Enterobacteriaceae Lecture

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All Enterobacteriaceae:

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• Ferment glucose
• Reduce nitrate to nitrites
• Oxidase pos (except Plesiomonas)
• Motile at body temp (except Klebsiella, Shigella, Yersinia)

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Microscopic Colony Morphology of Enterobacteriaceae

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• Gram negative cocco bacilli or rods
• Facultative anaerobic- thioglycolate broth-growth throughout the tube

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Macroscopic Morphology of Enterobacteriaceae

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Large moist, gray colonies

Some mucoid- Klebsiella

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Remarkable colony morphology on non-selective media

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Klebsiella (mucoid)

Proteus (swarm)

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What makes MacConkey agar selective

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bile salts

crystal violet

Inhibits most gram positive bacteria growth

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What make MacConkey a differential agar

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LF (Pink) and NLF (Clear)

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EMB selects for:

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• Gram neg bacteria
• Methylene blue and eosin inhibit gram positive

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EMB differentiates

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lactose fermentor

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Hektoen enteric agar is a:

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Selective differential

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HE agar is selective because:

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bile salts inhibit gram + and some gram =

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HE agar is differential:

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Sucrose and lactose:

Nonpathogens ferment lactose and sucrose (orange color)

Pathogens- green to blue color with hydrogen sulfate gas creating black ppt

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XLD agar (Xylose lysine deoxycholate) selective

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Sodium desoxycholate:

Inhibits gram +, some gram =

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Carbohydrates on XLD

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Sucrose, lactose in excess

Xylose with phenol red indicator

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Lysine XLD

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to detect lysine decarboxylase

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Thiosulfate XLD agar

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detect hydrogen sulfate

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Yellow Colonies XLD

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fermentors do not produce Lysine decarb

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Colorless or Red colonies on XLD are typically from what genus

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Shigella

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Red colonies with black center

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• Salmonella
• Initially yellow convert to red after lysine decarboxylate

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Virulence and Antigenic factor of Enterobacteriaceae

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Adherence- prevent removal

Toxins- food poisoning

Invasive enzymes- spreading factor

Serology: O (somatic antigen) heat stable, H (flagellar) heat stable, K antigens (capsular antigen) heat liable

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K antigen

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Capsular antigen heat liable

K1 E.coli, Vi antigen Salmonella typhi

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O antigen

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Somatic antigen

Heat stable

Located in the cell wall (LPS)

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H antigen

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Flagellar antigen

Heat stable

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Opportunistic pathogens

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Normal flora causing infection in non normal site:

Septicemia

wounds

UTI

meningitis

E. coli

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Primary Pathogens

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Salmonella spp

Shigella spp

Yersinia spp

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General E. coli characteristics

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• Dry pink colonies on Mac plates
• Beta-hemolysis on BAP
• Motile
• Sex pili, fimbriae
• Possess O,H, K antigens

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E. coli IMVC reaction

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++--

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Biochemical Reactions E. coli

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• Fermentation: glucose, lactose, trehalose, and xylose
• H2S =
• DNase =
• urease=
• PAD =
• Citrate =

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Five major categories of E. coli

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• EPEC enteropathogenic E. coli
• ETEC enterotoxigenic E. coli
• EIEC enteroinvasive E. coli
• EHEC enterohemorrhagic E. coi
• EaggEC enteroaggregative E. coli

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EPEC enteropathogenic E. coli

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• Multiple serotypes
• fecal-oral transmission
• infantile diarrhea
• Occasional nursery outbreaks in U.S.

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Enteropathogenic E. coli

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• Attaching and effacing lesion
• SSTT
• Injector system to inject bacterial proteins into host cell
• pedestal formation and effacement of microvilli, diarrhea

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EPEC: Type III secretion apparatus

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• Bundle forming pilus contact with intestinal cells which are destroyed
• Bacteria come in contact with epithelial cell
• Type III secretion apparatus injects effector molecules into the cell causing signal transduction
• Forms pedestal formation
• Effacement leads to diarrhea

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Enterotoxigenic ETEC

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• Diarrhea in infants and adults in tropics and subtropic
• Traveler's diarrhea
• Contaminated food and drink
• Colonize small intestines have adhesive fimbriae
• Release toxins

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Toxins ETEC

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Heat labile toxin (LT)- A and B subunits

B binds GM1

A adenyl cyclase

Leads to hypersecretion of fluid

Heat stable toxing (ST) stimulates guanylate cyclase increasing cGMP leading to hypersecretion

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Both toxins in ETEC

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are the reason for watery diarrhea; release of liquid from cells

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ETEC vaccines

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• Anti-fimbrial responses prevent attachment: Killed, attenuated, Purified CFs, live attenuated vector bacteria expressing CFs
• Mucosal response important
• Anti-LT response may offer short-term protection

