Enterobacteriaceae.

 

Lesson 1.

Theme: «Escherichiae. Klebsiella».

1. Escherichiae coli.

 

Genus Escherichiae subdivide into: common Escherichia coli and diarrheal Escherichia coli.

Common E.coli – normal resident inhabitant of colon.

Morphology of common E.coli.

E.coli is a Gram negative rods. It is motile by peritrichate flagella, though some strains may be nonmotile. Capsules are found in some strains. Spores are not formed.

Cultural characteristics of common E.coli.

It is a facultative anaerobe. Good growth occurs on ordinary media (MPA,MPB). On MacConkey s (Endo) medium, colonies are bright pink (lactose positive) due to lactose fermentation.

Biochemical reactions of common E.coli.

Glucose, lactose, maltose and many other sugars are fermented with the production of acid and gas.

The main positive functions of common E.coli for macroorganism:

·       Antagonistic function with respect to some pathogenic bacteria (Shigella, Salmonella, Vibrio).

·       Immunomodulator and immunostimulant function.

·       Participate in synthesis of vitamins (Vitamin B, K, nicotinic acid).

·       Participate in lipid and water-salt metabolism.

Common E.coli can cause different endogenous infections, if human organism have lowering of resistance.

Clinical forms of diseases which can cause common E.coli: cystitis, pyelonephritis, cholecystitis, meningitis, sepsis, pneumoniae, tonsillitis, appendicitis, etc.

Diarrheal E.coli have morphological, cultural and biochemical characteristics such as common E.coli. Diarrheal E.coli differ from common E.coli on based their antigenic structure.

Antigenic structure of diarrheal E.coli.

Serotyping of E.coli is based on three antigens – the somatic antigen O, the capsular antigen K and the flagellar antigen H. The antigenic pattern of a strain (serovars) is recorded as the number of the particular antigen it carries, as for example E.coli O111: K58: H2. In practical work  diarrheal E.coli identify by only O antigen (named as serogroup).

Diarrheal E.coli cause coli-infections (exogenous infections).

Source of infection: patient or bacteriocarrier.

The mode of infection: infection acquired by ingestion of contaminated water and food.

Four different types of diarrheagenic E.coli are now recognised enteropathogenic, enterotoxigenic, enteroinvasive and enterohemorrhagic (shigatoxigenic) E.coli.

                                                                                                Table 1.

Category	Serogroups	Clinical forms	Mechanism
1. EPEC (enteropathogenic E.coli)	O55, O86, O111	Diarrhea. Newborn and babies (on artificial feeding) are ill more often than other children.	1. Adhesion to cell of small intestine. 2. Multiply on the surface of cells. 3. Cytotoxic action
2. ETEC (enterotoxigenic E.coli)	O6, O8, O15	Its severity varies from mild watery diarrhea to fatal disease indistinguishable from cholera.	1. Adhesion and multiply on cell of small intestine. 2. production exoenterotoxin (cholera-like toxin).
3. EIEC (enteroinvasive E.coli)	O28, O143, O124	Clinically EIEC infection resembles shigellosis ranging from mild diarrhea to typical dysentery, and occurs, in children as well as adults.	1. Adhesion to cell of colon. 2. Invasion and multiply. 3. Ulcerative lesion.
4. EHEC (enterohemorrhagic E.coli)	O157, O26, O145	E.coli strains producing shiga-like toxin can give rise to diarrheal disease ranging in severity from mild diarrhea to fatal hemorrhagic colitis and hemorrhagic uremic syndrom particularly in young children and the elderly.	1. Adhesion to cell of colon. 2. Invasion. 3. Production shiga-like toxin.

Laboratory diagnosis.

Material for dignostics: feces.

1. Bacteriological method.

·        The feces is inoculated on Endo medium and incubated at 37⁰C.

·        Colonies appear after 18 – 24 hours and may be identified by  biochemical reactions sugar media ( for determination of genus – Escherichia) and antigenic structure (for determination: common or diarrheal E.coli) – agglutination test.

2. Klebsiella.

3 species. Klebsiella pneumoniae, Kl.ozaenae, Kl.rhinoscleromatis.

Morphology.

 Gram negative rods. They are nonmotile and nonsporing. They are capsulated (in vivo and in vitro).

Cultural characteristics.

Grow well on ordinary media (MPB, MPA). MPA – mucous colonies, MPB – turbidity.

Pathogenicity.

1. Adhesion.

2. Capsule – protects from phagocytosis.

3. R – plasmids (K.pneumoniae).

4. Endotoxins.

The main disease which can cause Klebsiella.

