Escherichia Coli in Diarrhoeal Disease

• Escherichia coli is a common member of the normal flora of the

large intestine.

• As long as these bacteria do not acquire genetic elements

encoding for virulence factors, they remain benign commensals.

• Strains that acquire bacteriophage or plasmid DNA encoding

enterotoxins (a toxin produced by bacteria which causes
vomiting and diarrhoea)or invasion factors become virulent and
can cause either a plain, watery diarrhoea or an inflammatory
dysentery or bacteraemia .

• These diseases are most familiar to endemic countries,

particularly among infants.

• Three groups of E coli are associated with diarrhoeal diseases

1 .
Escherichia Coli in Diarrhoeal Disease
• Escherichia coli strains that produce enterotoxins are called
enterotoxigenic E coli (ETEC).

• There are numerous types of enterotoxin. Some of these

toxins are cytotoxic, damaging the mucosal cells, whereas
others are merely cytotonic, inducing only the secretion of
water and electrolytes.

• A second group of E coli strains have invasion factors and

cause tissue destruction and inflammation resembling the
effects of Shigella. This group of enteroinvesive Escherichia
coli(EIEC).

• A third group of serotypes, called enteropathogenic E coli

(EPEC), are associated with outbreaks of diarrhoeal in
newborn; nurseries, but produce no recognisable toxins or
invasion factors. 2
• Enterotoxigenic
• Entero invasive
• Entero pathogenic

3
Clinical Manifestations

4
 Clinical Manifestations
• The diarrhoeal disease caused by
enterotoxigenic ETEC is characterised by a
rapid onset of watery, nonbloody diarrhoea of
considerable volume, accompanied by little
or no fever.

• Other common symptoms are abdominal

pain, malaise, nausea, and vomiting.

• Diarrhoea and other symptoms cease

spontaneously after 24 to 72 hours. 5
 Structure,
• ETEC organisms are Gram-negative, short rods
not visibly different from E coli found in the normal
flora of the human large intestine.

• Virulence-associated fimbriae are too small to be

seen by light microscopy.

• All ETEC contain plasmids (small independent

circle of DNA that replicate itself independently of
chromosomal DNA especially in bacterial cells), but
this is only distinguished using molecular biology
techniques to detect specific virulence-associated
genes on these plasmids. 6
Classification, and Antigenic Types
• Escherichia coli serotypes are specific O-group/H-antigen combinations.

• The H antigens are the flagella antigens, of which there are at least 56
types.

• Escherichia coli isolates may be nonmotile and nonflagellated and hence

H negative (H).

• H typing is important for E coli associated with diarrhoeal disease for two
reasons.

• First, a strain causing an outbreak or epidemic can be differentiated from

the normal stool flora by its unique O:H antigenic makeup.

• Second, most ETEC belong to specific serotypes O:H; this relationship

facilitates their identification .

7
 Enteric Infections
• Enterohemorrhagic E. coli (EHEC) occur largely as
a single serotype (O157:H7) causing sporadic cases
and outbreaks of hemorrhagic colitis characterized
by bloody diarrhoea.

• EHEC also may cause haemolytic uremic syndrome

(HUS), an association of haemolytic anemia,
thrombocytopenia, and acute renal failure.

• Enteroaggregative (EAEC) types exhibit a

characteristic aggregative pattern of adherence and
produce persistent gastroenteritis and diarrhoea
in infants and children in developing countries.
8
 Pathogenesis

• Escherichia coli diarrhoeal disease is

contracted orally by ingestion of food or water
contaminated with a pathogenic strain shed by
an infected person.

• ETEC diarrhoea occurs in all age groups, but

mortality is most common in infants,
particularly in the most undernourished or
malnourished infants in developing nations.

9
 Pathogenesis
• The pathogenesis of ETEC diarrhoea involves two
steps:

• (1) intestinal colonisation,

• (2) elaboration of diarrheagenic enterotoxin(s) ET

is actually a family of toxic peptides ranging from 18
to 50 amino acid residues in length.

• Those termed ETa can stimulate intestinal

guanylate cyclase, the enzyme that converts
guanosine 5'-triphosphate (GTP) to cyclic
guanosine 5'-monophosphate (cGMP).
10
 Pathogenesis

• Increased intracellular cGMP inhibits intestinal fluid

uptake, resulting in net fluid secretion.

• Those termed ETb do not seem to cause diarrhoea by

the reverse mechanism ie increase the (GTP) and
increase the fluid uptake in the intestine.

• One method for testing suspect E coli isolates for ET

production involves injection of culture supernatant fluids
into the stomach of infant mice and seeing whether
diarrhoea ensues.

• However Specific DNA gene probes and PCR assays

have been developed to test isolated colonies for the
presence of different genes encoding. 11
 Host Defences
• As in any orally transmitted disease, the first line of defence
against ETEC diarrhoea is gastric acidity and the ratio of
normal flora to the pathogens.

• Other non-specific defences are small-intestinal motility and

a large population of normal flora in the large intestine.

• Intestinal secretory immunoglobulin (IgA) directed against

surface antigens such as the cytotoxic factor antigens CFAs
and against other enterotoxins appears to be the key to
immunity from ETEC diarrhoea.

• Passive immune protection of infants by colostral (Milk)

antibody is important.

• Human breast milk also contains nonimmunoglobulin factors

(receptor-containing molecules) that can neutralize E coli 12
toxins and CFAs.
 Epidemiology
• Escherichia coli diarrhoeal disease of all types is transmitted from person
to person with no known important animal vectors. Unlike shigella

• The incidence of E coli diarrhoea is clearly related to hygiene, food

processing sophistication, general sanitation, and the opportunity for
contact.

• The geographic frequency of ETEC diarrhoea is inversely proportional to

the sanitation standards.

• Single-source outbreaks of ETEC diarrhoea involving contaminated

water supplies or food have been found in adults world wide.

• Adults travelling from temperate climates to more tropical areas typically

experience traveller's diarrhoea caused by ETEC.

• This phenomenon is not readily explained, but contributing factors are

low levels of immunity and an increased opportunity for infection.
13
 Diagnosis
• ETEC diarrhoea is characterised by copious watery
diarrhoea with little or no fever.

• The diarrhoeal stool yields a virtually pure culture of

E coli.

• Since the disease is self-limiting, virulence testing of

isolates and serotyping is impractical except in an
outbreak situation.

