Week 4 no week 3 Flashcards
What are the 6 main characteristics of enterobacterales and where are they found
they are an order of orgs sharing the same characteristics
- mostly in the intestine
-GNB
-Non spore forming
-oxidase negative
-Reduce nitrates to nitrites - ferment carbohydrates
-facultative anaerobes
What are two categories that enterobacter are divided into
Opportunistic
-Extraintestinal - UTI, Nocosomial, Meningitis
E.coli, Klebsiella, Enterobac, Serratia, Proteus, Citrobacter
Primary intestinal pathogens (enteric pathogens)
Salmonella, Shigella , Yersinia , E.coli**
-not part of normal flora
-in animals like poultry, pet turtles, pets
-humans are carriers
-must report all positive results to public health
Where does enterobac get colonized and how does it enter the body
Enter
-mouth to cause CNS infections
-via contaminated medical devices to infect surgical sites
-via contaminated intravenous catheter or fluids to cause bloodstream infections or pneumonias
-via urinary catheter to cause UTI or colonize colon, urethra
what are the Virulence factors for enterobac
- adheres to host cells
-produces toxin
-tissue invasion
-has plasmids that provide resistance genes
such as :
Ecoli, Pneumoniae, Oxytoca,
Extended Spectrum Beta Lactamases = ESBLs
Carbapenemase Resistant Enterobacteriaceae = CREs
What type of antigens are found on Enterobac
O (Somatic)
-heat stable
-on outer wall
H (flagellar)
-heat liable
-K capsular
-heat liable polysaccaride
-in encapsulated species like K1 of E coli, Vi of S Typhi
Where is Ecoli found
what are its virulence factors
its clinical significance
-normal in GI but can cause infections
-some strains are opportunistic
Virulence
-Pili attaches to urinary epithelial mucosa
-cytolysin/hemolysin kills effector cells and inhibits phagocytosis
-Aerobactin allow bacteria to chelate iron
-extra affinity
Clinical - ID via biochemical tubes/intrument
-UTI
-neonatal meningitis and septicemia - KI ag
-bacteremia in adults
-nosocomial and wound infections
Where is Klebsiella found
what does it cause
what is its distinguishing feature
- normal in GI tract of Humans/animals
Oppurtunisitc
Causes
-Pneumoniae, UTI, bacteremia - in immuno
-nosocomial outbreaks - resistance via ESBL and CPE
Features
-seen as mucoid due to presence of capsule that prevents phagocytosis and is associated with virulence
- non motile
can be ID via biochemical tubes and instrument
where is proteus found
what is its clinical significance
how is it ID
Found
-natural in GI tract H/M/E
-found in H as P. mirobilis and P vulgaris
Clinical Sig
-Opportunistic when from blood, urine, wounds and ears
-associated with nosocomial infections from urines
-swarming strain
ID
-via biochemical tubes, auto, CMI
-distinguishing feature PPD positive, Urea positive, H2S positive, has a smell
What are Providencia and Morganella
what are they found as
and their Clinical significance
Oppurtunisitc
-same tribe as Proteus
-normally found in GI of H
-ID via biochemical, auto
-like Proteus but it doesnt swarm and its H2S NEG
Providencia found as
- P. rettgeri - travelers diarrhea
-P. stuartii - burn unit outbreaks
-both known for nosocomial infections through urine
-tough to treat due to intrinsic resistance
Morganella found as
-M. morganii
-isolated from UTIs
-blood can cause neonatal sepsis
What is Serratia found as
Where is it found
what is its clinical significance
Found in
-GI tract of H but NOT A
-found in water sources
-S. marcescens, S. liquefaciens
Clinical Sig
-Opportunistic and nosocomial urine, wounds, resp, blood
