Gram (-) Rods Enterobacteriaceae (trans 1)

Question 1

ENTEROBACTERIACEAE

Answer 1

Members of Enterobacteriaceae. Although Vibrio and Yersinia are found in the intestines, they are not member of the Enterobacteriaceae family.

Question 2

ENTEROBACTERIACEAE - Characteristics

 Large, heterogenous group of small gram (-) rods
 Non-spore forming, facultative anaerobes (grow aerobically and anaerobically)
o Facultative: When grown in anaerobic environment it ferments carbohydrates for energy. But when given sufficient oxygen, will utilize the tricarboxylic acid cycle and the electron transport chain for energy production.
 Found in soil, water, decaying matter, and large intestines of humans and animals; also in insects

Answer 2

 Referred to as enteric bacilli or coliforms because of their normal habitat in humans
 Members (from Zinnser)
o Escherichia
o Enterobacter
o Klebsiella
o Salmonella
o Shigella
o Serratia
o Citrobacter
o Proteus

Question 3

ENTEROBACTERIACEAE - Characteristics

 Most are opportunistic and can cause infection when they are displaced from their normal habitat
 Responsible for the majority of nosocomial infections
 All are non-encapsulated (except Klebsiella and Enterobacter; capsules are large and regular in Klebsiella species, less so in Enterobacter species, and uncommon in the other species)
 All are motile with peritrichous flagella (except Klebsiella and Shigella)
 Catalase positive and oxidase negative(except Plesiomonas)

Answer 3

 All reduce nitrate to nitrite
 All are glucose fermenters, often with gas production
 Grow on peptone or meat extract media without the addition of sodium chloride or other supplements; grow well on MacConkey agar
 Destroyed easily by heat and by low concentrations of germicides and disinfectants.
 Can survive snow and ice for several months.

Question 4

ENTEROBACTERIACEAE - Virulence Factors

 Antigenic structures:
o Capsular Antigen (K): some are polysaccharides some are proteins; heat-labile
o Flagellar Antigen (H): used as a basis of antigenic typing (ex: Salmonella); denatured or removed by heat or alcohol; agglutinate with anti-H antibodies, mainly IgG
o Somatic Antigen (O): enhances the establishment of the organism in the host; heat-stable and alcohol-resistant. Antibodies to O antigens are predominantly IgM

 COLONIZATION FACTORS
o Capsule: Antiphagocytic
o Fimbriae/Pili: For attachment to host cells (E.coli)

Answer 4

Determinants of Pathogenicity:
o Endotoxin (Lipid A)
 A piece of the outer membrane lipopolysaccharide (LPS) of gram (-) bacteria
 Very toxic and is released when the bacterial cell undergoes lysis (destruction)
 Endotoxin is also shed in steady amounts from living bacteria
 Contributes to high mortality (90%) in patients with gram (-) bacteremia due to endotoxic shock
 Causes pooling of blood in the microcirculation w/c causes cellular hypoxia and metabolic failure due to inadequacy of blood in vital organs

o Enterotoxin
 Causes transduction of fluid into the lumen of the small intestine w/ subsequent diarrhea

o Shiga Toxins and Shiga-like Toxins (Verotoxins)
 Called “vero” because of its effects on vero tissue cells of African Green Monkeys
 Shigella: produces a toxin that interferes with protein synthesis Verotoxin-producing E. coli (VTEC) aka EHEC: causes diarrhea and Hemolytic Uremic Syndrome (HUS).
*Hemolytic: causes microcytic hemolysis
*Uremic: renal failure; occurs after a bout of EHEC diarrhea
 Some antibiotics may increase verotoxin production and precipitate HUS (ex Cotrimoxazole)

Question 5

Effects of septic shock

Answer 5

Question 6

BIOCHEMICAL TESTS

 Traditionally, biochemical tests and growth on certain culture media are used to identify gram (-) bacteria
 Some examples include; IMViC tests, growth characteristics on MacConkey, EMB, SSA, HEA.

Answer 6

1. IMViC tests
2. MacConkey agar
3. Triple Sugar Iron agar (TSI)
4. Eosin Methylene Blue agar (EMB)
5. Salmonella-Shigella agar
6. Hektoen Enteric agar (HEA)

Question 7

BIOCHEMICAL TESTS - IMViC tests

 Consists of four separate tests: Indole, Methyl red, Vogesproskauer, and Citrate utilization.
 Indole Test: Identifies organisms that can digest tryptophan to indole
(+) result: Red ring at the upper layer indicates the presence of indole which is detected by the addition of Kovac’s reagent (ex: E. coli)
(-) result: No red ring (ex: Klebsiella)

Answer 7

Indole test. Left: negative result; Right: Positive result

Question 8

BIOCHEMICAL TESTS - IMViC tests

**Methyl Red Test

• is a pH indicator that changes color depending on the pH of the environment. When used as a test, it detects glucose fermentation to acidic products.
  (+) result: Red color (ex: E. coli)
  (-) result: Yellow color (ex: Enterobacter, Klebsiella)

Answer 8

Methyl Red Test. Left: Positive result yields red color; Right: Negative result is yellow color.

