Ordo: Gram negative facultative anaerobic rods

Familia: Enterobacteriaceae

Lactose fermentation

Lactose fermenters Lactose non fermenters

Urease test Dextrose fermentation

Positive Negative With gas formation W/O gas formation

Klebsiella E.coli Urease test H2S-formation

Positive Negative Positive Negative

Proteus Salmonellae S. typhi Shigellae

(excl. S. typhi)

Lesser sugar sequence

Species Lactose Dextrose Sucrose Maltose Mannit
E. coli +G +G - +G +G
Klebsiella +G +G +G +G +G
Salmonella* - +G - +G +G
Proteus - +G V +G V
S. typhi - + - + +
Shigella V + - V V
Legend: * excl. Salmonella typhi
+ sugar is degraded - sugar is not degraded
G gas is produced V varies by particular strains or uncertain

Triple sugar iron medium (TSI)

TSI-reactions Representatives
Slope Butt Gas H 2S
(slant)
yellow Yellow + - Escherichia, Enterobacter,
Klebsiella
red Yellow - - Shigella, Serratia
red Yellow + + Salmonella, Proteus
red Red - - Pseudomonas

(1)DIFFERENTIATING MEDIA:
Allow the growth of all the enteral bacteria but each will have different growth
characteristics, as well as colony morphology due to their distinct biochemical
properties.

Eosine-methylenblue medium (EM):

• EM contains eosine and methylenblue dyes and lactose.
• Colonies of lactose fermenters appear to be blue-pruple
• Colonies of lactose non-fermenters appear to be colourless.

(2) SELECTIVE MEDIA:

Beside the above mentioned differentiating compounds selective media contain
selective inhibitors that will allow pathogens to grow. The growth of normal flora
will be normally inhibited. If members of normal flora grow, they will have different
colony morphology. Selective media are utilised if pathogens must be isolated from
patients’ samples also containing members of the normal flora.

Brilliant-green medium:
• Contains brilliant-green (partially inhibits growth of Proteus sp. and E. coli),
brilliant-sugar (lactose, dextrose, and sucrose), and Andrade-indicator
• Colonies of Salmonellae appear to be colourless
• Colonies of E. coli appear to be red if grown

Bismuth sulphite medium:

• Contains brilliant-green (as above), bismuth- and sodium-sulphite (to detect H2S-
production)
• Colonies of Salmonellae appear to be black

Desoxycholate-citrate medium (DC):

• Contains desoxycholic acid, sodium citrate, lead acetate, iron-ammoniac sulphate,
lactose and neutral red indicator
• Colonies of Shigellae appear to be colourless.
• Colonies of E. coli appear to be red if grown.
• Colonies of Proteus sp. appear to be black (H2S-production).

(3) ENRICHMENT MEDIA:

Enrichment media are used if number of pathogens is low in patients’ sample also
containing members of the normal flora. It is used to increase number of pathogens at
the expense of the normal flora.

Selenite enrichment broth:

• Contains selenite ion, which is more toxic to coliforms than to many Salmonella
and Shigella spp.

1
 Salmonella typhi

Disease association: typhoid fever (enteric fever)

Pathogenesis:
1. S.typhi is primarily infective for humans, acquisition from a human source.
2. The ingested salmonellae reach the small intestine epitheloid cells, then enter the
lymphatics and then the bloodstream.
3. By the blood they are carried to many organs : spleen, liver, kidney, lung &
intestine.
4. Multiply in the Peyer’s patches => hyperplasia and necrosis;
Hepatitis, focal necrosis of liver, inflammation of the gallbladder, periosteum, and
other organs.

Key points for the clinical diagnosis:

1. After an icubation period of 10-14 days, slowly progressing fever, which reaches
40-41 oC at the end of the first week of the disease.
2. Fever is often associate with headache, weakness, loss of appetite, coughing, sore
throat, muscle pain, and often with roseolae.
3. By the end of the first weak after the onset, disturbances of consciousness are
common.
4. There is hepatosplenomegaly and relative bradycardia on the physical
examination.
5. Aspecific laboratory findings involve marked neutropenia and eosinophilia
6. Complications: hepatitis, meningitis, pneumonia, arthritis, ostemyelitis, parotitis,
orchitis, perforation of the intestines.

