Enterobacteriaceae

Plesiomonas • All motile species have peritrichous flagella except Tatumella ptyseos
which is monotrichous.
➢ New member of Enterobacteriaceae
• All are catalase (+) except for Shigella dysenteriae.
➢ only oxidase positive member
• Some strains of Salmonella, Shigella, Escherichia, Enterobacter and
 General Characteristics Proteus possess fimbriae or pili.
• Grow on most media at 35°C within 24 hours
• Some may grow at 1 - 5°C (psychrophiles) especially Yersinia
enterocolitica and some strains of Serratia

Enterobacteriaceae divided into two broad categories

1. Opportunistic pathogens - part of the usual intestinal microbiota
- may produce infection outside their normal body sites (extraintestinal)
- produces significant virulent factors
2. Primary pathogens - not present as commensal biota in the GI tract of humans
- produce infections resulting from ingestion of contaminated food
and drink
✓ Salmonella spp. Yersinia spp.
✓ Shigella spp. Plesiomonas shigelloides

 Key characteristics of the family Enterobacteriaceae

• Facultative anaerobes, non-sporeforming
Classification of Enterics (Habitat)

Classification of Enterics (Lactose fermentation)

The Enterics

 part of the normal intestinal flora

 cause gastrointestinal disease

Colony Morphology
 Classification
 Subcategorized into Tribes
 Use of tribes in classifying the members – proposed by Ewing
 Virulence and Antigenic factor ✓ K1 antigen in Escherichia coli
 Virulence of Enterobacteriaceae genera is affected by their ability to: ✓ Vi antigen of Salmonella enterica subsp. enterica
serotype Typhi
• Adhere

• Colonize
Opportunistic Members of Enterobacteriaceae and Associated Infections
• Produce toxin

• Invade tissues

• Harbor plasmid that can provide antimicrobial resistance

• Virulence and Antigenic factor

Escherichia coli (colon bacillus)

Ö Major facultative inhabitant of the large intestine

Ö Indicator organism for fecal contamination of water and foods
Ö normally resides in the colon without causing disease
Ö may acquire virulence factors and cause disease

The Enterics have three major surface antigen

K antigen – capsular antigen or envelope antigen Colonial morphology:

▪ Heat-labile polysaccharide found only in encapsulated species ➢ MAC

✓ Klebsiella spp.
✓ Lactose-positive  Uropathogenic Escherichia coli
pink colonies with
surrounding area of
precipitated bile
salts
✓ EMB
✓ Green metallic
sheen

 Gastrointestinal Pathogens
Strains of Escherichia coli that cause diarrhea in man:
1. Enteropathogenic Escherichia coli (EPEC)
• does not produce enterotoxin
• associated with infantile diarrhea
• causes nursery outbreak diarrhea
• watery diarrhea with mucus but no blood
• serologic typing – to identify EPEC serotypes for
• epidemiologic studies

2. Enterotoxigenic Escherichia coli (ETEC)

 • produce enterotoxins
• major causes of traveller’s diarrhea 5. DAEC
• heat - labile toxin (LT) similar to cholera exotoxin (rice water stool) ➢ pediatric UTIs and diarrheal disease
• heat-stable toxin (ST) ➢ cystitis in children
• Infective dose: 106 - 1010 ➢ acute pyelonephritis in pregnant women EAEC
➢ cause diarrhea by adhering to the surface of the
intestinal mucosa
3. Enteroinvasive Escherichia coli (EIEC)
➢ peristent watery diarrhea
• bacillary dysentery in all age groups similar to shigellosis
➢ WBC and RBC are absent in stool
• Main virulence factor is encoded in a plasmid shared by Shigella spp.
and Escherichia coli
• Watery diarrhea is bloody with WBC, RBC and mucus 6. Enterohemorrhagic Escherichia coli (EHEC) serotype 0157:H7
• detect invasiveness using monolayer cell cultures with Hep-2 (Human o Hemolytic Uremic Syndrome (HUS) – most severe manifestation of
epithelial-2) cells EHEC
• Invasive E. coli is identified by Sereny Test (+) o Hemorrhagic colitis
o HUS - characterized by low platelet, hemolytic anemia and kidney
failure
4. Enteroadherent Escherichia coli
o Bloody diarrhea w/o WBC which distinguishes it from dysentery
2 kinds of disease
caused by Shigella spp. or EIEC
a. UTIs
o Appears colorless in MacConkey agar
b. diarrheal syndromes
7. Enterohemorrhagic Escherichia coli (EHEC) serotype 0157:H7
2 types of enteroadherent Escherichia coli (EAEC) Cytotoxins: Verotoxin I and II
a. DAEC – diffusely adherent Escherichia coli 1. Verotoxin I

b. EAEC – enteroaggregative Escherichia coli ✓ identical to the Shiga toxin (Stx) of Shigella dysenteriae
 ✓ damages Vero cells (African green monkey kidneys cells)
✓ neutralize by antibody against Stx
2. Verotoxin II
✓ Not neutralize by antibody against Stx