Question 9

BIOCHEMICAL TESTS - IMViC tests

**Voges-Proskauer Test

• Identifies organisms that digest glucose to acetylmethyl carbinol.
  *Organisms that are Methyl red (+) will be VP(-) and vice versa.
  (+) result: pink to red color (ex: Enterobacter, Klebsiella)
  (-) result: yellow color (ex: E. coli)

Answer 9

Voges-Proskauer Test. Left: Positive result yields red color; Right: Negative result is yellow color

Question 10

BIOCHEMICAL TESTS - IMViC tests

**Citrate utilization

• This is done in a Simmon Citrate agar (SCA). Tests organisms’ ability to use citrate as a source of carbon. It contains bromthymol blue which changes color from green to blue in an alkaline environment.
  (+) result: SCA turns blue (ex: Enterobacter, Klebsiella, Citrobacter)
  (-) result: SCA remains green (ex: E. coli)

Answer 10

Citrate utilization. Left: Negative result remains green; Right: Positive result turns blue

Question 11

BIOCHEMICAL TESTS - IMViC tests

Answer 11

IMViC results for some bacteria

Question 12

BIOCHEMICAL TESTS - MacConkey agar

 Is a selective and a differential media which selects for gram negative and differentiates between lactose (LF) and non-lactose (NLF) fermenters.
 Contains bile salts and crystal violet to inhibit gram positive organisms, and contains phenol red to detect lactose fermentation
 Lactose fermenters grow as pink colonies in Mac, while non-lactose fermenters are colorless.

Answer 12

E. coli in MacConkey agar which produces pink colonies

Question 13

BIOCHEMICAL TESTS - Triple Sugar Iron agar (TSI)

 One of the most commonly used media to differentiate between enteric bacteria.
 Contains sucrose, lactose and glucose in 10:10:1 ratio with phenol red as an indicator
 Detects an organisms ability to ferment glucose, lactose, produce gas and to produce Hydrogen sulfide (H2S)
 All enteric bacteria that can ferment lactose can ferment glucose, hence they will turn the butt and the slant yellow (A/A)
 Not all enteric bacteria that can ferment glucose are able to ferment lactose hence they can only turn the butt yellow. (K/A)
 Gas production is seen as bubbles or cracks in the agar
 This also has ferrous sulfate and if the bacteria forms H2S, this chemical will react with the iron to form ferrous sulfide seen as a black precipitate in the butt

Answer 13

TSI results for some bacteria. From left => right: Uninoculated control, Pseudomonas aeruginosa, Shigella sonnei, Salmonella typhi, Escherichia coli, Proteus mirabilis

Question 14

TSI results for some bacteria.

Answer 14

Question 15

BIOCHEMICAL TESTS - Eosin Methylene Blue agar (EMB)

 Is both a selective and a differential media which contains eosin and methylene blue to inhibit G (+) organisms and lactose to differentiate LF from NLF
 LF yields colored colonies while NLF has colorless colonies.
 Only E. coli can produce the characteristic greenish metallic sheen in EMB
 To differentiate Klebsiella and Enterobacter (since they have the same results in IMViC and TSI

Answer 15

E. coli in EMBA which produces green metallic sheen.

Question 16

BIOCHEMICAL TESTS - Salmonella-Shigella agar

 Specific for salmonella and shigella (suppresses the growth of other enteric and gram (+) organisms
 Differentiates between the two by H2S production, Salmonella grows with black centers while Shigella has colorless colonies.

Answer 16

LEFT: Shigella on SSA, note the transparent colonies produced; RIGHT: Salmonella on SSA, note the colorless colonies with black center.

Question 17

BIOCHEMICAL TESTS - Hektoen Enteric agar (HEA)

 Also used to differentiate between Salmonella and shigella.
 Shigella has blue-green colonies on HEA. Salmonella has black colonies on HEA.

Answer 17

LEFT: Shigella on HEA, note the blue-green colonies produced; RIGHT: Salmonella on HEA, note the transparent colonies with black centers.