Microbiological diagnosis

1. Clinical specimen:
- faeces: positive from the second or third weeks
- urine : positive from the second week
- bile, bone marrow aspirate
- blood for blood-culture : often positive in the first week
2. Direct smear has no value in the diagnosis.
3. Culture:
• Faeces is inoculated into an enrichment medium containing selenite (Leifson
medium) then is plated on differential and selective media.
• Samples are inoculated onto brilliant green and/or bismuth sulphite selective
media.
4. Biochemical identification: (refer to flow charts) Lactose (-), dextrose
fermentation w/o gas formation, H2S (+)
5. Serological identification:
• Slide agglutination with specific antibodies to show presence of S. typhi cells in
the culture.
• Blood serology:
=> Gruber-Widal reaction (Widal`s type tube agglutination): (Ag = 'H' as well
as 'O' antigens of the laboratory strain of S. typhi) to show presence and
establish titre of specific antibodies in the patients` sera.
 2
6. Phage typing: (for public health purposes only)
• Preparation of lawn culture from the isolated S. typhi.
• Inoculation of suspensions of different bacteriophages onto the surface of the
medium.
• Incubation.
• Reading the result: phages specific for the given isolate will lyse the bacterial
lawn giving rise to a plaque at the site of the inoculation of the bacteriophage.
From the lysis spectrum, isolate is characterised into one of the groups.

Treatment
• Antibiotics according to susceptibility tests: S. typhi strains are usually susceptible
to ampicillin, sulfomethoxazole + trimethoprim, or 3rd generation cephalosporins.
• Complications should be treated accordingly
• Chronic carriers may be treated with ampicillin alone, but in most cases
cholecystectomy must be combined with drug treatment

Prevention
• Aspecific sanitary measures and control of chronic carriers.
• Active immunisation with acetone killed bacterial suspension administered
parenterally is available for high-risk individuals. An orally administered vaccine
of a live avirulent mutant of S. typhi also provides significant protection.

“Carriers”
3 % of survivors of typhoid become permanent carriers, harboring the organisms in
the gallbladder, biliary tract or urinary tract.
 
Salmonellae responsible for enterocolitis (gastroenteritis)

Disease association:
1. Salmonellosis (gastroenteritis) in otherwise healthy individuals
2. Invasive infections (sepsis, meningitis) in very young, or very old, as well as
inmmunocompromised patients

(Any of the approx. 2,000 types of Salmonellae can be encountered to cause

enterocolitis, but S. typhimurium, S. enteritidis (over 1,500 serotypes) are most
frequently identified in clinical specimens.)

Pathogenesis:
1. The reservoir for human infection: poultry, pigs, rodents, cattle, pets
2. The organisms enter via the oral route, with contaminated food or drink
3. Inflammatory lesions of the small and large intestine

Key points for the clinical diagnosis:

1. After an incubation period of 6-48 hours, nausea, vomiting, headache, muscle pain
develop
2. Soon thereafter, there is a watery, or green-yellow diarrhoea with a parallel fever
(38-39 oC)

Microbiological diagnosis
1. Clinical specimen:
- faeces : positive from first week, and may remain + for several weeks
- blood for blood-culture: just in 2-4 % (+)
- CSF, and food leftover
2. Direct smear has no diagnostic value.
3. Culture: as shown with S. typhi.
4. Biochemical identification: (refer to flow-charts) Lactose (-), fermentation of
dextrose with gas formation, urease (-)
5. Serological identification: slide agglutination with type specific antibodies to
identify bacteria taken from culture
6. Phage typing: for public health purposes as shown with S. typhi.

