Chair of Medical Biology, Microbiology, Virology, and Immunology

PATHOGENIC ENTEROBACTERIACEAE

Lecturer as. O.V. Pokryshko

Classification of the Enterobacteriaceae

Genera

EscherichiaEscherichia ShigellaShigella

EdwardsiellaEdwardsiella SalmonellaSalmonella

CitrobacterCitrobacter KlebsiellaKlebsiella

EnterobacterEnterobacter HafniaHafnia

SerratiaSerratia Proteus Proteus

ProvidenciaProvidencia MorganellaMorganella

YersiniaYersinia ErwiniaErwinia

Shigellaa. Slender, gram-negative rod; non lactose-fermenting (except for S. sonnei)b. In contrast to E. coli: no H2S production, no lysine decarboxylation, no acetate utilizationc. Invasive (key to pathogenesis)d. In contrast to Salmonella: non-motile; no gas from glucose fermentation; no H2S productione. Toxin production limited to a few strains

f. All have O antigens-four groups (A-D)g. Differentiating species ( S. dysenteriae - no mannitol fermentation; S. boydii - C antigen group; S. flexneri - B antigen group; S. sonnei-orniltine decarbexylase production)h. Specimensi. Rectal swab from colonic ulcer is best for culture.j. Fecal specimen - must be immediately innoculated onto transport media or culture media.k. Sensitive to acids present in feces.

Morphologically dysentery bacilli correspond to the organisms of the family Enterobacteriaceae. Dysentery bacilli have no flagella and this is one of the differential characters between these organisms and bacteria of the coli-typhoid-paratyphoid group.

Morphology.

Dysentery bacilli

Intracellular Shigella

Colonies of dysentery bacilli on Ploskirev's medium

Cultivation.

Colonies of Salmonella on Mac-Conkey medium

Fermentative properties.

None of the species of dysentery bacilli liquefy gelatin nor produce hydrogen sulphide. They ferment glucose, with acid formation, with the exception of the Newcastle subspecies which sometimes produce both acid and gas during this reaction. With the exception of the Sonne bacilli, none of them ferment lactose.

SubgroupSubgroup Fermentation of Fermentation of

carbohydratescarbohydrates In

dole

pro

du

ctio

nIn

dole

pro

du

ctio

n

Cata

lase

Cata

lase

lacto

se

lacto

se

glu

cose

glu

cose

man

nite

man

nite

su

ccro

se

su

ccro

se

su

ccro

se

su

ccro

se

S. S. dysenteriae – dysenteriae – AA

–– ++ –– –– –– –– ––

S. fiexneri – S. fiexneri – BB

–– ++ ++ ++ –– –– ++

S. boydii – CS. boydii – C –– ++ ++ ++ –– ++ ––S. sonnei – DS. sonnei – D + +

slowlslowlyy

++ ++ –– ++slowlslowl

yy

–– ++

Shigellae Biochemical Properties

Test for determination of motility and producing hydrogen sulphide1. S. flexneri – nonmotile, no produce hydrogen sulphide; 2. Enterobacter cloacea – motile, no produce hydrogen sulphide; 3. Proteus mirabilis – motile, produce hydrogen sulphide.

Toxin production.

S. dysenteriae produce thermolabile exotoxin which displays marked tropism to the nervous system and intestinal mucous membrane. This toxin may be found in old meat broth cultures, lysates of a 24-hour-old agar culture, and in desiccated bacterial cells.

An intravenous injection of small doses of the exotoxin is fatal to rabbits and white mice. Such an injection produces diarrhoea, paralysis of the hind limbs, and collapse.

The dysentery exotoxin causes the production of a corresponding antitoxin. The remaining types of dysentery bacilli produce no soluble toxins. They contain endotoxins, which are of a gluco-lipo-protein nature, and occur in the smooth but not in the rough variants.

Thermolabile substances exerting a neurotropic effect were revealed in some S. sonnei strains. They were extracted from old cultures by treating the latter with trichloracetic acid.

Antigenic structure.

Dysentery bacilli are subdivided into 4 subgroups within which serovars may be distinguished. The antigenic structure of shigellae is associated with somatic O-antigens and surface K-antigens.

