Enterobacteriaceae
MLAB 2434 – Microbiology Keri Brophy-Martinez
Overview- Enterics
Family Enterobacteriaceae often referred to as “enterics”
Four major features:All ferment glucose (dextrose)All reduce nitrates to nitritesAll are oxidase negative, except
PlesiomonasAll except Klebsiella, Shigella and
Yersinia are motile
Microscopic and Colony Morphology
Gram negative bacilli or coccobacilli Non-spore forming Facultative anaerobe Colony morphology on BAP or CA of
little value, as they look the same large, moist and grey on SBA
Selective and differential media are used for initial colony evaluation ex. MacConkey, HE, XLD agars
Classification of Enterics
Due to the very large number of organisms in the Family Enterobacteriaceae (see Table 19-1, page 429), species are grouped into Tribes, which have similar characteristics
Within each Tribe, species are further subgrouped under genera
Virulence and Antigenic Factors of Enterics
Ability to colonize, adhere, produce various toxins and invade tissues
Some possess plasmids that may mediate resistance to antibiotics ESBL’s: Extended-Spectrum beta
lactamases• Inactivate extended spectrum
antibiotics like penicillins, cephalosporins and aztreonam
Virulence and Antigenic Factors of Enterics Many enterics possess antigens that can be
used to identify groups O antigen – somatic, heat-stable antigen
located in the cell wall, made by all bacteria, stimulates EARLY antibody production
H antigen – flagellar, made by bacteria with flagella, heat labile antigen,stimulates LATE antibody production
K antigen – capsular, heat-labile antigen, made by some bacteria
• Vi- capsular antigen of S. typhii
Clinical Significance of Enterics Enterics are ubiquitous in nature Except for few, most are present
in the intestinal tract of animals and humans as commensal flora; therefore, they are sometimes call “fecal coliforms”
Some live in water, soil and sewage
Clinical Significance of Enterics (cont’d) Based on clinical infections produced,
enterics are divided into two categories: Opportunistic pathogens – normally part
of the usual intestinal flora that may produce infection outside the intestine
Primary intestinal pathogens – not commensal flora, they produce infection from ingestion of contaminated food or water (Salmonella, Shigella, and Yersinia sp.)
Escherichia coli
Most significant species in the genus
Important potential pathogen in humans
Common isolate from colon flora
Escherichia coli (cont’d)
InfectionsWide range including meningitis,
gastrointestinal, urinary tract, wound, and bacteremia
Urinary Tract Infections• E. coli is most common cause of UTI and
kidney infection in humans• Usually originate in the large intestine• Able to adhere to epithelial cells in the
urinary tract
Escherichia coli (cont’d)
Septicemia & Meningitis• E. coli is one of the most common causes of
septicemia and meningitis among neonates; acquired in the birth canal before or during delivery
• E. coli also causes bacteremia in adults, primarily from a genitourinary tract infection or a gastrointestinal source
Escherichia coli (cont’d)
Gastrointestinal Infections • Enteropathogenic (EPEC) – primarily in
infants and children; outbreaks in hospital nurseries and day care centers; stool has mucous but not blood; identified by serotyping
Escherichia coli (cont’d)
• Enterotoxigenic (ETEC) – “traveler’s diarrhea”; watery diarrhea without blood; self-limiting; usually not identified, other than patient history and lactose-positive organisms cultured on differential media
• Enteroinvasive (EIEC) – produce dysentery with bowel penetration, invasion and destruction of intestinal mucosa; watery diarrhea with blood; do NOT ferment lactose; identified via DNA probes
Escherichia coli (cont’d)
• Enteroadherent E. coli• Enteroaggregative (EAEC)
• Cause diarrhea by adhering to the mucosal surface of the intestine; watery diarrhea; symptoms may persist for over two weeks
• Diffusely adherent(DAEC)• Associated with UTI’s and
diarrheal disease, esp. in children and pregnant women
Escherichia coli (cont’d)
• Enterohemorrhagic (EHEC serotype 0157:H7) • Origin
• Isolated in 1970• Undercooked hamburger, unpasteurized milk and
apple cider have spread the infection• Pathogenesis
• Circulating Shiga toxin binds to kidney endothelium causing an inflammatory response
• Macrophages and neutrophils damage the endothelium and glomerular basement membrane
• Associated with hemorrhagic colitis, TTP and hemolytic-uremic syndrome (HUS), which includes low platelet count, hemolytic anemia, and kidney failure; potentially fatal, especially in young children
Escherichia coli (cont’d)
Enterohemorrhagic (EHEC serotype 0157:H7) Clinical Symptoms
• Starts with a watery diarrhea then progresses to bloody diarrhea. No WBC’s are found in stool
• Laboratory Diagnosis• Does NOT ferment sorbitol• Identified by serotyping, latex tests
Escherichia coli (cont’d)
General CharacteristicsDry, pink (lactose positive) colony
