GRAMNEGATIVE ENTERIC GRAMNEGATIVE ENTERIC RODSRODS

FERMENTATIVEFERMENTATIVE

ENTEROBACTERIACEAEENTEROBACTERIACEAE

G- bacteriaG- bacteria

G- rodsG- rods

Family: ENTEROBACTERIACEAEFamily: ENTEROBACTERIACEAE

Largest and most heterogenous collection of Largest and most heterogenous collection of medically important G- bacillimedically important G- bacilli

32 genera, over 130 species32 genera, over 130 species

Classification based on:Classification based on:

- biochemical properties- biochemical properties

- antigenic structure- antigenic structure

- nucleic acid hybridization and sequencing- nucleic acid hybridization and sequencing

Less than 20 species responsible for over 95% of Less than 20 species responsible for over 95% of infectionsinfections

EnterobacteriaceaeEnterobacteriaceaeUbiquitous organisms found worldwide inUbiquitous organisms found worldwide in

- soil- soil - water- water -vegetation-vegetation

Some of them are part of normal intestinal Some of them are part of normal intestinal flora of most animalsflora of most animalsCause variety of human diseases:Cause variety of human diseases:

- up to 35% of all septicaemias- up to 35% of all septicaemias - over 70% of urinary tract infections- over 70% of urinary tract infections - diarrheas- diarrheas

Some organisms are strict patogens: Some organisms are strict patogens: Shigella, Salmonella typhi, Yersinia pestisShigella, Salmonella typhi, Yersinia pestis (always associated with disease)(always associated with disease)Members of normal intestinal flora able to Members of normal intestinal flora able to cause opportunistic infections:cause opportunistic infections:

Escherichia coli, Proteus mirabilis, Klebsiella Escherichia coli, Proteus mirabilis, Klebsiella pneumoniaepneumoniaeNormally commensal flora becoming Normally commensal flora becoming pathogenic after acquiring virulence factor pathogenic after acquiring virulence factor genes on plasmids, bacteriophages or genes on plasmids, bacteriophages or pathogenicity islands:pathogenicity islands:

E.coli E.coli associated with gastroenteritisassociated with gastroenteritis

COMMON MEDICALLY COMMON MEDICALLY IMPORTANT IMPORTANT ENTEROBACTERIACEAE:ENTEROBACTERIACEAE:Citrobacter fruendii, C. buseri, Citrobacter fruendii, C. buseri, Enterobacter cloaceae, Enterobacter cloaceae, Enterobacter aerogenes, Enterobacter aerogenes, Escherichia coli, Klebsiella Escherichia coli, Klebsiella pneumoniae, Klebsiella pneumoniae, Klebsiella oxytoca,Morganella morganii, oxytoca,Morganella morganii, Proteus vulgaris, Proteus mirabilis, Proteus vulgaris, Proteus mirabilis, Serratia marcescens, Shigella Serratia marcescens, Shigella flexneri, Shigella sonnei, flexneri, Shigella sonnei, Salmonella typhi, Salmonella Salmonella typhi, Salmonella enterica,Yersinia pestis, Yersinia enterica,Yersinia pestis, Yersinia enterocolitica, enterocolitica,

Y.pseudotuberculosisY.pseudotuberculosis

Enterobacteriaceae infectionsEnterobacteriaceae infections

can originate from can originate from

animal reservoir:animal reservoir: most most SalmonellaSalmonella and and YersiniaYersinia species species

human carrier:human carrier: Salmonella typhiSalmonella typhi and and ShigellaShigella species species

through through endogenous spreadendogenous spread of organisms of organisms in a susceptible patientin a susceptible patient

Structure and PhysiologyStructure and Physiology

Gram-negative bacilliGram-negative bacilliSize: 0.3 – 1.0 x 1.0 – 6.0 Size: 0.3 – 1.0 x 1.0 – 6.0 mmDo not form spores (non-sporogenous)Do not form spores (non-sporogenous)Motile (with peritrichous flagella) / non-motileMotile (with peritrichous flagella) / non-motileFast aerobic or anaerobic growth (facultative Fast aerobic or anaerobic growth (facultative anaerobes) on variety of nonselective and anaerobes) on variety of nonselective and selective mediaselective mediaFerment glucose, reduce nitrate, catalase+, Ferment glucose, reduce nitrate, catalase+, oxidase- (absence of cytochromeoxidase)oxidase- (absence of cytochromeoxidase)