• Confirmation is achieved by serotyping, serologic

identification of a specific cytotoxic factor antigen
CFA on isolates, and identification of genes 14
encoding these virulence factors.
Laboratory methods for isolation and
identification of ETEC.

15
 Control
• Escherichia coli diarrhoeal disease is best
controlled by preventing transmission and by
stressing the importance of breast-feeding of
infants, especially where ETEC is endemic.

• The best treatment is oral fluid and electrolyte

replacement (intravenous in severe cases).

• Antibiotics are not recommended because this

practice leads to an increased burden of
antibiotic-resistant pathogenic E coli and of
more life-threatening enteropathogens.
16
 Escherichia, Klebsiella, Enterobacter,Serratia,
Citrobacter, and Proteus
• The Gram-negative bacilli of the genera
Escherichia, Klebsiella, Enterobacter, Serratia,
Citrobacter, and Proteus are members of the
normal intestinal flora of humans and animals and
may be isolated from a variety of environmental
sources.

• With the exception of Proteus, they are sometimes

collectively referred to as the coliform bacilli
because of shared properties, particularly the ability
of most species to ferment the sugar lactose.
17
Escherichia, Klebsiella, Enterobacter,Serratia,
Citrobacter, and Proteus

• Many of these microorganisms used to be dismissed as

harmless commensals.
• Today, they are known to be responsible for major health
problems worldwide.
• A limited number of species, including
• E coli,
• Klebsiella pneumoniae,
• Enterobacter aerogenes,
• Enterobacter cloacae,
• Serratia marcescens,
• And P mirabilis, are responsible for most infections
produced by this group of organisms.

18
 Escherichia, Klebsiella, Enterobacter,Serratia,
Citrobacter, and Proteus

• The increasing incidence of the coliforms, Proteus, and

other Gram-negative organisms in diseases reflects in
part a better understanding of their pathogenic potential
but more importantly the changing ecology of bacterial
disease.

• The widespread and often indiscriminate use of

antibiotics has created drug-resistant Gram-negative
bacilli that readily acquire multiple resistance through
transmission of drug resistance plasmids (R factors).

• Also, development of new surgical procedures, health

support technology, and therapeutic regimens has
provided new portals of entry and compromised many
host defences. 19
 Clinical Manifestations
• As opportunistic pathogens, the coliforms
and Proteus take advantage of weakened
host defences to colonise and elicit a variety
of disease states .

• Together, the many disease syndromes

produced by these organisms are among the
most common infections in humans requiring
medical intervention.

20
Sites of colonisation and extraintestinal disease production
by the coliforms and Proteus.

21
 Salmonella
• Salmonellae are ubiquitous human and animal
pathogens, and salmonellosis, a disease that affects
many people worldwide each year, is common
throughout the world.

• Salmonellosis in humans usually takes the form of a self-

limiting food poisoning (gastroenteritis), but occasionally
manifests as a serious systemic infection (enteric fever)
which requires prompt antibiotic treatment.

• In addition, salmonellosis causes substantial losses of

livestock.

22
 Clinical Manifestations
• Three clinical forms of salmonellosis:
• (1) gastroenteritis,
• (2) septicaemia
• (3) enteric fevers.

23
 Clinical Manifestations
• (1) gastroenteritis :The incubation period for Salmonella gastroenteritis
(food poisoning) depends on the dose of bacteria.

• Symptoms usually begin 6 to 48 hours after ingestion of contaminated food

or water and usually take the form of nausea, vomiting, diarrhea, and
abdominal pain.

• Myalgia and headache are common; however, the cardinal manifestation

is diarrhea.

• Fever (38oC to 39oC) and chills are also common.

• At least two-thirds of patients complain of abdominal cramps.

• The duration of fever and diarrhea varies, but is usually 2 to 7 days.

24
 Clinical Manifestations

• (2) septicaemia :The septicaemia form of

salmonella infection can be an intermediate stage
of infection in which the patient is not experiencing
intestinal symptoms and the bacteria cannot be
isolated from faecal specimens.

• The severity of the infection and whether it remains

localized in the intestine or disseminates to the
bloodstream may depend on the resistance of the
patient and the virulence of the Salmonella isolate.
25
 Clinical Manifestations
• (3) Enteric fevers: enteric fevers are severe systemic forms of
salmonellosis.

• The best studied enteric fever is typhoid fever, the form caused by S
typhi, but any species of Salmonella may cause this type of disease.

• The symptoms begin after an incubation period of 10 to 14 days.

• Enteric fevers may be preceded by gastroenteritis, which usually

resolves before the onset of systemic disease.

• The symptoms of enteric fevers are non-specific and include fever,

anorexia, headache, myalgias, and constipation.

• Enteric fevers are severe infections and may be fatal if antibiotics are not
promptly administered

26
 Structure,

• Salmonellae are Gram-negative, flagellated, facultative anaerobic

bacilli possessing three major antigens:
• H or flagella antigen;
• O or somatic antigen;
• Vi antigen (possessed by only a few serovars).

• O antigens occur on the surface of the outer membrane and are

determined by specific sugar sequences on the cell surface.
• Vi antigen is a superficial antigen overlying the O antigen; it is
present in a few serovars, the most important being S typhi.

27
 Classification,
• Antigenic analysis of salmonellae by using
specific antisera offers clinical and
epidemiological advantages.

• Determination of antigenic structure permits one

to identify the organisms clinically and assign
them to one of nine serogroups

• H antigen also provides a useful epidemiologic

tool with which to determine the source of
infection and its mode of spread.
28
 Antigenic Types
• As with other Gram-negative bacilli, the cell envelope of salmonellae
contains a complex lipopolysaccharide (LPS) structure that is liberated
on lysis of the cell and, to some extent, during culture.

• The lipopolysaccharide moiety may function as an endotoxin, and may

be important in determining virulence of the organisms.

• This macromolecular endotoxin complex consists of three components,

• an outer O-polysaccharide coat,

• a middle portion (the R core),

• and an inner lipid A coat.

• Lipopolysaccharide structure is important for several reasons.

• First: is responsible for O antigen specificity; it may also help determine

29
the virulence of the organism.
 Antigenic Types
• Salmonellae lacking the Lipopolysaccharide are called rough because of the
rough appearance of the colonies; they are usually avirulent or less virulent
than the smooth strains which possess a full complement of
Lipopolysaccharide

• Second, antibodies directed against the R core (common enterobacterial

antigen) may protect against infection by a wide variety of Gram-negative
bacteria sharing a common core structure or may moderate their lethal
effects.

