Enterobacteriaceae: Salmonella

Ni Nyoman Nami Arthisari

PPDS-1 Mikrobiologi Klinik
Pembimbing: Dr. dr. Ida Sri Iswari, Sp.MK, M.Kes
 Overview of
Enterobacteriaceae
Talaro K.P., Chess B. 2012. Foundation in Microbiology. Ed 8. Pasadena. Mc Graw Hill.
Gram Negative Bacteria (bacilli)

Enterobacteriaceae
 Properties of the family
Enterobacteriaceae
• Members of the Enterobacteriaceae family are the most common gram-negative
bacteria responsible for human disease.
• They are aerobes and facultative anaerobic bacilli
• These two families (with Vibrionaceae) share the common property of fermenting
glucose, as well as having several diseases in common: gastroenteritis and wound
infections.
• Members of this family are ubiquitous, found worldwide in soil, water, and vegetation,
and are part of the normal intestinal flora of most animals, including humans (coliform
bacilli) .
• Tolerate a wide range of temperatures and pH, but are susceptible to gastric acid
exposure to a large inoculum is required for disease.
• They are generally motile, except Shigella and Klebsiella.

Simple nutritional requirements but require salt for growth (halophilic or “salt loving”),
they ferment glucose, and are oxidase negative, catalase + (except Shigella
dysenteriae type-1)
Murray PR, Baron EJ, Jorgensen JH, et al. 2007. Manual of Clinical Microbiology. 9th Edition. Washington DC : ASM Press.
Classification of Selected Species Within the Family Enterobacteriaceae
Culture Growth

 MacConkey Agar (MAC) is a

selective and differential
medium designed to isolate and
differentiate enterics based on
their ability to ferment lactose.
 Bile salts and crystal violet
inhibit the growth of Gram
positive organisms or other
commensal bacteria.
 Lactose provides a source of
fermentable carbohydrate
allowing for differentiation.
 Appearance on Media

Tille P.M. 2017. Bailey & Scott’s Diagnostic Microbiology: Bacterial Genetics, Metabolism, and Structure. Ed 14. Missouri. Elsevier.
 Virulence
● The epidemiologic (serologic) classification of the Enterobacteriaceae is based on three major
groups of antigens:

○ Somatic O polysaccharides (cell wall antigen)

○ K antigens in the capsule (type-specific polysaccharides) or fimbrial antigen

○ H proteins in the bacterial flagella.

● The heat-stable lipopolysaccharide (LPS) is a major cell wall antigen in the Enterobacteriaceae
consists of three components:

○ Outermost somatic O polysaccharide

○ A core polysaccharide (enterobacterial common antigen)

○ Lipid A

The lipid A component of LPS is responsible

for endotoxin activity
Murray PR, Baron EJ, Jorgensen JH, et al. 2007. Manual of Clinical Microbiology. 9th Edition. Washington DC : ASM Press.
Salmonella
 SALMONELLA

• Family : Enterobacteriaceae
• Genus : Salmonella
• Spesies : Salmonella spp
• Ubiquitous human and animal pathogens
• They colonize virtually all animals including poultry, birds,
livestock, reptiles, rodents, domesticated animals, and humans.
• Characteristics: Short, flagellated rods ; facultative anaerobes ;
ferment glucose; do not ferment lactose; catalase positive; oxidase
negative, culture on MacConkey Agar

Cornelissen C.N., Fisher B.D., Harvey R.A. 2001. Lippincott’s Illustrated Reviews: Microbiology. Ed 3. Philadelphia. Lippincott.
Mahon CR, Lehman DC, Manuselis G. 2019. Textbook of Diagnostic Microbiology. 6th Edition. China : Elsevier, Saunders
 S. enterica subsp. enterica
Classification of Salmonella serotype Paratyphi-
Salmonella Paratyphi A
(serogroup A)

Salmonella Paratyphi B
Previously, the genus Salmonella comprised three (serogroup B)
S. enterica subsp. enterica
biochemically discrete species:
(subspecies I)
○ S. enteritidis, S. choleraesuis, and S. typhi. Salmonella Choleraesuis
(serogroup C1)
S. enterica subsp. Salamae
(subspecies II)
Salmonella Typhi
S. enterica subsp. Arizonae (serogroup D).
(subspecies IIIa)
S. enterica
S. enterica subsp. diarizonae
Salmonella
(subspecies IIIb)
S. bongori
S. enterica subsp. houtenae
(subspecies IV)

S. enterica subsp. Indica

(subspecies VI).

