Enterobacteriaceae MLAB 2434 – Microbiology Keri Brophy-Martinez

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


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


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


• 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


• 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


• 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


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


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


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


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


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)


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


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 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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Enterobacteriaceae MLAB 2434 – Microbiology Keri Brophy-Martinez