• Third, the endotoxin component of the cell wall may play an important role in
the pathogenesis of many clinical manifestations.

• Endotoxins evoke fever, activate the serum complement, kinin, and clotting
systems, depress myocardial function, and alter lymphocyte function.

• Circulating endotoxin may be responsible in part for many of the

manifestations of septic shock that can occur in systemic infections.
30
 Pathogenesis
• Salmonellosis includes several syndromes (gastroenteritis, enteric
fevers, septicaemia, focal infections, and an asymptomatic carrier state) .
however, on occasion, any serotype can produce any of the syndromes.

• Particular serovars show a strong propensity to produce a particular

syndrome
• S typhi,
• S paratyphi-A,
• S schottmuelleri produce enteric fever;

• S. choleraesuis produces septicaemia or focal infections;

• S typhimurium
• S enteritidis produce gastroenteritis);

• In general, more serious infections occur in infants, in adults over the age
of 50, and in subjects with debilitating illnesses.
31
Pathogenesis of salmonellosis.

32
Scheme of the Pathogenesis of Salmonella
enterocolitis and diarrhoea

33
Invasion of intestinal mucosa by Salmonella.

34
 Invasion of intestinal mucosa by Salmonella.
• After invading the epithelium, the organisms multiply
intracellularly and then spread to mesenteric lymph nodes
and throughout the body via the systemic circulation; they
are taken up by the reticuloendothelial cells.

• The reticuloendothelial system confines and controls spread

of the organism.

• However, depending on the serotype and the effectiveness

of the host defences against that serotype, some organisms
may infect the liver, spleen, gallbladder, bones, meninges,
and other organs .

• Fortunately, most serovars are killed promptly in

extraintestinal sites, and the most common human
Salmonella infection, gastroenteritis, remains confined to the
intestine.
35
 Invasion of intestinal mucosa by Salmonella.

• Only strains that penetrate the intestinal mucosa are associated with the
appearance of an acute inflammatory reaction and diarrhoea ; the
diarrhoea is due to secretion of fluid and electrolytes by the small and
large intestines.

• Salmonella penetrate the intestinal epithelial cells but, phagosotised. Thus,

the extent of intercellular spread and ulceration of the epithelium is
minimal.

• Salmonella escape from the basal side of epithelial cells into the lamina
propria.

• Systemic spread of the organisms can occur, giving rise to enteric fever.

• Invasion of the intestinal mucosa is followed by activation of mucosal

adenylate cyclase; the resultant increase in cyclic AMP induces secretion.

• In addition, Salmonella strains elaborate one or more enterotoxin-like

substances which may stimulate intestinal secretion. 36
 Invasion of intestinal mucosa by Salmonella.
• These evoke an acute inflammatory response
and may also be responsible for damage to
the intestine.

• Because of the intestinal inflammatory

reaction, symptoms of inflammation such as
fever, chills, abdominal pain, leukocytosis, and
diarrhoea are common.

• The stools may contain polymorphonuclear

leukocytes, blood, and mucus. 37
 Invasion of intestinal mucosa by Salmonella.
• After invading the intestine, most salmonellae induce an
acute inflammatory response, which can cause ulceration.
• They may elaborate cytotoxins that inhibit protein synthesis.
• However, invasion of the mucosa causes the epithelial cells
to synthesize and release various pro-inflammatory
cytokines, including:
• IL-1,
• IL-6,
• IL-8,
• TNF-2,
• IFN-U, Interferon meu
• MCP-1 monocyte chemottractant Protein-1
• GM-CSF. Granucyte macrophage colony stimulating factors

38
 Host Defences

• Various host defences are important in resisting intestinal

colonisation and invasion by Salmonella .

• Normal gastric acidity (pH < 3.5) is lethal to salmonellae.

• In healthy individuals, the number of ingested salmonellae is

reduced in the stomach, so that fewer or no organisms enter
the intestine.

• Normal small intestinal motility also protects the bowel by

sweeping ingested salmonellae through quickly.

• The normal intestinal microflora protects against

salmonellae, probably through anaerobes, which liberate
short-chain fatty acids that are thought to be toxic to 39
salmonellae.
 Host Defences
• Alteration of the anaerobic intestinal flora by antibiotics
renders the host more susceptible to salmonellosis.

• Secretor or mucosal antibodies also protect the intestine

against salmonellae.

• When these host defences are absent or blunted, the host

becomes more susceptible to salmonellosis.

• For example, in AIDS, Salmonella infection is common,

frequently persistent and bacteraemic, and often resistant to
even prolonged antibiotic treatment.

• Relapses are common.

40
 Epidemiology
• Contaminated food is the major mode of transmission for
non-typhoidal salmonellae because salmonellosis is a
zoonosis and has an enormous animal reservoir.
• The most common animal reservoirs are
• chickens,
• turkeys,
• pigs,
• cows;
• dozens of other domestic and wild animals also harbour
these organisms.

• Because of the ability of salmonellae to survive in meats

and animal products that are not thoroughly cooked, animal
products are the main vehicle of transmission.

• The magnitude of the problem is demonstrated by the 41

following recent yields of salmonellae:
 Epidemiology
• The epidemiology of typhoid fever and other enteric
fevers primarily involves person-to-person spread
because these organisms lack a significant animal
reservoir transmitter.

• Contamination with human faeces is the major

mode of spread, and the usual vehicle is
contaminated water.

• Occasionally, contaminated food (usually handled

by an individual who harbours S typhi) may be the
vehicle.
42
 Epidemiology
• In typhoid fever and non-typhoidal salmonellosis, two other factors have
epidemiologic significance.

• First, an asymptomatic human carrier state exists for the agents of either form of
the disease.

• Approximately 3% of persons infected with S typhi and 0.1% of those infected with
non-typhoidal salmonellae become chronic carriers.

• The carrier state may last from many weeks to years.

• Thus, human as well as animal reservoirs exist.

• Second, use of antibiotics in animal feeds and indiscriminate use of antibiotics in

humans increase antibiotic resistance in salmonellae by promoting transfer of R
factors.