SubgroupSubgroup Species Species and and serotypeserotype

SubseSubse--rotyperotypess

Antigenic Antigenic formulaformula

Type Type antigeantigenn

Group Group antigeantigens ns

A.A. Does not Does not ferment ferment mannitemannite

S.dysenteriS.dysenteriaa

1-121-12

B.B. Ferments Ferments mannite as a mannite as a rulerule

S. flexneriS. flexneri 11, , 22, , 33, , 44, , 55,,

66, , X variantX variant Y variantY variant

1a, 1b, 1a, 1b, 2a, 2b,2a, 2b,

3a, 3b, 3a, 3b, 4a, 4b4a, 4b

I, II, III, I, II, III, IV, V, VIIV, V, VI

2, 3, 4, 2, 3, 4, 6, 7, 86, 7, 8

C.C. Ferments Ferments mannite as a mannite as a rulerule

S. boydii,S. boydii, 1- 1-1818

D.D. Ferments Ferments mannite, slowly mannite, slowly lactose and lactose and saccharosesaccharose

S. sonneiS. sonnei

Antigens

Classification.

Dysentery bacilli are differentiated on the basis of the whole complex of antigenic and biochemical properties. S. sonnei have four fermentative types which differ in the activity of ramnose and xylose and in sensitivity to phages and colicins.

Epidemiology and Pathogenesis of Shigellosis.

Humans seem to be the only natural hosts for the shigellae, becoming infected after the ingestion of contaminated food or water. Unlike Salmonella, the shigellae remain localized in the intestinal epithelial cells, and the debilitating effects of shigellosis are mostly attributed to the loss of fluids, electrolytes, and nutrients and to the ulceration that occurs in the colon wall.

Pathogenesis of shigellosis in humans

Shigella dysenteriae type 1 secreted one or more exotoxins (called Shiga toxins), which would cause death when injected into experimental animals and fluid accumulation when placed in ligated segments of rabbit ileum.

The mechanism whereby Shiga toxin causes fluid secretion is thought to occur by blocking fluid absorption in the intestine. In this model, Shiga toxin kills absorptive epithelial cells, and the diarrhea results from an inhibition of absorption rather than from active secretion.

To cause intestinal disease, shigellae must invade the epithelial cells lining of the intestine. After escaping from the phagocytic vacuole, they multiply within the epithelial cells. Thus, Shigella virulence requires that the organisms invade epithelial cells, multiply intracellularly, and spread from cell to cell by way of finger-like projections to expand the focus of infection, leading to ulceration and destruction of the epithelial layer of the colon.

Gross pathology of shigellosis

Histopathology of acute colitis following peroral infection with shigellae.

Immunity.

Immunity acquired after dysentery is specific and type-specific but relatively weak and of a short duration. For this reason the disease may recur many times and, in some cases, may become chronic. This is probably explained by the fact that Shigella organisms share an antigen with human tissues.

Laboratory diagnosis.

Reliable results of laboratory examination depend, to a large extent, on correct sampling of stool specimens and its immediate inoculation onto a selective differential medium. The procedure should be carried out at the patient's bedside, and the plate sent to the laboratory.

Rules the correct procedure of material collection :

carry out bacteriological examination of faeces before aetiotropic therapy has been initiated; collect faecal samples (mucus, mucosal admixtures) from the bedpan and with swabs (loops) directly from the rectum (the presence in the bedpan of even the traces of disin fectants affects the results of examination); inoculate without delay the collected material onto enrichment media, place them into an incubator or store them in preserving medium in the cold; send the material to the laboratory as soon as possible.

Bacteriological examination. Faecal samples are streaked onto plates with Ploskirev's

medium and onto a selenite medium containing phenol derivatives, beta-galactosides, which retard the growth of the attendant flora, in particular E. coli. The inoculated cultures are placed into a 37 °C incubator for 1S-24 hrs. The nature of tile colonies is examined on the second day.

Colourless lactose-negative colonies are subcultured to Olkenitsky's medium or to an agar slant to enrich for pure cultures. On the third day, examine the nature of the growth on Olkenitsky's medium for changes in the colour of the medium column without gas formation. Subculture the material to Hiss' media with malonate, arabinose, rhamnose, xylose, dulcite, salicine, and phenylalanine. Read the re sults indicative of biochemical activity on the following day. Shigellae ferment carbohydrates with the formation of acid

To determine the species of Shigellae, one can employ the following tests:

1.The agglutination test is performed first with a mixture of sera containing those species, and variants of Shigellae that are prevalent in a given area, and then the slide agglutination test with monoreceptor species sera.