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Enteroinvasive (EIEC)

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• Adults and children
• Dysentery: direction penetration, invasion, and destruction of intestinal mucosa
• Require higher amounts of inoculum
• watery diarrhea
• Non-motile
• Lactose =

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Enterohemorrhagic EHEC

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• Hemorrhagic diarrhea, colitis
• Hemolytic-uremic syndrome (HUS): low platelets, hemolytic anemia, kidney failure
• Watery diarrhea progressing to bloody diarrhea without pus: can be fatal, undercooked meats, unpasteurized milk, apple cider

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EHEC outbreaks typically due to:

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E. coli O157:H7

Sources: Undercooked beef

Contamination with cattle feces

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E. coli O157:H7 Toxins

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Produced by Shiga toxigenic E.coli (STEC)

• Verotoxin I (phage mediated) cytotoxin
• Verotoxin II

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Verotoxin I

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• Damages vero cells
• Also known as Shiga toxin
• toxin neutralized by shiga toxin antibodies

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Verotoxin II

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Not neutralized by Shiga toxin antibodies

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Screening for O:157H7

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On SMAC plate:

Sorbitol containing Mac Plate

Does not ferment sorbitol

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Shiga toxin

A/B structure

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• 5 copies of B subunit allow binding
• A subunit- enzymatic: N-glycosidase, inactivates ribosomal RNA, inhibits protein synthesis

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Shiga toxin damage to endothelial cell

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• Endothelial cell damage triggers a cascade of events
• Result in microvascular lesions with microthrombi
• Occlude arterioles and cappallaries

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Shiga toxin can also cause

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• Hemolytic anemia and low platelet count
• HUS in kidney: can result in acute renal failure

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EHEC: treatment and vaccines

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• Antibiotic therapy may worsen disease
• Monoclonal antibodies
• vaccines composed of: shiga toxoid, killed bacteria, attenuated bacteria

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Enteroaggregative (EAggEC)

Diffusely adherent (DAEC)

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• Adheres to the surface of the small intestine
• Watery diarrhea with symptoms lasting more than two weeks
• Commensal from colon with special adherence adaptations for urinary tract infections: cultue with Hep2 cells causing aggregative pattern

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Extraintestinal E. coli Infections: UTIs

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• Catheterization
• Patients with urinary tract defects

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Extraintestinal E.coli: Speticemia and Meningitis

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• Most common in neonates and very young children
• Infection just before or during delivery and when infections involve the amniotic fluid
• Capsule antigen K1: Meningitis

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Escherichia hermanii

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• Formerly Enteric group II
• Clinical significance still not fully established

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E. vulneris

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Newest member: wound infections

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Klebsiella, Enterobacter, Serratia, and Hafnia infections

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Normal flora and GI tract

Opportunistic and nosocomial infections

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Characterisitics of Klebsiella, Enterobacter, Serratia, and Hafnia

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• Klebsiella is non-motile
• Simmon's citrate
• H2S-
• PAD-
• Voges Proskauer +
• IMVC: --++

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K. pneumoniae characteristics:

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• Non-motile
• Most common isolate: lower respiratory tract infection of hospitalized patients
• Moist gray mucoid colonies
• Virulent factor (polysaccharide capsule)

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Klebsiella Capsule

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• Antiphagocytic
• resists antimicrobial absorption
• Mucoid appearance of colonies

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Kelbsiella Infections

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• Frequent cause of nosocomial infections
• Hospital acquired infectons
• Often antibiotic resistance: Ampicillin
• KPC strain

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K. oxytoca

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Indole +

Affeccts similar sites to K. pneumoniae

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K. ozaenae causes

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isolated from nasal secretions and cerebral abcesses

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K. rhinoscleromatis

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Africa and South america

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2 Enterobacter species

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E. cloacae

E. aerogenes

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Pantoea agglomerans

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• Formerly Enterobacter agglomerans
• Similar to Klebsiella pneumoniae
• Motile

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Cronobacter sakazakii was

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Formerly Enterobacter sakazakii

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Enterobacter spp IMVC

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--++

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Enterobacter Ornithine decarboxylase and Lysine decarboxylase

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ODC: +

LDC: + (not E. cloacae)

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Infection sites of Enterobacter

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• Wound
• Urine
• Blood
• CSF

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Serratia species:

ONPG

DNase

Antimicrobals

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+

+

Highly resistant to antimicrobals

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Serratia marcescens typical characteristics

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• Red pigment at RT
• Most clinically revelant
• Nosocomial infections of the urinary tract, respiratory tract, bacteremia

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3 other Serratia species

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Serratia liquefaciens

Serratia rubidaea- some form red pigment at RT

Serratia odorifera- dirty musty odor

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Pathogenicity Serratia

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• Opportunistic pathogens
• colonize URT and UT
• Causes about 2% of nosocomial infections