• Salmonellosis is a major public health problem because of its large and varied
animal reservoir, the existence of human and animal carrier states, and the lack of
a concerted nationwide program to control salmonellae.
43
 Diagnosis
• The diagnosis of salmonellosis requires bacteriologic isolation of the
organisms from appropriate clinical specimens.

• Laboratory identification of the genus Salmonella is done by

biochemical tests; the serologic type is confirmed by serologic testing.

• Faeces, blood, or other specimens should be plated on several

nonselective and selective agar media (blood, MacConkey, eosin-
methylene blue, bismuth sulfite, Salmonella-Shigella, and brilliant green
agars) as well as into enrichment broth such as selenite or
tetrathionate.

• Any growth in enrichment broth is subsequently subculture onto the

various agars.

• The biochemical reactions of suspicious colonies are then determined

on triple sugar iron agar and lysine-iron agar, and a presumptive
identification is made.
44
 Diagnosis

• Biochemical identification of salmonellae has been

simplified by systems that permit the rapid testing of 10-
20 different biochemical parameters simultaneously.

• The presumptive biochemical identification of Salmonella

then can be confirmed by antigenic analysis of O and H
antigens using polyvalent and specific antisera.

• Fortunately, approximately 95% of all clinical isolates

can be identified with the available group A-E typing
antisera.

• Salmonella isolates then should be sent to a central or

reference laboratory for more comprehensive serologic
testing and confirmation. 45
 Control
• Salmonellae are difficult to eradicate from the environment.

• However, because the major reservoir for human infection is poultry and livestock,
reducing the number of salmonellae harboured in these animals would significantly
reduce human exposure.

• All animal feeds should be treated to kill salmonellae before distribution, resulting in
a marked reduction in salmonellosis.

• Other helpful measures include changing animal slaughtering practices to reduce

cross-contamination of animal carcasses;

• protecting processed foods from contamination;

• providing training in hygienic practices for all food-handling personnel in

slaughterhouses,

• food processing plants, and restaurants;

• cooking and refrigerating foods adequately in food processing plants, restaurants,

46
and homes; and expanding of governmental enteric disease surveillance programs.
 Control
• Vaccines are available for typhoid fever
and are partially effective, especially in
children.

• Continued research in this area and

increased understanding of the
mechanisms of immunity to enteric
infections are of great importance.

47
 Control

• General salmonellosis treatment measures include replacing fluid loss by

oral and intravenous routes, and controlling pain, nausea, and vomiting.

• Specific therapy consists of antibiotic administration.

• Typhoid fever and enteric fevers should be treated with antibiotics.

• Antibiotic therapy of non-typhoidal salmonellosis should be reserved for the

septicaemia, enteric fever, and focal infection syndromes.

• Prolonged antibiotics are not recommended for uncomplicated Salmonella

gastroenteritis because they increase the number of antibiotic-resistant
strains.

48
 Shigella
• Gram-negative, facultative anaerobes of the genus Shigella are the
principal agents of bacillary dysentery.

• This disease differs from profuse watery diarrhea, as is commonly seen

in choleraic diarrhea or in enterotoxigenic Escherichia coli diarrhea, in
that the dysenteric stool is scanty and contains blood, mucus, and
inflammatory cells.

• In some individuals suffering from shigellosis, however, moderate

volume diarrhea is a prodrome (indicator) or the sole manifestation of
the infection.

• Bacillary dysentery constitutes a significant proportion of acute intestinal

disease in children of developing countries, and this infection is a major
contributor to stunted growth of these children.

• Shigellosis also presents a significant risk to travellers visiting endemic

areas, and sporadic food or water-borne outbreaks occur in developed
countries. 49
• S flexneri…diarrhoear
• S.sonnei…. Sporadic outbreaks in
developed countries
• S dysenterae…bacteremia…HUS,
arthritis,

50
 Shigella
• The pathogenic mechanism of shigellosis is complex,
involving a possible enterotoxic and/or cytotoxic
diarrheal, cytokine-mediated inflammation of the
colon, and necrosis of the colonic epithelium.

• The underlying physiological that initiates this

inflammatory cascade is the invasion of Shigella into
the colonic epithelium and the lamina propria.

• The resulting colitis and ulceration of the mucosa

result in bloody, mucoid stools, and/or febrile
diarrhea.
51
 Clinical Presentation
• Shigellosis has two basic clinical presentations:

• (1) watery diarrhoea associated with vomiting and

mild to moderate dehydration, and

• (2) dysentery characterised by a small volume of

bloody, mucoid stools, and abdominal pain (cramps
and tenesmus)

• Volunteer challenge studies show that shigellosis

can be evoked by an extremely small inoculums
(101-102 organisms), and the time of onset of
symptoms is somewhat influenced by the size of the
challenge and the immune status of the host.
52
 Clinical Presentation
• The most important point is that shigellosis
is an acute infection with onset of
symptoms usually occurring within 24-48
hours of ingestion of the etiologic agent.

• The average duration of symptoms in

untreated adults is 7 days, and the
organism may be cultivated from stools for
30 days or longer.

53
 Clinical Presentation
• The clinical features of shigellosis
are

• Watery diarrhoea occurs as a sole

clinical manifestation, in a majority
of patients infected with S sonnei.

• Diarrhoea is often of the dysentery

characterising infection with other
species of Shigella. 54
 Clinical Presentation
• Recently discovered enterotoxins secreted by
S. flexneri may contribute to the diarrhoeal
phase as the etiologic agents traverse the
small intestine.

• However, diarrhoea is most common in

patients who have colitis involving the
transverse colon or cecum.
• These patients evidence net water secretion
and impaired absorption in the inflamed colon.
55
 Clinical Presentation
Dysentery is also characterised by the daily loss of
200-300 ml of serum protein into the faeces.

• This loss of serum proteins results in depletion of

nitrogen stores that encourage malnutrition and
growth stunting.

• Depletion of immune factors also increases the risk

of concurrent, unrelated infectious disease and
contributes to substantial mortality.

56
Pathogenesis of shigellosis in humans.

57
 Pathogenesis of shigellosis in humans.
• Possible complications of shigellosis include bacteraemia,
convulsions and other neurological complications,
reactive arthritis, and haemolytic-uraemic syndrome.

• Bacteraemia occasionally accompanies S dysenteriae

serotype 1 infections in malnourished infants, but this
complication is uncommon in otherwise healthy individuals.

• Convulsions have been reported in up to 25% of Shigella

infections involving children under the age of 4 years.

