MT 111 LECTURE 12: GRAM-NEGATIVE ENTERIC BACILLI

SALMONELLA

 Gram-negative bacilli, facultative anaerobe, non-lactose fermenter, oxidase negative.

 Outer membrane makes the organism susceptible to drying.
 More than 2500 O serotypes
 Salmonella enterica
o Salmonella enterica enterica

 Salmonella choleraesuis  Salmonella enteritidis

 Salmonella paratyphi  Salmonella typhimurium
 Salmonella typhi

 Salmonella bongori
o Rarely isolated species
o It caused human enteritis in Sicily in 1982-1997

SALMONELLA TYPHI

 Facultative intracellular parasite. Gram-negative motile bacilli that do not ferment lactose.
 They have peritrichous flagella
 Produce only a small amount of hydrogen sulfide (H 2S)
 Capable of tolerating relatively large concentration of bile
 Antigenic structure:
o O or somatic antigen- outer polysaccharide of the cell wall
o H or flagellar antigen- for species identification; change periodically, protecting it from own antibodies
o Vi antigen- polysaccharide capsule that surrounds the O antigen thus protecting the bacteria from antibody attack on the O
antigen; for detection of carriers
 Virulence factors:
o Surface antigen
 Makes possible the attachment of organism to host receptor cells & to survive intracellularly
 Tolerant to acids in phagocytic vesicles
 Can survive in macrophages and spread from the intestine to other body sites
o Endotoxin
 May stimulate endogenous pyrogen release from macrophage & PMNs to produce fever
 Activates the chemotactic properties of the complement system that may cause localization of leukocytes
o Invasiveness
 Capacity of the bacteria to penetrate the epithelial cell into the subepithelial tissues
 Clinical Infection: Typhoid fever
o Incubation period: 7-14 days
o Portal of entry: mouth
o Transmission: contaminated food & water; transmitted only by humans, no known animal reservoir host
 Carriers are often the source of infection.
o Infecting dose: 100,000 bacilli: gastric acid = important host defense; ↓ in acidity→ low infecting dose
 Pathogenesis:
o Organism is ingested→ intestinal tract→ passes through in and between epithelial cells lining the ileocecal area (multiplies and
remains viable after engulfment by macrophages)→ as a response to this engulfment, mononuclear cell will go to the area where
the organism is→ reticuloendothelial system (multiplies intracellularly)- clinical signs of sepsis, causes hyperplastic changes in
mesenteric lymphoid tissue like necrosis, hemorrhage and perforation of intestinal wall→ re-enters the bowel, passing through the
liver→ gallbladder→ intestine
o Found in stool spx once excreted out from the body.
o Takes 2-3 weeks or longer if without treatment; shorter if with treatment
 o Gradual increase of the fever at the same time pulse rate will also increase→ after the second week when patient experiences
melena, temperature suddenly drops and pulse rate also rapidly increases but it also goes down→ fever will again rise after it goes
down and finally pulse rate goes down
o Common symptoms:
 Spiking fever, enlarged spleen, leukopenia, and hemorrhages of the skin→ rose spots, ulcerated Payer’s patches
o Convalescent patients continue to secrete bacilli 3 weeks to 3 months after apparent recovery:
 3-12 months = temporary carriers; More than 12 months= chronic carriers (typhoid Mary)
 3% of typhoid fever patients become chronic carriers
o Carrier rate- higher among women, esp. those with previous gallbladder disease
o Typhoid Mary- one who has infected more than 1,000 persons