-hard to treat very resistant to AB - nurseries, CIU, burn units
ID
-produce red pigment called prodigiosin
-biochemial - LLF, ONPG pos, DNAse Pos
Where is Pantoea found
Clinical Sig
found as
-P. agglomerans
-natural in A/H
-YELLOW PIGMENT
Clinical
-wound, UTI bacteremia
-outbreaks can occur due to contaminated IV fluids
What is Salmonella enterica
-enteric pathogen
-cause enteric disease in elderly, infants, IMC
-disease occurs 12-72 hours after ingestion
Sources except Typhi and Paratyphi
-NORMAL in reptiles, birds, turtles
-spread person to person
-importer food handling
-undercooked chicken and eggs
-contaminated veggies
-unpasteurized dairy
-pet treats
-peanut butter
What are the virulence factors and antigenic structures associated with Salmonella enterica
Virulence
-Fimbriae (help with adherence)
-resistance to gastric acids; can invade gallbladder and peyers patches in bowel
-enterotoxin
Antigenic structure
-O ag somatic
-H ag - flagellea
-Capsular Vi (virulence) also in typhi
what are the clinical infections Salmonella enterica can cause with the 3 categories of disease *****
1-Gastroenteritis (vomiting, diarrhea)
-10^6 high effective dose
-occurs 8-72 hours after digestion starting with belly ache leading to watery diarrhea
-can occur with/out bactermia
-if you have sickle or other hemolytic diseases , ulcerative colitis you are more prone
2-Bacteremia and Extraintestinal infections
-common Choleraesuis, Typhimurium and Paratyphi
-ones that cause fever (nontyphoidal fever), intermittent bacteremia
3 -Enteric fever and typhoid fever
-life threatening with prolonged fever/bacteremia
-multisystem (lymph nodes, blood, liver, spleen) caused when the serotypes Typhi and Paratyphi are invovled
-necrosis of peyers patches and gallbladder =perforation of bowel
-tropical places, bad hygiene , no running water
4- CARRIER STATE after salmonella infection is in the gall bladder
how is salmonella enterica ID’d and treated
ID
via biochemical tube NLF, H2S positive
Treatment
-AST testing
-anti diarrhea meds are not recommended as it may encourage invasion
-fluid replacement if dehydrated
-gall bladder removal
Where is Yersinia enterocolitica found and what are its virulence factors
Found
-NOT in H NF
-NF in pigs, dogs and cats
-undercooked food, vac pack meat, contaminated water
Virulence
-attaches via fimbria to invade intestinal mucosa and spread to lymphatic tissue (mesenteric lymphadenitis)
-survives in cool temps so refrigeration is not effective
-Post transfusion sepsis associated with transfusion of contaminated packed RBC
What is the clinical significance of Yersinia enterocolitica
Enterocolitis (acute enteritis)- most common
-headache, abdominal pain, diarrhea
-bloody stool
-children 1-5
Appendicitis like syndrome
-older kids and immunocom adults
-tummy ache and fever
Arthritis
-happens after gastrointestinal episodes
-Erythema nodosum - red itchy/burny nodules
What is the lab diagnosis of Yersinia enterocolitica and how is it treated
-GNB - bipolar staining like Yersinia pestis
-biochem testing
-use CIN media for primary isolation makes a bullseye due to mannitol fermentation
Treated
-dont treat unless bacteremia
Where is Yersinia pestis –Bubonic Plague found
-NOT enteric pathogen only related
-transmitted to H via flea or rat bite or p/p if they have pneumonia
-incubates 1-3 days for pneumonic form and 2-6 days for bubonic or glandular form
-endemic
-associated with prairie dogs in southwest US
What are the virulence factors of Yersinia pestis –Bubonic Plague
and what is it clinical significance