• Both high fever and a family history of seizures are risk

factors for a convulsive episode.

58
 Host Defence
• Shigellae are remarkably infectious enteric
pathogenes that can cause disease after the
ingestion of as few as 101 organisms.

• Nonetheless, shigellosis is normally an acute, self-

limiting disease that exemplifies the regenerative
capacity of the intestinal epithelium.

• Shigella virulence probably reflects both the

efficient uptake by the follicle associated epithelium
cells and the amplifying effect of the inflammatory
cascade generated by apoptic macrophages.
59
 Host Defence
• In endemic areas, shigellosis is essentially a
childhood disease, and the incidence decreases
drastically in the indigenous population over 5 years
of age.

• Controlled volunteer challenge studies in adults

also indicate that prior infection with S. flexneri
protects against re-infection with the homologous
serotype (70% efficacy).

• Serotype-specific immune protection against

shigellosis suggests that antibody recognising the
O-polysaccharide of lipopolysaccharide (LPS)
protects against clinical symptoms. 60
 Host Defence

• Ingested bovine colostrum containing antibody

recognising the O-polysaccharide of S flexneri 2a
passively protects volunteers challenged with the
homologous Shigella serotype.

• These observations have encouraged the development

of a number of parenteral and mucosally administered
O-polysaccharide vaccines that are currently in safety
and/or efficacy trials.

• These vaccines offer the possibility of effective control of

shigellosis independent of the needed improvements in
the public health infrastructure of developing countries,
but licensure and delivery of practical Shigella vaccines
remains a distant prospect. 61
 Epidemiology
• Humans are the primary reservoir of
Shigella species, with captive
subhuman primates as accidental
hosts.

• In developing countries with prevailing

conditions of inadequate sanitation
and overcrowded housing, the
infection is transmitted most often by
the excreta of infected individuals via
direct faecal-oral contamination.
62
 Epidemiology

• Flies may contribute to spread from faeces

to food.

• The most common species, S dysenteriae

and S flexneri, are also the most virulent.

• In developed countries, sporadic common-

source outbreaks, predominantly involving
S sonnei, are transmitted by uncooked
food or contaminated water.
63
 Epidemiology
• The latter outbreaks usually involve semi-public
water systems such as those found in camps,
trailer parks, and local reservations.

• Direct faecal-oral spread can also occur in

institutional environments such as child day-care
centres, hospitals, and nursing homes.

• Homosexual men are also at increased risk for

direct transmission of Shigella flexneri infections,
and chronic, illness complicating HIV infection.
64
 Diagnosis Clinical

• Patients presenting with watery diarrhoea and fever

should be suspected of having shigellosis.

• The diarrhoeal stage of the infection cannot be

distinguished clinically from other bacterial, viral, and
protozoan infections.

• Nausea and vomiting can accompany Shigella diarrhoea,

but these symptoms are also observed during infections
with nontyphoidal salmonellae and enterotoxigenic E
coli.

• However bloody, mucoid stools are highly indicative of

shigellosis, but the differential diagnosis should include
Salmonella enteritidis, Yersinia enterocolitica,
Campylobacter species, and Entamoeba histolytica. 65
 Diagnosis Clinical

• Although blood is common in the stools of

patients with amebiasis, it is usually dark
brown rather than bright red, as in Shigella
infections.

• Microscopic examination of stool smears

from patients with amoebiasis should
reveal erythrophagocytic trophozoites in
the absence of PMN, whereas bacillary
dysentery is characterized by sheets of
PMN.

66
 Laboratory Diagnosis
• Although clinical signs may evoke the
suspicion of shigellosis, diagnosis is
dependent upon the isolation and
identification of Shigella from the
faeces.

• Positive cultures are most often

obtained from blood-tinged plugs of
mucus in freshly passed stool
specimens obtained during the acute
phase of disease. 67
 Laboratory Diagnosis
• Rectal swabs may also be used to culture
shigellae if the specimen is processed
rapidly or is deposited in a buffered
glycerol saline holding solution.

• Isolation of shigellae in the clinical

laboratory typically involves an initial
streaking for isolation on
differential/selective media with aerobic
incubation to inhibit the growth of the
anaerobic normal flora.
68
 Laboratory Diagnosis
• Commonly used primary isolation media include
MacConkey, Hektoen Enteric Agar, and Salmonella-
Shigella (SS) Agar.

• These media contain bile salts to inhibit the growth

of other Gram-negative bacteria and pH indicators
to differentiate lactose fermenters (Coliforms) from
non-lactose fermenters such as shigellae.

• A liquid enrichment medium (Hajna Gram-negative

broth) may also be inoculated with the stool
specimen and subcultured onto the
selective/differential agarose media after a short
growth period.
69
 Laboratory Diagnosis
• Following overnight incubation of primary
isolation media at 37° C, colorless, non-lactose-
fermenting colonies are streaked and stabbed
into tube slants of Kligler's Iron Agar or Triple
Sugar Iron Agar.
• In these differential media, Shigella species
produce an alkaline slant and an acid butt with
no bubbles of gas in the agar.
• This reaction gives a presumptive identification,
and slide agglutination tests with antisera for
serogroup and serotype confirm the
identification. 70
 Treatment

• Although severe dehydration is uncommon in shigellosis,

the first consideration in treating any diarrheal disease is
correction of abnormalities that result from isotonic
dehydration, metabolic acidosis, and significant
potassium loss.

• The oral rehydration treatment developed by the World

Health Organization has proven effective and safe in the
treatment of acute diarrhea, provided that the patient is
not vomiting or in shock from severe dehydration.

• In the latter case, intravenous fluid replacement is

required until initial fluid and electrolyte losses are
corrected. intestinal stasis and may promote bacterial
invasion, prolonging the febrile state. 71
 Treatment
• With proper hydration, shigellosis is generally a
self-limiting disease, and the decision to prescribe
antibiotics is predicated on the severity of disease,
the age of the patient, and the likelihood of further
transmission of the infection.

• Effective antibiotic treatment reduces the average

duration of illness from approximately 5-7 days to
approximately 3 days and also reduces the period
of Shigella excretion after symptoms subside.

• Absorbable drugs such as ampicillin (2 g/day for 5

days) are likely to be effective when the isolate is
sensitive.
72
 Treatment

• Trimethoprim (8 mg/kg/day) and

sulfamethoxazole (40 mg/kg/day) will
eradicate sensitive organisms quickly from the
intestine, but resistance to this agent is
increasing.