Structure and Physiology – cont´dStructure and Physiology – cont´dLactose fermenting strains (Endo Agar): Lactose fermenting strains (Endo Agar): Escherichia, Klebsiella, Citrobacter, Serratia and Escherichia, Klebsiella, Citrobacter, Serratia and EnterobacterEnterobacter

Resistant to bile-salts: Salmonella, ShigellaResistant to bile-salts: Salmonella, Shigella

Some have prominent capsules (Klebsiella)Some have prominent capsules (Klebsiella)

Others surrounded by a diffusible slime layerOthers surrounded by a diffusible slime layer

Antigens: Antigens:

somatic O polysaccharide + lipid A (endotoxin)somatic O polysaccharide + lipid A (endotoxin)

flagellar H proteins (heat-labile)flagellar H proteins (heat-labile)

capsular K proteins or polysaccharides (heat-labile)capsular K proteins or polysaccharides (heat-labile)

Antigenic structureAntigenic structure

Pathogenesis and ImmunityPathogenesis and Immunity

FACTORS OF VIRULENCEFACTORS OF VIRULENCE::

EndotoxinEndotoxin

CapsuleCapsule

Antigenic Phase VariationAntigenic Phase Variation

Type III Secretion SystemsType III Secretion Systems

Sequestration of Growth FactorsSequestration of Growth Factors

Resistance to Serum KillingResistance to Serum Killing

Antimicrobial resistanceAntimicrobial resistance

EndotoxinEndotoxinActivity based on Lipid A released at cell lysisActivity based on Lipid A released at cell lysis

Activation of complementActivation of complementRelease of cytokinsRelease of cytokinsLeukocytosisLeukocytosisThrombocytopeniaThrombocytopeniaDisseminated intravascular coagulationDisseminated intravascular coagulationFeverFeverDecreased peripherial circulationDecreased peripherial circulationShockShockDeathDeath

CapsuleCapsule

Protects bacteria from phagocytosis by Protects bacteria from phagocytosis by hydrophilic capsular antigens which repel hydrophilic capsular antigens which repel the hydrophobic phagocytic cell structure.the hydrophobic phagocytic cell structure.

These antigens interfere with the binding These antigens interfere with the binding of antibodies to bacteria and are poor of antibodies to bacteria and are poor immunogens or complement activators.immunogens or complement activators.

!!! Development of specific anti-capsullar !!! Development of specific anti-capsullar antbodiesantbodies

Antigenic Phase VariationAntigenic Phase Variation

Protects bacteria from antibody-mediated Protects bacteria from antibody-mediated cell deathcell death

= genetic control of expression of capsular = genetic control of expression of capsular and flagellar antigensand flagellar antigens

Alternative expression of H and K antigensAlternative expression of H and K antigens

Type III Secretion SystemsType III Secretion Systems

Salmonella, Escherichia, Shigella, Yersinia Salmonella, Escherichia, Shigella, Yersinia have common effector system for have common effector system for delivering their virulence genes into delivering their virulence genes into targeted eukaryotic cells.targeted eukaryotic cells.Type III Secretion System consists of Type III Secretion System consists of approx. 20 proteins that facilitate secretion approx. 20 proteins that facilitate secretion of bacterial virulence into the host cell.of bacterial virulence into the host cell.In the absence of TIIISS the bacteria loose In the absence of TIIISS the bacteria loose their virulence.their virulence.

Sequestration of Growth FactorsSequestration of Growth Factors

Nutritional scavengers of bacteria growing in vivo.Nutritional scavengers of bacteria growing in vivo.

Iron – bound in heme proteins (haemoglobin, Iron – bound in heme proteins (haemoglobin,

myoglobin) or inmyoglobin) or in

iron-chelating proteins (transferrin, iron-chelating proteins (transferrin,

lactoferrin)lactoferrin)

Bacteria counteract the binding by producing own Bacteria counteract the binding by producing own competitive iron-chelating compounds (siderophore competitive iron-chelating compounds (siderophore enterobactin)enterobactin)

Iron can also be released from host cells by Iron can also be released from host cells by hemolysins produced by bacteria.hemolysins produced by bacteria.