Mahon CR, Lehman DC, Manuselis G. 2019. Textbook of Diagnostic Microbiology. 6th Edition. China : Elsevier, Saunders
 Salmonella -
Morphology
• Gram-negative bacilli (1–3 µm in size)
• Motile  peritrichous flagella (Salmonella Gallinarum and
Salmonella Pullorum are exceptions)
• They do not form spores and capsules.
• Some strains of salmonellae may produce fimbriae
• Most strains of Salmonella Paratyphi A and few strains of
Salmonella Paratyphi B, Salmonella Typhi, and Salmonella
Typhimurium are non fimbriated.

Mahon CR, Lehman DC, Manuselis G. 2019. Textbook of Diagnostic Microbiology. 6th Edition. China : Elsevier, Saunders
 Virulence factors
1. Plasmid Virulen
Spv locus
pef operon
Gen tlpA
Gen traT, rck
rsk
2. Toksin
Endotoxin  lipid A from LPS
Eksotoxin  Cytotoxin, Enterotoxin, Salmolysin
3. Fimbriae
4. Flagella
Phase 1 flagellin (code by gene fliC)
Phase 2 flagellin (code by gene fliB)

 Role of fimbriae in adherence in initiating intestinal infection and colonisation

 Ability to traverse intestinal mucosa.
 Enterotoxin produced by certain Salmonella strains that cause gastroenteritis.

Mahon CR, Lehman DC, Manuselis G. 2019. Textbook of Diagnostic Microbiology. 6th Edition. China : Elsevier, Saunders
 Antigenic structures

● Salmonellae possess antigens similar to antigens of other enterobacteria.

● The somatic O antigens and flagellar H antigens are the primary antigenic structures used in
serologic grouping of salmonellae.
● A few strains may possess capsular (K) antigens, designated Vi antigen.

Mahon CR, Lehman DC, Manuselis G. 2019. Textbook of Diagnostic Microbiology. 6th Edition. China : Elsevier, Saunders
 • Heat-stable O • Heat- labile Flagellar antigen • Heat-labile Vi antigen
O Ag

Vi Ag
H Ag
antigen proteins. is a surface
• The H antigens of salmonellae polysaccharide
Lipopolysaccharide
occur in one of two phases capsular antigen
located in the outer • Phase 1 (Specific phase) • Found in Salmonella
membrane of the cell
• Occur only in a few Typhi and a few strains
wall. serotypes of Salmonella
• Determine the immunologic Choleraesuis.
identity of the particular • Preventing
serotype. phagocytosis of the
• Agglutinate only with organism.
homologous antisera. • The Vi antigen often
• Phase 2 (Non specific phase) blocks the O antigen
• Occur among several during serologic typing
strains. but may be removed by
• Shared by numerous heating.
serotypes
• React with heterologous
antisera.

Mahon CR, Lehman DC, Manuselis G. 2019. Textbook of Diagnostic Microbiology. 6th Edition. China : Elsevier, Saunders
 Clinical Infection
Gastroenteritis
Etiology and Pathogenesis Clinical manifestation
• Contaminated food. • The symptoms appear in 8 to 36 hours
• The Salmonella gastroenteritis are usually • Nausea, vomiting, fever, and chills,
strains found in animals (S. enterica accompanied by watery diarrhea and
subsp. Enterica). abdominal pain.
• Source: • Self-limiting. Symptoms usually
• Insufficiently cooked poultry, milk, eggs, disappear within a few days, with few or
and egg products as well as to handling no complications except:
pets (calves and poultry at petting zoos). • Patients with sickle cell disease and
• Contaminated Cooking utensils. other hemolytic disorders, ulcerative
• Direct transmission from person to colitis, and malignancy
person. • Very young children, elderly adults, and
• The infective dose necessary to initiate patients with other underlying disease.
the disease103–106 bacteria.

Mahon CR, Lehman DC, Manuselis G. 2019. Textbook of Diagnostic Microbiology. 6th Edition. China : Elsevier, Saunders
 Enteric fever

Etiology Clinical manifestation

• Contaminated food • Prolonged fever
• Infective dose: ingestion of food or water • Bacteremia
contaminated with salmonellae 103–106 • Involvement of the reticuloendothelial
bacilli system, (liver, spleen, intestines, and
• Enteric fever caused by Salmonella Typhi mesentery)
is known as typhoid fever • Dissemination to multiple organs
• Typhoid fever develops in approximately 9 • The clinical manifestations of
to 14 days paratyphoid fevers are similar to typhoid
• Other enteric fevers include paratyphoid fever but are less severe, and the
fevers, which may be due to Salmonella fatality rate is lower.
serotypes Paratyphi A, B, and C and
Salmonella serotype Choleraesuis.