2. The coagglutination test which allows to determine the specificity of the causative agent by a positive reaction with protein A of staphylococci coated with specific antibodies. On a suspected colony put a drop of specific sensitized protein A of Staphylococcus aureus, then rock the dish and 15 min later examine it microscopically for the appearance of the agglutinate (these tests may also be carried out on the second day of the investigation with the material from lactose-negative colonies).

3. Direct and indirect immunofluorescence test.

IFT: IFT: Salmonella enterica serovar TyphimuriumSalmonella enterica serovar Typhimurium inside (green) inside (green) and on the surface (blue) of human intestinal epithelial cells. and on the surface (blue) of human intestinal epithelial cells. Actin is labelled in red.Actin is labelled in red.

4. The indirect haemagglutination (IHA) test with erythrocyte diagnosticums with the titre of 1:160 and higher is performed. The test. is repeated after at least seven days. Diagnostically important is a four-fold rise in the anti body litre, which can be elicited from the 10th-12th day of the disease. To distinguish between patients with subclinical forms of the disease and Shigella carriers, identify immunoglobulins of the G class.5. ELISA. For the epidemiological purpose the phagovar and colicinovar of Shigellae are also identified.6. To determine whether the isolated cultures belong to the genus Shigella, perform the keratoconjunctival test on guinea pigs. In contrast to causal organisms of other intestinal infections, the dysentery Shigellae cause marked keratitis.

7. An allergic test consisting in intracutaneous injection of 0.1 ml of dysenterin is applied in the diagnosis of dysentery in adults and children. Hyperaemia and a papule 2 to 3.5 cm in diameter develop at the site of the injection in 24 hours in a person who has dysentery. The test is strictly specific.

8. An allergy intracutaneous test with Tsuverkalov's dysenterine is of supplementary significance. It becomes positive in dysentery patients beginning with the fourth day of the disease. The result is read in 24 hrs by the size of the formed papula. The test is consid ered markedly positive in the presence of oedema and skin hyperaemia 35 mm or more in diameter, moderately positive if this diameter is 20-34 mm, doubtful if there is no papula and the diameter of skin hyperaemia measures 10-15 mm, and negative if the hyperaemic area is less than 10 mm.

9. The nature of the isolated culture may be determined in some cases by its lysis by a polyvalent dysentery phage

Treatment of Shigellosis

Intravenous replacement of fluids and electrolytes;

antibiotic therapy (ampicillin frequently is not effective, and alternative therapies include sulfamethoxazole / trimethoprim and, the quinolone antibiotics such as nalidixic acid and ciprofloxacin)

Dysentery control is ensured by a complex of general and specific measures; (1) early and a completely effective clinical, epidemiological, and laboratory diagnosis; (2) hospitalization of patients or their isolation at home with observance of the required regimen; (3) thorough disinfection of sources of the disease; (4) adequate treatment of patients with highly effective antibiotics and use of chemotherapy and immunotherapy; (5) control of disease centres with employment of prophylaxis measures; (6) surveillance over foci and the application of prophylactic measures there; (7) treatment with a phage of all persons who were in contact with the sick individuals; (8) observance of sanitary and hygienic regimens in children's institutions, at home and at places of work, in food industry establishments, at catering establishments, in food stores.

Morphology. Cholera vibrios are shaped like a comma or a curved rod measuring 1-5 mcm in length and 0.3 mcm in breadth

They are very actively motile, monotrichous, nonsporeforming, noncapsulated, and Gram-negative.

Vibrio Cholerae

Scanning electron micrograph V. cholerae

Gram’s stain

Colonies of V. cholerae on bismuth-sulphit-agar

Cultivation.

Colonies of V. cholerae on blood agar

Fermentative properties.

The cholera vibrio liquefies coagulated serum and gelatin; it forms indole and ammonia, reduces nitrates to nitrites, breaks down urea, ferments glucose, levulose, galactose, maltose, saccharose, mannose, mannite, starch, and glycerine (slowly) with acid formation but does not ferment lactose in the first 48 hours, and always coagulates milk. The cholera vibrio possesses lysin and ornithine decarboxylases and oxidase activity. B. Heiberg differentiated vibrios into biochemical types according to their property of fermenting mannose, arabinose, and saccharose.

