ENTEROBACTERIACEAE

Family Enterobacteriaceae - often referred to as "enterics"

1. g-rods (or coccobacilli)
2. all ferment glucose
3. all reduce nitrates to nitrites
4. all are cytochrome oxidase (-)
5. all are motile except Klebsiella, Shigella, and Yersinia; if motile, the flagellar arrangement is peritrichous
6. Grow well on MacConkey's agar

EPIDEMIOLOGY:
 Inhabit a wide variety of niches (human & animal GIT, various environmental sites)
 Natural habitat: the intestinal tract of humans and animals
 Some are agents of zoonoses
 Other species (Salmonella spp., Shigella spp., Yersinia enterocolitica) only inhabit the bowel at the time they
are causing infection; acquired by ingestion of contaminated foods or water

Two Categories of Members of Family Enterobacteriaceae

1. Opportunistic pathogens
- often a part of the normal intestinal flora; outside their habitat, they may produce serious extra intestinal opportunistic infection
2. Primary intestinal pathogens
- true pathogens; not commensal flora in the gastrointestinal tract

Virulence and Antigenic Factors

- ability to colonize, adhere, and invade tissues; production of toxins; presence of plasmids that mediate resistance to antimicrobials
- antigens that can be used for identifying different serologic groups
1. O (somatic antigen) heat-stable located in the cell wall
2. H (flagellar antigen) heat-labile found in the flagellum
3. K (capsular antigen) heat-labile capsule K1 antigen of E. coli; Vi antigen of Salmonella typhi

Classification of the Family Enterobacteriaceae

Tribe
Genus Species
Escherichieae Escherichia coli, blattae, vulneris, fergusonii, hermannii
Shigella dysenteriae, flexneri, boydii, sonnei
Edwardsielleae Edwardsiella tarda, hoshinae, ictaluri
Salmonelleae Salmonella
Subgroup I (most serotypes)
typhi, choleraesuis, paratyphi A, gallinarum, pullorum
Subgroup II
Subgroup III (Arizona)
Subgroup IV
Subgroup V
Citrobacteriaceae Citrobacter freundii, diversus, amalonaticus
Klebsielleae Klebsiella pneumoniae, ozaenae, oxytoca, rhinoscleromatis, planticola,
terrigena
Enterobacter aerogenes, cloacae, agglomerans, sakazakii, gergoviae, dissolvens,
 Hafnia alvei
Serratia marcescens, liquefaciens, rubidaea, fonticola, odorifera,
Proteeae Proteus mirabilis, vulgaris, penneri, myxofaciens
Morganella morganii
Providencia alcalifaciens, stuartii, rettgeri, rustigianii
Yersinieae Yersinia pseudotuberculosis, pestis, enterocolitica, frederiksenii,
kristensenii
Erwinieae Erwinia amylovora, carotovora

**Mnemonics-Enterobacteriaceae 
Think MESSY SPECK!
Morganella Serratia
Escherichia Proteus
Shigella Enterobacter
Salmonella Citrobacter
Yersinia Klebsiella

Laboratory Diagnosis
A. Collection and transport media: Cary-Blair, Amies, Stuart media
B. Culture media
1. nonselective media : BA, Choc
- on Choc or BA, enteric bacteria produce large, grayish, smooth colonies
- on BA, colonies may be hemolytic (usually beta) or nonhemolytic
2. selective media : Composition LF NLF
MacConkey selective bile salts and crystal violet pink to red colorless
differential lactose; neutral red

EMB selective Eosin Y & methylene blue dyes colored colonies colorless,
differential lactose & sucrose translucent

XLD selective sodium dosoxycholate yellow Salm= red w/

differential lactose, sucrose, xylose, phenol red (w/ or w/o black center
lysine (for lysine decarboxylation) black center)
Na thiosulfate & ferric ammo citrate Shig=colorless
(H2S = black) or red

SS selective bile salts, Na citrate, brilliant green pink-red Salm=colorless

differential lactose, neutral red (w/ or w/o w/ black center
Na thiosulfate & ferric chloride black center)
(H2S= black) Shig=colorless

HE selective bile salts bright orange to green to

(Hektoen enteric) differential lactose, sucrose, bromthymol blue salmon-pink blue-green
Na thiosulfate, ferric ammo citrate
(H2S= black)
* do not autoclave, do not overheat

Selenite F broth enrichment

(selective) sodium selenite
lactose

MacConkey agar
- selective, differential medium; selects for Enterobacteriaceae and other g-rods; inhibits g+ organisms

EMB (Eosin Methylene Blue) agar

- selective, differential medium useful in the isolation and identification of g- enteric bacteria; inhibits g- bacteria

XLD (Xylose Lysine Desoxycholate) agar

- selective, differential primary plating medium used to isolate Salmonella and Shigella from stool and other specimens
containing mixed flora
- inhibits g+cocci and some lactose-fermenting g-rods; less selective than SS and HE but permits better recovery of Shigella

SSA (Salmonella Shigella Agar

- selective, differential medium used to select for Salmonella and some strains of Shigella from stool specimens
- inhibits g+ and many lactose-fermenting g-rods normally found in feces

HEA (Hektoen Enteric Agar

- selective, differential medium used for direct isolation of enteric pathogens from feces and for indirect isolation from
selective enrichment broth
- contains bile salts in concentrations that not only inhibit g+bacteria but also retard the growth of many g-organisms that
are part of the normal intestinal flora

Selenite F broth
- enrichment broth for recovery of low nos. of Salmonella & some strains of Shigella from stool &other spns w/ mixed flora
- sodium selenite inhibits growth of many g-rods & enterococci but permits recovery of Salmonella & some Shigella species
- selenite is most effective at a neutral pH; reduction of selenite during growth of bacteria produces alkaline products, so
lactose has been included; LF produce acid which neutralizes alkaline products and returns the medium to a neutral pH
- one should subculture the broth to enteric media after it has incubated 8-12 hrs to avoid overgrowth with normal flora

C. Biochemical Principles and Reactions on Conventional Media

***Lactose fermentation
- lactose is a disaccharide consisting of glucose and galactose connected by a galactoside bond
- two enzymes necessary for a bacterium to take up lactose
B-galactoside permease - transport enzyme; facilitates entry of lactose through the bacterial cell wall
B-galactosidase - hydrolyzes lactose into glucose and galactose

B-galactoside permease B-galactosidase

LF + +
NLF -
late / slow LF - +
RAPID LACTOSE FERMENTERS

Enterobacter aerogenes Klebsiella pneumoniae Escherichia coli

LATE LACTOSE FERMENTERS

Citrobacter spp. Serratia marcescens

NON- LACTOSE FERMENTERS

Proteus mirabilis Pseudomonas aeruginosa

Triple Sugar Iron (TSI) or Kligler Iron Agar (KIA)

