uninoculated appearance: Clear, dark purple
B. MacConkey Agar (MAC)
o Slightly selective medium for gram (-) bacteria
o Supports the growth of lactose fermenters with a red or pink colored colonies because of the pH indicator;
non-lactose fermenters will appear COLORLESS.
o Sugar: Lactose (responsible for differential property)
o pH indicator: Neutral red
o Inhibitors: bile salts and Crystal violet; responsible for the selectivity
o Differentiates Lactose Fermenters from Non-lactose Fermenters
o Uninoculated appearance: Clear, Light pink
2. Differential, Moderately Selective Media
 Turbidity: moderately selective media does not involve autoclaving in its preparation
A. Salmonella Shigella Agar (SSA)
o Moderately selective medium; primarily for Salmonella organisms
o Promotes the growth of gram (-) enteric organisms; only the true pathogens are suported by this medium
o Sugar: Lactose
o pH indicators: Neutral red, Ferric ammonium citrate and sodium thiosulfate
 Ferric ammonium citrate and sodium thiosulfate→ hydrogen sulfide indicators and it is also the responsible for its
differential property
o Inhibitors: sodium citrate, brilliant green and bile salts→ makes the agar moderately selective
o Shigella: Colorless colonies without black centers (do not produce H2S)
o Salmonella: Colorless colonies with black centers (produces H2S)
o Uninoculated appearance: Turbid, light orange
B. Hektoen-Enteric agar (HE/HEK)
o Moderately selective media; primarily for both Salmonella and Shigella organisms
o Sugar: Lactose, Sucrose and Salicin
o pH indicators: acid fuchsin and bromthymol blue
o H2S indicators: ferric ammonium citrate and sodium thiosulfate (differential property)
o Inhibitors: bile salts→ responsible for its selective property
o Shigella: Green colonies without black center (do not produce H2S)
o Salmonella: Blue-green colonies with black center (do not produce H2S)
o Uninoculated appearance: Turbid, dark green
o Colonies of Shigella and Salmonella are colorless but since the colorless colonies of these two are present on top of a dark green
colored culture medium that is why it is appearing green colonies without black center for Shigella and black center for Salmonella.
C. Xylose Lysine Deoxycholate Agar (XLD)
o Moderately selective medium; primarily isolation of Shigella organisms
o Sugars: Lactose, Sucrose and Xylose
 The incorporation of xylose in the XLDA makes it specific for Shigella organisms because all enteric
organism except Shigella ferments Xylose
 When shigella organisms are grown in XLD agar they will give a different colonial morphology because they
do not ferment xylose.
o pH indicators: Phenol red
o H2S indicators: sodium thiosulfate and ferric ammonium citrate→ (differential property)
o Inhibitors: sodium deoxycholate→ selective property
o Shigella: Red colonies without black centers
o Salmonella: Red colonies with black centers
o Uninoculated appearance: Turbid, bright red
3. Highly Selective Media
A. Bismuth Sulfite Agar (BSA)
o Highly selective medium for isolation of Salmonella typhi
o Sugar: Glucose
o pH Indicator: bismuth sulfite
o H2S indicator: ferrous sulfate→ (differential property)
o Inhibitor: brilliant green
 It inhibits the growth of all gram (-) enteric organisms except for Salmonella typhi
o Characteristic Colonial Morphology for S. typhi: Jet-black colonies with silver metallic sheen
o Uninoculated appearance: Turbid, light green
B. Cefsulodin Irgasan Novobiocin Agar (CIN)
o Antibiotics; supports the growth of Yersinia Enterocolitica
o Appearance: Bull’s-eye colonies
o pH indicator: Neutral red
o Colorless colonies with red/burgundy central dot
o Uninoculated appearance: Turbid, Reddish-pink color
C. Sorbitol MacConkey Agar (SMaC)
o Isolation of EHEK/VTEK
o Other name for the Escherichia coli 0157:H7 serotype is EHEK/VTEK; an organism of interest because it causes massive
traveler’s diarrhea
o All different strains or serotypes of Escherichia coli ferments sorbitol. Only EHEK/VTEK does not ferment sorbitol
o Uninoculated appearance: Turbid, Light pink