Mahon CR, Lehman DC, Manuselis G. 2019. Textbook of Diagnostic Microbiology. 6th Edition. China : Elsevier, Saunders
 Pathogenesis

Ingested The febrile

organism Bloodstream to The organisms episode
resistant to gastric liver, spleen, multiply becomes more
acids  reaching Lymphatic during this
and bone intracellularly
the proximal end of system and release.
marrow, then then released
the small mesenteric
engulfed by into the The organisms
intestine invade lymph nodes.
mononuclear bloodstream for may be isolated
and penetrate the phagocytes. the second time. easily from the
intestinal mucosa. blood.
• constipation >>
diarrhea

The ability of Salmonella to survive in

macrophages is very important for the
occurrence of systemic dissemination. Other
source: brought by macrophage to RES.

Mahon CR, Lehman DC, Manuselis G. 2019. Textbook of Diagnostic Microbiology. 6th Edition. China : Elsevier, Saunders
 Clinical Significance

Typhoidal Typhoid fever

Salmonella Intestinal
infection
Nontyphoidal
Extraintestinal
infection

Mahon CR, Lehman DC, Manuselis G. 2019. Textbook of Diagnostic Microbiology. 6th Edition. China : Elsevier, Saunders
 ● Strains of nontyphoidal Salmonella usually cause :

○ Intestinal Infection: accompanied by diarrhea, fever, abdominal cramps.

(Primary Infections)
○ Extraintestinal infection: bactermia, urinary tract infection, endocarditis,
meningitis, osteomyelitis, especially in immunocompromised persons.
(Secondary infections)
● The serotypes most commonly associated: Typhimurium, Paratyphi,
Choleraesuis

Mahon CR, Lehman DC, Manuselis G. 2019. Textbook of Diagnostic Microbiology. 6th Edition. China : Elsevier, Saunders
Cornelissen C.N., Fisher B.D., Harvey R.A. 2001. Lippincott’s Illustrated Reviews: Microbiology. Ed 3. Philadelphia. Lippincott.
 The organisms invade the gallbladder
and Peyer’s patches of the bowel.
The gallbladder becomes the
During the second and third • “Rose spots” appear during the foci of long-term carriage of the
weeks of the disease, the second week of fever. organism
patient generally • Involvement of biliary system sites
experiences sustained fever Occasionally reseeding the
initiates GI symptoms (reinfection). intestinal tract and shedding
with prolonged bacteremia.
the organisms in the feces.
• The organism now exists in large
numbers in the bowel - isolated from
the stool.

Possible complication:
• Necrotizing cholecystitis
• Necrosis of the Peyer’s patches leading to hemorrhage and
perforation of the bowel.
• Pneumonia, thrombophlebitis, meningitis, osteomyelitis,
endocarditis, and abscesses.

Mahon CR, Lehman DC, Manuselis G. 2019. Textbook of Diagnostic Microbiology. 6th Edition. China : Elsevier, Saunders
 Timeline for detection

Talaro K.P., Chess B. 2012. Foundation in Microbiology. Ed 8. Pasadena. Mc Graw Hill.

 Carrier state
 Individuals who recover from infection may harbor the organisms
in the gallbladder, which becomes the site of chronic carriage.
 Such individuals excrete the organisms in their feces either
continuously or intermittently; nevertheless, they become an
important source of infection for susceptible persons.
 The carrier state may be terminated by antimicrobial therapy if
gallbladder infection is not evident  Otherwise,
cholecystectomy has been the only solution to the chronic state of
enteric carriers.

Mahon CR, Lehman DC, Manuselis G. 2019. Textbook of Diagnostic Microbiology. 6th Edition. China : Elsevier, Saunders
 Diagnosis
 Specimen
• Blood culture must be taken repeatedly (results are often positive in the first week of
the disease).
• Bone marrow cultures
• Urine culture results may be positive after the second week.
• Stool specimens also must be taken repeatedly
 In enteric fevers, positive results from the second or third week
 In enterocolitis, positive results during the first week
• A positive culture of duodenal drainage establishes the presence of salmonellae in the
biliary tract in carriers.

Brooks G.L et al. Jawetz, Melnick, & Adelberg’s Medical Microbiology. Ed 27. New York. 2016. Lange.
 Bacteriologic Methods for
Isolation of Salmonellae
• They are aerobic and facultatively anaerobic; they grow at an optimum temperature
of 37ºC in a pH of 6–8
• Nutrient agar and blood agar (18–24 hours of incubation):
• gray white moist colonies with smooth convex surface
• S. Paratyphi B  large mucoid colonies (the production of loose polysaccharide
slime)
• MacConkey agar  pale colorless colonies because they do not ferment lactose
• Deoxycholate citrate agar (48 hours or more ) they produce colonies with a black
center
• Wilson and Blair’s bismuth sulfite agar is the medium of choice for Salmonella spp
 produce jet black colonies surrounded by a metallic sheen (production of H2S)
• XLD (xylose, lysine deoxycholate agar)
• pink colonies with black centers as a result of H2S production.
• H2S-negative Salmonella serotypes produce red colonies without black centers