- used to detect: 1. fermentation of glucose, lactose and sucrose
2. production of gas from the fermentation of sugars
3. production of hydrogen sulfide (H2S) gas
- critical to understanding how this medium works is the fact that glucose is present in a much lesser amount than are sucrose
and lactose
- organisms use the simplest carbohydrate, glucose, first; once this is consumed, they attack the more complex
carbohydrate, lactose; if they lack the appropriate enzymes, they move on to protein catabolism
- there is sufficient lactose in the medium to prevent breakdown of peptone, provided that the medium is read at the
appropriate time
- poured on a slant which sets up reaction chambers within the tube; slant (aerobic) and butt (anaerobic)
- to inoculate TSI agar, pick a well-isolated colony with an inoculating needle and stab the butt (all the way to the bottom)
at the base of the slant; streak the slant by "fish tailing" as the needle is being pulled out of the butt
- the cap should be slightly loose to provide sufficient air for peptone catabolism
- reaction patterns are written with the slant results first, followed by the butt reaction, separated by a backslash
(slant rxn / butt rxn)

- it is important that the reactions be read within an 18-24 hr incubation period

--- if read earlier, orgs only able to to ferment glucose may appear to be lactose-fermenters
--- if read later, lactose-fermenters may consume the more complex carbohydrates and begin to catabolize
 peptones with the slant reverting to red

- a yellow slant and a red butt (A/K) may indicate the failure to stab butt or inoculation of medium with g+ organism
- a black butt should be read as acid even though the yellow color may be obscured
- H2S indicator system is not as sensitive as the lead acetate or SIM

Reactions on TSI or KIA

1. No fermentation K/K (alkaline/alkaline) or K/NC (alkaline/no change)
- typical of organisms that are not members of the Enterobacteriaceae (nonenteric bacilli)
- unable to ferment glucose or lactose, but they can degrade the peptones present in the medium, resulting in the
production of alkaline byproducts and changing the indicator to a deep red color

2. Fermentation of glucose only A/A (acid/acid) 8-12 hours; K/A (alkaline/acid) 18-24 hours
- typical of true enteric pathogens

8-12 hrs (A/A)

- TSI and KIA contain glucose in a 0.1% concentration; the acid produced from this is enough to change the
indicator to yellow throughout the medium in this time frame
- reading the results after less than 12 hours of incubation gives the false appearance of an organism capable of
fermenting glucose and lactose; for this reason, KIA or TSI must be incubated for 18-24 hours

18-24 hrs (K/A)

- after 18-24 hours, the glucose concentration is depleted in the slant and the butt
- the organism begins oxidative degradation of the peptones in the slant, producing ammonia; this causes a pH
rise and the slant reverts to red
- fermentation (anaerobic) of glucose in the butt produces larger amounts of acid, overcoming the alkaline effects
of peptone degradation; therefore the butt remains acid (yellow)

3. Fermentation of glucose, lactose, and/or sucrose

- typical of most opportunistic enterics; they attack glucose first, and then lactose (sucrose)
- acid production from the fermentation of the additional sugars is sufficient to keep both the slant and the butt
acid (yellow) when examined at the end of 18-24 hours

4. Gas production
- indicated by formation of bubbles, splitting of the media in the butt, complete displacement of the media from
the bottom of the tube

5. Hydrogen sulfide (H2S) production K/A H2S or A/A H2S

bacterium (acid environment) + Na thiosulfate = H2S gas (colorless)

H2S + ferric ions = ferrous sulfide (black precipitate)

- H2S is a colorless gas, therefore the second indicator is necessary to visually detect its production

Indole Production
- indole is a degradation product of the amino acid tryptophan
- organisms with tryptophanase can deaminate tryptophan to indole, pyruvic acid and ammonia
- when p-dimethylaminobenzaldehyde (Ehrlich or Kovac reagent) is added to a tryptophan broth culture, any indole
produced by the organism reacts with the aldehyde portion of dimethylaminobenzaldehyde to form a red color

- inoculate broth and incubate for 24 hours at 35 C

- add 5 drops of Kovac rgt to the medium and look for the appearance of red color in the reagent layer or at the interface of
the reagent and broth
- if Ehrlich rgt is used, add 1 mL of xylene or ether to the broth culture, shake, and then add 5 drops of the rgt
- Kovac rgt is generally used in testing for members of Enterobacteriaceae,
and Ehrlich when testing for non-glucose-fermenting g-rods and anaerobes
- media used in testing for indole production : tryptophan broth (1%)
SIM (sulfide indole motility) agar
MIO (motility indole ornithine) medium
***A spot test using filter paper saturated with para-dimethylaminocinnamaldehyde rgt is also used for indole determination
Citrate Utilization test (Simmon's citrate agar)
- determines whether the organism can utilize sodium citrate as sole source of carbon for metabolism
- the alkaline pH that results from use of citrate turns the indicator in the medium from green to blue
- inoculated slants should be incubated at 35 C for up to 4 days and monitored daily for the expected color change
- it is important to keep the inoculum light, since dead organisms can be a source of carbon, producing a false+ reaction

Urease Production test

- urea agar and broth detect an organism's ability to hydrolyze urea
- urea is hydrolyzed to form CO2, water, and ammonia; the ammonia then reacts with components in the medium to form
ammonium carbonate; this compound causes a rise in pH causing the production of pink color
- when using urea agar, streak slant and incubate at 35 C for 18-24 hours
- rapid urese producers such as Proteus species turn the entire tube pink and may be detectable in a few hours
- slow urease producers such as Klebsiella only turn the slant pink

Motility test
- to determine whether the organism is motile or non-motile
- test medium has low agar concentration of 0.4% or less (semisolid), to allow free spread of organism
- use an inoculating needle to stab (single stab) the medium
- be careful to remove the needle along the initial stab line; do not stab the medium clear to tube's bottom
- incubate the inoculated medium at 35 C overnight
- movement away from the stab line or a hazy appearance throughout the medium indicates a motile organism
- nonmotile organisms, which lack flagella grow only along the stab line and the surrounding medium remains clear
- Shigella and Klebsiella are always nonmotile, and certain Yersinia sp. show motility at RT but not at 35 C

- media used : Motility test medium

Motility test medium with TTC (triphenyltetrazolium chloride)
--- bacteria will reduce TTC to red formazan pigment; medium shows reddening where there is growth
- SIM
- MIO

Decarboxylase tests
- determine whether the bacterial species possess enzymes capable of decarboxylating (attacking the carboxyl group of an
amino acid) specific amino acids in the test medium

lysine, ornithine, arginine + decarboxylase enzyme = specific amine products + CO2

