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Ρούτσιας Γιάννης, MD, PhD Επικ καθηγητης Ανοσολογίας/Μικροβιολογιας

FAMILY ENTEROBACTERIACEAE

Escherichia Klebsiella Enterobacter Salmonella Shigella Proteus Providencia

Citrobacter Morganella Serratia

IDENTIFICATION

MORPHOLOGY AND IDENTIFICATION

Short Gram negative rods May form chains In culture : circular, convex, smooth, with distinct

edges Growth characteristics : carbohydrate fermentation

patterns and activity of amino acid decarboxylases and enzymes are used in biochemical differentiation

IMViC REACTIONS

ACRONYM for the following tests: Indole Methyl Vogues-Proskauer Citrate

Patterns exhibited by groups of organisms

helpful in further differentiation

IMViC REACTIONS

Indole: Kovak's reagent

(+) E. coli Proteus vulgaris

(-) Klebsiella Enterobacter Proteus mirabilis

Indole:

Culture on differential media: allows rapid presumptive identification

Lactose fermented rapidly Escherichia coli : metallic sheen; motile; flat; non-

viscous colonies Enetrobacter aerogenes : raised colonies; no

metallic sheen; often motile; more viscous growth Klebsiella pneumonia : very viscous; mucoid growth;

nonmotile

Lactose fermented slowly Edwardsiella, Serratia, Citrobacter, Arizona,

Providencia, Erwinia

( cont. culture on differential media )

Lactose not fermented Shigella species: Nonmotile; no gas from dextrose Salmonella species: Motile; acid and usually gas from dextrose Proteus species: “ Swarming” on agar; urea rapidly hydrolyzed

(smell of ammonia )

MOTILITY, FLAGELLA

MOTILITY Escherichia coli Enterobacter Salmonella Proteus Providencia Morganella Serratia Citrobacter

PATHOGENIC FACTORS

ENDOTOXINS PART OF THE GRAM

NEGATIVE CELL WALL (LPS)

Release various mediators of inflammation IL-1 IFN TNF

Trigger humoral and enzymatic mechanisms intrinsic fibrinolytic kinin

WAYS OF CAUSING DISEASE: ATTACHMENT

ABILITY TO COLONIZE & ADHERE TO A RELEVANT REGION IN THE BOWEL

EXAMPLES: EPEC

(enteropathogenic E. coli): pili

ETEC (enterotoxigenic E. coli): CFA

WAYS OF CAUSING DISEASE: TOXIN PRODUCTION

ENTEROTOXIN alter metabolic

activity of intestinal epithelial cells

EXAMPLES: ETEC Shigella

dysenteriae Salmonella Aeromonas

WAYS OF CAUSING DISEASE: TOXIN PRODUCTION

CYTOTOXIN disrupt the structure

of individual epithelial cells

NEUROTOXIN

EXAMPLES: EHEC Shigella spp. Salmonella

Shigella spp.

WAYS OF CAUSING DISEASE: INVASION

AFTER ADHERENCE GAIN ACCESS TO

INTRACELLULAR ENVIRONMENT

REACH DEEPER TISSUES ACCESS

NUTRIENTS FOR GROWTH

AVOID HOST IMMUNE SYSTEM

EXAMPLES: EIEC & EHEC Shigella spp.

ENTERIC INFECTIONS: UPSETTING OF FLUID AND ELECTROLYTE BALANCE / NON-INFLAMMATORY

MAJOR SYMPTOMS watery diarrhea no fecal leukocytes no fever

EXAMPLE/S ETEC

(enterotoxigenic E. coli)

Vibrio cholerae

ENTERIC INFECTIONS: PENETRATION W/ SUBSEQUENT ACCESS TO THE BLOODSTREAM (ENTERIC FEVER)

MAJOR SYMPTOMS signs of systemic

infection headache, malaise,

sore throat fever

EXAMPLE/S Salmonella typi

ENTERIC INFECTIONS: INVASION AND POSSIBLE CYTOTOXIN PRODUCTION / INFLAMMATORY DYSENTERY

MAJOR SYMPTOMS dysenteric-like

diarrhea mucus, blood &

leukocytes fever

EXAMPLE/S EIEC

(enteroinvasive E. coli)

Salmonella enteritidis

Shigella spp.