VibrioVibrio

FermentatiFermentation on

within 24 within 24 hrs hrs

Sheep

ery

thro

cyte

hem

oly

sisS

heep e

ryth

rocy

te h

em

oly

sis

Lysis b

y sp

ecifi

c O-1

sub

gro

up p

hag

es

Lysis b

y sp

ecifi

c O-1

sub

gro

up p

hag

es

Ag

glu

tinatio

n b

y O

-1 ch

ole

ra se

rum

Ag

glu

tinatio

n b

y O

-1 ch

ole

ra se

rum

Sen

sitivity

to p

oly

mix

in B

Sen

sitivity

to p

oly

mix

in B

sach

aro

sesa

charo

se

man

nose

man

nose

ara

bin

ose

ara

bin

ose

Vibrio Vibrio cholerae cholerae biovar biovar choleraecholerae

AA AA –– –– ++ ++ ++

Vibrio Vibrio cholerae cholerae biovar El Torbiovar El Tor

AA AA –– ++ ++ ++ ––

Vibrio Vibrio cholerae cholerae biovar biovar ProteusProteus

AA AA –– ++ –– –– ––

Vibrio Vibrio cholerae cholerae biovar biovar albensisalbensis

АА –– –– –– –– –– ––

Toxin production. an exotoxin (cholerogen) which is

marked by an enterotoxic effect the endotoxin also exerts a powerful

toxic effect fibrinolysin hyaluronidase collagenase mucinase lecithinase neuraminidaseproteinases

Mechanism of action of cholera enterotoxin according to Finkelstein. Cholera toxin approaches target cell surface. B subunits bind to oligosaccharide of GM1 ganglioside. Conformational alteration of holotoxin occurs, allowing the presentation of the A subunit to cell surface. The A subunit enters the cell. The disulfide bond of the A subunit is reduced by intracellular glutathione, freeing A1 and A2. NAD is hydrolyzed by A1, yielding ADP-ribose and nicotinamide. One of the G proteins of adenylate cyclase is ADP-ribosylated, inhibiting the action of GTPase and locking adenylate cyclase in the "on" mode.

Cholera toxin activates the adenylate cyclase enzyme in cells of the intestinal mucosa leading to increased levels of intracellular cAMP, and the secretion of H20, Na+, K+, Cl-, and HCO3- into the lumen of the small intestine.

Antigenic Determinants of Vibrio cholerae

Cholera is undoubtedly the most dramatic of the

water-borne diseases. The cholera vibrios are transmitted from sick

persons and carriers by food, water, flies, and contaminated hands.

Pathogenesis and diseases in man.

Cholera is characterized by a short incubation period of several hours to up to 6 days (in a disease caused by the El Tor vibrio it lasts three to five days), and the disease symptoms include general weakness, vomiting, and a frequent loose stool. The stools resemble rice-water and contain enormous numbers of torn-off intestinal epithelial cells and cholera vibrios. The major symptom of cholera is a severe diarrhea in which a patient may lose as much as 10 to 20 L or more of liquid per day. Death, which may occur in as many as 60% of untreated patients, results from severe dehydration and loss of electrolytes.

Phases in the development of the disease:

1. Cholera enteritis (choleric diarrhoea) which lasts 1 or 2 days. In some cases the infectious process terminates in this period and the patient recovers.

2. Cholera gastroenteritis is the second phase of the disease. Profuse diarrhoea and continuous vomiting lead to dehydration of the patient's body and this results in lowering of body temperature, decrease in the amount of urine excreted, drastic decrease in the number of mineral and protein substance, and the appearance of convulsions. The presence of cholera vibrios is revealed guite frequently in the vomit and particularly in the stools which have the appearance of rice water.

3. Cholera algid which is characterized by severe symptoms. The skin becomes wrinkled due to the loss of water, cyanosis appears, and the voice becomes husky and is sometimes lost completely. The body temperature falls to 35.5-34° C. As a result of blood concentration cardiac activity is drastically weakened and urination is suppressed.

Immunity

acquired after cholera is high-grade but of short duration and is of an anti-infectious (antibacterial and antitoxic) character. It is associated mainly with the presence of antibodies (lysins, agglutinins, and opsonins). The cholera vibrios rapidly undergo lysis under the influence of immune sera which contain bacteriolysins.

Laboratory diagnosis.

A strict regimen is established in the laboratory. Examinations are carried out in accordance with the general rules observed for particularly hazardous diseases.