lysine + lysine decarboxylase = cadaverine (amine) + CO2

ornithine + ornithine decarboxylase = putrescine

arginine + arginine dihydrolase = citrulline + ornithine + OD = putrescine

- Moeller decarboxylase base medium contains glucose, peptone, pH indicators bromcresol purple and cresol red,
specific amino acid; the uninoculated medium is purple
- glucose is important because decarboxylases are inducible enzymes produced in an acid pH
- all members of the Enterobacteriaceae are glucose+, providing a growth stimulus
- a control tube containing only the base medium without the amino acid is tested along with the test organism to determine
the viability of the organism and to determine whether sufficient acid is produced
- both tubes are inoculated with the test organism, are overlaid with a layer (4-5 mm) of sterile mineral oil, which creates
anaerobic conditions to avoid oxidative deamination of available protein that can cause a false + rxn
- should be incubated at 35 C for up to 4 days and should be checked daily
- for decarboxylation to take place, two conditions must be met; an acid pH and an anaerobic environment
- fermenters (Enterobacteriaceae) will ferment glucose, turning the control and all decarboxylase tubes yellow
---for these organisms, as the pH drops, the hydrogen ion concentration becomes optimal for decarboxylase activity
---in the decarboxylase tubes
--if the org produces the specific decarboxylase, conversion of the amino acid to amines raises the pH,
reversing the yellow to purple (+ result)
--if the organism does not possess the specific decarboxylase, the medium remains yellow (- result)
---while the control tube remains yellow
- nonfermenters do not produce the initial yellow color change, and utilization of the amino acid is indicated when the
amino acid-containing tube becomes deeper purple than the control
MIO (Motility, Indole, Ornithine) test
- a semisolid agar medium used to detect motility and production of indole and ornithine decarboxylase
- useful in differentiating Klebsiella species from Enterobacter / Serratia species
- motility is shown by clouding of the medium or spreading of growth from the line of inoculation
- indole production is detected by the addition of Kovac's rgt; (+) pink or red color
- ornithine decarboxylation is indicated by a purple color throughout the medium
---since MIO is a semisolid medium, it does not have to be overlaid with mineral oil to provide anaerobic conditions

ONPG (Orthonitrophenyl galactopyranoside) test

- determines whether the organism is a slow/late LF or a true NLF
- slow or late LF appear as nonfermenting colonies on EMB, MacConkey, TSI/KIA after 18-22 hours of incubation,
raising suspicion that the species is an intestinal enteric pathogen
- the ONPG is structurally similar to lactose, except that glucose is replaced with ONPG, and ONPG is more easily
transported through the bacterial cell wall
- B-galactosidase acts on the ONPG (a colorless compound), cleaves it into galactose and orthonitrophenol, and the
compound turns yellow; it remains colorless if the organism is a NLF

LIA (Lysine Iron Agar) slant

- tubed agar slant; contains lysine as the specific amino acid, glucose, ferric ammonium citrate, and sodium thiosulfate
- measures three parameters
1. lysine decarboxylation butt changes from purple to yellow (glucose fermentation), then back to purple
2. lysine deamination slant turns to a plum or reddish-purple (from the original purple color)
3. H2S production any blackening in the butt

- most useful in conjunction with TSI in screening stool specimens for the presence of enteric pathogens
- helpful in differentiating Salmonella (lysine +) from Citrobacter (lysine-)
- also useful in differentiating Proteus, Morganella, and Providencia species from the rest of the members of
Enterobacteriaceae; this group of enterics deaminate (attack the NH2 group instead of the carboxyl group) amino acids

- uninoculated medium appears purple

- inoculated by stabbing the butt twice and streaking the slant with an inoculating needle
- cap should be left slightly loose, since oxygen is required for detection of deamination
- reactions should be read after 18-24 hours of incubation at 35 C; may be incubated for up to 48 hours if needed
- not as sensitive as other media for H2S detection; typically, H2S-producing Proteus sp. may appear negative
- can be used only with organisms that can ferment glucose; not a true replacement for the Moeller decarboxylase tests

- Three lysine utilization patterns: deamination occurs in the slant (aerobic), decarboxylation occurs in the butt (anaerobic)
1. Purple / Purple (K/K)
- organism decarboxylates lysine but cannot deaminate it
- initially, the org ferments glucose, causing production of acid and changing indicator in the butt to yellow
- it then decarboxylates lysine to produce cadaverine (alkaline product) causing pH indicator to change back to purple
2. Purple / yellow (K/A)
- organism fermented the glucose but was unable to deaminate or decarboxylate the lysine
3. (Bordeaux red/yellow)
- organism deaminated lysine but could not decarboxylate it
- the yellow butt is caused by glucose fermentation

MR - VP (Methyl Red & Voges-Proskauer) tests

- lactose degradation results in glucose and galactose; glucose is metabolized producing pyruvic acid
- to further degrade pyruvic acid, there are two pathways that enterics take:
1. mixed acid fermentation pathway MR (+)
2. butylene glycol / butanediol fermentation pathway VP (+)
- MR-VP tests detect the end products of glucose fermentation; each test detects products from a different pathway
- the MR-VP broth is inoculated and incubated overnight
- then it is split equally into two parts, one part for the MR test, the other for the VP test

Methyl Red test

- if glucose is metabolized by the mixed acid fermentation pathway, acidic products are produced which results in a low pH
- red color develops after addition of the indicator methyl red
glucose pyruvic acid mixed acid fermentation (pH 4.4) red color with MR indicator
Voges-Proskauer test
- carbon compounds are degraded to butylene glycol and acetoin, which is further reduced to diacetyl
- diacetyl in the presence of KOH and alpha-naphthol forms a red complex
- the pH remains at a relatively neutral level
glucose pyruvic acid acetoin diacetyl + KOH + alpha-naphthol red complex
butylene glycol
Malonate test
- determines whether the organism is capable of utilizing sodium malonate as its sole source of carbon
- indicator changes to blue (+ result)

Carbohydrate fermentation tests

- determine the ability of a microorganism to ferment a specific 1% conc of carbohydrate incorporated into a basal medium
- maltose, lactose, sucrose, rhamnose, raffinose, arabinose, adonitol, dulcitol, mannitol, sorbitol

Phenylalanine deaminase test

- determines whether the organism has the enzyme that deaminates phenylalanine to phenylpyruvic acid
- if phenylpyruvic acid is present, addition of a ferric chloride reagent results in a green color
- helpful in initial differentiation of Proteus, Morganella, and Providencia (PAD+) from the rest of the Enterobacteriaceae(-)

phenylalanine phenylalanine deaminase phenylpyruvic acid + FeCl3 green

Nitrate reduction test

- determines whether the organism has the ability to reduce nitrate to nitrite and further reduce nitrite to nitrogen
- organism is inoculated into a nutrient broth with nitrogen
- after 24 hours of incubation, sulfanilic acid and N,N-dimethyl-1naphthylamine (NNDN) is added
- a red color indicates the presence of nitrite
- if no color develops, this may indicate that nitrate has not been reduced (- result), or that nitrite has been further reduced
to nitrogen (+result) which the reagents will not be able to detect; zinc dust must then be added
- zinc dust reduces nitrate; development of a red color after the addition of zinc confirms a true negative test result

K nitrate nitrate reductase nitrite + (sulfanilic acid & NNDN) red (+ result)
colorless + Zinc dust colorless (+result)
colorless + Zinc dust red (- result)

Potassium cyanide test

- determines the ability of the organism to live and grow in KCN
- Citrobacter freundii (+), Salmonella (-)
- not performed in clinical laboratories because of its potential lethal effects on personnel

Ingredients Mtd of Uninoculated Reactions

Inoculation Medium (+) (-)

TSI stab & streak red

Indole transparent red
Citrate streak green blue green
Urease streak pink
Motility stab
Control decarb purple
Ornithine decarb purple purple yellow
Lysine decarb purple purple yellow

LIA
ONPG yellow
MR red
VP red
PAD phenylalanine, ferric chloride green
Malonate broth Na malonate, glucose green blue green or
bromthymol blue yellow (glu fermentation)
Nitrate reduction broth with nitrogen red
sulfanilic acid, NNDN red
Oxidase (CO) purple
 Opportunistic Members of the Family Enterobacteriaceae