Escherichia coli

Κροσσοι:pili ,fimbriae

Μαστίγια: flagella

Escherichia coli

Escherichia coli

Escherichia coli

HABITAT/ RESERVOIR

normal bowel flora soil, water, vegetation female genital tract

TRANSMISSION endogenous ingestion nosocomial

Escherichia coli

AVIRULENT strains part of the normal flora cause contiguous

infection opportunistic

PATHOGENIC strains EPEC

(enteropathogenic) ETEC

(enterotoxigenic) EIEC

(enteroinvasive) EHEC

(enterohemorrhagic) EAEC

(enteroaggregative)

Escherichia coli EXTRAINTESTINAL INFECTION

VIRULENCE FACTORS endotoxin capsule pili

DISEASE & INFECTION UTI bacteremia nosocomial infections most common cause

of Gram negative nosocomial infections

Escherichia coli EIEC (enteroinvasive E. coli)

VIRULENCE FACTORS cause disease similar to

shigellosis invades intestinal

mucosal epithelial cells

DISEASE & INFECTION Dysentery

(necrosis, ulceration, bowel inflammation )

in young children poor sanitation

Escherichia coli ETEC (enterotoxigenic E. coli)

VIRULENCE FACTORS Heat labile exotoxin

(LT)- stim Heat stable enterotoxin

(ST)

DISEASE & INFECTION Traveller’s &

childhood diarrhea profuse watery

stools

Escherichia coli EAEC (enteroaggregative E. coli) VIRULENCE

FACTORS probably binding by

pili Heat stable-like and

hemolysin-like toxins

actual mechanism not known

DISEASE & INFECTION Watery diarrhea

w/c may be prolonged

transmission not well understood

Escherichia coli EHEC (enterohemorrhagic E. coli)

VIRULENCE FACTORS toxin similar to

Shiga toxin (by S. dysenteriae)

associated w/ certain serotypes such as E. coli O157:H7 (from undercooked ground beef or raw milk)

DISEASE & INFECTION Hemorrhagic colitis

(inflammation & bleeding of the large bowel mucosa)

Hemolytic Uremic Syndrome (toxin mediated damage to kidneys)

Escherichia coli EPEC (enteropathogenic E. coli)

VIRULENCE FACTORS bundle forming pilus

mediate attachment

resulting in changes in cell surface (such as loss of microvilli)

DISEASE & INFECTION Diarrhea in infants

in developing or low-income nations

can cause chronic diarrhea

Salmonella spp.

Salmonella spp.

Salmonella spp.

VIRULENCE FACTORS factors that protect

organisms from stomach acids

attachment & phagocytosis

TRANSMISSION ingestion of

contaminated food products poultry, eggs

direct fecal-oral spread in children

Salmonella

DISEASES typhoid fever (enteric

fever), septicemia, gastroenteritis

asymptomatic carriage

animals as reservoir except typhoid & paratyphoid fevers

Laboratory Diagnosis - isolation - 1st week of illness:

best specimen is blood

- 3rd week of illness : best specimen is stool - epidemiologic studies and complete ID : serologic typing

Salmonella WHO IS AT RISK ?

anyone consuming foods with large numbers of Salmonella esp. children

Salmonella typhi & paratyphi

HABITAT not part of normal

flora only found in

humans at time of infection

MODE OF TRANSMISSION person-to-person

spread by fecal-oral route

ingestion of food / water contaminated w/ human excreta

Other Salmonella spp.

HABITAT disseminated in

nature & various animals

MODE OF TRANSMISSION ingestion of contaminated

food products from animals (poultry or dairy)

direct person-to-person transmission by fecal-oral route

in health care settings

Shigella Shigella dysenteriae

Shigella

Shigella sonnei

Shigella

Culture : facultative anaerobes but grow best aerobically : convex, circular, transparent colonies with intact edges Growth characteristics : all ferment glucose; unable to ferment lactose Antigenic structure : somatic O antigens which are lipopolysaccharides ( more than 40 serotypes )

Shigella

HABITAT: - limited to the

intestinal tracts of humans and other primates

MODE OF TRANSMISSION - person-to-person

spread by fecal-oral route

overcrowded areas poor sanitary condition

Shigella spp.