-adaptation to survive intracellularly with anti-phagocytic capsule, exotoxins, endotoxins, coagulase and fibrinogen
Clinical
Bubonic plague
-cause of black death
-macrophages take up org, multiply and release
-spread through lymph system forming hot lymph nodes or buboes spreading to organs
-90% if untreated
Pneumonic plague
-2dary to bubonic plague
-into bloodstream and resp tract
-if untreated leads to septicimia -100% fatal if untreated
Germ warfare/bioterrorism
how is Yersinia pestis handled in the lab and treated
-report to public health
-GNB with bipolar staining with safety pin appearance
-NLF
-ID via biochemical and auto
treated
-vaccine available
where is shigella found and how is it transmitted
-p/p; oral fecal through poor sanitation, overcrowding
-contaminated food sources
-need low dose
-highly communicable
what are the virulence factors for Shigella
-able to adhere to and invade mucosal cells, escape from phagocytic vesicles, intercellular spread
-SHIGA TOXIN - inhibits protein synthesis- associated with HUS
-has O and K ag (need to boil the suspension to remove k)
-NOT MOTILE so no H ag
Why is shigella clinically significant
-after 1-2 being exposed you are diseased
-asymp or fever, tenesmus
-Causes classical bacillary dysentery (blood, mucous, and pus in poop)
acute inflammatory colitis and blood diarrhea because the intestinal epithelial cells have been penetrated and ulcers have formed
which strains of Shigella are associated with what and how to ID
S. sonnei Serogroup D- watery diarrhea
Predominant strain found in N.A.
S. dysenteriae Serogroup A –bloody stool (Shiga toxin) within hours, associated with hemolytic uremic syndrome (HUS)
S. flexneri Serogroup B – Most common cause of the endemic form of shigellosis
Endemic form cause of most shigellosis-related death
S. boydii Serogroup C– dysentery
ID via
biochemical testing, Vitek NOT MALDI
-serology LATEX TESTING GOR GROUPS A-D
Enterohemorrhagic E. coli (EHEC) orShiga Toxin-producing E. coli (STEC)
What is most important one
-E COLI 0157:H7
-produces cytotoxins (verotoxins 1,2)
-serotoxin VI is similar to Shiga toxin of S dysteneraiae - shiga toxin
-cow vaccine developed
how is Escherichia coli 0157:H7 transmitted
-dairy and beef cattle
-contaminated food or water
-if ground beef isnt handled properly
-apple cider, bean sprouts, lettuce, mayonnaise
what is the clinical significance of Escherichia coli 0157:H7
-diarrhea can be bloody
-low or NO fever
-no leuks in stool (different from Shigella)
-Hemolytic uremic syndrome (HUS)
- RBC prematurely destroyed and clog the kidneys causing Microangiopathic hemolytic anemia , thrombocytopenia, acute renal disease
how do we test for Escherichia coli 0157:H7
-NON MOTILE, sorbitol negative so use MAC with sorbitol (no color)
-serology with 0157:H7 antiserum
Enterotoxigenic E.coli (ETEC)
caused by:
virulence :
treated /clinical:
Caused:
-poor hygiene, contaminated food/water
Virulence factor:
-pili (colonization), toxins that increase water and lytes secretion
-heat liable or heat stable toxin production
Clinical
-travelers diarrhea- DUKORAL
-infant bacterial diarrhea
Enteropathogenic E.coli (EPEC)
caused by:
virulence :
treated/clinical :
Caused:
-outbreaks in nurseries and daycare
Virulence :
-pili- mucosal bowel attachment
-does NOT produce enterotoxin or Shiga toxin
-NOT invasive
Clinical
-infantile diarrhea
Enteroinvasive E. coli (EIEC)
caused by:
virulence :
treated/clinical :
-Watery diarrhea, fever but can progress to dysentery like symptoms, bloody stool with mucus
Virulence