I. Genus Escherichia
Escherichia coli
- first described by Theodore Escherich in 1885
- most significant species in the genus; common isolate from the colon flora
- potential pathogen; gastrointestinal, meningeal (newborns), urinary tract, wound, bacteremic infections
- most strains are motile and possess both sex pili and adhesive fimbriae
- has O, H, and K antigens
--- E. coli O groups show cross-reactivity with similar antigens in other enterics, notably with Shigella
--- typing for H antigen is useful in completing the serogrouping of a particular strain
--- capsular K antigen (suggesting a virulence property) masks the O antigen during bacterial agglutination by specific
antiserum; K1 antigen is identical to that of group B Neisseria meningitidis

 E. coli-Associated Diarrheal Diseases:

 E. coli that cause diarrhea are extremely common worldwide
 These are classified by the characteristics of their virulence properties and each group causes disease by a
different mechanism
 The small or large bowel epithelial cell adherence properties are encoded by genes on plasmids
 The toxins often are plasmid- or phage-mediated
 Enteropathogenic E. coli (EPEC): E. coli 0111a,0111b:H2
Important cause of diarrhea in infants
Previously associated with outbreaks of diarrhea in nurseries in developed countries
EPEC adhere to the mucosal cells of the small bowel
Chromosomally mediated factors promote tight adherence
There is loss of microvilli (effacement), formation of filamentous actin pedestals
or cup-like structures, and, occasionally, entry of the EPEC into the mucosal cells
The duration of the EPEC diarrhea can be shortened and the chronic diarrhea cured by antibiotic
treatment

 Enterotoxigenic E coli (ETEC): E. coli 078:H11, 078:H12

Common cause of "traveler's diarrhea" Belly Delhi, Montezumas revenge
Very important cause of diarrhea in infants in developing countries
ETEC colonization factors specific for humans promote adherence of ETEC to
epithelial cells of the small bowel
Some strains of ETEC produce a heat-labile exotoxin that is under the genetic control of a plasmid
Subunit B attaches to the GM1 ganglioside at the brush border of epithelial cells
of the small intestine and facilitates the entry of subunit A into the cell, where the latter activates adenylyl
cyclase
This markedly increases the local concentration of cyclic adenosine
monophosphate (cAMP), which results in intense and prolonged
hypersecretion of water and chlorides and inhibits the reabsorption of sodium
The gut lumen is distended with fluid, and hypermotility and diarrhea ensue, lasting for several days
LT is antigenic and cross-reacts with the enterotoxin of Vibrio cholerae
LT stimulates the production of neutralizing antibodies in the serum of persons
previously infected with enterotoxigenic E coli
Persons residing in areas where such organisms are highly prevalent are likely
to possess antibodies and are less prone to develop diarrhea on
reexposure to the LT-producing E. coli
Some strains produce the heat-stable enterotoxin STa (under the genetic control
of a heterogeneous group of plasmids)
STa activates guanylyl cyclase in enteric epithelial cells and stimulates fluid
secretion; STa-positive strains also produce LT
Plasmids carrying the genes for enterotoxins (LT, ST) also may carry genes for
the colonization factors that facilitate the attachment of E. coli strains to intestinal epithelium

 Enterohemorrhagic E. coli ( EHEC)/Shiga-toxin producing E. coli (STEC): O157:H7

Produce cytotoxic toxins Shiga-like toxin 1, Shiga-like toxin 2 (similar to Shiga toxin of Shigella
dysenteriae type 1 but antigenically and genetically distinct)
Associated with hemorrhagic colitis, hemolytic uremic syndrome (ARF, microangiopathic hemolytic
anemia, thrombocytopenia)
O157:H7 is the most common
 Many cases of hemorrhagic colitis and its associated complications can be prevented by thoroughly
cooking ground beef/ apple juice/ hamburger

 Enteroinvasive E.coli (EIEC):E. coli 0124:H30

Produces a disease very similar to shigellosis
The disease occurs most commonly in children in developing countries and in travelers to these countries
They are nonlactose or late lactose fermenters and are nonmotile
EIEC produce disease by invading intestinal mucosal epithelial cells

 Enteroaggregative E. coli (EAEC):Ag 164 O, 100 K Ag, 50 H Ag

Causes acute and chronic diarrhea (> 14 days in duration) in persons in developing countries
These organisms also are the cause of food-borne illnesses in industrialized countries
They are characterized by their characteristic pattern of adherence to human cells
EAEC produce ST-like toxin and a hemolysin

Other E. coli Infections

UTI - most common cause of urinary tract and kidney infections in humans
- virulence factors of urethrogenic E. coli
adhesins organism can adhere to the epithelial cells lining the urinary tract
hemolysins kill leukocytes, inhibit phagocytosis and chemotaxis
aerobactin iron chelator
Septicemia and meningitis
- one of the most common causes of septicemia and meningitis in neonates; acquired during passage in infected
birth canal or through contamination of the amniotic fluid; K1 antigen is the virulence factor in neonatal menigitis
- in adults, bacteremia results from a genitourinary tract or gastrointestinal infection

Escherichia hermannii / E. coli atypical or enteric group II

- yellow-pigmented colonies; isolated from spinal fluid, wounds, blood; raw milk and beef

Escherichia vulneris
- may also produce yellow-pigmented colonies (more than half of the strains); isolated from wounds

Laboratory Diagnosis
1. Smear : gram (-) bacillus
2. Culture in
a. BLM: lactose fermenters
b. EMB: colonies with greenish metallic sheen
c. MAC: pink to brick red colonies
d. XLD: yellow (fermenters) red (nonfermenters)
e. BAP: some strains are beta hemolytic
3. Biochemical Test
a. TSI: A/A, gas (+), H2S (-)
b. IMViC: ++--
c. Urease: (-)
d. LD: (+)
 e. Acetate: (+)
f. ONPG: (+)
g. MUG (4-methylumbelliferyl-B-glucoronide): +
4. Serotyping

Tribe Klebsielleae
- Genera: Klebsiella, Enterobacter, Serratia, Hafnia
- usually found in the intestinal tract of humans and animals or free-living in soil, water, and plants
- associated with a wide variety of opportunistic and nosocomial infections (pneumonia, wound, UTI)

II. Genus Klebsiella

- usually found in the GIT of humans and animals
- absence of motility distinguishes Klebsiella species from other members of the family Enterobacteriaceae

Klebsiella pneumoniae ( Friedlander’s bacillus/ Bacillus mucosus capsulatus)

- most commonly isolated species in the genus
- has a polysaccharide capsule that contributes to virulence; also responsible for the moist,
mucoid colonies
- clinical infections:
lower respiratory tract infections in hospitalized patients, newborns, elderly, and seriously
ill patients on respirators wound infections, UTI, bacteremia
- nosocomial outbreaks in newborn nurseries
- Majority of clinical isolates/ occurs in older alcoholic men