Mahon CR, Lehman DC, Manuselis G. 2019. Textbook of Diagnostic Microbiology. 6th Edition. China : Elsevier, Saunders
FIG. 19.13 H 2 S– producing colonies of salmonellae growing
FIG. 19.14 Salmonella growing on CHROMagar Salmonella
on xylose-lysine-desoxycholate (XLD) agar. (Courtesy
differential agar. (Courtesy BD Diagnostic Systems, Sparks, MD.)
American
Society for Clinical Laboratory Science, Education and
Research
Fund, Inc., 1982.)
 Salmonella - The
biochemical features

 In almost every isolate, they do not ferment lactose

 Indole (-), Voges-Proskauer test (-) , phenylalanine deaminase (-), and urease
(-)
 Most produce H2S (except Salmonella Paratyphi A)
 They do not grow in medium containing potassium cyanide
Salmonella - Biochemical
reactions
 Serologic Methods
● Agglutination test

○ Known sera and unknown culture are mixed on a slide Clumping can be observed within a few minutes.

○ Commercial kits available to agglutinate and serogroup salmonellae by their O antigens: A, B, C1, C2, D, and E.
● Tube dilution agglutination test (Widal test)

○ Low specificity (50-70%). Can not differentiate active/ previous infection or even vaccination.

○ At least two serum specimens, obtained at intervals of 7–10 days, are needed to prove a rise in antibody titer.

■ A titer against the O antigen of greater than 1:320 and against the H antigen of greater than 1:640 is considered positive.

■ High titer of antibody to the Vi antigen occurs in some carriers.

○ Alternatives to the Widal test include rapid colorimetric and EIA methods.
● Tubex test

○ Imunochromatography test to detect IgM to antigen O:9 as main determinant antigen in LPS S typhi
● TyphiDot

○ Detect IgM dan IgG to outer membrane antigen protein of S.Typhi

● Nucleic Acid Amplification Tests

Brooks G.L et al. Jawetz, Melnick, & Adelberg’s Medical Microbiology. Ed 27. New York. 2016. Lange.
 Salmonella - Other properties
• Susceptibility to physical and chemical agents, the bacilli are
killed at:

• temperature of 55ºC in 1 hour or at 60ºC in 15 minutes.

• 0.2% mercuric chloride or 5% phenol in 5 minutes

• Boiling, chlorination of water, and pasteurization of milk

• They survive for weeks in polluted water and soil, and for months
in ice.
• Cultures may be viable for years if prevented from drying.

Mahon CR, Lehman DC, Manuselis G. 2019. Textbook of Diagnostic Microbiology. 6th Edition. China : Elsevier, Saunders
 Treatment

■ Uncomplicated enterocolitis:
● No need antibiotic, It is believed to prolong the carrier state.
● Only supportive treatment (Fluid replacement therapy).
● Antidiarrheal agents are also restricted
■ Encourage adherence and further invasion.
○ Antimicrobial treatment of Salmonella enteritis in neonates is important.
■ Choice: chloramphenicol, ampicillin, and trimethoprim-
sulfamethoxazole.
● Susceptibility testing
● In most carriers, the organisms persist in the gallbladder (particularly if
gallstones are present) and in the biliary tract.
○ Some chronic carriers have been cured by ampicillin alone, but in most
cases cholecystectomy must be combined with drug treatment.

Brooks G.L et al. Jawetz, Melnick, & Adelberg’s Medical Microbiology. Ed 27. New York. 2016. Lange.
 Treatment (Typhoid fever or Enteric fever)

Infection NOT acquired in Asia:

Ciprofloxacin 400 mg IV/750 mg po q12h or

Levofloxacin 750 mg IV/po q24h

Treat for 7-10 days

Sanford Guide. 2019. Antimicrobial Therapy.

 Salmonella - Prevention and Control

● safe drinking water, proper food hygiene, and sanitary disposal of excreta
● Immunization

○ persons with intimate exposure (e.g., household contact) to S. Typhi cases or carrier

○ travelers to countries associated with an increased risk of exposure to S.Typhi;

○ microbiology laboratory personnel working

REFERENCES

 Mahon C, Lehman D. Textbook of Diagnostic Microbiology. 6th

ed. St. Louis, Missouri: Elsevier Saunders; 2019.
 Talaro K.P, Chess B. Foundations in Microbiology. 10 th ed. New
York: McGraw-Hill Education; 2018.
 Ryan K.J, George R. Sherris Medical Microbiology. 7th ed. New
York: McGraw-Hill Education; 2019
 Tille, Patricia M. Bailey & Scott’s Diagnostic Microbiology. 14th
ed. St. Louis, Missouri: Elsevier; 2017.