Resistance to Serum KillingResistance to Serum Killing

Virulent organisms capable for producing Virulent organisms capable for producing systemic infections are often resistant to systemic infections are often resistant to serum killing:serum killing:

Bacterial capsuleBacterial capsule

Factors preventing binding of complement Factors preventing binding of complement components to the bacteria andcomponents to the bacteria and

subsequent complement-mediated subsequent complement-mediated clearenceclearence

Escherichia coliEscherichia coliNormal flora of intestineNormal flora of intestine

Pathogenic effect depends Pathogenic effect depends on E. coli strainon E. coli strain

ETEC (enterotoxigenic)ETEC (enterotoxigenic)

EPEC (enteropathogenic)EPEC (enteropathogenic)

EIEC (enteroinvasive)EIEC (enteroinvasive)

EHEC (enterohemorrhagic)EHEC (enterohemorrhagic)

HUS (hemolytic-uremic-HUS (hemolytic-uremic-syndrome)syndrome)

UTI – have P pilliUTI – have P pilli

Meningitis, pneumoniaMeningitis, pneumonia

Escherichia coliEscherichia coliETECETEC – labile and/or stable toxins causes – labile and/or stable toxins causes deregulation of intestinal cells and a volumi- nous deregulation of intestinal cells and a volumi- nous watery diarrhea (traveller´s diarrhea)watery diarrhea (traveller´s diarrhea)

EPECEPEC – binds to intestinal cells with bundle- – binds to intestinal cells with bundle- forming pilli and causes cupping of intestinal forming pilli and causes cupping of intestinal epithelial cells, altered function, diarrhea.epithelial cells, altered function, diarrhea.

EIEC EIEC – Shigella-like: bind to and invade M cells – – Shigella-like: bind to and invade M cells – lateral spread to enterocytes. Cause bloody lateral spread to enterocytes. Cause bloody diarrhea.diarrhea.

EHEC EHEC – bind to enterocytes by BFP, produce – bind to enterocytes by BFP, produce shigatoxin (causes bloody diarrhea) HUS – shigatoxin (causes bloody diarrhea) HUS – hemolytic uremic syndrom (hamburger disease)hemolytic uremic syndrom (hamburger disease)

Escherichia coliEscherichia coliE. coli with K1 capsule – newborn meningitisE. coli with K1 capsule – newborn meningitis

Pneumonia: aspiration of GIT organismsPneumonia: aspiration of GIT organisms

Urinary tract infectionsUrinary tract infections

Immunity : strain-specific. Protective Immunity : strain-specific. Protective antibodies against toxins, pilli, adhesins antibodies against toxins, pilli, adhesins

Epidemiology: contaminated food, carriers, Epidemiology: contaminated food, carriers, endogenous infections – displacement from endogenous infections – displacement from GITGIT

Diagnostics: culture+microscopy, lactose +, Diagnostics: culture+microscopy, lactose +, biochemical properties, serotyping (O,H,K).biochemical properties, serotyping (O,H,K).

SalmonellaSalmonellaS. typhiS. typhi

S. paratyphiS. paratyphi

S. typhimuriumS. typhimurium

S. entericaS. enterica

Salmonella typhiSalmonella typhi

Diseases caused: typhoid Diseases caused: typhoid fever (blood-born fever (blood-born infection involving infection involving multiple organs, starting multiple organs, starting as obstipationas obstipation

Carriers of salmonellaCarriers of salmonella

Salmonella typhiSalmonella typhi

PathogenesisPathogenesis: Salmonella typhi invades : Salmonella typhi invades through M cells in the small intestine – transport through M cells in the small intestine – transport through lymphatics --- bloodstream --- phagocy- through lymphatics --- bloodstream --- phagocy- tosis of organisms – grow in macrophages of tosis of organisms – grow in macrophages of the liver, spleen and other organs.the liver, spleen and other organs.