Question 18

ESCHERICHIA COLI

Answer 18

Characteristics
 Facultative inhabitant of the large intestine
 Part of the normal flora but can be pathogenic both within and outside of the GI tract (but more pathogenic when outside of its normal habitat)
 Responsible for virtually all the clinically significant infections caused by the genus
 UTI is the most common extra-GIT nosocomial infection caused by E. coli
 Glucose and lactose fermenter
 Produces both acid and gas during fermentation of carbohydrates
 Reduces Nitrates to Nitrites to produce energy
 Lacks cytochrome oxidase (oxidase negative)
 May be beta-hemolytic on BAP

Question 19

ESCHERICHIA COLI - Virulence Factors

Antigenic Structures:
o Serologic Typing is based on O Antigen, H Antigen and when applicable, the K Antigen.
o Antigenic profile is useful for epidemiologic studies:
 O157:H7 produces Shiga-like Toxin causing hemorrhagic colitis and HUS
 O78:H11 strains are enterotoxigenic

Answer 19

Determinants of Pathogenicity:
o Polysialic Acid Capsule (K1 Ag)
 Enables the organism to resist killing by neutrophils
 Aids in survival of organisms in the blood & CSF
 Strains possessing this are more likely to cause neonatal sepsis
o S Fimbriae
 Predilection for binding to vascular endothelium and ventricles of the brain.
o Other fimbriae
 Binding to other host tissues
o Verotoxin (Shiga-like toxin)
 VTEC is associated with diarrhea, hemorrhagic colitis and HUS
o Enterotoxins: Target organ is the small intestine
 Heat Labile enterotoxin (LT) : Stimulates adenyl cyclase in the mucosa which increases permeability, resulting in the loss of fluids and electrolytes.
 Heat Stable Enterotoxin (ST) : activates guanylate cyclase in the mucosa inhibiting Na and Cl reabsorption

Question 20

ESCHERICHIA COLI - Clinical Manifestations

Answer 20

Intestinal Disease
 Transmission of intestinal disease is commonly by the fecal–oral route, with contaminated food and water serving as vehicles
 At least five types of intestinal infections that differ in pathogenic mechanisms have been identified. All are basically the same organism, differing only by the acquisition of specific pathogenic traits.
o EnteroToxigenic (ETEC): Traveler’s diarrhea
o EnteroPathogenic (EPEC): Infantile (inPantile)diarrhea
o EnteroHemorrhagic(EHEC): HUS
o Enteroinvasive (EIEC): Dysentery-like syndrome
o Enteroaggregative (EAEC): Watery diarrhea

Question 21

ESCHERICHIA COLI - ENTEROTOXIGENIC E. COLI (ETEC)

 Common cause of traveler’s diarrhea.
 Transmission: fecal–oral (contaminated food or water) or by person-to-person contact
 ETEC colonizes the small intestine (pili facilitate the binding of the organism to the intestinal mucosa)
 Enterotoxins cause prolonged hypersecretion of chloride, ions and water by the intestinal mucosal cells, while inhibiting the reabsorption of sodium. The gut becomes full of fluid, resulting in significant watery diarrhea that continues over a period of several days.

Answer 21

o Heat-stable toxin (ST): Causes an elevation in cellular (cGMP) levels by activating guanylate cyclase in the mucosa, inhibiting Na and Cl reabsorption
o Heat-labile toxin (LT): causes elevated (cAMP) by stimulating adenyl cyclase in the mucosa which increases permeability, resulting in the loss of fluids and electrolytes. LT is closely related in structure and function to cholera toxin.

Question 22

ESCHERICHIA COLI - ENTEROTOXIGENIC E. COLI (ETEC)

Answer 22

Process of pathogenicity of Enterotoxigenic E.coli.

Question 23

ESCHERICHIA COLI - ENTEROPATHOGENIC E. COLI (EPEC)

Answer 23

• Cause of diarrhea in infants, especially in locations with poor sanitation.
• The newborn becomes infected perinatally
• There is destruction of the microvilli in the small intestine.
• EPEC is not invasive; does not cause bloody diarrhea
• Toxins are not elaborated by EPEC strains.
• Watery diarrhea results, which, on rare occasions, may become chronic

Question 24

ESCHERICHIA COLI - ENTEROPATHOGENIC E. COLI (EPEC)

 It binds to cells in the large intestine and produces attaching and effacing lesions
 Produces exotoxins (Shiga-like toxins 1 or 2), resulting in a severe copious, bloody diarrhea called as hemorrhagic colitis
 Serotype 0157:H7 is associated w/ outbreaks of hemolytic uremic syndrome (HUS) characterized by fever, acute renal failure, microangiopathic hemolytic anemia, and thrombocytopenia in children ages 5 to 10 years
 Primary reservoir: cattles
 Possibility of infection can be decreased by thoroughly cooking ground beef and pasteurizing milk
 Raw hamburgers are a culprit in the US

Answer 24

Note: HEMOLYTIC-UREMIC SYNDROME, abbreviated HUS, is a disease characterized by hemolytic anemia (anemia caused by destruction of red blood cells), acute kidney failure (uremia), and a low platelet count (thrombocytopenia). It predominantly, but not exclusively, affects children. Most cases are preceded by an episode of infectious, sometimes bloody, diarrhea acquired as a foodborne illness or from a contaminated water supply and caused by E. coli O157:H7. Certain antibiotic may stimulate further verotoxin production and thereby increase the risk of HUS. However there is also tentative evidence that some antibiotics like quinolones may decrease the risk of hemolytic uremic syndrome.