 Diagnosis:
o Widal test
 Detects agglutinating antibodies against the O and H antigens of Salmonella typhi.
 If homologous antibody is present in patient's serum, it will react
with respective antigens in the tube 1st Week 2nd Week 3rd Week
 10 test tubes (can contain H or O antigens) with 0.5 mL saline per Widal’s Test - + +
tube→ 1mL of patient’s serum is added (1:10 dilution)→ 0.5 mL
Blood Culture + + -
transferred from tube to tube until 9th tube (1:2560 dilution)→
0.5mL is discarded and 10th tube is for control Stool Culture - - +
 Titer of the patient serum is the highest dilution of the serum Urine +
sample that gives a visible agglutination.
 Consider as clinically significant if: O titer- agglutination in 1:100; H titer- 1:200
 1:160= current infection; the reciprocal of the dilution is the antibody titer; in 1:160, 160 antibody units/mL of serum
 Positive during the second week presuming that patient already produces antibodies
 Limitations of Widal:
 Time consuming to find antibody titer and often times when diagnosis is reached, it is too late to start an antibiotic regimen.
 Should be interpreted in the light of baseline titers in a healthy local population
 May be falsely positive in patients who have had previous vaccination or infection with Salmonella typhi.
 Besides cross-reactivity with other Salmonella species, the test cannot distinguish between a current infection and a previous
infection or vaccination against typhoid.
 False negative results may be associated with early treatment, with hidden organisms in bone and joints, and with relapses of
typhoid fever. Occasionally the infecting strains are poorly immunogenic.
 False negative Widal tests may be due to antibody responses being blocked by early antimicrobial treatment or following a
typhoid relapse
 Severe hypoproteinemia may also prevent a rise in O and H antibody titers
 The antibody levels found in a healthy population however, may vary from time to time and in different areas, making it
difficult to establish a cut-off level of baseline antibody in a defined area and community.
 In low typhoid endemic areas, weak and delayed O and H antibody responses limit the usefulness of the Widal test. Variations
also exist between laboratories in the performance and the reading of Widal tests which compromise further the reliability of
the test (seldom used).
o Blood culture- 80% of patients show (+) blood culture during the first and second weeks of illness
o Stool culture- Positive on the first day and negative on the next two weeks; positive during the third week
o Urinary co-agglutination test- Positive during the third week
o Stool (using Tetrathionate broth as enrichment medium) and Blood (using Thioglycollate broth/Trypticase soy broth). Both are
inoculated into Bismuth sulfite agar (jet-black colonies with metallic sheen) and biochemical tests performed.
 Triple Sugar Iron- alkaline slant and acid butt without gas and with a small amount of H 2S
 Indole (-), Methyl red (+), Voges-Proskauer (-), Citrate (-), Urease (-), Sulfite Indole Motility (+)
 Prevention:
o Isolation of carriers
o Active immunization (confer only 50-80% protection)
 Acetone killed typhoid vaccine- combined with Cholera (PHIL)
 Primary vaccination: adults= 1 cc subcutaneously; 6 mos.-10 yrs. = 0.5 cc. subcutaneously
  Live attenuated oral Typhoid Ty 21a
 3 oral doses given every other day; gives 1 yr. protection
o Proper sewage treatment
o Chlorination of water
o Routine stool cultures of food handlers
o Handwashing