Typhoid fever – organisms released from lysed Typhoid fever – organisms released from lysed macrophages reseed the bloodstreammacrophages reseed the bloodstream

CarriersCarriers harbor bacteria in tissues (gall bladder) harbor bacteria in tissues (gall bladder) and pass microorganisms in the stoolsand pass microorganisms in the stools

Immunity:Immunity: protective antibodies to O-antigen protective antibodies to O-antigen (LPS), (LPS), vaccinationvaccination

Salmonella typhiSalmonella typhiEpidemiology:Epidemiology: human-human spread, water human-human spread, water contaminated with feces (drinking water)contaminated with feces (drinking water)

Diagnostics:Diagnostics: culture (blood, endo, DC-agar, culture (blood, endo, DC-agar, Wilson-Blair agar, McConkey agar)Wilson-Blair agar, McConkey agar)

G- bacilli with flagellaG- bacilli with flagella

Serotyping (O,H,Vi antigens)Serotyping (O,H,Vi antigens)

THERAPY:THERAPY:

chloramphenicolchloramphenicol

PROTECTION:PROTECTION:

live vaccine (oral)live vaccine (oral)

Salmonella entericaSalmonella enterica

causes gastroenteritiscauses gastroenteritis

Organisms bind to entero- Organisms bind to entero- cytes by pilli in the small cytes by pilli in the small intestine and cause ruffles on intestine and cause ruffles on the cell surface. Bacteria the cell surface. Bacteria invade the cells, migrate invade the cells, migrate through the cells into the through the cells into the lamina propria of small lamina propria of small intestine causing an intense intestine causing an intense inflammation.inflammation.

This leads to malabsorption This leads to malabsorption and diarrhea. Disease is and diarrhea. Disease is usually self-limiting in healthy usually self-limiting in healthy people people

Salmonella entericaSalmonella entericaImmunity: antibody to LPS O-antigen can Immunity: antibody to LPS O-antigen can be protective, but protection is strain-spe- be protective, but protection is strain-spe- cificcificEpidemiology: Common pathogenic strains Epidemiology: Common pathogenic strains are acquired by food contaminated with are acquired by food contaminated with feaces from infected animals or humans. feaces from infected animals or humans. Most animal sources: eggs and poultry.Most animal sources: eggs and poultry.More than 1500 different serotypes can More than 1500 different serotypes can cause diarrhea in humanscause diarrhea in humansMost frequent: Salmonella enterica Most frequent: Salmonella enterica serotype enteritidisserotype enteritidis

Salmonella entericaSalmonella entericaDiagnostics: stool samp- Diagnostics: stool samp-

les plated solid medium les plated solid medium ----------------------------------------------

(Blood, Endo, DC-agar(Blood, Endo, DC-agar

McConkey)McConkey)

Microscopy: G- rodsMicroscopy: G- rods

Serotyping: O and H an-Serotyping: O and H an-

tigenstigens

Biochemical characteri-Biochemical characteri-

sation (API, Enterotest)sation (API, Enterotest)

ShigellaShigella

S. dysenteriaeS. dysenteriae

S. flexneriS. flexneri

S. sonneiS. sonnei

Causes dysentery : Causes dysentery : frequent, low-volume frequent, low-volume stools containing stools containing inflammatory cells and inflammatory cells and bloodblood

30 or more portions a day30 or more portions a day

HIGHLY INFECTIVE!!!!HIGHLY INFECTIVE!!!!

10 bacteria initiate disease10 bacteria initiate disease

ShigellaShigella

Pathogenesis:Pathogenesis: Descending infection from Descending infection from small intestine to large intestine (main site). small intestine to large intestine (main site). Organisms bind to M cells and invade lamina Organisms bind to M cells and invade lamina propria, then invade neighboring enterocytes propria, then invade neighboring enterocytes from the bottom. Bacteria grow and induce from the bottom. Bacteria grow and induce actin polymerization that pushes them actin polymerization that pushes them laterally into neighboring cells, where laterally into neighboring cells, where continue to spread causing cell-death and continue to spread causing cell-death and inflammationinflammation

Immunity: Immunity: little innate protection. little innate protection. Convalescent antibodies to O-antigen (LPS)Convalescent antibodies to O-antigen (LPS)

ShigellaShigellaEpidemiology:Epidemiology: Highly infectious, fecal-oral Highly infectious, fecal-oral spread. Human are the only reservoirspread. Human are the only reservoir

Diagnosis:Diagnosis: culture+microscopy culture+microscopy

G- rods without flagella, do not grow on WB, G- rods without flagella, do not grow on WB, serotyping (O antigen)serotyping (O antigen)

NO VACCINE AVAILABLENO VACCINE AVAILABLE

YersiniaYersiniaY. pestis :Y. pestis :