8. Uropathogenic Escherichia coli (UPEC) serotype

o Most common cause of UTI in human
 Extraintestinal infection
Neonates: Common characteristic of Tribe Klebsielleae
 septicemia • IMViC - - + +
 meningitis • Growth in Potassium cyanide broth
• Do not produce H2S, LF, Mucoid
Virulence factor: • Motility is variable
➢ K1 antigen • Few hydrolyzed urea slowly

✓ Immunochemically identical to the capsular antigen of

Neisseria meningitidis group B
Other Escherichia species:
Escherichia hartmannii
-CSF, wounds, blood
Escherichia vulneris – infected wounds
Escherichia albertii – diarrheal disease in children
Klebsiella pneumoniae subsp. pneumoniae
 Bacillus mucosus capsulatum, Friedlander’s bacillus
 Possess large polysacharride capsule
➢ Protects organism against:
✓ Phagocytosis
✓ Antimicrobial absorption
✓ Responsible for colonial characteristics:
✓ moist
✓ mucoid
Enterobacter, Cronobacter and Pantoea  Cronobacter (Enterobacter) sakazakii typically produces a yellow pigment

Enterobacter spp.  IMViC - - + + TSI – A/A + gas

 Colony morphology of many of the species resembles Klebsiella when  documented as a pathogen in neonates causing meningitis and bacteremia,
growing on often coming from powdered infant formula

 MAC agar (LF)  It has also been isolated from cultures taken from brain abscesses and
respiratory and wound infections
 Common characteristics
• Growth in potassium cyanide broth
Genus Serratia
• IMViC - - + +
• In contrast to Klebsiella, Enterobacter spp. usually produce ornithine Serratia marcescens Serratia liquefaciens Serratia rubidea
decarboxylase Serratia odorfera Serratia plymuthica Serratia ficaria
• Lysine decarboxylase is produced by most species but not by E.
Serratia entomophilia Serratia fonticola
gergoviae or E. cloacae

Pantoea spp. General Characteristics

 Pantoea (Enterobacter) agglomerans gained notoriety with a nationwide  Motile GN rods, NLF but sucrose fermenters
outbreak of septicemia resulting from contaminated intravenous fluids  Differentiated from other members of the
 Designated early on as E. agglomerans complex, it includes members that tribe by their ability to produce Dnase
are:  produce a non-water soluble red to pink
• lysine-, ornithine-, and arginine-negative or “triple decarboxylases- pigment (prodigiosin)
negative” ➢ Serratia rubidaea
 Enterobacter, Cronobacter and Pantoea ➢ Serratia marcescens
➢ Serratia plymuthica
Cronobacter spp.
 ➢ Pigment production is typically a characteristic in strains of  However, two distinct biotypes are recognized:
environmental origin
• H. alvei
Serratia marcescens • H. alvei biotype 1 – grows in the beer wort of breweries and has not
 species most significant clinically been isolated clinically

 Found frequently in nosocomial infections of the: • Hafnia has been isolated from many anatomic sites in humans and in
the environment
• urinary or respiratory tract
 Hafnia has been linked to gastroenteritis and is occasionally isolated from
• bacteremic outbreaks in nurseries and cardiac surgery and stool cultures
burn units
 A delayed positive citrate reaction is a major characteristic of Hafnia
 Contamination of antiseptic solution used for joint injections has
resulted in an epidemic of septic arthritis