OTHER SALMONELLA

 Similar in morphology and antigenic structure to Salmonella typhi

 Ferments glucose and other sugars with gas production
 All produce large amounts of hydrogen sulfide (H 2S) except for Salmonella para A
 Determinants of pathogenecity: endotoxin & enterotoxin
 Clinical Infections:

SALMONELLA GASTROENTERITIS


Most common type of Salmonella infection

Source of has been attributed primarily to poultry, milk, eggs and egg products

Insufficiently cooked eggs and domestic fowl such as a chicken, turkey and duck are common sources of infections

Transmission: Cooking utensils such as knives, pans, and cutting boards used in preparing the contaminated meat can spread the
bacteria to other food
 Occurs when a sufficient number of organisms contaminate food that is maintained under inadequate refrigeration
 Infecting dose: 106 or 1,000,000 bacteria
 Occurs in multiple sources of contamination, including foods (most commonly poultry & poultry products) (organism can enter
the egg while the egg shell is formed in the chicken); food handlers; exotic pets (turtles & snakes)
 Symptoms begins 18-24 hrs. after ingestion
 Pathogenesis:
 Commonly, Salmonella enteritidis causes Salmonella gastroenteritis
 Absorbed to epithelial cells in terminal portion of the small intestine→ bacteria penetrate cells and migrate to lamina propria
layer of the ileocecal region→ multiply in the lymphoid follicles→ to reticulum endothelial hyperplasia and hypertrophy
(PMN will go to the area and confine infection to gastrointestinal drug) → inflammatory response which can also mediate
release of prostaglandins→ stimulates cyclic AMP so that there will be active fluid secretion to lose diarrheal stools
 Symptoms that appear within 8-36 hours after ingestion- nausea, vomiting, chills & fever, water diarrhea, abdominal pain &
leukocytosis; most are self-limiting
 Patients with sickle cell disease and other hemolytic disorders, ulcerative colitis and malignancy are more susceptible
o Enteric fever
 Similar to typhoid fever but milder & mortality rate lower; caused by Salmonella paratyphi A, B & C
o Bacteremia
 Salmonella bacteremia with and without extraintestinal foci of infection caused by non-typhoidal
Salmonella
 Characterized by prolonged fever and intermittent bacteremia
o Septicemia
 Salmonella cruising the bloodstream→ infect the lungs, brain or bone
 Most often of nosocomial origin; does not involve the GIT
 Triad of high remittent fever, chills & hypotension
 Due to Salmonella choleraesuis
o Diagnosis:
 Specimen: Gastroenteritis→ Stool; Septicemia and Enteric fever→ Blood
 Culture medium of choice: Brilliant Green Agar (pink-colored colonies with an intense red background)
 Biochemical Tests:
 Triple Sugar Iron: alkaline slant/acid butt with gas with H2S except for Salmonella para A (no H2S)
 Indole (-), Methyl red (+), Voges-Proskauer (-), Citrate (+), Urease (-), Sulfite Indole Motility (+)
SHIGELLA
Shigella Shigella Shigell Shigell
 Species designation determined by O antigen dysenteria flexneri a boydii a
o Group A (Shigella dysenteriae)- most pathogenic e sonnei
o Group B (Shigella flexneri)- recently associated with Fermentation
- + + +
outbreaks among homosexual males; are now seen in young Mannitol
adult males Ornithine
o Group C (Shigella boydii)- endemic in India decarboxylas - - - +
o Group D (Shigella sonnei)- most common in the United e
States ONPG - - - +
 Gram negative, non-motile bacilli
 Non-lactose fermenter except for some serotypes of Shigella sonnei
 All ferment glucose with acid production only except for certain types of Shigella flexneri that produces gas
 Virulence Factors:
o Endotoxin and genes for adherence, invasion, and intracellular replication
o Permeability barrier of outer membrane (smooth lipopolysaccharide)
o Exotoxin (Shiga toxin) that disrupts protein synthesis and produces endothelial damage
o Enterotoxin- can cause diarrhea
 Resistance:
o Fragile organisms; most common disinfectant is lethal for Shigella
o Can survive in water at room temperature for over 6 months
o Susceptible to the various effects of physical and chemical agents such as disinfectants and high concentration of bile
o Because they are susceptible to the acid pH in stools, they should be plated immediately.
 Antigenic structure: O Ag (divides them into 4 groups) and K antigen
 Clinical Infection: Bacillary dysentery (Shigellosis/Shigella enterocolitis)
o Primarily a pediatric disease with most infections in children 6 months to 10 years of age
o Infecting dose: 100-200 bacilli (smaller than other Salmonella)
o Incubation period: 1-4 days
o Transmission: through the 4 F’s: Fingers, Food, Feces, and Flies
 Pathogenesis:
o Characterized by acute inflammation of the wall of the large intestine and ileum
o Sudden onset of lower abdominal pain, tenesmus, diarrhea & fever; stools are liquid
& scant; after the first few bowel movement; they contain mucus, pus &
occasionally blood
o Carriers: 1-4 weeks
 Diagnosis:
o Best specimen: Rectal swab of an ulcer taken by sigmoidoscopy
o Stool- use bits of pus or blood tinged mucus from freshly passed stools; needs fresh
specimen because they are sensitive to the acid in stools, needs transport medium
o Culture medium of choice: Glycerol M Phosphate (Stool) and Modified Stuart’s (Rectal Swab)→ Enrichment medium, once
growth→ inoculate in Salmonella Shigella Agar (colorless)/Hektoen Enteric agar (green)/Xylose Lysine Desoxycholate (red)→
Biochemical Tests (TSI, IMViC, Urease, SIM)
o Triple Sugar Iron: alkaline slant/acid butt without gas and without H 2S
o Indole (-), Methyl red (+), Voges-Proskauer (-), Citrate (-), Urease (-), Sulfite Indole Motility (-)
o Only the 4 groups can be identified but not the specific species, Carbohydrate fermentation test is used
 Prevention:

o Hand washing with soap o Breastfeeding of young infants and children

o Ensuring the availability of safe drinking water o Safe handling and processing of food
o Safely disposing of human waste o Control of flies
ESCHERICHIA COLI

 Gram-negative coccobacilli with variable motility

 Ferments glucose, lactose & other sugars with the production of acid & gas (Salmonella and Shigella are non-
lactose fermenters)
 Produce indole from tryptophan and cannot use citrate as a sole carbon source
 Virulence factors:

o Endotoxin o Exotoxins (Shiga-like toxins)

o Permeability barrier of outer membrane o Invasive capacity
o Adhesins (colonization factor antigen)

o
Enterotoxin (can produce diarrhea)
 Common gram-negative organism found in the gastrointestinal tract of humans; normal habitat of GI tract
 Fecal contamination is usually detected when Escherichia coli is seen in water
 Uropathogenic Escherichia coli
o Most common cause of urinary tract infection in humans
 Both a true pathogen and an opportunist. Some cases when found in the gastrointestinal tract, it can cause infection while
when found also in other sites of the body like urinary tract, it can also cause UTI
o Primary virulence factor associated with the ability of Escherichia coli to cause UTI is the production of pili, cytolysins and
aerobactins.
o Gastrointestinal Pathogens

Enterotoxigenic Escherichia coli (ETEC) Traveler’s diarrhea (watery diarrhea)

Enteroinvasive Escherichia coli (EIEC) Bloody diarrhea in areas of poor hygiene (Dysentery)
Enterohemorrhagic Escherichia coli/Verotoxin- O157:H7 (bloddy diarrhea), Hemorrhagic colitis,
producing Escherichia coli (EHEC/VTEC) Hemolytic uremic syndrome
Enteropathogenic Escherichia coli (EPEC) Infantile diarrhea
Enteroadherent Escherichia coli (EAEC) Persistent pediatric diarrhea and patients infected with
HIV
o
Other diseases
 Septicemia
 Pneumonia
 Neonatal meningitis
 Diagnosis:
o Specimens include urine, blood, pus, spinal fluid, sputum, or other material as indicated by the infection.
o Culture medium of choice: Eosin Methylene Blue or MacConkey agar
o Specific analysis for a specifics strain can be used especially for the specific serotype of gastrointestinal infection
o Stool→ Enrichment medium→ MacConkey, Eosin Methylene Blue
o Urine, Blood, Sputum, CSF→ MacConkey, Eosin Methylene Blue→ TSI, IMViC, Urease, SIM
o Eosin Methylene Blue- Greenish-metallic sheen; MacConkey- pink-colored colonies
o Triple Sugar Iron: acid slant/acid butt with gas without H2S
o Indole (+), Methyl red (+), Voges-Proskauer (-), Citrate (-), Urease (-), Sulfite Indole Motility (+ or -, depending on serotype)
Summary:
Salmonella typhi Other Salmonella Shigella Escherichia coli
Triple Sugar Iron alkaline slant-acid butt alkaline slant-acid butt alkaline slant-acid butt acid slant-acid butt
without gas and with a with gas with H2S except for without gas and without with gas without H2S
small amount of H2S Salmonella para A (no H2S) H 2S
Indole - - - +
Methyl red + + + +
Voges-Proskauer - - - -
Citrate - + - -
Urease - - - -
Sulfur Indole + + - +/- (depends)
Motility