• Ciprofloxacin (1 g/day for 3 days) is effective

against multiple drug resistant strains, but this
antibiotic is not approved for use in children
less than 17 years of age because there is a
theoretical risk of cartilage damage.
73
 Control
• As is the case with other intestinal infections, the
most effective methods for controlling shigellosis are
provision of safe and abundant water and effective
faeces disposal.

• These public health measures are, at best, long

range strategies for control of enteric infections in
developing countries.

• The estimated five million deaths annually attributed

to diarrhoeal disease in these countries, in addition
to the malabsorption and growth stunting among
survivors, require more immediate and practical
approaches.
74
 Control
• The most effective intervention strategy to minimise morbidity
and mortality would involve comprehensive media and personal
outreach programs consisting of the following components:

• (1) education of all residents to actively avoid faecal

contamination of food and water and to encourage hand
washing after defecation;

• (2) encourage mothers to breast-feed infants;

• (3) promote the use of oral rehydration therapy to offset the

effects of acute diarrhoea;

• (4) encourage mothers to provide convalescent nutritional care

in the form of extra food for children recovering from diarrhoea
or dysentery. 75
 Nosocomial Infections
• Nosocomial Infections: The coliforms and Proteus are
responsible for most resent nosocomial (hospital-
acquired) infections in the world.

• Estimates of nosocomial infections in world hospitals

suggest that estimated 40 million annual admissions,
had at least one nosocomial infection.

• Thus, the coliforms and Proteus probably are

responsible for most hospital-acquired infections.

• Aside from the enormous cost measured in human life,

nosocomial infections prolong the duration of
hospitalisation by an average of 4 days and increase the
cost of medical care by $4.5 billion a year.
76
 Nosocomial Infections
• The aetiology of nosocomial infections has markedly
changed during past decades.

• Streptococci were the major nosocomial pathogens

in the preantibiotic era.

• However, following the introduction and use of

sulphonamides and penicillin, Staphylococcus
aureus has become the predominant pathogen.

• Aerobic gram negative rods e.g(pseudomonas

aeroginosa) gained prominence as nosocomial
pathogens with widespread use of aminoglycosides
and first generation cephalosporins. 77
 Nosocomial Infections
• Subsequent widespread use of broad spectrum
cephalosporins was associated with changes
in the frequency and aetiology of nosocomial
infections earlier with the trend towards certain
gram-positive pathogens.

• For example, in nosocomial bloodstream

infections marked increases in the incidence of
coagulase-negative staphylococci, S. albus,
enterococci, and Candida albicans infections
occurred.
78
 Nosocomial Infections
• The highest numbers of nosocomial infections
NI occur in surgical and medicine services.
• Among surgical patients, highest rates of
nosocomial infections occur with surgery on the
stomach (21%)
• and bowel (19%),
• Craniotomies=skull studies (18%),
• coronary artery bypass graft procedures (11%)
• and other cardiac surgery (10%).
• High rates also are observed with burn (15%)
79
 Nosocomial Infections
• In order of decreasing frequency, the major sites
of nosocomial infection are the urinary tract,
surgical sites, bloodstream, and lower
respiratory tracts.
• The coliforms and Proteus were responsible for
• 46% of urinary tract
• and 24% of surgical site infections,
• 17% of the bacteraemia,
• and 30% of the pneumonias

80
 Nosocomial Infections
• Escherichia coli, the predominant nosocomial pathogen, is the
major cause of infection in the urinary tract and is common in
other body sites.

• Staphylococcus aureus and Pseudomonas aeruginosa

followed by Enterobacter and Klebsiella are currently the most
common pathogens in nosocomial pneumonias,

• Coagulase-negative staphylococci have replaced E. coli as

the predominant pathogen in blood stream infections.

• The major causes of surgical site infections are S. aureus,

coagulase- positive staphylococci, and enterococci proteus
spp.
81
• Urinary…escherichia coli
• Surgical…. S. aureus, coagulase- positive
staphylococci,
• Blood stream…… Coagulase-negative
staphylococci
• Respiratory sites…. Staphylococcus
aureus and Pseudomonas aeruginosa

82
 Nosocomial Infections
• Other coliform bacilli and Proteus have been
incriminated in various hospital-acquired
infections.
• Klebsiella,
• Enterobacter,
• and Serratia species are frequent causes of
bacteraemia at some medical centres and also
are frequently involved in infections associated
with respiratory tract manipulations, such as
tracheostomy and procedures using
contaminated inhalation therapy equipment. 83
 Nosocomial Infections
• Klebsiella and Serratia species commonly cause
infections following intravenous and urinary
catheterization and infections complicating burns.

• Proteus species frequently cause nosocomial

infections of the urinary tract, surgical wounds, and
lower respiratory tract.

• Less frequently, Proteus species cause bacteraemia,

most often in elderly patients.

• outbreaks of bacteraemia can be caused by

contaminated commercial fluids for intravenous
injections, involving Enterobacter cloacae, 84
Enterobacter agglomerans, and Citrobacter freundii.
 Nosocomial Infections
• The role of Citrobacter species in human disease is not as
great as that of the other coliforms and Proteus.
• Citrobacter freundii and Citrobacter diversus (C koseri)
have been isolated predominantly as superinfecting agents
from urinary and respiratory tract infections.
• Citrobacter septicaemia may occur in patients with multiple
predisposing factors;
• Citrobacter species also cause meningitis,
• septicaemia,
• and pulmonary infections in neonates and young children.
• Neonatal meningitis produced by Citrobacter diversus,
while uncommon, is associated with a very high frequency of
brain abscesses, death, and mental retardation in survivors.
• Although E coli and group B streptococci cause most cases
of neonatal meningitis, the most common cause of brain
abscesses in neonatal meningitis is P mirabilis.(proteus) 85
 Nosocomial Infections
• Immunocompromised patients often develop non-hospital-acquired
infections with coliforms.

• For example, group B streptococci and E coli are responsible for most cases
of neonatal meningitis, with the latter accounting for about 40 percent of
cases.

• Infections seen in cancer patients with solid tumours or malignant blood

diseases frequently are caused by E coli, Klebsiella, Serratia, and
Enterobacter species.

• Such infections often have lethal course.