Serratia odorifera contains two biogroups

➢ Emits a dirty, musty odor resembling that of rotten potatoes
➢ S. odorifera biogroup 1 is isolated predominantly from the
respiratory tract and is positive for sucrose, raffinose, and ornithine
• biogroup 1 may be indole-positive (60%)
➢ S. odorifera biogroup 2 is negative for sucrose, raffinose, and
ornithine and has been isolated from blood and CSF
• biogroup 2 may also be indole-positive (50%)
S. liquefaciens, S. rubidaea, and S. fonticola have also been isolated from human
sources
Proteus
General Characteristics
Hafnia sp.  Widely disseminated in the environment
 Genus Hafnia is composed of one species, H. alvei  Part of normal intestinal microbiota
 Opportunistic pathogens • Swarming of Proteus is inhibited by:
 Proteus mirabilis is the most common clinical isolate • 5 % agar
 phenylalanine deaminase (+) • Salt –free culture media
➢ only member of Enterobacteriaceae that synthesize this enzyme • PEA
 all produce a bluish gray confluent surface growth or translucent sheet of • CLED (cystine lactose electrolyte difficient)
growth on moist blood agar that gives off a “burnt gun powder” or “ burnt
• Diene’s phenomenon
chocolate” odor
 P. mirabilis and P. vulgaris are widely recognized human pathogens
Antigenic Structure
 Both species hydrolyze urea and produce H2S, although some strains of P.
vulgaris are negative for H2S  All possess O, H and K antigens
 P. mirabilis is differentiated from P. vulgaris by the indole and ornithine  Proteus vulgaris
decarboxylase tests
➢ same antigenic structure as Rickettsia
➢ P. mirabilis is indole-negative but ornithine-positive
➢ O antigens (OX-19, OX-2) of some strains
➢ P. vulgaris is indole-positive but ornithine-negative
✓ detect the rickettsial antibodies in the Weil-Felix test
 Both species, particularly P. mirabilis, can produce “swarming” colonies on
 Proteus mirabilis
non-selective media, such as SBA and CHOC
➢ Source of O antigen (OX-K) that detect the rickettsial antibodies in the
 Swarming is a result of a tightly regulated cycle of differentiation from
Weil-Felix test
standard vegetative cells (swimmers) to hyperflagellated, elongated,
polyploid cells (swarmers) capable of coordinated surface movement
 P. vulgaris ferments sucrose and gives an acid/acid reaction in TSI agar Clinical Infection

 Diene’s phenomenon  Majority of human infections are caused by Proteus

• Different strains of Proteus when inoculated on a culture media  leading cause of community-acquired UTI
swarm towards each other but do not merge and are separated by a  major cause of nosocomial infections
narrow demarcation line between them
 urease activity of Proteus mirabilis Genus Morganella
➢ leads to struvite kidney stones (calculi)

Morganella morganii

✓ Morganella spp. is motile but does not swarm

  Providencia alcalifaciens
• Most commonly found in the feces of children with diarrhea but its
role as a cause of diarrhea has not been proven

Edwardsiella tarda is the only recognized human pathogen

Edwardsiella
Providencia
 Members of this genus are:
 Providencia rettgeri
• Negative for urea
• UTI w/ occasional outbreaks in health care units
• IMViC + + - -
• Diarrheal disease among travellers
• Lysine decarboxylase-positive
• TSI K/A + gas + H2S
 Providencia stuartii
• Outbreaks in burn units
Edwardsiella tarda
• UTI
• isolated from humans and associated with
 • diarrhea  All species grow on Simmons citrate medium and give positive reactions in
the methyl red test
• wound infections
 inhabitants of the GI tract and are associated with hospital-acquired
• bacteremia
infections, most frequently UTI
• isolated from the environment and many cold blooded and
 C. freundii can be isolated in diarrheal stool cultures, and although it is a
warm blooded animals
known extra-intestinal pathogen, its pathogenic role in intestinal disease is
• NLF and H2S (+) – differentiates it from Escherichia coli not established
 Citrobacter

Citrobacteriaceae  C. freundii has been associated with infectious diseases acquired in hospital
settings; UTI, pneumonias, and intraabdominal abscesses have been reported
 Earlier classifications of the family Enterobacteriaceae included Citrobacter
within the tribe Salmonelleae, which formerly consisted of the genera  C. freundii has been associated with endocarditis in IV drug abusers
Salmonella, Citrobacter, and Arizona
 However, changes in the classification and nomenclature have caused the
Citrobacter freundii
reclassification of genus Citrobacter into its own tribe, Citrobacteriaceae, and
of Arizona as a subspecies of Salmonella  80% produces H2S

Citrobacter  Some strains (50%) fail to ferment lactose

 Genus Citrobacter consists of at least 11 species that all have been isolated  Colonies on primary selective media - mistaken for Salmonella when
from clinical specimens isolated from stool cultures

3 species most often isolated:

• Citrobacter freundii
• Citrobacter koseri
• Citrobacter braakii
 Most hydrolyze urea slowly and ferment lactose slowly, producing colonies
on MAC agar that resemble those of E. coli
Primary Intestinal Pathogens of the Family Enterobacteriaceae  The biochemical features for the genus include the following:
• In almost every case, they do not ferment lactose
• Negative for indole, Voges-Proskauer, phenylalanine deaminase,
and urease
• Most produce H2S – a major exception is Salmonella Paratyphi A,
which does not produce H2S
• Do not grow in medium containing potassium cyanide