Laboratory Diagnosis: Specimen: pus/ sputum

1. Smear : G(-) bacteria
2. Culture: BLM, EMB, MAC, XLD
a. Produce large, moist, mucoid colonies that have the tendency to coalesce, (+) string test
3. Biochemical test:
a. TSI: A/A, gas (+), H2S(-)
b. IMViC: (-+++)
c. Urease, LD,KCN,VP (+)
i. Butanediol fermenters, acetoin production
d. Quellung reaction (+)

- inactive strains of K. pneumoniae:

Klebsiella ozaenae - isolated form nasal secretions and cerebral abscesses
Klebsiella rhinoscleromatis - isolated from those w/ rhinoscleroma (infection of the nasal cavity; intense swelling
and malformation of the entire face and neck)
Klebsiella oxytoca
- identical to K. pneumoniae except for its production of indole
- causes infections similar to those of K. pneumoniae

Klebsiella group 47
- isolated from urine, respiratory tract and blood
a. Klebsiella ornithinolytica indole (+); ornithine decarboxylase (+)
b. Klebsiella planticola

**Mnemonics 
5A’s of KlebsiellA
- Aspiration pneumonia
- Abscess in lungs and liver
- Alcoholics
- Di-A-betics
- “curr-A-nt jelly” sputum

III. Genus Enterobacter

- colony morphology resembles that of Klebsiella when growing on MacConkey agar
- motile , G(-) bacillus, rapid LF <18 hours of incubation
- All are OD (+) except E. agglomerans
- All are LD (+) E. sakazakii & E. taylorae
 E. aerogenes (common in Philippines)
  E. agglomerans (common in Philippines)
 E. cloacae (common in USA)
 E. taylorae
 E. gergoviae
 E. sakazakii (isolated in Japan)

Biochemical Test:
1. TSI: A/A, Gas(+), H2S(+)
2. IMViC: (--++)
3. VP,Citrate, KCN: (+)
4. Urease: (+/-)

*Enterobacter agglomerans / Pantoea agglomerans

- may produce a yellow-pigmented colony
- outbreak of septicemia due to contaminated IV fluids

Enterobacter sakazakii
- yellow-pigmented
- pathogen in neonates (meningeal and bacteremic infections)
- isolated from brain abscesses, respiratory and wound infections

Enterobacter gergoviae
- found in respiratory samples; rarely isolated from blood cultures

Enterobacter hormaechei
- isolated from blood, wounds, sputum

IV. Genus Citrobacter

- formerly classified under the tribe Salmonelleae; reclassified into its own tribe Citrobacteriaceae
- most Citrobacter species ferment lactose, producing colonies on MacConkey that resemble those of E. coli
- motile, biochemically resembles Salmonella

Citrobacter freundii
- associated with infectious diseases acquired in hospital settings: UTI, pneumonia, intraabdominal abscesses, endocarditis
- colonial morphology may be easily mistaken for that of Salmonella, as most C. freundii are H2S(+) and
some strains (50%) fail to ferment lactose
- C. freundii lysine(-); urease(+); ONGP (+) TSI: A/A or K/A Gas +, H2S + (late LF)
Salmonella lysine(+); urease(-); ONGP (-) IMVC: - + - +

Citrobacter diversus
- causes nursery outbreaks of neonatal meningitis and brain abscesses
* H2S(-), Indole (+)

Citrobacter amalonaticus
- frequently found in feces (not a causative agent of diarrhea); also isolated from blood and wounds

Notes:
Citrobacter freundii: H2S(+), Lysine (-)
Edwardsiella: H2S(+), Lysine (+), Indole (+)
Arizona: H2S(+), Lysine (+), Indole (-), Malonate & Gelatin (+)
Salmonella: H2S(+), Lysine (+), Indole (-), Malonate & Gelatin (-)

V. Genus Serratia
- opportunistic pathogens associated with nosocomial outbreaks
- ferment lactose slowly (ONPG+), except S. fonticola
- differentiated from the tribe by the ability to produce extracellular DNase
- resistant to a wide range of antimicrobials
- prodigiosin is a pink to red pigment produced by S. marcescens and S. rubidaea, esp when cultures are left at RT
  S. marcescens common opportunistic pathogen in hospitalized
patients
 Serratia causes pneumonia, bacteremia, and endocarditis—
especially in narcotics addicts and hospitalized
patients
 ~10% of isolates form the red pigment (prodigiosin) that has
long characterized Serratia marcescens
 S. marcescens is often multiply resistant to aminoglycosides
and penicillins
Infections can be treated with third-generation cephalosporins

Serratia marcescens
- the species usually considered clinically important
- frequently found in hospital-acquired infections of the urinary and respiratory tract and in bacteremic outbreaks in
nurseries, cardiac surgery and burn units (75-90 %)
- ability to produce DNAse, Lipase, Gelatinase and by resistance to colistin & cephalothin
- motile , NLF
- chromogenic and produce-soluble red pink pigment (prodigiosin)

Biochemical Test
1. TSI: K/A or A/A, Gas (+), H2S (-)
2. IMViC: (--++)
3. DNASe: (+)
4. LD, OD: (+)
5. Malonate (-)

Serratia rubidaea
- have also been isolated from human sources

Serratia liquefaciens
- have also been isolated from human sources

Serratia odorifera
- gives off a dirty, musty odor like potatoes
- contains two biogroups: biogroup 1 - isolated from the respiratory tract
- ornithine+, sucrose+, may be indole+ (60%)

biogroup 2 - isolated from blood and CSF

- ornithine-, sucrose-, may also be indole+ (50%)

VI. Genus Hafnia

- composed of one species H. alvei; delayed citrate (+) reaction is a major characteristic
- late LF
- two distinct biotypes
Hafnia alvei - isolated from a number of anatomical sites; occasionally isolated from stool cultures
Hafnia alvei biotype 1 - grows in beer wort of breweries

Tribe Proteeae
- Genera: Proteus, Morganella, Providencia
- normal intestinal flora; opportunistic pathogens
- all are NLF; differentiated from other enterics by being PAD(+) and lysine deaminase
(+)
VII. Genus Proteus
Proteus mirabilis and Proteus vulgaris
- recognized as human pathogens
- isolated from urine, wound, ear and bacteremic infections
- easily identified in the lab because of their characteristic swarming
colonies on nonselective media such as BA
- produce a distinct odor of "burned chocolate"
 - both are H2S(+), and urease(+)
- actively motile @ 37 OC
- G(-) pleomorphic rods, possess the O,H and K Ag

Proteus mirabilis K/AG + H2S IMVC: - + v v motility(+)

Indole (-) Ornithine (+) PADT(+) urease(+)

Proteus vulgaris K/AG + H2S IMVC: + + - - motility(+)

Indole (+) ornithine (-) PADT(+) urease(+) sucrose (+)

Proteus penneri (formerly a P. vulgaris strain)

- a newly recognized species; also swarms on nonselective media

VIII. Genus Morganella

- has only 1 species Morganella morganii (formerly Proteus morganii)
- implicated in diarrheal illness (role as etiologic agent is being further examined)
- can cause UTI; isolated from other human body sites
H2S(-) K/A G(+) IMVC: + + - - (NLF)
PADT (+) urease(+) ornithinie(+)

IX. Genus Providencia

- resistant to antimicrobials
K/A IMVC: + + - +
PADT(+) urease(+)