with surrounding pink area on MacConkey
Other Escherichia species Escherichia hermannii – yellow
pigmented; isolated from CSF, wounds and blood
Escherichia vulneris - yellow pigmented; wounds
Escherichia coli (cont’d)
Characteristics• Ferments glucose, lactose, trehalose, &
xylose• Positive indole and methyl red tests• Does NOT produce H2S or phenylalanine
deaminase• Simmons citrate negative• Usually motile• Voges-Proskauer test negative• Fimbriae• O, H, K antigens
Klebsiella, Enterobacter, Serratia & Hafnia sp. Usually found in intestinal tract Wide variety of infections, primarily
pneumonia, wound, and UTI General characteristics:
Some species are non-motile Simmons citrate positive H2S negative Phenylalanine deaminase negative Some weakly urease positive MR negative; VP positive
Klebsiella species
Usually found in GI tract K. pneumoniae is mostly commonly
isolated species Possesses a polysaccharide capsule,
which protects against phagocytosis and antibiotics AND makes the colonies moist and mucoid
Has a distinctive “yeasty” odor Frequent cause of nosocomial
pneumonia
Klebsiella species (cont’d)
Significant biochemical reactions• Lactose positive• Most are urease positive• Non-motile
Enterobacter species
Comprised of 12 species; E. cloacae and E. aerogenes are most common
Isolated from wounds, urine, blood and CSF
Major characteristics Motile Simmons citrate positive MR negative; VP positive
Enterobacter species(cont’d)
Serratia species
Seven species, but S. marcescens is the only one clinically important
Frequently found in nosocomial infections of urinary or respiratory tracts
Implicated in bacteremia, septicemia, cardiac surgery, and burn units
Fairly resistant to antibiotics
Serratia species (cont’d)
Major characteristicsFerments lactose slowlyProduce characteristic pink or red
pigment, especially when cultures are left at room temperature
S. marscens on nutrient agar →
Hafnia species
Hafnia alvei is only species Has been isolated from many
anatomical sites in humans and the environment
Occasionally isolated from stools Delayed citrate reaction is major
characteristic
Proteus, Morganella & Providencia species All are normal intestinal flora Opportunistic pathogens Deaminate phenylalanine All are lactose negative
Proteus species
P. mirabilis and P. vulgaris are widely recognized human pathogens
Isolated from urine, wounds, and ear and bacteremic infections
Both produce swarming colonies on non-selective media and have a distinctive “burned chocolate” odor
Both are strongly urease positive Both are phenylalanine deaminase
positive
Proteus
Lactose negative
Proteus species (cont’d)
A: exhibits characteristic “swarming”
B: shows urease positive on right
Morganella species
Morganella morganii is only species
Documented cause of UTI Isolated from other anatomical
sites Urease positive Phenylalanine deaminase
positive
Providencia species
Providencia rettgeri is pathogen of urinary tract and has caused nosocomial outbreaks
Providencia stuartii can cause nosocomial outbreaks in burn units and has been isolated from urine
Both are phenylalanine deaminase positive
Citrobacter species
Citrobacter freundii associated with nosocomial infections (UTI, pneumonias, and intraabdominal abscesses)
Ferments lactose and hydrolyzes urea slowly
Methyl red positive, Simmons citrate positive
Resembles Salmonella sp.
Salmonella
Produce significant infections in humans and certain animals
Infections caused by ingestion of food or water contaminated with either human or animal feces
Salmonella (cont’d)
Salmonella on MacConkey
Salmonella (cont’d)
Lactose negative Negative for indole, VP,
phenylalanine deaminase, and urease
Most produce H2S Do not grow in potassium
cyanide
Salmonella
Virulence Factors Fimbriae- help in attachment Enterotoxin Anitgenic Structures
• O and H structures are used for serologic grouping
• Vi- helpful in ID of Salmonella typhi
Salmonella (cont’d)
Clinical Infections Acute gastroenteritis or food poisoning
• Source = handling pets, insufficiently cooked eggs and chicken,milk, and contaminated cooking utensils
• Occurs 8 to 36 hours after ingestion• Symptoms include vomiting, chills, watery
diarrhea and abdominal pain• Requires a high microbial load for infection• Self-limiting in healthy individuals
(antibiotics and antidiarrheal agents may prolong symptoms)
Salmonella (cont’d)
Typhoid and Other Enteric Fevers• Prolonged fever• Bacteremia• Involvement of the RE system,
particularly liver, spleen, intestines, and mesentery
• Dissemination to multiple organs• Occurs more often in tropical and
subtropical countries
Salmonella (cont’d)
Typhoid fever S. Typhii causative organism
• Invades intestinal mucosa causing constipation• Gains entrance into lymphatic system and bloodstream
then into the liver, spleen and bone marrow where they are phagocytized by PMN’s. They multiply in the PMN’s and are eventually released into the blood stream.