Classification of Salmonella
• 3 biochemically discrete spp.
• Salmonella enteritidis
• Salmonella cholerasuis
Salmonella
• Salmonella typhi
 Members of the genus Salmonella produce significant infections in humans
and in certain animals 1. 2 species designation of Salmonella

 Salmonella serotypes are typically found in cold-blooded animals as well as in Salmonella enterica
rodents and birds, which serve as their natural hosts ➢ type species of genus Salmonella
 Salmonellae are gram-negative, facultatively anaerobic bacilli that Salmonella bongori
morphologically resemble other enteric bacteria
➢ Rarely isolated from human
 On selective and differential media used primarily to isolate enteric
pathogens (e.g., MAC), salmonellae produce clear, colorless, non-lactose- Within the species S. enterica are six subspecies:
fermenting colonies ✓ S. enterica subsp. enterica (also called subspecies I)
 Colonies with black centers are seen if the media (e.g., HE or XLD) contain ✓ S. enterica subsp. salamae (subspecies II)
indicators for H2S production
✓ S. enterica subsp. arizonae (subspecies IIIa)
 ✓ S. enterica subsp. diarizonae (subspecies IIIb) ▪ Heat stable
✓ S. enterica subsp. houtenae (subspecies IV)
✓ S. enterica subsp. indica (subspecies VI) 2. H antigen – flagellar antigen
• All former species are now serotypes of Salmonella enterica subsp. enterica ▪ only bacteria that are motile will possess this antigen
➢ e.g. Salmonella enterica subsp. enterica serotype Typhi or simply ▪ Heat-labile
written as Salmonella Typhi (serotype is capitalized and not italized
2 phases:
• Members of the former genus Arizona, now subspecies IIIa of S. enterica, are
Phase 1 (specific phase)
found in infections with symptoms identical to those of Salmonella infections
and may be transmitted to humans from pet turtles, snakes, and fish • shared by a few organisms
• determine immunologic identity
• agglutinate only with homologous antisera
Phase 2 (nonspecific phase)
• shared by many organisms
• react heterologous antisera
3. Vi antigen – capsular antigen
▪ Heat-labile polysaccharide found only in encapsulated species
▪ Prevents phagocytosis
✓ Salmonella serotype Typhi
Antigenic structures
✓ Salmonella serotype Choleraesuis
1. O antigen – the most external component of the lipopolysaccharide (LPS)
of gram negative bacteria ✓ Salmonella

▪ somatic antigen Clinical Infections

▪ Antibodies to O antigen are predominantly IgM  In humans, salmonellosis may occur in several forms, as follows:
  Acute gastroenteritis or food poisoning characterized by vomiting and • Salmonella strains associated with gastroenteritis are usually strains
diarrhea found in animals; most such strains are members of S. enterica subsp.
enterica
 Typhoid fever, the most severe form of enteric fever, caused by
Salmonella serotype Typhi, and enteric fevers caused by other • Source of the infection has been attributed primarily to poultry, milk,
Salmonella serotypes (e.g., Salmonella Paratyphi and Choleraesuis) eggs, and egg products as well as to handling pets
 Non-typhoidal bacteremia • Insufficiently cooked eggs and domestic fowl, such as chicken, turkey,
and duck, are common sources of infection
 Carrier state following Salmonella infection
• Salmonellosis is a common cause of GI tract infections
• Cooking utensils, such as knives, pans, and cutting boards used in
Mode of transmission
preparing the contaminated meat, can spread the bacteria to other
• Typhoid fever food

- human carrier is the sole source of the organism , Salmonella • Direct transmission from person to person has been reported in
serotype Typhi institutions

• Nontyphoidal salmonellosis • Salmonella gastroenteritis occurs when a sufficient number of

organisms contaminate food that is maintained under inadequate
- is transmitted by ingestion of contaminated food (usually eggs,
refrigeration, allowing growth and multiplication of the organisms
poultry and beef products) and water; fecal-oral route
• The infective dose necessary to initiate the disease, 106 bacteria
- aside from the human source, animals and animal products are the
major sources  Enteric Fevers