• Individuals who are immunosuppressed by therapy (e.g., cancer patients or

transplant recipients) or by congenital defects of the immune system may
develop Klebsiella, Enterobacter, and Serratia infections.

• Many additional factors such as diabetes, trauma, and chronic lung disease
may predispose to infection by coliforms and other microbes
86
 Community-Acquired Infections
• The coliform organisms and Proteus species are major causes of diseases
acquired outside the hospital; many of these diseases eventually require
hospitalisation.

• Escherichia coli causes approximately 85 percent of cases of urethrocystitis

(infection of the urethra and bladder),

• about 80 percent of cases of chronic bacterial prostatitis,

• and up to 90 percent of cases of acute pyelonephritis (inflammation of the renal

pelvis and parenchyma).

• Approximately one half of females have had a urinary tract infection by their late
twenties due to E coli from their faecal flora.

• Proteus, Klebsiella, and Enterobacter species are among the other organisms
most frequently involved in urinary tract infections.

• Proteus, particularly P mirabilis, is believed to be the most common cause of

infection-related kidney stones, one of the most serious complications of
unresolved or recurrent bacteriuria.
87
• Urethrocystis
• Proctatis
• Pyelonephritis….renal pelvis
• Proteus mirabilis with kidney stones

88
 Community-Acquired Infections
• Klebsiella was first recognized clinically as an agent of pneumonia.

• Klebsiella pneumoniae accounts for a small percentage of pneumonia

cases; however, extensive damage produced by the organism results in
high case fatality rates (up to 90 percent in untreated patients).

• Klebsiella rhinoscleromatis is the agent of rhinoscleroma, a chronic

destructive granulomatous disease of the respiratory tract that is
endemic in Eastern Europe and Central America.

• Klebsiella ozaenae, a rare cause of serious infection, is classically

associated only with ozena, an atrophy of nasal mucosal membranes
with a mucopurulent discharge that tends to dry into crusts; however,
recent studies indicate that the organism may cause various other
diseases including infections of the urinary tract, soft tissue, middle ear,
and blood.

89
 Coliforma and proteus Structure
•

90
 Antigens Structure
• The generalized structure and antigenic
composition of coliform bacilli, as well as of
Proteus and other members of the family
Enterobacteriaceae, are referred to as H, K, and
O antigens.

• The coliforms and Proteus are divided into

serotypes on the basis of combinations of these
antigens; different serotypes may have different
virulence properties or may preferentially
colonize and produce disease in particular body
habitats.

91
 Major Surface Antigens
• K antigens (capsule antigens) are components of the polysaccharide capsules.
Certain K antigens (e.g., K88 and K99 of E coli) are pilus-like proteins.

• The K antigens often block agglutination by specific O antisera.

• In the past, K antigens routinely were differentiated into A, L, and B groups on the
basis of differences in their lability to heat; however, these criteria are subject to
difficulties that make the distinction tenuous questionable.

• Some Citrobacter serotypes produce Vi (virulence) antigen, a K antigen also found in

Salmonella typhi.

• Species of Proteus, Enterobacter, and Serratia apparently have no regular K

antigens. (pes)

• However, the K antigens are important in the pathogenesis of some coliforms.

• A diffuse slime layer of variable thickness (the M antigen) also may be

produced but, unlike the K antigens, it is nonspecific and is serologically
cross-reactive among different organisms.
92
 Antigens Structure
• The H antigen determinants are flagella
proteins. Escherichia coli, Enterobacter,
Serratia, Citrobacter, and Proteus organisms
are peritrichous (i.e., they have flagella that
grow from many places on the cell surface).

• Klebsiella species are nonmotile and

nonflagellated and thus have no H antigens

93
 Toxins
• Enterotoxin=poisonous substances that has marked effect upon
the gastrointestinal tract causing vomiting diarrhoea and
abdominal pain.

• Enterotoxigenic strains of Klebsiella, Enterobacter,

Serratia,Citrobacter, and Proteus also have been isolated from
infants and children with acute gastroenteritis.

• The enterotoxins of at least some of these organisms are of the

heat-labile and heat-stable types and have other properties in
common with the E coli toxins.

• However, the importance of the coliforms and Proteus, other

94
than E coli, in enteric infections is questionable
 Pathogenesis
• Production of disease by coliforms or Proteus species in
extraintestinal sites often involves specific serotypes of the
organisms and special virulence factors.

• For example, respiratory tract infections by K pneumoniae

predominantly involve capsular types of virulence factors.

• E coli are found with high frequency in urinary tract and

other extraintestinal infections.

• These observations suggest that different serotypes may

have specific pathogenicities.

• An alternative explanation is that such strains may simply be

the most prevalent types in the normal gut flora.
95
 Pathogenesis
• The enzyme urease, produced by Proteus, and to a lesser extent by
Klebsiella species, is thought to play a major role in the production
of infection-induced kidney urinary stones

• Urease hydrolyses urea to ammonia and carbon dioxide.

• Alkalinisation of the urine by ammonia can cause magnesium

phosphate and calcium phosphate to become supersaturated and
crystallize out of solution to form, respectively, struvite and apatite
stones. Magnesium phosphate isnt soluble in alkaline;

• Bacteria within the stones may be refractory to antimicrobial

therapy.

• Large stones may interfere with renal function.

• The ammonia produced by urease activity may also damage the

epithelium of the urinary tract.
96
 Host Defences
• It cannot be overemphasised that coliforms (except
for E coli in enteric diseases) and Proteus species
are unlikely to cause disease unless the local or
generalized host defences fail in some way.

• The normal gastrointestinal flora, which includes E

coli and, frequently, other coliforms and Proteus
species in small numbers, is important in preventing
disease through bacterial competition.

• Prolonged antibiotic therapy compromises this

defence mechanism by reducing susceptible
components of the normal flora, permitting
nosocomial coliform strains or other bacteria to
colonise or overgrow. 97
 Host Defences
• The organisms may breach anatomic barriers through third-
degree burns etc,

• ulcers associated with solid tumors of the skin and mucous

membranes,

• intravenous catheters,

• and surgical or instrumental procedures on the biliary,

gastrointestinal, and genitourinary tracts.

• The lungs may be violated by instrumentation, as in tracheal

intubation,

• or even by aerosols from contamination which carry

organisms to the terminal alveoli. 98
 Epidemiology
• The epidemiology of coliform and Proteus infections is
complex and involves multiple reservoirs and modes of
transmission.