• Finally, they invade the gall bladder and other parts of the intestinal tract to initiate GI symptoms.
Found in contaminated food originating from infected individuals
Other causes, improper disposal of sewage, poor sanitation, lack of modern H2O systems
Develops 9-14 days after ingestion with symptoms of fever, malaise, anorexia, myalgia, continuous dull headache, rash
Severe
Salmonella (cont’d)
Salmonella BacteremiaCarrier State
• Organisms shed in feces• Gallbladder is the site of organisms
(removal of gallbladder may be the only solution to carrier state)
Shigella species
Closely related to the Escherichia Fragile organisms All species cause bacillary
dysentery, not normal GI flora S. dysenteriae (Group A) S. flexneri (Group B) S. boydii (Group C) S. sonnei (Group D)
Shigella (cont’d)
Characteristics Non-motile Do not produce gas from glucose Do not hydrolyze urea Do not produce H2S on TSI On differential, selective media appear
as clear, NLF Lysine decarboxylase negative Citrate negative Possess O and some have K antigens
lack the H antigen
Shigella (cont’d)
Clinical Infections Cause dysentery (bloody stools, mucous,
and numerous WBC)• Endotoxin- LPS• Exotoxin- Shiga toxin
Confined to GI tract,septicemia rare Humans are only known reservoir Oral-fecal transmission, person-person Fewer than 200 bacilli are needed for
infection in healthy individuals= takes a low infective dose
Shigella (cont’d)
Yersinia species
Consists of 11 named species Yersinia pestis
Causes plague, which is a disease primarily of rodents; transmitted by rat fleas
Two forms of plague• bubonic- flea bite, buboes• Pneumonic- secondary to bubonic,
organisms multiply in bloodstream and resp. tract
Characteristic bubo
Yersinia gram stain
Yersinia species
Yersinia enterocolitica Most common form of Yersinia Found worldwide Found in pigs, cats and dogs Human also infected by ingestion of
contaminated food or water Some infections result from eating
contaminated market meat and vacuum-packed beef
Is able to survive refrigerator temperatures (can use “cold enrichment” to isolate)
Mainly causes acute gastroenteritis with fever, headaches
Stool may be bloody Can mimic appendicitis
Laboratory Diagnosis of Enterics Collection and Handling
If not processed quickly, should be collected and transported in Cary-Blair, Amies, or Stuart media
Isolation and IdentificationSite of origin must be consideredEnterics from sterile body sites
are highly significantRoutinely cultured from stool
Laboratory Diagnosis of Enterics (cont’d) Media for Isolation and Identification of
Enterics Most labs use BAP, CA and a
selective/differential medium such as MacConkey
On BA, produce large, greyish, smooth colonies; can be β-hemolytic or nonhemolytic
On MacConkey, lactose positive are pink; lactose negative are clear and colorless
Laboratory Diagnosis of Enterics (cont’d)
For stools, highly selective media, such as Hektoen Enteric (HE), XLD, or SS is used along with MacConkey agar
CIN- selects for Yersenia, hold at room temp.
Identification Most labs use a miniaturized or
automated commercial identification system, rather than multiple tubes inoculated manually
Laboratory Diagnosis of Enterics (cont’d)
Identification (cont’d)All enterics are
• Oxidase negative• Ferment glucose• Reduce nitrates to nitrites
Laboratory Diagnosis of Enterics (cont’d) Common Biochemical Tests
Lactose fermentation and utilization of carbohydrates
Triple Sugar Iron (TSI)ONPGGlucose metabolism
• Methyl red• Voges-Proskauer
Laboratory Diagnosis of Enterics (cont’d) Common Biochemical Tests
(cont’d)Miscellaneous Reactions
• Indole• Citrate utilization• Urease production• Motility• Phenylalanine deaminase• Decarboxylase tests
Identification Clues
• Lactose Fermenter• Think: E. coli, Klebsiella, Enterobacter, possibly
Citrobacter• Lactose Fermenter with mucoid colony
• Think: Klebsiella, Enterobacter• H2S +
• Think: Proteus, Salmonella, Citrobacter• Nonmotile
• Think: Klebsiella, Shigella• Voges- Proskauer +
• Think: Klebsiella, Enterobacter, Serratia
Screening Stools for Pathogens Because stools have numerous
microbial flora, efficient screening methods must be used to recover any pathogens
Fecal WBC Fecal pathogens are generally lactose-
negative Enteric pathogens include Salmonella,
Shigella, Aeromonas, Campylobacter, Yersinia, Vibrio, and E. coli 0157:H7
References
Engelkirk, P., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic Microbiology . Baltimore, MD: Lippincott Williams and Wilkins.
Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.
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