Question 25

ESCHERICHIA COLI -ENTEROINVASIVE E.COLI (EIEC)

Answer 25

 Causes dysentery-like syndrome w/ fever & bloody stool
 Has plasmid-encoded virulence factors identical to those of Shigella spp. that allow the invasion of epithelial cells and intercellular spread by use of actin-based motility
 Produces hemolysin (HlyA)

Question 26

ESCHERICHIA COLI - ENTEROAGGREGATIVE E. COLI (EAEC)

Answer 26

 Adherence to the small intestine is mediated by aggregated adherence fimbriae
 The adherent rods resemble stacked bricks resulting to shortening of microvilli
 EAEC strains produce a heat-stable toxin that is plasmid-encoded
 Causes traveler’s diarrhea and persistent watery diarrhea in children and patients infected with HIV

Question 27

ESCHERICHIA COLI

Answer 27

Summary of E. coli strains, resulting syndrome, and therapy.

Question 28

ESCHERICHIA COLI - Clinical Manifestations

Answer 28

 Urinary Tract Infection
o E. coli is the leading cause of both nosocomial and community-acquired UTI including cystitis and pyelonephritis
o Women are particularly at risk because their urethras are shorter, straighter and wider; also the short proximity to the anus; wiping of tissue paper from south going north 
 Neonatal Meningitis
o E. coli is a major cause of disease occurring within the first month of life
o The K1 capsular antigen is particularly associated with such infections
 Nosocomial Pneumonia
o Most patients are 50y/o with chronic diseases
 Gram-Negative Sepsis and Endotoxic Shock
o E. coli is the most frequent cause
 Peritonitis in Ruptured Viscus - a ruptured viscus is a hole in a hollow organ in the abdomen

Question 29

ESCHERICHIA COLI - Laboratory Diagnosis

Answer 29

 Gram stain: gram (-) short rods
 Culture: MacConkey Agar: pink to brick red colonies

Question 30

ESCHERICHIA COLI - Laboratory Diagnosis

E. coli fermented in MacConkey agar showing pink to brick red colonies

Answer 30

E.coli in EMB showing greenish metallic sheen colonies

Question 31

ESCHERICHIA COLI - Biochemical Tests

Answer 31

Triple Sugar Iron Test (TSI): A/A Gas (+) H2S (-)

**IMViC reaction: + + - -

Question 32

ESCHERICHIA COLI - Treatment

Answer 32

 Urinary tract infection (UTI):
o Ciprofloxacin, Trimethoprim/Sulfamethoxazole
 Local or systemic disease:
o Ampicillin, Cefotaxime, Aminoglycoside, Ciprofloxacin, Trimethoprim/Sulfamethoxazole
 Meningitis in infants:
o Cefotaxime

Question 33

KLEBSIELLA PNEUMONIAE
 Also known as Friedlander’s Bacillus
 Gram (-), lactose fermenting, non-motile encapsulated bacilli
 Part of the normal flora of the GIT

Answer 33

Has O and K antigens
o O antigen
 Most external part of the cell wall lipopolysaccharide
 Are resistant to heat and alcohol
 Detected by bacterial agglutination
 Predominantly detected by IgM antibodie
o K antigen
 External to O antigens, present only in some enterobacteriaceae
 May interfere with O antisera agglutination, hence may mask the presence of O antigens
 Used in serologic typing

Question 34

KLEBSIELLA PNEUMONIAE - Virulent Factors

Determinants of Pathogenicity
o Large Capsule
 Anti-phagocytic
 Encapsulated strains are more virulent
o Endotoxin
o Enterotoxin-producing strain
 Isolated from patients with Tropical Sprue

Answer 34

Note: Tropical Sprue

• Tropical sprue is a malabsorption disease commonly found in the tropical regions
• Characterized by abnormal flattening of the villi and inflammation of the lining of the small intestine, acute or chronic diarrhea, weight loss, and malabsorption of nutrients
• Treatment: Antibiotics (Tetracycline or Co-trimoxazole) and supplementation with B12 and Folic acid

Question 35

KLEBSIELLA PNEUMONIAE - Clinical Manifestations

 Primary Community Acquired Pneumonia in the immunocompromised
 Nosocomial Pneumonia
o found in the elderly with underlying medical problems (DM, alcoholism, bronchopulmonary problems)
o Produces a thick non putrid bloody sputum (Currant Jelly Sputum)
o Necrotizing consolidation and abscess formation (more common with Klebsiella)
 UTI, wound infection, bacteremia and meningitis