Plague – bubonic – potentially fatalPlague – bubonic – potentially fatal

- pneumonic – highly contagious,- pneumonic – highly contagious,

lethallethal

Y. pseudotuberculosis:Y. pseudotuberculosis:

mesenteric lymphadenitis (pseudoappen-mesenteric lymphadenitis (pseudoappen-

dicitis) dicitis)

Y. enterocolitica:Y. enterocolitica:

gastroenteritis, mesenteric lymphadenitisgastroenteritis, mesenteric lymphadenitis

Yersinia pestisYersinia pestis

Yersinia enterocoliticaYersinia enterocolitica

Yersinia pseudotuberculosisYersinia pseudotuberculosis

Family: VibrionaceaeFamily: VibrionaceaeGenus: VibrioGenus: Vibrio

FamilyFamily: Vibrionaceae: VibrionaceaeVibrio choleraeVibrio cholerae

Disease caused: CHOLERA Disease caused: CHOLERA (voluminous watery diarrhea, (voluminous watery diarrhea, life-threat through dehydrationlife-threat through dehydration

Pathogenesis: ingestion of Pathogenesis: ingestion of fecally contaminated water or fecally contaminated water or food. Flagellar motility + muci- food. Flagellar motility + muci- nase – penetration to epithe- nase – penetration to epithe- lium of small intestine. Toxin-lium of small intestine. Toxin-corregulated pilli – attachment, corregulated pilli – attachment, toxin production causes dere- toxin production causes dere- gulation of crypt-cells by ADP-gulation of crypt-cells by ADP-ribosylating Gs protein of ade- ribosylating Gs protein of ade- nylate cyclase system.Efflux of nylate cyclase system.Efflux of water + ions into the lumenwater + ions into the lumen

Vibrio choleraeVibrio cholerae

Vibrio choleraeVibrio choleraeImmunity: Antibody to cholera toxin greatly Immunity: Antibody to cholera toxin greatly reduces the severity. VACCINATIONreduces the severity. VACCINATIONEpidemiology: Virulent organisms – O1 or Epidemiology: Virulent organisms – O1 or O139 antigens. O1 serotypes AB (Inaba), AC O139 antigens. O1 serotypes AB (Inaba), AC (Ogama) ABC (Hikojima). 2 biovars: classical (Ogama) ABC (Hikojima). 2 biovars: classical and El Tor.and El Tor.Organisms live in salt water attached to algae Organisms live in salt water attached to algae copepods or shells of crustaceans. If copepods or shells of crustaceans. If conditions become unfavorable – dormant and conditions become unfavorable – dormant and unculturableunculturableContamination of water sources - epidemiesContamination of water sources - epidemies

Vibrio choleraeVibrio cholerae, serotype O1 and , serotype O1 and O139 virulence factorsO139 virulence factors

Family: Family: CampylobacteriaceaeCampylobacteriaceaeSpiral G- bacteria with Low C-G DNA ratio, inable to Spiral G- bacteria with Low C-G DNA ratio, inable to

ferment or oxidize carbohydrates, with ferment or oxidize carbohydrates, with microaerophilic growth requirements, motilemicroaerophilic growth requirements, motile

Campylobacter Campylobacter C. jejuni, C. ileiC. jejuni, C. ilei ArcobacterArcobacter

Family: unnamedFamily: unnamedWalinellaWalinellaHelicobacter Helicobacter H. pyloriH. pyloriFlexispiraFlexispira

Genus: Genus: CampylobacterCampylobacter

Campylobacter jejuniCampylobacter jejuni

Helicobacter pyloriHelicobacter pylori

Helicobacter pyloriHelicobacter pylori

Pseudomonas aeruginosaPseudomonas aeruginosa

Straight or slightly curved G- bacilli, motile Straight or slightly curved G- bacilli, motile (polar flagella), 0.5 to 1.0 x 1.5 – 5.0 (polar flagella), 0.5 to 1.0 x 1.5 – 5.0 m.m.

Contain cytochromeoxidaseContain cytochromeoxidase

Produce different pigments (pyocyanin – Produce different pigments (pyocyanin – blue), fluorescein –yellow and pyorubin- blue), fluorescein –yellow and pyorubin- red/blue). red/blue).

Main cause of nosocomial infectionsMain cause of nosocomial infections

Pseudomonas aeruginosaPseudomonas aeruginosa