Species of Klebsiella	Diseases	Material for diagnostics
K.pneumoniae	1. Pneumonia, bronchopneumonia. 2. Meningitis, sepsis, pyelonephritis, cystitis. 3. Enterocolitis, enteritidis.	1. Sputum. 2. Liquor, blood, urine. 3. Feces.
K.ozaenae	Ozaenae – disease characterised by damage (atrophy) of mucous membrane of nose and foul smelling nasal discharge.	Nasal discharge.
K.rhinoscleromatis	Rhinoscleroma – chronic granulomatous hypertrophy of the nose.

Laboratory diagnosis.

1. Bacteriological method.

2. Microscopic method (Burri-Gins stain).

3. Serological method – complement fixation test, reaction passive hemagglutination, agglutination test.

Lesson 2.

Theme: «Salmonella. Shigella.»

1. Salmonella.

Salmonellae currently comprise above 2000 serotypes or species, all of them potentially pathogenic. For practical purposes, they may be divided into two groups:

1) The enteric fever group consisting of the typhoid and paratyphoid bacilli that are exclusively or primarily human parasites;

2) The food poisoning group, which are essentially animal parasites but which can also infect, human beings, produsing gastroenteritis, septicemia or localised infections.

Salmonella typhi,, Salmonella paratyphi A, Salmonella paratyphi B (S.schottmuelleri), Salmonella paratyphi C (S.hirschfeldii) – agents of enteric fever.

Morphology.

Gram negative rods. They are motile with peritrichate flagella, but noncapsulated and nonsporing.

Cultural characteristics.

Salmonellae facultatively anaerobic, growing readily on simple media (MPA, MPB). On Endo medium colonies are colourless (lactose negative) due to the absence of lactose fermentation. On bismuth sulphite medium, jet black colonies with a metallic sheen are formed due to production of H₂S.

Biochemical reactions. S.typhi ferment some sugars, forming acid. S.paratyphi A and B ferment some sugars forming acid and gas. Indole is not produced.

Antigenic structure.

1) O-antigen (somatic) – phospholipid-protein-polysaccharide complex.

2) H-antigen. This antigen present on the flagella is a heat labile protein.

3) K-antigen.

4) Vi-antigen (surface antigen) – found in some species. The Vi-antigen affords a method of epidemiological typing of the S.typhi strains based on specific Vi-bacteriophage.

Classification and nomenclature.

Classification within the genus is on antigenic characterisation based on the Kauffmann – White scheme. This scheme depends on the identification, by agglutination, of the structural formulae of the O and H antigens of the strains (Table 1).

Table 1.     

Kaufmann – White scheme.

Serogroups	Antigen O	Antigen H    (Phase I)	Antigen H    (Phase II)
Group A (O2)
S.paratyphi A	1, 2, 12	a	(1, 5)
Group B (O4)
S.schottmuelleri	1, 4, (5), 12	b	1, 2
S.abony	1, 4, (5), 12, 27	b	e, n, x
S.typhimurium	1, 4, (5), 12	i	1, 2
S.heidelberg	1, 4, (5), 12	r	1, 2
Group C1, C2, C3 (O7, 8)
S.hirschfeldi	6, 7 (Vi)	c	1, 5
S.choleraesuis	6, 7	(c)	1, 5
S.tompson	6, 7	c	1, 5
S.newport	6, 8	e, h	1, 2
Group D1        (O9, 12)
S.typhi	9, 12 (Vi)	d	-
S.enteritidis	1, 9, 12	g, m	(1, 7)
Group E1 (O3, 10)
S.oxford	3, 10	a	1, 7
S.anatum	3, 10	e, h	1, 6
S.london	3, 10	l, v	1, 6

Pathogenicity.

1. Pili – facilitates adhesion of Salmonella to the host cell surface.

2. Invasion – they enter the lymphoid follicles of the ileum.

3. Endotoxin.

4. R – plasmids.

Enteric fever.

The term enteric fever includes typhoid fever caused by S.typhi and paratyphoid fever caused by S.paratyphi A, B, and C. The clinical course may vary from a mild undifferentiated pyrexia (ambulant typhoid) to a rapidly fatal disease. The onset is usually gradual, with headache malaise, anorexia a coated tongue and abdominal discomfort with either constipation or diarrhea, rose-rash (roseola) on skin.

Source of infection: patient or bacteriocarrier – for enteric fever, cattle and hen – for S.schottmuelleri.

The mode of infection: acquired by ingestion of contaminated food (milk, egg, meat).

Stages of pathogenesis of enteric fever.