Answer 35

Currant jelly sputum in nosocomial pneumonia

Question 36

KLEBSIELLA PNEUMONIAE - Laboratory Diagnosis

 Gram Stain: Gram-negative rod
o Presence of a large capsule is suggestive of Klebsiella
 Specimen: Blood, urine, pus, spinal fluid, and sputum
Culture:
o Blood agar plate
o MacConkey agar: Pink mucoid colonies
 TSI: A/A, Gas (+), H2S (-)

Answer 36

K. pneumoniae on MacConkey Agar

Question 37

KLEBSIELLA PNEUMONIAE - Laboratory Diagnosis

 Urease test: delayed urease positive (days after)
 EMBA: Brown mucoid colonies

Answer 37

LEFT: TSI test, note space at bottom of tube and crack (presence of gas)

RIGHT: EMBA, shows growth of brown, darkcentered, mucoid colonies (smaller than Enterobacter).

Question 38

IMViC results for Klebsiella pneumoniae

Answer 38

Question 39

KLEBSIELLA PNEUMONIAE - Treatment

Answer 39

 3rd Generation Cephalosporins
 Aminoglycosides
 Fluoroquinolones
 May produce Beta Lactamase which inactivates Ampicillin
and Carbenicillin but not Cephalosporins

Question 40

GENUS ENTEROBACTER

 Gram-negative, lactose fermenting, motile bacilli
 Inhabits soil and water; large intestine of man and animals

Answer 40

Species that cause illness in man
o E. cloacae
o E. aerogenes
o E. agglomerans
o E. gergoviae
o E. sakazaki
o E. taylorae

Question 41

GENUS ENTEROBACTER - Virulence Factors

Answer 41

 Antigenic Structures:
o 53 O antigens and 57 H antigens are used for typing E. cloacae
 Determinants of Pathogenicity:
o E. cloacae causes majority of infections followed by E. aerogenes and E. agglomerans
o Expresses Extended Spectrum Beta-Lactamases (ESBL) - Allowing resistance to Penicillin (PCN) including Ceftazidime and Ampicillin
o Produces a potent Cephalosporinase that inactivates 1st Generation Cephalosporins

Question 42

GENUS ENTEROBACTER - Laboratory Diagnosis

Answer 42

 EMBA: Pink colonies with central dark purple dot
 MacConkey Agar: Pink colonies
 TSI: A/A Gas (+) H2S (-)
 IMViC: - - + +

Question 43

GENUS ENTEROBACTER - E. aerogenes results

Answer 43

TOP LEFT: E. aerogenes in EMBA which produces pink colonies with central dark purple dot; TOP RIGHT: TSI which produces acid, acid butt, gas (+), H2S (-); BOTTOM: MacConkey agar which produces pink colonies.

Question 44

GENUS ENTEROBACTER - TREATMENT

Answer 44

 Aminoglycosides or Fluoroquinolone or both
 Piperacillin–Tazobactam
 2nd or 3rd Generation Cephalosporin
 Prophylaxis
o Sterility during procedures
o Handwashing

Question 45

GENUS SHIGELLA

4 Major O antigen serogroups:
o Shigella dysenteriae
o Shigella flexneri
o Shigella boydii
o Shigella sonnei

Answer 45

 Major cause of Bacillary dysentery (Shigellosis)
o Signs and symptoms include severe abdominal cramps, low volume stool with blood and mucus
 Non motile, gram (-) rod, non-lactose fermenter (clear colonies on EMB and MacConkey)
 Ferments glucose, does NOT produce gas or H2S on TSI
 Can survive >6 months in water at room temperature BUT most common disinfectants will kill it

Question 46

GENUS SHIGELLA - Virulence Factors

Determinants of Pathogenicity:
o O antigen with smooth LPS structure
 Ability to survive the passage through host defenses
o Invasiveness:
 Penetrate mucosa and epithelial cells of the colon by endocytosis
 Escape the endocytic vesicle and multiply inside the cell, protected from macrophages
 Shigellae polymerizes actin at one pole, propelling the bacterium through the cytoplasm and into adjacent cells
 NOTE: As the cells die, mucosal abscess forms which causes diarrhea with blood and mucus along with painful abdominal cramps

Answer 46

o Shiga Toxin
 Isolated from S. dysenteriae type 1. (Toxicity results in development of hemorrhagic colitis and HUS)
 Cytotoxic and interferes with protein synthesis
 Shigella multiply in a non-invasive manner in the jejunum and produce toxin resulting in an activated secretory process.
 Toxin blocks absorption of electrolytes, glucose and amino acids from the lumen of the intestine.
 The second phase is tissue invasion of the large intestine producing microvascular damage with bloody diarrhea (Currant Jelly Stool)