-invades enterocytes in colon and destroys intestinal mucosa
- similar to Shigella can be NON MOTILE
how do we workup enteric pathogens
-Large grey NH or BH-Ecoli
-collect stool specimen early into the illness so the org that caused it is still there in large numbers if collected when youre recovering then it can be negative
-needs to be processed in 2hours
-transport media when delayed Cary Blair
-if you have enteric fever like typhoid it will be recovered from blood not stool
-NO GRAM on stool
-culture on enteric media - MAC, SMAC, HEK, CIN, Campylobacter and Selenite broth
-NOT on BA, CHOC OR ANAO2 due to excess NF in stool can be overgrown
-Biochemical tubes
-MALDI
-REPORT to public health and send to their lab
What do we look for when using MAC agar
-LF or NLF
-selective for GNBs but not all that grow on it are Enterobacteriaceae
-selective and differential
-Bile salt and CV inhibit GP - SELECTIVE
-Lactose and neutral red indicator DIFFERENTIAL based on lactose fermentation
What is in Salmonella Shigella (SS) Agar
-Bile salts, sodium citrate, ferric citrate and brilliant green -SELECTIVE to removed non sig GNB
-Neutral red is DIFFERENTIAL also indicator since it detects lactose fermentation
-H2S indicator
What is in Hektoen Agar
DIFFERENTIAL - CHO fermentation and H2S production ; 3 carbs Lac, Suc, Salicin
SELECTIVE - bile salts inhibit GP
INDICATOR - bromothymol blue
DETECTION OF H2S - sodium thiosulphate and ferric ammonium citrate
Fermenter - yellow -orange
Non fermenter - green-blue
What is in MacConkey with Sorbitol (SMAC)
-just like MAC but instead of lactose it has sorbitol
-differentiates between non sorbitol fermenting EHEC or E. coli 0157:H7)
fermenter- SF- pink
NON sorbitol fermenter NSF - no color
What is in Cefsulodin, Irgasan, Novobiocin (CIN)
Selectively isolate Yersinia enterocolitica from stool specimens
SELECTIVE- Antimicrobics: C, I, N
INDICATOR Neutral red, mannitol
Yersinia- small dark red center with clear margin (“bull’s eye)
Methods of Identification of unknown enteric pathogens
Maldi-tof - Known limitations: can’t tell the difference between E. coli and Shigella
Vitek2 GN ID card
API 20E
Biochemical tubes
PCR
Confirmation/typing using antisera E.g. E. coli O157
what does the oxidase tube contain and what is the interpretation
Purpose: distinguish between the GNB in the Enterobacteriaceae family (oxidase -) and Pseudomonas (oxidase +)
Reagent: 6% tetramethyl phenylenediamine OR Dimethyl-p-phenylenediamine oxalate reagent
Interpretation: If oxidized by the cytochrome oxidase system, they turn purple (positive)
what does the Oxidation –Fermentation (O-F) contain and what is the interpretation
Purpose: Determines if organism is capable of
metabolizing CHO aerobically (oxidative), anaerobically (fermentative) or not at all
* All Enterobacteriaceae are all positive for both
Ingredients: glucose (can have other sugars e.g. maltose), peptone, bromothymol blue
Set up: Run in duplicate, stab both tubes with organism, cover one with oil
what does the Nitrate tube contain and what is the interpretation
Purpose: To determine if an organism can reduce nitrate to nitrites or nitrogen gas (ammonia). Enterobacteriaceae reduce nitrates to nitrites
Set up: (Check for bubbles in Durham tube before inoculating).
2 Reagents : Dimethyl-α-naphthylamine in glacial acetic acid, Sulfanilic acid in glacial acetic acid (Nitrate A and Nitrate B)
Interpretation : observe for the presence of gas in the Durham tube before adding reagents
If it turns red after the addition of reagents: Nitrates reduced to nitrites.