VIRULENCE FACTORS adherence invasion escape from

phagocytic vesicles intercellular spread shiga toxin

(interferes with protein synthesis )

DISEASE & INFECTION Dysentery

acute inflammatory colitis

bloody diarrhea tenesmus, bloody

mucoid stools S. sonnei

watery diarrhea

Shigella: Laboratory Diagnosis Specimens : fresh stool, mucus flecks, rectal

swabs for culture : serum ( 10 days apart to demonstrate rise in

antibody titer )

Culture : streaked on differential media (MacConkeys’ agar ) and selective ( Salmonella-Shigella agar ) Serology : rise in antibody titer

Shigella: Prevention and Control

Mass chemoprophylaxis Eliminate organisms by :

sanitary control of water, food and milk; sewage disposal; fly control

isolation of patients and disinfection of excreta detection of subclinical cases and carriers,

particularly food handlers

Vibrio cholerae

Vibrio cholerae

Vibrio cholerae

Vibrio cholerae

MORPHOLOGY Short (0.5 μm by 1.5 to

3.0 μm), gram-negative rods, comma-shaped

Has a single, thick,sheathed flagellum

CULTURE Produces convex,

smooth, round,opaque, granular colonies

Most vibrios grow well at 37oC on thiosulfate-citrate-bile-sucrose (TCBS) agar

* The cause of epidemic asiatic cholera

Vibrio cholerae

CULTURE Positive oxidase test

differentiates V.cholera from other vibrios

Ag STRUCTURE Has O lipopoly-

saccharides that confer serological specificity

BIOLOGIC CLASSIFICATION

Serotypes: Ogawa and Inaba

Biotypes: classical and El Tor (produces a hemolysin, (+) Voges-Proskauer, resistant to polymyxinB)

Vibrio cholerae

VIRULENCE FACTORS

Heat-labile enterotoxin consisting of 2 subunits (A & B)

Subunit A increases levels of intracellular cyclic AMP hypersecretion of water & electrolytes

TRANSMISSION OF INFECTION

Person-to person contact involving individuals with mild or early illness

Transmitted by water and food

Vibrio cholerae

LAB DIAGNOSIS Specimens for culture

consists of mucus flecks from stools

Dark-field or phase-contrast microscopy may show rapidly motile vibrios

Rapid growth in peptone agar, blood agar ,or TCBS agar

May be identified thru

slide agglutination test using anti-O antiserum

Vibrio cholerae

CONTROL MEASURES Education and improvement of sanitation Repeated injection of a vaccine confers

limited protection Chemoprophylaxis with antimicrobial drugs

may have a place in control

CAMPYLOBACTER

CAMPYLOBACTER

CAMPYLOBACTER

MORPHOLOGY : - slender, Gram negative, helically curved rods - morphologic forms : spirals, S shapes, commas,

coccoid - infected tissues: comma shaped - after isolation : filamentous or coccoid - cork-screw darting type of motility : best observed with

phase contrast or darkfield mic. - single unipolar or bipolar flagellum

CAMPYLOBACTER

ANTIGENIC STRUCTURE: - possess lipopolysaccharide and flagellar antigen - C. fetus : has a microcapsule or S layer ( anti-

phagocytic )

EPIDEMIOLOGY: - commensals of the intestinal tract of wild and domesticated animals * C. jejuni – found in poultry, dogs, cats, sheep, and cattle

CAMPYLOBACTER

MODE OF TRANSMISSION : 1.consumption of contaminated milk, water and food 2. fecal-oral route LABORATORY DIAGNOSIS : 1. Specimen : diarrheal stool 2. Smears : Gram stain shows comma shaped rods 3. Culture : require low oxygen tension and increased

carbon dioxide levels for growth

CAMPYLOBACTER

INFECTION: Large outbreaks of human infection traced to

contaminated milk, water, food C.jejuni frequently causes acute diarrheal diseases in

travelers visiting developing countries C.fetus causes frequently fatal septicemic illness in

debilitated patients, pregnant women and in the newborn

HELICOBACTER PYLORI

HELICOBACTER PYLORI

HELICOBACTER PYLORI

HELICOBACTER PYLORI

MORPHOLOGY: - In tissues, gram-negative, curved organisms - In culture, more rodlike, bizarre U-shaped and circular

cells - Has a tuft of sheathed polar flagella

HELICOBACTER PYLORI

Motile, and a strong producer of urease

VIRULENCE FACTORS: Strong association between presence of H.pylori in the

gastric antrum and duodenal ulceration Toxins and LPS may damage mucosal cells LABORATORY DIAGNOSIS: 1. Mucosal biopsies – routine stains will demonstrate

gastritis

H. pylori

LABORATORY DIAGNOSIS (CONT) 2. Mucosal biopsies - Giemsa or special silver stain

can show curved or spiraled organism 3. Culture: grows on media containing whole or lysed

blood : grows at 37oC in standard CO2 incubators :produces circular, translucent,colonies 4. Rapid tests to detect urease activity –for presumptive

identification in specimens

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