Question 47

GENUS SHIGELLA - Clinical Manifestations

 S. sonnei causes 80% of infections followed by S. flexneri which has been associated with outbreaks among sexually active men who have sex with men.
 Shigella dysenteriae causes the most serious infections, including HUS similar to that caused by EHEC.
 S. dysnteriae type 1 produces Shiga toxin which is capable of resulting in HUS in susceptible individuals
 Spread by flies, fingers, food, feces, and water contaminated by infected individuals who shed organisms in the feces.
 Low infective dose: only 10-100 organisms are required to establish an infection
o This makes secondary cases within a household common, especially in poor sanitary conditions. Spread by 4Fs (Flies, Finger, Food, Feces)

Answer 47

 Bacillary Dysentery
o Initially with fever and watery diarrhea
o Changes on second day to frequent small volume stools with blood and mucus
o Severe form: will have fever, chills, convulsions, abdominal cramps, tenesmus, and bloody stools.
o Mortality is due to dehydration and electrolyte imbalance.
o Most disease is seen in the age group of 1-10 years of age.
o On recovery, most people remain chronic carriers and have recurrent bouts of infection

Question 48

GENUS SHIGELLA - Laboratory Diagnosis

 Specimen:
o Rectal swab of ulcer taken by sigmoidoscopy (best
specimen)
o Fecal specimen: Place in Buffered transport medium since Shigella is sensitive to acids in fecal material
 IMViC: ( - + - - )
 Culture:
o MacConkey agar: white colonies
o EMBA: non-colored colonies
o Salmonella-Shigella Agar: transparent colonies
o Hektoen Agar: Blue green colonies
 TSI: K/A Gas (-), H2S (-)

Answer 48

TOP: Shigella on EMB (left) and MacConkey (right)

BOTTOM: Shigella on SSA (left) and Hektoen Agar (right)

Question 49

GENUS SHIGELLA - Treatment

 Consider state of hydration
o IV fluid (NSS) for severe dehydration at 20ml/kg over 1 hour
o ORS for mild to moderate dehydration
 Ampicillin/Amoxicillin: Doxycyclin for sensitive isolates
 TMP-SMX: Doxycyclin when sensitivity is unknown or the px is allergic to Penicillin.
 Azithromycin
 Ofloxacin/Ciprofloxacin

Answer 49

GENUS SHIGELLA - Epidemiology and Control

 Adequate sanitization
 Detection and treatment of carriers
 Carriers are not to participate in food preparation
 Proper sewage disposal and chlorination of water
 Breastfeeding through the first year of life (IgA)
 Candidate vaccine in advanced development stages
o Conjugate vaccine composed of O-antigen polysaccharides from Shigellae
o Live attenuated vaccine

Question 50

GENUS SALMONELLA

 Gram (-) rod with peritrichous flagella, motile
 Non lactose fermenter
 Produces acid during glucose fermentation
 Produces H2S
 Tolerates a large concentration of bile
 O and H antigens are used for serotyping
 The genus Salmonella contains two species, S. bongori and S. enterica, the latter of which is divided into 6 subspecies; enterica, salamae, arizonae, diarizonae, houtenae and indica.

Answer 50

 Salmonella enterica
o All strains affecting humans grouped under this species
o Approximately 2,500 serotypes/serovars
 Has an accepted nomenclature
o Example: S. enterica subspecies enterica serotype Typhimurium => shortened to Salmonella Typhimurium (capitalized because it is serovar

Question 51

GENUS SALMONELLA

Salmonella serotypes of clinical significance

Answer 51

o S. Paratyphi A (Serogroup A)
o S. Paratyphi B (Serogroup B)
o S. Cholerasuis (Serogroup C1)
o S. Typhi (Serogroup D)
o Salmonella serotypes Entritidis and Typhimurium

Question 52

GENUS SALMONELLA - Virulent Factors

Determinants of Pathogenicity:
o Facultative Intracellular Parasite
 Survives intracellularly in phagocytes but also has the ability to grow in extracellular environment
 In macrophages, survival is due to production of protein that protects against defensins (antibacterial substance in macrophages)
o O antigenic side chain
 More resistant to complement mediated killing of normal serum
o Vi antigen in S. Typhi
 Prevents phagocytosis by PMNs
o Type 1 or Mannose Binding Fimbriae
 For attachment to host cells

Answer 52

o Invasiveness
 Virulent Salmonella penetrate cells of epithelial lining of small bowel into the submucosa where they are taken up by macrophages
 The organisms are carried to the Reticuloendothelial system (RES) where they multiply and spread to other body sites
 Bowel is reinfected through the liver and gall bladder
o Endotoxin
 Responsible for fever
o Cytotoxin
 Associated with the outer bacterial membrane
 Important in cellular invasion and destruction