If NO colour, add zinc dust –if it turns red then nitrates not reduced, no colour then nitrates reduced to nitrogen gas (N2)
This test is can be done on an API strip (in the glucose well after the result is recorded; observe for the presence of gas)
what does the Triple Sugar Iron tube contain and what is the interpretation
Purpose: Detects the fermentation of glucose (0.1%), lactose (1%), and/or sucrose (1%), detects gas production (H2, CO2), and H2S production (ferrous sulfate and sodium thiosulfate)
pH indicator: Phenol red
Set up: stab butt, streak slant, loose caps, incubate 35C in O2
Interpretation: observe for the presence of gas, H2S and colour change in the slant and butt
A/A=all sugars fermented
ALK/ALK=no CHO fermented AEROBIC
ALK/A = only glucose fermented
add g if you see gas or H2S if black
what does the SIM (H2S, Indole, Motility) tube contain and what is the interpretation
Purpose: Used in the identification of Enterobacteriaceae –determines 3 reactions (tests)
Set up: Inoculate by stabbing straight down (and back up). Incubate O2 at 35°C
Reagent: Kovac’s reagent
Interpretation: observe for motility and H2S (black) before adding reagent. Indole: Observe for red at top (after addition of Kovac’s)
if all black then MOTILE AND H2S
what does the Urea tube contain and what is the interpretation
Purpose: determine if the organism produces urease (to break down urea)
Set up: Inoculate slant with zigzag streak, loosen cap, Incubate O2 at 35C
Interpretation: observe for intense pink colour (positive) – the entire tube does not always go pink
You can check against an un-inoculated tube
what does the Decarboxylase tube contain and what is the interpretation
Purpose: to determine if an organism has the ability to decarboxylate an amino acid
useful for identification of gram negative bacilli –especially Enterobacteriaceae
Tubes contain the selected amino acid (not control tubes), glucose, peptones and 2 indicators: bromocresol purple and cresol red.
Set up: test must be set up with decarboxylase control (does not contain the amino acid),
Inoculate the liquid medium just below the surface, cover each tube with oil WHY?
Interpretation: purple is a positive test
Control tube must be valid (yellow)
what does the Citrate tube contain and what is the interpretation
Purpose: to determine if the organism uses citrate as a sole source of carbohydrate. Used in the identification of many organisms including Enterobacteriaceae
Ingredients: sodium citrate, ammonium salts (source of nitrogen), Bromothymol Blue indicator
Set up: Lightly inoculate by streaking the slant.
Incubate tubes with loose caps at 35°C in O2
Interpretation:
growth and or change to blue: positive
no growth and no change in colour: negative
what does the Phenylalanine Deaminase (PAD/PPD) tube contain and what is the interpretation
Purpose: Useful in identification of Proteus, Morganella and Providencia species.
Set up: streak slant with a zigzag pattern
Reagents: Ferric chloride
Interpretation: Presence of a dark green colour after the addition of reagent (positive)
what does the MR (Methyl Red) tube contain and what is the interpretation
Purpose: Detects the presence of mixed acids from the breakdown of glucose. Useful in the identification and differentiation of Enterobacteriaceae
Set up: Inoculate liquid (not too heavy) media below surface. Incubate in O2 at 35°C (ideal 48 hours)
Reagents : Methyl red indicator
Interpretation: Red colour: positive
what does the VP (Vogues Proskauer) tube contain and what is the interpretation
Purpose: Useful in the identification of Enterobacteriaceae
Set up: Inoculate liquid media below surface
Reagents: alpha naphthol and Potassium hydroxide
Interpretation: After adding reagents, observe for pink colour within 10 minutes (positive). Ecoli is NEG and E aerogenes is POS
Additional information: Media is the same as that used for Methyl Red
READ the SOP before adding reagents –you need to split the tube contents from MR
This test is on the API strip –same reagents but add them in the opposite order
what does the ONPG tube contain and what is the interpretation
Purpose: ONPG is used to detect β-galactoside permease negative, β galactosidase positive organisms, used to determine if the organism is a “slow” or “late” lactose fermenter.
Set up: Place disc in 0.5 mL saline in tube, inoculate and incubate in O2 @35°C
Interpretation: any shade of yellow is positive
NLF will be negative
LF and LLF organisms will be yellow
