1. Chapter 16 - Enterobacteriaceae
MLAB 2434 – Clinical Microbiology
Cecile Sanders & Keri Brophy-Martinez

2. 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

3. 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)

4. 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

5. 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

6. 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

7. 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.

8. Escherichia coli Most significant species in the genus
Important potential pathogen in humans
Common isolate from colon flora

9. Escherichia coli (cont’d)
Characteristics
Dry, pink (lactose positive) colony with surrounding pink area on MacConkey

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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

18. Klebsiella species (cont’d)
Significant biochemical reactions
Lactose positive
Most are urease positive
Non-motile

19. Enterobacter species
Comprised of 12 species; E. cloacae and E. aerogenes are most common
Isolated from wounds, urine, blood and CSF
Major characteristics
Colonies resemble Klebsiella
Motile
MR negative; VP positive

20. Enterobacter species (cont’d)

21. 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

22. 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 →

23. 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

24. Proteus, Morganella & Providencia species
All are normal intestinal flora
Opportunistic pathogens
Deaminate phenylalanine
All are lactose negative

25. 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

26. Proteus species (cont’d)
A exhibits characteristic “swarming”
B shows urease positive on right

27. Morganella species Morganella morganii is only species
Documented cause of UTI
Isolated from other anatomical sites
Urease positive
Phenylalanine deaminase positive

28. 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

29. Citrobacter species
Citrobacter freundii associated with nosocomial infections (UTI, pneumonias, and intraabdominal abscesses)
Ferments lactose and hydrolyzes urea slowly
Resembles Salmonella sp.

30. Salmonella
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)

31. Salmonella (cont’d)
Salmonella on MacConkey

32. 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

33. 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)

34. 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

35. 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)

36. 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)

37. 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

38. 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

39. Shigella (cont’d)

40. 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

41. 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

42. Yersinia species Yersinia pseudotuberculosis
Pathogen of rodents, particularly guinea pigs
Septicemia with mesenteric lymphadenitis, similar to appendicitis
Motile at 18 to 22 degrees C

43. 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

44. 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

45. 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

46. Laboratory Diagnosis of Enterics (cont’d)
Identification (cont’d)
All enterics are
Oxidase negative
Ferment glucose
Reduce nitrates to nitrites

47. 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

48. Laboratory Diagnosis of Enterics (cont’d)
Common Biochemical Tests (cont’d)
Miscellaneous Reactions
Indole
Citrate utilization
Urease production
Motility
Phenylalanine deaminase
Decarboxylase tests

49. 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

50. 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)