Question 53

GENUS SALMONELLA - Sequence of events in the pathogenicity of Salmonella

Answer 53

Question 54

GENUS SALMONELLA - Clinical Manifestations

 Organisms enter via oral route through contaminated food or drink (Fecal-Oral)
 Salmonella serovar Typhi may involve chronic carriers
 Pet turtles have been implicated as sources of infection
 Individuals in crowded institutions may also be involved in Salmonella epidemics

Answer 54

1. Enteric Fever (Typhoid Fever)
2. Gastroenteritis/Enterocolitis/Salmonellosis
3. Septicemia

Question 55

GENUS SALMONELLA - Clinical Manifestations

Enteric Fever (Typhoid Fever)
 Produced by S. Typhi
 Paratyphi A and B can cause it but symptoms are milder and mortality is lower
 Ingested from contaminated food and water

Answer 55

 The ingested organisms reach the small intestine from which they enter the lymphatics and the bloodstream. After an incubation period of 10-14 days

Question 56

GENUS SALMONELLA - Clinical Manifestations

Enteric Fever (Typhoid Fever): First week - Second week

First week
o Lethargy, remittent fever, malaise, body pains
o Patient gets constipated instead of diarrhea
o Organisms penetrate intestinal wall and infect regional lymph system, invade the blood stream, other parts of RES
o Ingested by monocytes, not killed and undergo multiplication

Second week
o Reenters the bloodstream causing prolonged bacteremia
o Infection of the biliary tree and other organs
 Hepatitis
 Focal necrosis of liver
 Inflammation of gall bladder, periosteum, and the lungs may occur
o Patient is severely ill
 With sustained fever
 Tender abdomen with Rose Spots
 Diarrhea begins during 2nd to 3rd week of illness
o Reinfection of intestinal tract from the gall bladder, may cause necrosis of Peyer’s patches

Answer 56

Enteric Fever (Typhoid Fever): Third week

o Patient is exhausted and febrile but continues to show improvement if no complications occur
o Complications:
 Intestinal perforation
 Severe bleeding
 Cholecystitis
 Pneumonia
 Abscess
o Convalescent or chronic carriers are the sole source of organisms
o Majority of chronic carriers are older women with gall bladder disease

Question 57

GENUS SALMONELLA - Clinical Manifestations

Gastroenteritis/Enterocolitis/Salmonellosis

 Caused primarily by serovars Enteritidis and Typhimurium
 Characterized by
o Nausea
o Vomiting
o Diarrhea (usually nonbloody)
o These develop within 48 hours of ingesting contaminated water or food (poultry, eggs)

Answer 57

 Fever and abdominal cramping are common
 Generally self-limiting (48 to 72 hours)
 Convalescent carriage of organisms may persist for a month or more

Question 58

GENUS SALMONELLA - Clinical Manifestations

Answer 58

Septicemia
 Caused by Salmonella Cholerasuis
 Fever, chills, anorexia, anemia occur. Gastroenteritis is minor.
 There is early invasion of the bloodstream following oral infection
 There is little to no intestinal manifestations
 Focal lesions may develop in any tissue producing osteomyelitis, pneumonia, pulmonary abscess, meningitis or endocarditis

Question 59

GENUS SALMONELLA

Answer 59

Clinical diseases caused by Salmonellae

Question 60

GENUS SALMONELLA - Laboratory Diagnosis

 Specimen:
o Enteric Fever (B U S)
 Blood/bone marrow culture: 1st week
 Urine culture: 2nd week
 Stool culture: 3rd week
o Enterocolitis
 Stool culture: 1st week
o Septicemia
 Blood culture: 1st week

Answer 60

 Biochemical Tests
o IMViC : ( - + - + )
o EMB Agar: Grey mucoid colonies
o McConkey Agar: colorless colonies (non-lactose fermenter)
o Salmonella-Shigella agar: colorless colonies with black centers (Fisheye colonies)
o Hektoen agar: transparent colonies with black centers
o TSI: red slant, yellow butt, Gas (-), H2S (+)

Question 61

GENUS SALMONELLA - Laboratory Diagnosis

LEFT: Gray mucoid colonies on EMBA; RIGHT: Colorless colonies on MacConkey agar.