• Klebsiella, Enterobacter, Serratia, Citrobacter, and Proteus

species live in water, soil, and occasionally food and, in
many cases, form part of the intestinal flora of humans and
animals.

• Escherichia coli is believed not to be free living, and its

presence in environmental samples is taken as indicating
recent faecal contamination.

• In fact, water quality is determined by the presence of the

rapid lactose fermenting E coli, Klebsiella, and Enterobacter
(coliform counts) and E coli (faecal coliform counts) using
special selective media. 99
 Epidemiology
• Certain properties of the coliforms may be important in the epidemiology
of hospital-acquired infections.

• Coliform bacteria other than E coli frequently are found in tap water or
even distilled or deionised water.

• They may persist or actively multiply in water associated with respiratory

therapy or haemodialysis equipment.

• Klebsiella, Enterobacter, and Serratia species, like Pseudomonas

species, may exhibit increased resistance to antiseptics and
disinfectants.(KES)

• The same group of coliforms has a selective ability over other common
nosocomial pathogens (including E coli, Proteus species, Pseudomonas
aeruginosa, and staphylococci) to proliferate rapidly at room temperature
in commercial parenteral fluids containing glucose.

100
 Diagnosis
• Because the coliforms and Proteus can cause many types of
infection, the clinical symptoms rarely permit a diagnosis.

• Culturing and laboratory identification are usually required.

• Selected characteristics that are useful in the differentiation of

coliform bacilli and Proteus species found in human clinical
specimens are shown in Table below.

• The organisms have simple nutritional requirements and

grow well on mildly selective media commonly used for
members of the Enterobacteriaceae, but not on some
moderately and highly selective enteric plating media
(Salmonella-Shigella, bismuth sulphite, and brilliant green
agar). 101
 Diagnosis
• Extraintestinal specimens such as urine, purulent material from
wounds or abscesses, sputum, and sediment from cerebrospinal fluid
should be plated for isolation on blood agar and a differential medium
such as MacConkey or eosin-methylene blue agar.

• The finding of more than 105 organisms/ml in clean voided midstream

urine is often taken as "significant bacteriuria."

• However, in acutely symptomatic females and with other types of

specimens i.e., those obtained by catheterization or suprapubic
aspiration (is a surgically-created connection between the urinary
bladder and the skin which is used to drain urine ...from either sex, a
more appropriate threshold, particularly in the presence of pus cells
and the absence of epithelial cells, might be more than 10 2 colonies of
a known uropathogen/ml.

• Because urine is a good growth medium for many microbes,

specimens should be refrigerated (4oC) if transport to the laboratory is
delayed longer than 30 minutes, unless a urine transport container
with preservative is used. 102
103
 Faecal specimen
• Isolation of certain coliforms or Proteus species from fecal
specimens may be facilitated by adding a moderately
selective medium such as desoxycholate –citrate agar(DCA).

• selenite broth for enrichment of enterotoxigenic strains from

faeces in MacConkey broth and agar is useful for screening
EHEC (commonly E. coli O157:H7) on which lactose-positive
colonies are considered suspicious for the organism.

• Unless the physician specifically requests that the laboratory

to look for the possibility of E. coli as an enteropathogen,
tests for pathogenic strains, including toxin assays,
serotyping, and serogrouping, will not be done
104
 Blood specimen
• In cases of suspected bacterimia, replicate bottles
(one cultured aerobically, the other anaerobically)
containing 25 to 100 ml of appropriate medium
Nutrient broth/ MacConkey broth inoculated with 10-
ml portions of blood.

• It is usually necessary to take multiple specimens,

both before and after antibiotic therapy is started.

• It is important to take specimens after antibiotic

treatment is started so that therapeutic failure can be
recognized while the bacteraemia may still be
amendable to more aggressive medical or surgical
treatment. 105
 Control
• Prevention of coliform and Proteus infections, particularly those that are
hospital acquired, is difficult and perhaps impossible.

• Sewage treatment, water purification, proper hygiene, and other control

methods for enteric pathogens will reduce the incidence of E coli
enteropathogens.

• However, these control measures are rarely available in less developed

regions of the world.

• Breast-feeding is an effective means of limiting outbreaks of

enteropahogens in infants.

• Aggressive infection control committees in hospitals can do much to

reduce nosocomial infections through identification and control of
predisposing factors, education and training of hospital personnel, and
limited microbial surveillance.

106
 Control
• Selective decontamination of the digestive tract with a suitable
nonabsorbable antimicrobial regimen may be useful during
outbreaks caused by nosocomial coliforms and Proteus.

• Meticulous hand washing after each patient contact a highly

effective means of reducing the transmission of nosocomial
pathogens is infrequently or poorly performed by some hospital
personnel.

• In a study conducted in an intensive care unit following an

educational campaign on the importance of hand washing, the
compliance was
• 17 percent for physicians,
• 100 percent for nurses,
• 82 percent for respiratory technicians,
• and 88 percent for diagnostic services personnel.

107
 Control
• Active or passive immunization against coliforms and
Proteus species is not practiced.

• However, vaccines or hyperimmune sera for the six

common Gram negative pathogens (E coli, Klebsiella,
Enterobacter, Serratia, Pseudomonas aeruginosa, and
Proteus) probably would have a major impact on
morbidity and mortality from nosocomial infections.

• In a trial, the mortality was reduced markedly in a

group of patients with Gram-negative bacteraemia
who had been given antiserum against a mutant E coli
with an exposed lipopolysaccharide core. 108
 Control
• Ampicillin, sulfonamides, cephalosporins,
tetracycline, trimethoprim-sulfamethoxazole,
nalidixic acid, ciprofloxacin, and nitrofurantoin
have been useful in treating urinary tract
infections by coliforms and Proteus species.

• Gentamicin, amikacin, tobramycin,

ticarcillin/clavulate, imipenem, aztreonam, and
a variety of third-generation cephalosporins
may be effective for systemic infections;
however, laboratory tests for drug
109
susceptibility are essential.
 Control
• Measures commonly used to control epidemics
of antibiotic resistant Gram-negative bacilli
have included:
• (1) closing the unit to new admissions until
control of the outbreak is underway;
• (2) reinforcing hand-washing practices;
• (3) gown and glove isolation, often combined
with isolation of patients in separate quarters;
and
• (4) restricting the use of the antibiotic to which
the offending clone is resistant 110