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H. alvei

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• delayed citrate reaction
• Beer wort
• Environmental and human samples

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Proteus, Morganella, Providencia characteristics

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• Normal intestinal fluid
• Causes opportunistic infections (often nonsocomial)
• NLF
• PAD+
• ODC +

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Proteus mirabilis

Proteus vulgaris

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• Isolated from urine, wounds, ear and blood infection
• Swarms media
• burnt chocolate odor
• H2S +
• Urease +

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P. mirabilis

Indole

ODC

TSI

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Indole =

Ornithine decarboxylase +

K/A H2S

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P. vulgaris

Indole

ODC

TSI

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Indole +

ODC +

Sucrose fermentor + A/A with H2S

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Proteus Species Virulence

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UTI with indwelling catheters

• Fimbriae, hemolysin, flagella, IgA protease, urease
• Ammonia production increases pH
• Precipitation of Calcium and magnesium form crystals
• Produce bladder and kidney stones; hallmark of proteus

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Morganelli morgani infection

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• Commonly found in the environment
• Uncommon cause of infection
• Resistant to many beta lactams
• Associated with snakebite infections

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5 species of Providencia

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• Stuartii
• rettgeri
• alcalifaciens
• rustigianii
• heimbachae

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Providencia stuartii characteristics

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Most common

• Nosocomial burn unit outbreaks
• Isolated in urine culture
• No swarming
• High resistance to antimicrobals

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Providencia rettgeri infection

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• Urinary tract pathogen
• Occasionally nosocomial outbreaks

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Providencia Pathogenesis: P. stuartii

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• Predilection for catheterized urine
• Persists in urine due to adhesin (mannose-resistant Klebsiella-like hemagglutin)
• Can proceed to invade bloodstream and cause sepsis

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Providencia Pathogenesis:

P. alcalifaciens

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Associated with diarrhea

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Salmonella Species General Characteristics

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Gram-negative rods, facultatively anaerobic

Clear, colorless, NLF, colonies with black centers:

• Negative: Indole, VP, PAD, Urease
• No growth in potassium cyanide

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2 species of Salmonella spp

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Salmonella bongori

Salmonella enterica:subsp

• enterica
• arizonae
• diarizonae
• houtenae
• indica
• salame

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Salmonella enterica general characteristics

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Many serotypes

Includes isolates from humans and warm blooded animals

Salmonella enterica subsp serotype Typhimurium

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Salmonella Subgroup I

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human infections

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Most serious Salmonella species infection

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S. typhi

S. choleraesuis

S. paratyphi

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Differentiating Salmonella subgroups I

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LDC rules out paratyphi

ODC rules out S. typhi

Trehalose fermentation rules out S. choleraesuis

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Salmonella enterica classified as either:

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Typhoid

Non-typhoid

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Non-typhoid Salmonella

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Diarrhea, inflammatory

Caused by many serotypes

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Typhoid

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Systemic syndrome enteric fever

Caused by S. typhi and S. paratyphi

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Non-typhoid salmonella aquired by

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Ingestion:

Associated with eggs and diary products

raw meat, fruits, and vegetables

contaminated preparation area

Animal host, self limiting, no antibiotics

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Non-typhoid Salmonellosis

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Watery diarrhea, abdominal pain, fever, blood pus mucus in stool

No antibiotics

Persistant diarrhea

Immunocompromised host

<2yrs in sub-saharan Africa common cause of bacteremia

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S. typhi

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Acquired by ingestion

No animal host

not self-limiting

antibiotics are indicated

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S. typhi causes a sytemic syndrome

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"typhoid fever"

Diarrhea is not prominent feature of typhoid fever

Responsible for substantial morbidity and mortality

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Salmonella enterica serovar Typhi

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• Causes typhoid fever
• Intestine salmonella invade, enter lymphatic systems, enter blood stream
• Mortality due to intestinal hemorrhage and perforation
• Has Vi antigen=capsule
• Lacking genes present in other salmonella

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S. typhi Epidemiology

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• Found only in humans
• Contracted via ingestion of contaminated food or water
• Asymptomatic excretion from bladder colonization

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S. typhi carriers

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• Associated with gall stones
• Progression to infection to carrier ill defined
• Bile induces responses in bacteria

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S. typhi immunity

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• Natural infection results in protective immunity
• Vacccines: oral live attenuated strain Ty21a
• 24 doses well tolerated
• Cross protection with S. paratyphi
• Parental- non for children <2yrs

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Salmonella Paratyphi A

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• Causes enteric fever
• Human specific
• predominant typhoidal pathogen in some regions
• Often multiply antibiotic resistant

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Pathogenesis of Salmonellosis

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Invasion of epithelial cells; starts in small intestines (non-typhoid stop here)

TTSS

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Salmonella invasion

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Causes transient disruption of the host cell membrane