Providencia rettgeri - pathogen of the urinary tract; has caused occasional nosocomial outbreaks

Providencia stuartii - nosocomial outbreaks in burn units; isolated from urine cultures

Providencia alcalifaciens - usually found in feces of children w/ diarrhea (role as a cause of diarrhea has not been proven)

X. Genus Edwardsiella
- composed of three species, but E. tarda is the only recognized human pathogen; NLF, H2S(+)
- Edwardsiella tarda is an opportunist, causing bacteremic and wound infections; pathogenic role in diarrhea remains a controversy
K/AG + H2S IMVC: + + - - urease (-)

Primary Intestinal Pathogens

I. Genus Salmonella
- Daniel Salmon discoverer
- g-rods; produce clear, colorless, NLF colonies
- colonies with black centers are seen if media contain indicators for H2S production

- lactose(-), indole (-), VP(-), PAD(-), urease(-); most produce H2S in TSI
- all species are actively motile by peritrichous flagella except S. pullorum & S gallinarum
- inhabit the gastrointestinal tracts of animals; humans acquire the infection by ingesting organisms in contaminated
animal food products or insufficiently cooked poultry, milk, eggs, and dairy products
- other species, S.typhi and S. paratyphi, are found only in humans; they have no known animal reservoirs;
infections are transmitted by human carriers
- the genus is classified into 7 subgroups with designated subspecies
subgroup I
- most serious pathogens for humans, causing enteric fevers
- have similar biochemical characteristics except S. typhi, S. partyphi, S. choleraesuis
subgroup II, III, IV
- usually found in cold-blooded animals, rodents, and birds, which are their natural hosts
***Arizona - used to belong to its own genus; reclassified into Salmonella subgroup III
- may cause symptoms identical to Salmonella infections
- may be transmitted to humans from pet turtles, snakes and fish

Virulence Factors:
- fimbriae (for adherence); ability to traverse intestinal mucosa; enterotoxin (causes gastroenteritis)

Antigenic Structures: Based on Kaufmann- White Scheme

1. heat-stable somatic O antigens
- LPS located in the outer membrane of the cell wall
- there are many different O antigens; more than one O antigen may be found in a particular strain
- designated by arabic numbers
2. heat-labile flagellar H antigens
- treatable with ethanol or acid
- may occur in two phases
a. phase 1 / specific phase - occur only in a small no. of serotypes; agglutinate only w/ homologous antisera
- determine the immunologic identity of the paticular serotype
b. phase 2 / nonspecific phase - occur among several strains; react with heterologous antisera
- shared by numerous serotypes
3. heat-labile capsular K antigen, designated as Vi antigen (coined from the term virulence)
- found in S. typhi and a few other strains of Salmonella subgroup I; prevents phagocytosis
- most often blocks the O antigen during serologic typing; may be removed by heating

Clinical Infections:
- humans acquire the infection by ingesting the organisms in food, water, and milk contaminated w/ human or animal excreta
- salmonellosis may occur in several forms
1. an acute gastroenteritis or food poisoning characterized by vomiting and diarrhea
2. typhoid fever, the most severe form of enteric fever, caused by S. typhi;
other enteric fevers are caused by other Salmonella species (i.e. S. paratyphi, S. choleraesuis)
3. nontyphoidal bacteremia
4. carrier state that follows Salmonella infection
1. Salmonella gastroenteritis
- one of the most common forms of "food poisoning"; results from ingestion of the organisms
through contaminated food
- strains associated with this are usually those found in animals (in the US, serotypes of S.
enteritidis)
- sources of infection: poultry, milk, eggs, and egg products as well as to handling pets
- insufficiently cooked eggs and domestic fowl, such as chicken, turkey, and duck, are common
sources of infection
- cooking utensils such as knives, pans, and cutting boards used in preparing the contaminated
meat can spread the contamination to other food
- direct transmission from person to person
- gastroenteritis occurs when a sufficient number of organisms contaminate food that is
maintained under inadequaterefrigeration, thus allowing growth and multiplication of
the organisms
- the infective dose necessary to initiate the disease (106 bacteria) is higher than that required for
shigellosis
- symptoms may appear 8 to 36 hours after ingestion of contaminated food: nausea, vomiting,
fever and chills, accompanied by watery diarrhea and abdominal pain
- most cases are self-limiting; symptoms disappear usually within a few days, with little or no
complications
- those who suffer from sickle cell disease, ulcerative colitis, and malignancy seem to be more
susceptible
- infection may be more severe in the very young, the elderly, and those suffering from other
underlying disease
- antimicrobial therapy is usually not indicated in uncomplicated cases
- in cases of dehydration, fluid replacement therapy may be indicated
- dissemination may occasionally occur; in such cases, antimicrobial therapy is required
- antimicrobials of choice: chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole

2. Typhoid fever and other enteric fevers

- clinical features: prolonged fever, bacteremia, involvement of the RES (particularly liver, spleen, intestines, and
mesentery), dissemination to multiple organs
- enteric fever caused by S. typhi; febrile disease that results from the ingestion of food contaminated with the organisms
originating from infected individuals or carriers
- S. typhi does not have a known animal reservoir; humans are the only source of infection
- improper sewage disposal, poor sanitation, & lack of modern water system caused outbreaks when orgs reach a water source
- water purification and treatment, and pasteurization of milk diminished the incidence of typhoid in industrialized countries
- carriers, particularly food handlers, are important sources of infection
- direct transmission through fomites is also possible
- lab workers in the micro lab have contacted typhoid fever while working with the organisms
- develops approx 9-14 days after ingestion of the organisms; onset of symptoms depends on the no. of organisms ingested
- during the 1st week of the disease, px develops fever, malaise, anorexia, lethargy, myalgia, continuous dull frontal headache
- when the organisms are ingested, they seem to be resistant to gastric acids and, on reaching the proximal end of the small
intestine, they invade and penetrate the intestinal mucosa; at this time, px experiences constipation rather than diarrhea
- the organisms gain entrance into the lymphatic system and are sustained in the mesenteric lymph nodes
- they eventually reach the blood stream and are further spread to the liver, spleen and bone marrow, where they are
immediately engulfed by mononuclear phagocytes
- the organisms multiply intracellularly; later they are released to the blood stream for the second time
- the febrile episode becomes more evident during this release of the organisms into the circulatory system
- at this time, the organisms may easily be isolated from the blood

- during the 2nd and 3rd weeks of the disease, px experiences sustained fever with the prolonged bacteremia
- the organisms invade the gallbladder and Peyer's patches of the bowel; they also reach the intestinal tract via biliary tract
- "rose spots" (blanching, rose-colored papules around the periumbilical region) appear during the 2 nd week of fever

- involvement of the biliary system sites initiates gastrointestinal symptoms as the organisms reinfect the intestinal tract
- the organism now exists in large numbers and may be isolated from the stool
- gallbladder becomes the foci of long-term carriage, occasionally reseeding the intest tract and shedding the orgs in the feces