Treatment
• Supportive therapy, replacement of fluid and electrolytes.
• The vast majority of enterocolitis cases do not require an antibiotic treatment. In
fact, in salmonella enteritis, antimicrobial therapy may prolong clinical symptoms
and excretion of Salmonellae.
• Antimicrobial treatment of Salmonella infection of neonates, as well as invasive
salmonella infections is important. Drugs of choice are ampicillin,
sulfomethoxazole + trimethoprim, or a 3rd generation cephalosporin, but
susceptibility tests are important
 
Shigella dysenteriae, flexneri, boydii, sonnei

Disease association: bacillary dysentery

Infections are almost limited to the gastrointestinal tract !

Pathogenesis:
1. The essential pathologic process is invasion of the mucosal epithelial cells by
induced phagocytosis, escape from the phagocytic vacuole, multiplication and
spread within the epithelial cell cytoplasm
2. Microabscessus in the wall of the large intestine and terminal ileum lead to
necrosis, bleeding and formation of a pseudomembrane
3. S. dysenteriae type I. Produces exotoxin = like E. coli verotoxin
 inhibits sugar and amino acid absorption in the
small intestine
 bleeding, haemolysis, HUS
 “neurotoxin” => coma, meningismus

Key points for the clinical diagnosis:

1. After an incubation period of 2-4 days, fever develops with diffuse abdominal
discomfort and sensitivity to palpation especially above the sigma and at the
coecal area
2. Bloody diarrhoea with tenesmus (urging, painful, non-productive impulse of
defecation)
3. In severe cases with general (toxic) symptoms: hyperpyrexia, shock, convulsions,
meningeal signs

4. Microbiological diagnosis
1. Clinical specimen: faeces, food leftover
2. Direct smear has no diagnostic value
3. Culture:
• Eosine-methylenblue agar ⇒ small, round-shaped, colourless colonies
(lactose -)
• Desoxycholate-citrate agar ⇒ containing desoxycholic acid, sodium
citrate, lead acetate, ferrous ammonium sulfate, lactose, and neutral red
indicator
• Shigellae: small, round-shaped, colourless colonies (lactose -, H2S-).
• E. coli: small, round-shaped, redish colonies, medium appears to be
turbid around colonies (lactose+, H2S-).
• Proteus sp: mediumturns to black (lactose -, H2S+).
4. Biochemical identification: (refer to flow charts) lactose (-), but S. sonnei is
ONPG+ (slow fermenter), fermentation of dextrose w/o gas production, H2S (-)
5. Serological identification: slide agglutination with type specific antibodies to
identify bacteria taken from culture
6. Phage typing: for public health purposes.

Treatment and prevention

1. Antibiotics: sulfamethoxazole + trimethoprim, ciprofloxacin, ampicillin,
tetracycline, and chloramphenicol
2. Correct sanitation and food hygiene. No specific measures of prevention are
available.

Klebsiella spp

Disease association:
 may be member of the normal flora of the gut and
in small number of the throat
 cause enteritis in infancy, urinary tract infections,
cholecystitis, cholangitis, peritonitis, pneumonia, otitis, wound infections, sepsis
and meningitis
 K. pneumoniae → Friendländer pneumonia
(fulminant)
 K. ozaenae → ozena
 K. rhinoslceromatis → rhinoscleroma.

Microbiological diagnosis

1. Clinical specimen: faeces, pus, urine, sputum, punctates, blood-culture, CSF

2. Direct smear: in the sediment of CSF appears as Gram negative, short rods with
large and regular capsule

3. Culture:
• Eosine-methylenblue agar: large (2-3 mm in diameter), mucoid,
glistering, raised, deep purple coloured colonies.
• Simple agar medium: large, (2-3 mm in diameter), mucoid, glistering,
colourless colonies.

4. Biochemical identification: (refer to flow charts) Lactose (+), urease (+), indol (-),
H2S (-), KCN-test (+), Voges-Proskauer test (+), methyl red test (+), citrate
utilisation (+)

5. Serological identification: Neufeld`s capsule swelling (Quellung) reaction to

identifiy serotypes with the specific antibodies directed against capsular antigens.