• Humans acquire the infection by ingesting the organisms in food, water, and  The clinical features of enteric fevers include:
milk contaminated with human or animal excreta
• Prolonged fever
 Gastroenteritis
• Bacteremia
• One of the most common forms of “food poisoning”
• Involvement of the reticuloendothelial system, particularly the
• Results from the ingestion of the organisms through contaminated liver, spleen, intestines, and mesentery
food
• Dissemination to multiple organs
  Enteric fever caused by Salmonella Typhi is known as typhoid fever ✓ Adults – experience transient bacteremia during episodes of
gastroenteritis or develop symptoms of septicemia without
• Salmonella Typhi does not have a known animal reservoir; humans
gastroenteritis
are the only known source of infection
Carrier state
• Other enteric fevers include paratyphoid fevers, which may be due to
Salmonella serotypes Paratyphi A, B, and C and Salmonella serotype ➢ Individuals who recover from infection may harbor the organisms in the gall
Choleraesuis bladder
• Clinical manifestations of paratyphoid fevers are similar to typhoid ➢ Excrete the organisms in feces
fever but are less severe, and the fatality rate is lower
➢ May be terminated by
• Typhoid fever develops approximately 9 to 14 days after ingestion of
✓ antimicrobial therapy
the organisms
✓ Cholecystectomy – only solution to the carrier state
• The onset of symptoms depends on the number of organisms
ingested; the larger the inoculum, the shorter the incubation period Shigella
• Organisms seem to be resistant to gastric acids General characteristics
• On reaching the proximal end of the small intestine, they  slender, aerobic, non- motile, non-encapsulated, gram-negative rods
subsequently invade and penetrate the intestinal mucosa
 generally non-lactose fermenters and do not produce H2S (distinguished
 Bacteremia from Escherichia coli as lactose fermenters)
• Salmonella bacteremia, caused by non-typhoidal Salmonella, is  do not produce gas from glucose (anaerogenic) except for certain types of
characterized primarily by prolonged fever and intermittent Shigella flexneri
bacteremia
 all species can cause bacillary dysentery and are the major causes of it
• Serotypes most commonly associated with bacteremia are
Typhimurium, Paratyphi, and Choleraesuis  Never considered as part of the normal intestinal flora

• Salmonella infection has been observed among two different groups:

✓ Young children – experience fever and gastroenteritis with Factors contributing to virulence
brief episodes of bacteremia ➢ smooth lipopolysaccharide (LPS) structure – may be the one responsible
for the organism’s ability to resist gastric acidity
 ➢ Invasiveness • if invasive: pus cells, red blood cells and macrophages are seen
➢ Shiga toxin – interferes with protein synthesis and is neurotoxic,
cytotoxic and enterotoxic
Classification of Shigella according to serogrouping

Clinical infection
➢ bacillary dysentery or Shigellosis

Clinical manifestations
1. acute toxigenic gastroenteritis
- diarrhea with profuse, watery stools and fever of a self -limited course
2. acute tissue-invasive gastroenteritis
- diarrhea with tenesmus, bloody or blood streaked and mucoid stools,
vomiting, fever, abdominal cramps and abdominal tenderness
Yersinia
Only 3 species are pathogenic for humans:
Laboratory diagnosis
 Yersinia pestis
 Specimen:
 Yersinia enterocolitica
• rectal swab of ulcer taken by sigmoidoscopy (best specimen)
 Yersinia pseudotuberculosis
• feces – usual specimen
- facultatively intracellular
*specimens must be placed quickly on isolation media or transport
medium because they are susceptible to the acid pH of stool - primarily animal pathogens

a. stool examination
• if toxigenic, no abnormal findings
Yersinia pestis  Schwartzman phenomenon (presence of black areas of hemorrhages
all over the body)
Morphology
 Highly fatal
 Facultative anaerobic, nonmotile,
gram-negative coccobacillus , Pneumonic plague (the Black Death; Pandemic plague)
short, encapsulated  Usually arises from septic embolization to the lungs
 Shows marked bipolar staining  With bloody, bacteria-rich, highly infectious sputum
with Wayson’s stain, staining the
polar bodies blue and giving the  Transmitted by inhalation and is highly contagious
cells a “safety pin” appearance
Disease Produced: Plague Biochemical Identification of Gram-negative Bacteria
 Yersinia pestis is primarily pathogenic to rodents 1. IMViC
 humans are accidental hosts only 2. ONPG (Ortho – Nitrophenyl – β – D – galactopyranoside test
a. Mode of transmission (to humans) 3. TSI agar
• Via bites of infected rat fleas (Xenopsylla cheopis) 4. Oxidation – Fermentation test
• By handling the carcass of an infected animal 5. Gelatin liquefaction
• Via infected aerosol droplets from person to person 6. Motility
Clinical forms 7. Nitrate and nitrite reduction
Bubonic plague 8. Urease
 Characterized by infected and swollen lymph nodes (called buboes) 9. Dnase
 occur most commonly in the groin and less frequently in the axillary 10. Malonate utilization test
and cervical nodes
Septicemic plague
 Prominent finding is disseminated intravascular coagulation (DIC)