Date of disease	Stages	Material for diagnostics
1. Incubation period      (6-14 days)	Digestive stage – transit of microbe from oral cavity to small intestine with food. Invasion stage – bacteria penetrate to the lamina propria of submucosa, then mesenteric lymph nodes.
2. 1 week	Bacteremia – bacteria multiply in the blood. Intoxicating: ·         Destruction of salmonella ·         Endotoxin go out from bacteria.	1. Hemoculture (blood from ulnar vena – 10 ml) 2. Urineculture (urine). 3. Coproculture (feces).
3. 2 week	Parenchymal diffussion – bacteria multiply in the organ (liver, spleen, lung, skin, bone marrow, kidney). Secretory stage – secretion of salmonella with bile, feces, urine, breast milk.	1. Serum for agglutination test (Widal reaction). 2. Coproculture. 3. Urineculture. 4. Hemoculture (20 ml) 5. Roseoloculture (lymph from roseola). 6. Lactoculture (breast milk from mother). 7. myeloculture (bone marrow) – after death of the patient.
4. 3 week	Secretory stage. Allergological stage – necrosis of small intestine →appear ulcer. Ulseration of the bowel leads to the two major complications of the disease – intestinal perforation and hemorrhage.	1. Coproculture. 2. Urineculture. 3. Serum  (Widal reaction).
5. 4 week	Convalescence.	1. Coproculture. 2. Urineculture.

  

Laboratory diagnosis.

1. Bacteriological method. Identification bacteria by biochemical and antigenic properties (agglutination test). Blood inoculate on liquid media (Rappaport media). Feces, urine and other materials for diagnostics inoculate on Endo medium and bismuth sulphite agar.

2. Serological method (Widal reaction).

Prophylaxis.

1. Heat killed typhoid bacillus vaccine.

2. Salmonellosis.

The main agent. Salmonella typhimurium.

 

Human infection results from the ingestion of contaminated food. The most frequent sources of salmonella food poisoning are poultry, meat, milk, and milk products. Of great concern are eggs and egg products.

Food contamination may also result from the droppings of rats or other small animals. Gastroenteritis may occur without food poisouing as in cross infection in hospitals (direct contact – by hands of medical personnel medical apparatus etc.

Clinically the disease develops after a short incubation period of 24 hours or less with diarrhea, vomiting, abdominal pain and fever.

3. Shigella.

The causative agents of bacillary dysentery belong to the genus shigella. Shigella are classified into four species:

·        Shigella dysenteriae

·        Shigella flexneri

·        Shigella boydii

·        Shigella sonnei

Morphology.

Shigellae are short, Gram negative rods. They are nonmotile, nonsporing and noncapsulated.

Cultural characteristics.

They are aerobes and facultative anaerobes. They grow on ordinary media but less readily than other enterobacteria. Colonies on MacConkey (Endo) agar are colourless due to the absence of lactose fermentation.

Biochemical reactions.

Glucose is fermented with the production of acid, witho ut gas.

Antigenic structure.

·        O-antigen (somatic)

·        K-antigen

Pathogenicity.

1. Adhesion (pili).

2. Invasive property (penetrate and multiply in colonic mucosa) – plasmids coding.

3. Exotoxin:

·        Enterotoxic action

·        Cytotoxic action

·        Neurotoxic action

4. Endotoxin.

5. R-plasmids.

Shigellae cause bacillary dysentery. Infection occurs by ingestion. The minimum infective dose is low, as few as 10-100 bacilli being capable of initiating the disease, probably because they survive gastric acidity better than other enterobacteria.

The main clinical features are frequent passage of loose, scanty feces containing blood and mucus, along with abdominal cramps and tenesmus. Fever and vomiting may be present.

The source of infection: patient or bacteriocarrier.

The mode of infection: acquired by ingestion of contaminated water and food.

Laboratory diagnosis.

Material for diagnostics: feces.

1. Bacteriological method. Identification bacteria by biochemical and antigenic properties.

2. Reaction passive hemagglutination, immunofluorescence test – for determination of shigella antigen in the feces.

                                                

Lesson 3.

Theme: «Vibrio. Campylobacter. Helicobacter».

1. Vibrio cholerae.

 

3 agents: Vibrio cholerae biovar classical, Vibrio cholerae biovar El-Tor, Vibrio cholerae biovar O-139 «Bengal».

Morphology.

The cholera vibrio is Gram negative, curved rods. Organization in cells – comma shaped. They are nonsporing and noncapsulated. Monotrichous.

Cultural characteristics.

The cholera vibrio is strongly aerobic. Growth is better in alkaline medium (optimum pH 8,5 -  9,0). On alkaline agar, after overnight growth colonies are moist, translucent, round discs with a bluish tinge in transmitted light.