Answer 61

LEFT: Colorless colonies with black centers on SSA; RIGHT: Transparent colonies with black centers on Hektoen Agar

Question 62

GENUS SALMONELLA - Laboratory Diagnosis

Agglutination Test: known sera + unknown culture. Clumping is a positive test

• Typhi Dot Test
• Widal Test : Detects the rise of antibodies against Typhoid during the 2nd or 3rd week of Typhoid infection; not being used anymore

Answer 62

Indicative results of Typhi dot test

Question 63

GENUS SALMONELLA - Treatment

Answer 63

• Uncomplicated
– Chloramphenicol : 3-4 grams/day PO x 14 days
– Cotrimoxazole : 160/800mg : BID x 14 days
– Amoxicillin : 4-6 grams /day x 14 days
• Severe S/S
– Ciprofloxacin : Tab 500mg BID x 10 days
– Ofloxacin : Tab 400mg BID x 10 days
– Ceftriaxone : 2 g IV infusion OD x 7 days - Safe for the pregnant and children

Question 64

GENUS PROTEUS

• The 2 frequently isolated pathogens are Proteus mirabilis and Proteus vulgaris

Antigenic Structure and Other Characteristics
• Possesses O, H, and K antigens
• Certain P vulgaris strains (Ox-19, Ox-2, Ox K) share antigens w/ Rickettsia and these are utilized in the Weil- Felix Test for dx of Rickettsial infections.
• H2S (+) with Gas in TSI, Urease (+)
• Non lactose fermenting
• Actively motile at 37°C producing a translucent sheet of growth on non selective media like BAP (Swarming)
• Proteus can be distinguished from other enterics by their ability to produce Phenylalanine Deaminase.

Answer 64

P. vulgaris
• Ferments glucose and sucrose resulting in an A/A Gas (+),
H2S (+) in TSI
• Since it ferments both glucose and sucrose, in TSI test a lot of acid is produced so butt and slant are yellow plus black butt due to H2S
P. mirabilis
• Ferments glucose only resulting in a K/A Gas (+), H2S (+) in TSI
• In TSI test, not enough acid is produced to turn the slant yellow because of the very small amount of glucose fermented

Question 65

GENUS PROTEUS - Clinical Manifestations

Answer 65

• Proteus mirabilis accounts for the majority of human infections seen in this group.
• 2nd leading cause of Community Acquired UTI and a major cause of nosocomial infections.
• UTI is characterized by an alkaline pH due to the production of urease (which splits urea into CO2 and ammonia which is alkaline)
• The increase in pH causes precipitation of Ca and Mg salts resulting in the formation of infected struvite stones
• IV line infection leading to bacteremia / sepsis

Question 66

GENUS PROTEUS - Laboratory Diagnosis

Urease test:
o identifies those organisms that are capable of hydrolyzing urea to produce ammonia and carbon dioxide (rapidly urease positive)
o It is primarily used to distinguish ureasepositive Proteus from other Enterobacteriaceae

Answer 66

Urea agar slants were inoculated as follows: (a) uninoculated, (b) Proteus mirabilis (rapidly urease positive), (c) Klebsiella pneumoniae (delayed urease positive), (d) Escherichia coli (urease negative)

Question 67

GENUS PROTEUS - TREATMENT

• P. mirabilis is sensitive to Ampicillin & Cephalosporin
• Aminoglycosides

Answer 67

• Cotrimoxazole
• Resistant to Tetracycline

Question 68

GENUS CITROBACTER

• Isolated from the environment, feces of man and animals.
• Implicated in a wide variety of human infections (UTI to Neonatal Meningitis)

Answer 68

Taxonomy and Other Characteristics
• C. freundii, C. diversus, C. amalonaticus
• Possess O, H, and K Antigens. Some possess the Vi Antigen of Salmonella ( Antiphagocytic )
• H2S (+)

Question 69

GENUS CITROBACTER

Clinical Manifestations
• Majority of infections are caused by C. diversus
• Opportunistic and can infect any body site. Majority are in the urinary tract.
• C. diversus has been identified as a cause of neonatal meningitis, brain abscess and neonatal septicemia.
• Enterotoxigenic C. freundii - diarrhea

Answer 69

Treatment
• Most isolates are sensitive to Aminoglycosides, Tetracycline and Chloramphenicol

Question 70

GENUS SERRATIA

• Widely distributed in soil and water. Associated w/ a large number of plants and animals.
• Majority of human disease is caused by one species – Serratia marcescens
• Produces Extracellular Deoxyribonuclease (DNAse), lipase and gelatinase
• O and H antigens are important epidemiologic markers

Answer 70

Clinical Manifestations
• Almost ALL Serratia infections are associated with underlying disease, immunosuppressive treatment, or mechanical manipulation of the patient.
• 90% are Hospital acquired
• Serratia marcescens is the most frequently isolated organism
• Associated with nosocomial outbreaks of UTI, wound infections, pneumonia and septicemia.
• Contaminated respiratory equipment and lapses in catheterization technique are the major factors in the dev’t of Serratia infection. Treatment
• Amikacin, Gentamycin, Chloramphenicol, Ciprofloxacin, Cotrimoxazole
• Resistant to Colistin and Cephalotin