6. Phage typing: for public health purposes only.

Resistance aginst antibiotics:

7. β -lactamase production is common and transmissible plasmid encoded
• simple β -lactamase production results in resistance against ampicillin,
amoxicillin, and 1st , 2nd generation cephalosporins
• extended spectrum β -lactamase (ESBL) production results in
resistance against penicillins, 1st, 2nd, 3rd generation cephalosporins
8. Secondary resistance to any of the antimicrobial drugs that are effective against
Klebsiella spp. may develop
Treatment and prevention
Antibiotics: based on the susceptibility tests. β -lactame + β -lactamase inhibitor
combination, 3rd generation cephalosporins, carbapenems, monobactams,
fluoroquinolones and amynoglycosides have marked antibacterial effect against
Klebsiella spp, but variation in susceptibility is great. No specific measures of
prevention are readily available.


Escherichia coli

Disease association: member of the normal flora of the gut, but may cause infections
if outside the gut, the manifestation of which depends on the site of infection (refer to
textbook):
1. Urinary tract infections,
2. Coli associated diarrhoeal diseases (EPEC, ETEC, EHEC, EIEC, EAEC) ,
3. Wound infections and sepsis,
4. Meningitis.

Microbiological diagnosis
1. Sample to be taken: faeces, urine, pus, blood-culture, CSF, water and food (coli
count)
2. Direct smear: in the sediment of CSF appears as Gram negative short rods without
capsule.
3. Culture:
• Eosine-methylenblue agar: intermediate sized, glistering,
iridescent ,"sheen", blue-purple coloured colonies.
• Simple agar medium: intermediate sized, glistering,
iridescent ,"sheen", colourless colonies.
4. Biochemical identification: (refer to flow charts) Lactose (+), urease (-), indol (+),
Voges Poskauer test (-), methyl red reaction (+), citrate utilisation (-), nirate
reduction to nitrite (+), H2S (-)
5. Serological identification: slide agglutination with specific antibodies against the
'O'-, 'K'-, and 'H'-antigens.
• 'O'-ag = outermost part of the bacterial LPS, consisting of
polysaccharide, resistant to heat and alcohol, and usually detected by bacterial
agglutination. anti-O antibodies are mainly IgM in nature.
• 'K'-ag= external to 'O'-ag, polysaccharide in E.coli, may be associated
with virulence (K1 - meningitis in infancy, some other types - attachment to
epithelial cells prior urinary tract invasion)
• 'H'-ag = located on the flagella and can be denatured or removed by
heat and alcohol. Anti-H antibodies are mainly IgG in nature.
6. Phage typing: for public health purposes only.
Resistance against antibiotics
1. β -lactamase production is common and transmissible plasmid encoded
• simple β -lactamase production results in resistance against ampicillin,
amoxicillin, and 1st , 2nd generation cephalosporins
• extended spectrum β -lactamase (ESBL) production results in
resistance against penicillins, 1st, 2nd, 3rd generation cephalosporins
2. Secondary resistanc to any of the antimicrobial drugs that are effective against E.
coli may develop

Treatment and prevention

Antibiotics: based on the susceptibility tests. ampicillin, 2nd, 3rd generation
cephalosporins, fluoroquinolones and amynoglycosides have marked antibacterial
effect against E. coli, but variation in susceptibility is great.
No specific measures of prevention are readily available.

Genus: Yersinia

Y. pestis
plague, "black death"
Pathogenesis :
1. Reservoir : rodents / rat is the most common /
2. antropo-zoonosis transmitted by bites of fleas (vector )
Flea feeds on a rodent infected with Y.pestis  ingested organism multiply in the
gut of the flea & with the help of the coagulase blocks its proventriculus ( so no
food can pass through )
3. The hungry flea bites  blood aspiration  regurgitation into the wound
multiplication in the monocytes ( killed by PMN) lymphatic vessels 
enlarged lymph nodes ( haemorrhagic inflammation )  necrosis ;fluctuant
4.  blood stream  meningitis
 pleuro-pericarditis
 pneumonia
5. Coughing  droplets , speading from human to human

Key points of the clinical diagnosis:

1. Prodromial symptoms: weakness, discomfort, fever, headache
2. Enlarged, painful, inflammed regional lymph nodes especially at the inguinal
region (bubonic plague)
3. Generalisation of infection: haemorrhagic pneumonia (lung plague),
haemorrhagic meningitis, sepsis

Microbiological diagnosis
1. Sample to be taken: punctate of the enlarged lymph nodes ("buboes"), sputum,
blood-culture, CSF
2. Direct smear:
Giemsa’stain, specific immunofluorescent staining
Wayson`s stain: small rounded 1.5-2.0 µ m sized rods with a striking bipolar
(hairpin-like) appearance.
3. Culture: on blood agar medium at 28 oC, for 48 hours. Colonies are tiny, round
shaped, viscous and grey.
Definite identification of cultures is best done by IF.
4. Biochemical tests:
• Lactose (-), but ONPG (+), NO3- → NO2- (+), Urease (-), oxidase (-)
5. Serological identification:
• Slide and tube agglutination with specific antibodies (test serum) to
identify cultures
6. Blood serology:
• Passive haemagglutination (ag adsorbed to sheep RBCs) to show
specific antibodies in patients` sera
• Tube agglutination (ag = formalin ianactivated Y. pestis) to show
specific antibodies in patients` sera

2
Treatment
Antibiotics: streptomycine is the drug of choice, alternatively chloramphenycol,
tetracylines and sulfonamides are given. Streptomycine + tetracyclines are powerful
combination to treat plague. Treatment should be started as soon as possible.

Prevention
• Active immunization: with fomalin killed Y. pestis.
• Chemoprofilaxis: tetracyclines.

Y. enterocolitica & pseudotuberculosis

Disease association:
Y. enterocolitica  gastroenteritis
Y.pseudotuberculosis gastroenteritis + mesenteral lymphadenitis, sepsis

Pathogenesis :
1. Reservoirs : rodents, domestic animals
2. Ingestion  multiplication in the gut mucosa ( ileum )  inflammation,
ulceration  BLOODY DIARRHOEA
3. Complications : lympatic vessels  mesenterial lymphadenitis
Immuncomplex formation  arthritis, spondylosis
ankylopetica, erythema nodosum
Rarely  septic case ( pneumonia, meningitis )
Laboratory diagnosis
1. Sample to be taken: faeces, blood for blood culture, materials obtained at surgical
exploration.
2. Direct smear: not contributory to the diagnosis.
3. Culture:
• On blood agar at 28 oC, aerobically, for 24 hours → colonies are tiny,
round shaped, greyish.
• "cold enrichment" (faeces is incubated in buffered, pH 7.6, saline at 4
o
C, for 2 weeks) then inoculated onto MacConkey agar, or eosine-
metyleneblue medium → colonies are tiny, round shaped and colourless.
4. Biochemical identification:
• lactose (-), but ONPG (+), urease (+), oxidase (-)
5. Serological identification:
• Slide agglutination to identify cultures with specific antibodies (test
serum) based on the 'O' -antigen .
6. Blood serology:
 • Tube agglutination (ag = formalin inactivated bacteria) to show
specific antibodies in patients` sera.
Treatment
In mild diarrhea disease is self-limited, the benefit of antibiotic therapy is unknown.
In severe cases (sepsis, meningitis), antibiotic treatment should be started as soon as
possible with 3rd generation cephalosporins in combination with aminoglycosides or
with fluoroquinolones.
Prevention No specific preventive measures are available.
 1
Ordo: Gram negative facultative anaerobic rods
Familia: Vibrionaceae
Genus: Vibrio

Major pathogenic species: V. cholerae

Disease association: endemic and pandemic cholera

Pathogenesis: pathogenic only for humans!

