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Chapter 16 - Enterobacteriaceae. MLAB 2434 – Clinical Microbiology Cecile Sanders & Keri Brophy-Martinez. Chapter 16 - Enterics. Family Enterobacteriaceae often referred to as “enterics” Four major features: All ferment glucose (dextrose) All reduce nitrates to nitrites

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Chapter 16 enterobacteriaceae l.jpg

Chapter 16 - Enterobacteriaceae

MLAB 2434 – Clinical Microbiology

Cecile Sanders & Keri Brophy-Martinez

Chapter 16 enterics l.jpg
Chapter 16 - Enterics

  • Family Enterobacteriaceae often referred to as “enterics”

  • Four major features:

    • All ferment glucose (dextrose)

    • All reduce nitrates to nitrites

    • All are oxidase negative

    • All except Klebsiella, Shigella and Yersinia are motile

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Microscopic and Colony Morphology

  • Gram negative bacilli or coccobacilli

  • Non-spore forming

  • Colony morphology on BAP or CA of little value, as they look the same, except for Klebsiella

  • Selective and differential media are used for initial colony evaluation (ex. MacConkey, HE, XLD agars)

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Classification of Enterics

  • Due to the very large number of organisms in the Family Enterobacteriaceae (see Table 16-11), species are grouped into Tribes, which have similar characteristics (Table 16-1, page 466)

  • Within each Tribe, species are further subgrouped under genera

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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

  • Many enterics possess antigens that can be used to identify groups

    • O antigen – somatic, heat-stable antigen located in the cell wall

    • H antigen – flagellar, heat labile antigen

    • K antigen – capsular, heat-labile antigen

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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

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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 – Salmonella, Shigella, and Yersinia sp.

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Escherichia coli

  • Most significant species in the genus

  • Important potential pathogen in humans

  • Common isolate from colon flora

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Escherichia coli (cont’d)

  • Characteristics

    • Dry, pink (lactose positive) colony with surrounding pink area on MacConkey

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Escherichia coli (cont’d)

  • 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

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Escherichia coli (cont’d)

  • Infections

    • Wide range including meningitis, gastrointestinal, urinary tract, wound, and bacteremia

    • 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

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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

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Escherichia coli (cont’d)

  • Enterohemorrhagic (EHEC serotype 0157:H7) – associated with hemorrhagic diarrhea and hemolytic-uremic syndrome (HUS), which includes low platelet count, hemolytic anemia, and kidney failure; potentially fatal, especially in young children; undercooked hamburger, unpasteurized milk and apple cider have spread the infection; does NOT ferment sucrose; identified by serotyping

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Escherichia coli (cont’d)

  • Enteroaggregative (EaggEC) – cause diarrhea by adhering to the mucosal surface of the intestine; watery diarrhea; symptoms may persist for over two weeks

  • Urinary Tract Infections

    • E. coli is most common cause of UTI and kidney infection in humans

    • Usually originate in the large instestine

    • Able to adhere to epithelial cells in the urinary tract

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    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 hermannii – yellow pigmented; isolated from CSF, wounds and blood

  • Escherichia vulneris - wounds

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    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

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    Klebsiella species

    • Usually found in GI tract

    • Four major species

    • 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

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    Klebsiella species (cont’d)

    • Significant biochemical reactions

      • Lactose positive

      • Most are urease positive

      • Non-motile

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    Enterobacter species

    • Comprised of 12 species; E. cloacaeand E. aerogenes are most common

    • Isolated from wounds, urine, blood and CSF

    • Major characteristics

      • Colonies resemble Klebsiella

      • Motile

      • MR negative; VP positive

    Enterobacter species cont d l.jpg
    Enterobacter species (cont’d)

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    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 bacteremic outbreaks in nurseries, cardiac surgery, and burn units

    • Fairly resistant to antibiotics

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    Serratia species (cont’d)

    • Major characteristics

      • Ferments lactose slowly

      • Produce characteristic pink pigment, especially when cultures are left at room temperature

        S. marscens on

        nutrient agar →

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    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

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    Proteus, Morganella & Providencia species

    • All are normal intestinal flora

    • Opportunistic pathogens

    • Deaminate phenylalanine

    • All are lactose negative

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    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

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    Proteus species (cont’d)

    • A exhibits characteristic “swarming”

    • B shows urease positive on right

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    Morganella species

    • Morganella morganii is only species

    • Documented cause of UTI

    • Isolated from other anatomical sites

    • Urease positive

    • Phenylalanine deaminase positive

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    Providencia species

    • Providencia rettgeri is pathogen of urinary tract and has caused nosocomial outbreaks

    • Providenicia stuartii can cause nosocomial outbreaks in burn units and has been isolated from urine

    • Both are phenylalanine deaminase positive

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    Citrobacter species

    • Citrobacter freundii associated with nosocomial infections (UTI, pneumonias, and intraabdominal abscesses)

    • Ferments lactose and hydrolyzes urea slowly

    • Resembles Salmonella sp.