In 1% peptone water, growth occurs in about six hours as a fine surface pellicle.

Biochemical reactions.

Heiberg classified vibrios into six groups based on the fermentation of mannose, sucrose and arabinose. Two more groups were added later. Cholera vibrios belong to Group I (Table 3).

                                                                                                Table 3.

Heiberg grouping of vibrios.

Group	Fermentation of mannose	Fermentation of Sucrose	Fermentation of Arabinose
I	A (+)	A (+)	-
II	-	+	-
III	+	+	+
IV	-	+	+
V	+	-	-
VI	-	-	-
VII	+	-	+
VIII	-	-	+

Antigenic structure.

O-antigen (somatic)

H-antigen (flagella)

Based on the major (specific) somatic (O) antigen, Genus Vibrio was classified into serogroups, 150 of which are currently known. Vibrio cholerae classical and V.cholerae El-Tor belong to serogroups O-1, which include three serovars: Ogawa, Inaba and Hikojima. Serovar «Bengal» belong to serogroup O-139.

Pathogenicity.

1. Adhesion (pili).

2. Cholera exoenterotoxin (choleragen). The toxin molecule consists of one A and B subunits. The B (binding) unit attach to the ganglioside receptors on the surface of jejunal epithelial cells. The A (active) subunit causes prolonged activation of cellular adenylate cyclate and accumulation of cAMP, leading to outpouring into the small intestinal lumen, of large quanfities of water and electrolytes and the consequent watery diarrhea.

Cholera – is an acute diarrheal disease.

The source of infection: patient or vibriocarrier.

The mode of infection: infection is acquired through fecally contaminated water or food.

The main clinical forms.

1. Choleraic enteritis and gastroenteritis

·        Profuse painless watery diarrhea.

·        Copious effortless vomiting.

Stools may be from 3 to 10 times (mild cases of disease) and from 200 to 300 times (serious illness). As a result massive loss of fluid and electrolytes from patient.

2. Choleraic algid (without treatment)

·        Fall of temperature (below 36,6⁰).

·        Common complications: muscular cramps, renal and cadiac failure, pulmonary edema, etc.

Laboratory diagnosis.

1. Bacteriological method (main).

2. Genetic method – PCR.

3. Express method: immunofluorescence test (direct), reaction passive hemagglutination.

Treatment.

The treatment of cholera consists essentially of the prompt and adequate replacement of lost fluid and electrolytes (by special solutions which injection per os and intravenous).

Specific prophylaxis.

1. Vaccine V.cholerae corpuscular inactivated.

2. Chemical vaccine (mono- and bivalent).

2. Campylobacter.

 

The Genus Campylobacter contains spirally curved Gram negative rods. They are typically comma shaped but may occur as «S». They are nonsporing and motile with a single polar flagellum at one or both poles.

Cultural characteristics.

Growth occurs under microaerophilic conditions, 5% oxygen concentration being optimal. They grow on nutritional media with blood.

Campylobacters of medical importance are the following:

·        Causing diarrheal disease (gastroenteritis, enterocolitis): C.jejuni, C.coli.

·        Causing extraintestinal infection: C.fetus.

Campylobacter jejuni.

Medically, this is the most important campylobacter species as it causes attacts of diarrhea worldwide. The infection is zoonotic, the source being food or animal origin, especially raw milk. It is part of the normal intestinal flora of domestic animals and birds, and is shed in their feces.

 

Campylobacter coli.

C.coli causes an infection clinically indistinguishable from that due to C.jejuni. C.coli is commonly found in intestinal tract of healthy pigs.

Campylobacter fetus.

The source of infection: sheep and cattle.

It is a very important veterinary pathogen.

Human infection occurs by ingestion of contaminated food: milk and meat.

C.fetus may lead to bacteremia, sepsis and meningitis.

Helicobacter pylori.

They were originally named Campylobacter pylori. They morphological, cultural characteristics such as C.coli and C.jejuni.

Today, helicobacters colonise the stomachs of half the human population of the world.

The sole source of H.pylori is the human gastric mucus. The axact mechanism of transmission is not clear, but it is likely to be oral-oral or fecal-oral mechanisms.

H.pylori causes:

·        Gastritis

·        Ulcer of the stomach

·        Cancer of the stomach

Laboratory diagnosis:

1. Microscopic method – use Gram stain.

2. Bacteriological method.

3. Serological method: CFT, immunofluorescence test, EIA).

4. For H.pylori – urease test. H.pylori is strongly urease positive.