1. Oral route ( 10 – 10 organisms) adherence to the epithelial cells of the small
intestine, multiplication here
2. Enterotoxin production:
subunit B => facilitate the entry of subunit A into the cell
subunit A => cAMP  , inhibition of Na – pump
 NaCl + water hypersecretion
“rice-water”, 15-20 L/day diarrhoea
 dehydration, acidosis, shock

Laboratory diagnosis

Clinical specimen: mucous flecks from stools, drinking water, food

Direct smear:
 not distinctive, but dark field, or phase contrast
microscopy may show rapid movement of V. cholerae cells.
 Smear taken from broth culture and stained by
Gram shows a comma-shaped, curved rod 2-4 µ m in length.
Culture:
• grows well and rapidly on peptone agar, or on blood agar supposed pH
is set to near 9.0 (alkalic)
• selective medium: TCBS (thiosulphate-citrate-bile-sucrose +
bromthimol-blue indicator) medium, at pH near 9.0, 37 oC, 18 hours.
Colonies: convex, smooth, round shaped, yellow.
• grows readily on agar containing 6 % NaCl

Biochemical identification:
• Oxidase+ (which is very important, because it differentiates it from
Enterobacteriaceae)
• decomposes glucose, mannose, and sucrose without gas production
• lactose (-), arabinose (-)
• indol (+)

Serological identification: slide agglutination with specific anti-'O' / O1  O 139/

test serum (antibodies)
O 1, O 139 => cholera
O 2- O 138 => non-severe enteritis
O 1 has two subtypes : Ogawa, Inaba
two byotypes : classic, El-Tor ( 1961 great epidemie , long term carrier
stage )
 2
Treatment of cholera
1. Most importantly, water and electrolyte replacement therapy should be started as
soon as possible to correct severe dehydration and electrolyte loss. Ways of
rehydration:
• intravenous infusions:
• ORF (oral rehydration fluid):
• 3,5 g NaCl, 2,5 g NaHCO3 and 1,5 g KCl and 20,0 g glycose to each
litre of boiled tap water or
• 1 teaspoon of NaCl and 4 teaspoons of glucose
•
A patient in severe condition requires aprrox. 25-30 litres of infusion for the
entire treatment!

2. Antibiotic therapy: tetracyclines. Note: plasmid controlled secondary resistance

against different drugs including tetracyclines has already been detected!

Prevention
• Active immunisation with lipopolysaccharides of V. cholerae or dense suspension
of inactivated V. cholerae cells confers only limited protection (50 %).
The WHO vaccination certificate for cholera is only valid for 6 months.
• Chemoprofilaxis with tetracyclines may have a role in prevention.

Aspecific measures: education and improvement of sanitation of food and water.

Boiling of drinking water and disinfecting of vegetables and fruits in endemic areas.

Misc. bacteria
Proteus
Agar-plate → swarming (no individual colonies are seen, bacteria spread through the
entire surface of the plate)

Serratia
Agar-plate → red water-insoluble pigment produced but only in dark (note "sweating
blood", "bleeding bread or bleeding altar-bread")
Non-pathogenic Escherichia coli
 virulence plasmid or/and parts of gene with conjugation
or transduction
pathogenic Escherichia coli

Enteropathogen E. coli / EPEC / : 60 Mda plasmid

Eae cromosomal gene
 O26: K60, O55 : K59,
O86:K61, O111:K58
 dyspepsia,
usually self-limited watering
diarrhea
 under 1 year

Enteroinvasive E. coli /EIEC /: 140 Mda plasmid / like shigellae /

 O 28a, O124, O136, O143
 dysenteria-like disease
 children in developing countries

Enterotoxigenic E.coli / ETEC/ : heat –labil toxin LT ~ like cholera

plasmid
Heat-stabil toxin ST
 watery diarrhea, “ traveller’s
disease “

Enterohemorrhagic E. coli / EHEC/: verotoxin plasmid

Eae cromosomal gene
Lysogenic conversion
 O 157 : H7  haemorrhagic
colitis (HUS, TTP, sepsis)

Enteroaggregative E. coli / EAggEC / : enteroadhesive 60MDa

plasmid
enterotoxin plasmid
 haemorhagic diarrhea

Enteroadhesive E.coli / EAEC/: enteroadhesive factor plasmid

 traveller’ disease