Effects on actin dynamics resulting in cytoskeletal alerations: SopE and SopE2 activate GTPases; SipA, SipC bind actin

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Type III secretion of Salmonella

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• Causes vacuolar remodeling, prevents lysosomal fusion
• Mutliplication within vacuole
• Bacteria escape and infect other cells (method unknown)
• Some bacteria get through the other side

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Salmonella type III secretion systems encoded on

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PAIs (pathogenecity islands)

SPI 1 -TTSS bacterial uptake

SPI 2 -TTSS intracellular survival and immune evasion

Adherence fimbriae

Vi Capsule (S. typhi only)

Typhi and para typhi have many pseudogenes

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Salmonella lymph nodes

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spread to mesenteric lymph nodes and throughout the body via lymphatics

Taken up by reticuloendothelial cells; some serotypes go onto spleen liver

Most are gastrointestinal

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Salmonella Antigenic structures

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• O and H antigen
• H antigen: Phase I (Id serotype) and II (nonspecific)
• Capsular antigen Vi similar to K capsule antigen- always prevents phagocytosis

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Shigella

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• Closely related to E. coli
• Not normal flora
• Causes: shigellosis, dysentery
• 4 species casuse disease in humans divided into 4 sero groups
• Each group may have multiple serotypes

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Group A

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S. dysenteriae

most serious infection

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Group B

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S. flexneri

Second most common isolate in the U.S.

Associated in gay men and in young adults

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Group C

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More common in dveloping countries

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Group D

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Most common isolate in the U.S.

S. sonnei

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Characteristics of Shigella species

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• Non-motile
• Generally NLF
• Does not produce gas from glucose (except S. flexneri)
• Urea -
• H2S -
• LDC -
• S. sonnei +ONPG and ODC/S. flexneri -

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Dysentery

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Local inflammation, fever, chills, shedding of intestinal lining, mucus, blood, ulcer formation, tenesmus (possible rectal prolapse)

Usually nonfatal and self-limiting in developed countries

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Inoculum for infection of Shigella

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10-200 organism:

Fecal-oral route

High risk: day

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Infection of Shigella

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Results from penetration of the mucosal epithelium

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Shigella spp is the major cause of watery diarrhea and dysentery in what area of the world

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Developing

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Causes persistent diarrhea, protein, loss malnutrition in

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infants and children

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What is the only reservoir for shigella

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humans

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What part of the body does Shigellosis occur? It is considered

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large intestines

Invasive and inflammatory

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Infection of shigella occurs

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via ingestion

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What cells does shigella invade?

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M cells

Passes through lamina propria

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Shigellosis induces what from what cell to release inflammatory meidators

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apoptosis of macrophages

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Shigellosis can also involve the

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intestinal lymphoid tissue

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What helps Shigella move from cell to cell on the basolateral side

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Macrogphages; can quickly escape the vacuole and spread to adjoining cells

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Management of Shigella infection

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• Maintain hydration
• Antibiotics are effective
• Watch for complications
• No vaccine yet

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Most virulent/infective Shigella

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S. dysenteriae

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S. dysenteriae toxin

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Shiga toxin

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S. dysenteriae Shiga toxin

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• Cleaves 28S ribosomal rRNA
• severe colitis
• neurological complications
• hemolytic uremic syndrome

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S. dsyenteriae reactions:

Mannitol fermentation

ONPG

ODC

Serogroup

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-

V

-

A

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S. flexneri reactions:

Mannitol fermentation

ONPG

ODC

Serogroup

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+

-

-

B

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S. boydii reactions:

Mannitol fermentation

ONPG

ODC

Serogroup

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+

V

-

C

Front 131

S. sonnei reactions:

Mannitol fermentation

ONPG

ODC

Serogroup

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+

+

+

D

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General Characteristics of Yersinia

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• Gram Negative
• Motility at 25 deg
• Not cultured

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Three pathogenic Yersinia spps

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Y. pestis

Y. entercolitica

Y. pseudotuberculosis

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Y. pestis colony morphology

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Grey-white translucent

safety pin coccobacilli

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Y. pestis biochemical

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Flocculent growth in broth

Non-motile at 25 deg

Catalase positive

Oxidase, Urease, Nitrate negative

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What agar helps to differentiate Yersinia

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CIN (motility)

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Differentiate Y. enterocolitica

and Y. pseudotuberculosis

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Y.e: ODC, Sucrose +

Y.p: ODC, sucrose=

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Pathogen for Edwardsiella

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E. tarda

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E. tarda profile

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Urea =

LDC +

H2S +

Indole +

Citrate =

Bacteremia wound infecitons

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Most common Citrobacter

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C. freundii

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Identifying Citrobacter

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Weak urease activity

ferment lactose

grow on simmons citrate

MR +