- serious complications: necrosis of the gallbladder leading to necrotizing cholecystitis

necrosis of the Peyer's patches leading to hemorrhage and perforation of the bowel
pneumonia, thrombophlebitis, meningitis, osteomyelitis, endocarditis, and abscesses
- other enteric fevers include paratyphoid fevers, which may be due to S. paratyphi serovar A (another strict human
pathogen), S. paratyphi serovar B, and S. paratyphi serovar C
- clinical manifestations are similar to those of typhoid fever but are less severe, and the fatality rate is lower

WEEK PRESENTATION CULTURE SOURCE

1 Stepwise fever, anorexia, malaise, relative bradycardia and BLOOD, bone marrow
bacteremia
2 Abdominal pain, bloating, constipation, rose spots, URINE, rose spots, bone marrow
hepatosplenomegaly, jaundice
3 Bleeding, ileitis, pneumonia STOOL, bone marrow
Week of Complication
4 Recovery or death Bone marrow
POST Chronic carrier state Bile, stool, bone marrow
Salmonella cerebritis: Typhoid psychosis

3. Salmonella bacteremia
- w/ or w/o extraintestinal foci of infection caused by nontyphoidal Salmonella, is characterized primarily by prolonged
fever and intermittent bacteremia
- most commonly associated strains are S. typhimurium, S. paratyphi A and B, and S. choleraesuis

4. Carrier state
- individuals who recover from the infection may harbor the organisms in the gallbladder, which becomes the site of
chronic carriage
- such individuals excrete the organisms in their feces either continuously or intermittently
- the carrier state may be terminated by antimicrobial therapy if gallbladder infection is not evident
- cholecystectomy has been the only solution to the chronic state of enteric carriers
Laboratory Diagnosis
1. Smear
2. Culture
a. BLM, Selenite F
b. EMB, Mac
c. SSA: colorless - black (H2S)
d. NA: maple leaf colonies with irregular colonies
e. XLD: slightly pink to white opaque colonies surrounded by a brilliant red medium
f. BSA (Bismuth Sulfite Agar) best medium for S. typhi
3. Biochemical for S. typhi
a. TSI: K/A, gas (-), H2S in small amount
b. IMViC: (-+-+)
c. Serotyping (rapid slide agglutination test)
d. FAT, ELISA
e. Tube Dilution Agglutination Test (Widal Test):
i. Serum agglutinins rise sharply during the second and third weeks of Salmonella Typhi infection
ii. The Widal test to detect these antibodies against the O and H antigens has been in use for decades
iii. At least two serum specimens, obtained at intervals of 7–10 days, are needed to prove a rise in
antibody titer
iv. Serial dilutions of unknown sera are tested against antigens from representative salmonellae
v. False-positive and false-negative results occur
vi. The interpretive criteria when single serum specimens are tested vary, but a titer against the O antigen
of > 1:320 and against the H antigen of > 1:640 is considered positive
vii. High titer of antibody to the Vi antigen occurs in some carriers
viii. Results of serologic tests for salmonella infection must be interpreted cautiously because the possible
presence of cross-reactive antibodies limits the use of serology
ix. The test is not useful in diagnosis of enteric fevers caused by salmonella other than Salmonella Typhi

II. Genus Shigella

- named after the Japanese microbiologist Kiyoshi Shiga, who first isolated the organism in 1896
- belongs to the tribe Escherichieae; very closely related to the genus Escherichia
- not members of the normal gastrointestinal flora
- all species can cause bacillary dysentery but they vary in epidemiology, mortality rate, and severity of disease produced
- dysentery is characterized by the presence of blood, mucus, and pus in the stool; occurs in an epidemic dimension
- marked by penetration of intestinal epithelial cells following attachment of the organisms to mucosal surfaces
- local inflammation, shedding of intestinal lining, and formation of ulcers (flask-shaped) follow the epithelial penetration
- clinical manifestations vary from asymptomatic to severe forms of the disease
- initial symptoms (high fever, chills, abdom cramps, pain, w/ tenesmus) appear 24 to 48 hours after ingestion of orgs
- the orgs w/c multiplied in the SI, move toward the colon where they may be isolated 1-3 days after the infection develops
- bloody stools containing mucus and numerous leukocytes follow the watery diarrhea, as the organisms invade the
colonic tissues and cause an inflammatory reaction
- infants w/ shigellosis may transiently lose the reflexive closing of the anal canal w/ external pressure as from a cotton
swab; loss of this anal wink reflex in an infant w/ diarrhea is a soft clue that Shigella may be the etiologic agent
- humans are the only known reservoir of Shigella; no animal reservoir has been identified
- transmission may occur by direct person-to-person contact, and spread via the fecal-oral route, with carriers as the source
- may also be transmitted by flies, fingers, and food or water contaminated by infected persons
- personal hygiene plays a major role in its transmission
- Shigella dysentery usually indicates improper sanitary conditions and poor personal hygiene
- young children in day care centers, people living in crowded and less than adequate housing, and people who participate
in anal-oral sex are most likely affected
- those 1 year of age and younger are the most susceptible
- highly communicable because of the low infective dose required to produce the disease (fewer than 200 bacilli)
- nonmotile; H2S (-); lysine decarboxylase (-); generally appear as clear, NLF colonies
- unlike Escherichia, Shigella do not utilize acetate as a source of carbon
- fragile organisms; susceptible to the various effects of physical and chemical agents
- because they are susceptible to the acid pH of stool, feces suspected of containing Shigella should be plated immediately
onto lab media to increase recovery of the organism
- all Shigella species possess O antigens, and certain strains may possess K antigens
- Shigella K antigens, when present, interfere with the detection of the O antigen during serologic grouping
- the K antigen is heat-labile and may be removed by boiling the organism in a cell suspension
- genus consists of four species that are biochemically similar
- Shigella species are also divided into four major O antigen groups and must be identified by serologic grouping
- there are several serotypes within each species (except S. sonnei which has only one serotype)
- four species and their respective serologic groups:
S. dysenteriae Type 1 (Shiga Bacillus) group A
S. dysenteriae t Type 2 (Schmits Bacillus) group A
S. flexneri ( Flexners Bacillus, Hiss and Russells bacillus group B
S. boydii (Newcastle Manchester bacillus) group C
S. sonnei (Duval’s Bacilus) group D

**Mnemonic-Shigella Transmission
4 Fs
Food, Fingers, Feces, Flies

Shigella sonnei (Sonne-Duvall’s bacillus)

- unique; ornithine(+); slow lactose fermenter (pink colonies on MacConkey after 48 hours of incubation); ONPG(+)
- at 18-24 hrs of incubation, it has a lactose(-) appearance on MacConkey; after 48 hrs, it has a lactose(+) appearance
- in the US, S. sonnei is the predominant isolate, followed by S. flexneri
- infection is usually a short, self-limiting disease characterized by fever and watery diarrhea
IMVC: - + - -

Shigella flexneri (Strong’s Bacillus)

- leading isolate in "gay bowel syndrome" in homosexual men

Shigella dysenteriae type 1(Dysentery bacillus)