Test specimens are collected from stools, vomit, organs obtained at autopsy, water, objects contaminated by patient's stools, and, in some cases, from foodstuffs. Certain rules are observed when the material is collected and transported to the laboratory, and examination is made in the following stages.

1. Stool smears stained by a water solution of fuchsin are examined microscopically. In the smears, the cholera vibrios occur in groups similar to shoals of fish.

2. A stool sample is inoculated into 1 per cent peptone water and alkaline agar. After 6 hours incubation at 37°C the cholera vibrios form a thin pellicle in the peptone water, which adheres to the glass. The pellicle smears are Gram stained, and the culture is examined for motility. A slide agglutination reaction is performed with specific agglutinating O-serum diluted in a ratio of 1 in 100.

Vibrio cholerae (stool smear)

The organisms are then transferred from the peptone water onto alkaline agar for isolation of the pure culture. If the first generation of the vibrios in peptone water is not visible, a drop taken from the surface layer is re-inoculated into another tube of peptone water. In some cases with such re-inoculations, an increase in the number of vibrios is achieved.

The vibrio culture grown on solid media is examined for motility and agglutinable properties. Then it is subcultured on an agar slant to obtain the pure culture.

3. The organism is identified finally by its agglutination reaction with specific O-serum, determination of its fermentative properties (fermentation of mannose, saccharose, and arabinose), and its susceptibility to phagolysis.

Colonies of Vibrio cholerae of font varying opacity (increasing from top right, left bottom right)  pseudocoloured to accentuate differences in gray-scale intensity. Of varying opacity (increasing from top left to top right, to bottom left to bottom right) pseudocoloured to accentuate differences in

grey-scale intensity. 

The following procedures are undertaken for rapid diagnosis: (1) dark field microscopy of the stool; (2) stool culture by the method of tampons incubated for 16-18 hours in an enrichment medium with repeated dark field microscopy; (3) agglutination reaction by the method of fluorescent antibodies; (4) bacterial diagnosis by isolation of cholera vibrios (the faecal mass is seeded as a thin layer into a dish containing non-inhibiting nutrient agar and grown for 4-5 hours, the vibrio colonies are detected with a stereoscopic microscope, and the culture is tested by the agglutination reaction with O-serum on glass; (5) since neuraminidase is discharged by the cholera vibrios and enters the intestine, a test for this enzyme is considered expedient as a means of early diagnosis (it is demonstrated in 66-76 per cent of patients, in 50-68 per cent of vibrio carriers, and occasionally in healthy individuals).

Treatment.

antibiotics of the tetracycline group (tetracycline, sigmamycin), amphenicol, and streptomycin are prescribed at first intravenously and then by mouth.

pathogenetic therapy is very important: control of dehydration, hypoproteinaemia, metabolic disorders, and the consequences of toxicosis, acidosis in particular, by infusion of saline (sodium and potassium) solutions, infusion of plasma or dry serum, glucose, the use of warm bath, administration of drugs which improve the tone of the heart and vessels.

Prophylaxis.

Cholera patients and vibrio carriers are the source of the disease. Individuals remain carriers of the El Tor vibrio for a lengthy period of time, for several years. Vibrios of this biotype are widely distributed in countries with a low sanitary level. They survive in water reservoirs for a long time and have been found in the bodies of frogs and oysters. Infection may occur from bathing in contaminated water and fishing for and eating shrimps, oysters, and fish infected with El Tor vibrio.The following measures are applied in a cholera focus:

1. detection of the first cases with cholera, careful registration of all sick individuals, immediate information of health protection organs;

2. isolation and hospitalization, according to special rules, of all sick individuals and carriers, observation and laboratory testing of all contacts;

3. concurrent and final disinfection in departments for cholera patients and in the focus;

4. protection of sources of water supply, stricter sanitary control over catering establishments, control of flies; in view of the possibility of El Tor vibrio reproducing in water reservoirs under favourable conditions (temperature, the presence of nutrient substrates) systematic bacteriological control over water reservoirs has become necessary, especially in places of mass rest and recreation of the population in the summer;

5. strict observance of individual hygiene; boiling or proper chlorination of water, decontamination of dishes, hand washing;

6. specific prophylaxis: immunization with the cholera monovaccine containing 8 thousand million microbial bodies per 1 ml or with the cholera anatoxin. Chemoprophylaxis with oral tetracycline is conducted for persons who were in contact with the sick individual or for patients with suspected cholera.