    Salmonella l.jpg

    • Produce significant infections in humans and certain animals

    • On differential selective agar, produces clear, colorless, non-lactose fermenting colonies with black centers (if media contains indicator for hydrogen sulfide)

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    Salmonella (cont’d)

    • Salmonella on MacConkey

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    Salmonella (cont’d)

    • Lactose negative

    • Negative for indole, VP, phenylalanine deaminase, and urease

    • Most produce H2S

    • Do not grow in potassium cyanide

    • Large and complex group of organisms; grouped by O, H, and Vi (for virulence) antigens

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    Salmonella (cont’d)

    • Clinical Infections

      • Acute gastroenteritis or food poisoning

        • Source = handling pets, insufficiently cooked eggs and chicken, and contaminated cooking utensils

        • Occurs 8 to 36 hours after ingestion

        • Requires a high microbial load for infection

        • Self-limiting in health individuals (antibiotics and antidiarrheal agents may prolong symptoms)

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    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

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    Salmonella (cont’d)

    • Salmonella Bacteremia

    • Carrier State

      • Organisms shed in feces

      • Gallbladder is the site of organisms (removal of gallbladder may be the only solution to carrier state)

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    Shigella species

    • Closely related to the Escherichia

    • All species cause bacillary dysentery

    • S. dysenteriae (Group A)

    • S. flexneri (Group B)

    • S. boydii (Group C)

    • S. sonnei (Group D)

    Shigella cont d l.jpg
    Shigella (cont’d)

    • Characteristics

      • Non-motile

      • Do not produce gas from glucose

      • Do not hydrolyze urea

      • Do not produce H2S on TSI

      • Lysine decarboxylase negative

      • ONPG positive (delayed lactose +)

      • Fragile organisms

      • Possess O and some have K antigens

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    Shigella (cont’d)

    • Clinical Infections

      • Cause dysentery (bloody stools, mucous, and numerous WBC)

      • S. sonnei is most common, followed by S. flexneri (“gay bowel syndrome”)

      • Humans are only known reservoir

      • Oral-fecal transmission

      • Fewer than 200 bacilli are needed for infection in health individuals

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    Yersinia species

    • Consists of 11 named species

    • Yersinia pestis

      • Causes plague, which is a disease primarily of rodents; transmitted by fleas

      • Two forms of plague, bubonic and pneumonic

      • Gram-negative, short, plump bacillus, exhibiting “safety-pin” or “bipolar” staining

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    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

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    Yersinia species

    • Yersinia pseudotuberculosis

      • Pathogen of rodents, particularly guinea pigs

      • Septicemia with mesenteric lymphadenitis, similar to appendicitis

      • Motile at 18 to 22 degrees C

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    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 Identification

      • Site of origin must be considered

      • Enterics from sterile body sites are highly significant

      • Routinely cultured from stool

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    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 MacConkey, lactose positive are pink; lactose negative are clear and colorless

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    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

  • Identification

    • Most labs use a miniaturized or automated commercial identification system, rather than multiple tubes inoculated manually

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    Laboratory Diagnosis of Enterics (cont’d)

    • Identification (cont’d)

      • All enterics are

        • Oxidase negative

        • Ferment glucose

        • Reduce nitrates to nitrites

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    Laboratory Diagnosis of Enterics (cont’d)

    • Common Biochemical Tests

      • Lactose fermentation and utilization of carbohydrates

      • Triple Sugar Iron (TSI)

      • ONPG

      • Glucose metabolism

        • Methyl red

        • Voges-Proskauer

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    Laboratory Diagnosis of Enterics (cont’d)

    • Common Biochemical Tests (cont’d)

      • Miscellaneous Reactions

        • Indole

        • Citrate utilization

        • Urease production

        • Motility

        • Phenylalanine deaminase

        • Decarboxylase tests

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    Screening Stools for Pathogens

    • Because stools have numerous microbial flora, efficient screening methods must be used to recover any pathogens

    • Enteric pathogens include Salmonella, Shigella, Aeromonas, Campylobacter, Yersinia, Vibrio, and E. coli 0157:H7

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    Screening Stools for Pathogens (cont’d)

    • Most labs screen for Salmonella, Shigella, and Campylobacter; many screen for E. coli 0157:H7

    • Fecal pathogens are generally lactose-negative (although Proteus, Providencia, Serratia, Citrobacter and Pseudomonas are also lactose-negative)