- remains the most virulent species, with significant morbidity and high mortality
- mortality rate of 5-10%, and perhaps even higher, particularly among undernourished children during epidemic outbreaks
- in developing countries, S. dysenteriae type 1 and S. boydii are the most common isolates
- in dysentery caused by S. dysenteriae type 1, patients experience more severe symptoms
- bloody diarrhea that progresses to dysentery may appear within a few hours to a few days
- patients suffer from extremely painful bowel movements, which contain predominantly mucus and blood
- in young children, abdominal pain is quite intense, and rectal prolapse may result from excessive straining
- although Shigella toxin has been implicated for the signs of the disease, connection between toxin symptoms remains
unclear; however, toxin levels produced by S. dysenteriae type 1 are higher than those produced by other Shigella species
IMVC: v + - - ornithine(-)

Complications:
- severe cases of shigellosis may become life-threatening as extraintestinal complications develop
1. ileus, an obstruction of the intestines, with marked abdominal dilatation, possibly leading to toxic megacolon
2. bacteremia
3. seizures, which may occur during any Shigella strain infection
4. HUS (hemolytic uremic syndrome), a complication exclusively associated with S. dysenteriae type 1 shigellosis

III. Genus Yersinia

- named for Alexander Yersin, a French microbiologist who isolated the plague bacillus in 1894

Yersinia pestis (Plague bacillus) / Yersinia pseudotuberculosis subsp pestis

- g-, short, plump rod
- when stained with methylene blue or Wayson stain, it shows intense staining at each end of the bacillus, referred to as
"bipolar staining," which gives it a "safety-pin" appearance
- may be isolated on routine culture medium; although it grows at 37 C, it has a preferential growth temp of 25-30 C
IMVC: - + - - ornithine(-)
- causative agent of plague
- a life-threatening disease of rodents transmitted to humans by bites of fleas
- rats are the natural hosts for the vectors that transmit the disease, and humans are accidental hosts
- vectors are fleas (Xenopsylla cheopis) that normally infest the brown and black rats
- this ancient disease still exists in areas where reservoir hosts are found
- there have been three pandemics of plague
1. started near Egypt in 542 AD, ravaged Europe for 50 years, killing 100 million people
2. started in the 14th century; this pandemic, named the Black Death, killed 25 million Europeans,
1/4 of the total population at that time
3. started in Burma and spread to many parts of the world; started in the 1890's and is just now subsiding
- in humans, plague can occur in two forms
1. bubonic / glandular plague
- usually results from the bite of an infected insect vector; characteristic symptoms appear 2-5 days after infection
- symptoms include high fever with painful regional lymph nodes as buboes begin to appear
2. pneumonic plague
- occurs secondary to the bubonic plague when organisms proliferate in the blood stream and respiratory tract
- subsequent epidemic outbreaks may arise from the respiratoy transmission of the organisms
- fatality rate is high if patients remain untreated

Yersinia enterocolitica
- most commonly isolated species of the genus
- morphologically resembles other Yersinia species; g- coccobacilli with bipolar staining
- also grows on routine isolation media, such as BA and MacConkey agar; has an optimal growth temp of 25-30 C
- appropriate cultures on a specific Yersinia media at 25 C should be performed in diarrheal outbreaks of unknown etiology
- motility is clearly noted at 25 C but not at 35 C
IMVC: v + - - ornithine(+)

- Cold enrichment has provided better recovery of Y. enterocolitica

- fecal samples suspected of containing this organism are inoculated on isotonic saline and kept at 4 C for 1-3 weeks
- Cefsulodin Irgasan Novobiocin (CIN) agar
- a selective medium to detect the presence of Y. enterocolitica
- employs cefsulodin, irgasan, novobiocin, bile salts, and crystal violet as the inhibitory agents
- inhibits normal colon organisms better than MacConkey agar, for better recovery Y. enterocolitica from feces
- this selective medium has been modified, mannitol has been added to the medium (differential property)
- fermentation of mannitol results in a drop in pH around the colony causing the pH indicator neutral red to turn red
- nonfermentation of mannitol produces a colorless, translucent colony

- produces an infection that mimics appendicitis; cause of diarrhea in a number of community outbreaks
- found in a wide variety of animals, including domestic swine, cats and dogs; pig is a natural reservoir
- infection may be acquired from contact with household pets
- cultures from environmental reservoirs, such as water from streams, have yielded the organism
- human infections have also been reported following the ingestion of contaminated food and possibly water
- other sources of infection include contaminated food, such as market meat and vacuum-packed beef
- a major concern regarding the potential risk of transmitting infection with this organism is its ability to survive in cold
temperatures; food refrigeration becomes an ineffective preventive measure

- infections manifest in several forms

1. acute enteritis - most common form of the infection
2. an appendicitis-like syndrome - severe abdominal pain which is concentrated in the right lower quadrant
3. arthritis
4. erythema nodosum - inflammatory rxn char by tender, red nodules in the legs and arms

Yersinia pseudotuberculosis
- appears as a typical-looking plague bacillus
- may be differentiated from Y. pestis by its motility at 18-22 C, production of urease, and ability to ferment rhamnose
IMVC: - + - - ornithine(-)

- like Y. pestis, it is a pathogen primarily of rodents, particularly of guinea pigs

- in addition to farm and domestic animals, birds are also natural reservoirs; turkey, geese, pigeons, doves, and canaries
- causes a disease characterized by caseous swellings called pseudotubercles
- often fatal in animals
- human infections, which are rare, are associated with close contact with infected animals or their fecal material
or ingestion of contaminated drink and foodstuff
- when the organisms are ingested, they spread to the mesenteric lymph nodes, producing a generalized infection
- clinical manifestations: septicemia accompanied by mesenteric lymphadenitis (similar to appendicitis)
 BIOCHEMICAL REACTIONS OF MEDICALLY SIGNIFICANT ENTEROBACTERIACIAE
TESTS CITROBACTER ENTEROBACTER ESCHERICHIA KLEBSIELLA MORGANELLA PROTEUS PROVIDENCIA SALMONELLA SERRATIA SHIGELLA
H2S V - - - - + - V - -
INDOLE V - + V + V + - - V
MOTILITY + + V - + + + + + -
TSI K/A A/A A/A K/A A/A A/A K/A K/A K/A K/A K/A A/A K/A
GAS + + + V V V V V V -
GLUCOSE + + - + + + + + + +
SUCROSE V - - - - + - V - -
VP - + - + - - - - + -
CITRATE + + - + - V + V + -
LYSINE - V + + - - - + + -
PHENYLALANINE - - - - + + + - - -
ORNITHINE V + V - + V - + + V
UREASE - - - V + + V - - -
ARGININE V V - - - - - V - -
DNASE - - - - - - - - + -
**MNEMONICS 

TSI REACTIONS
A/A (Sun Over Desert)
SEEK (Serratia, Escherichia, Enterobacter, Klebsiella) Seek Shelter in the hot desert

A/A H2S + (Sun Over Desert)

CAP ( Citrobacter, Arizona, Proteus) Wear a cap to protect yourself from the geyser (H2S +)

K/A H2S+ ( Moon over Desert)

CASE (Citrobacter, Arizona, Salmonella, Edwardsiella) A case of fire crackers going off smells like Sulphur (H2S +)

K/A (Moon over Desert)

SCiPPY (Shigella, Citrobacter, Providencia, Proteus, Yersenia) Sk(c)ippy Cayote howls at the moon