Quiz 3

Methyl red, citrate test, new bacteria, etc.

Methyl red test

* +=convert glucose → pyruvate → mixed organic acids =red color
* indicator is methyl red, turns red at ph 4.5 or less
* IMViC media for MR/VP test
* Differential media

T/F organisms tested + in MR tested + in VP

F, anything negative in MR are positive in VP

VP test (Voges- Proskauer test)

* += pink, glucose→ Pyruvate→ acetoin (intermediate)→ butanediol pathway (2,3-butanediol)
* differential media
* Indicator: alpha-naphthol and potassium hydroxide

IMViC stands for

I= Indole

M= Methyl red

V= Voges- Proskauer

C= citrate

Citrate test

* media contains: citrate (carbon source) and inorganic ammonium salts (nitrogen source)
* Tests for citrate permease: converts citrate to Pyruvate
* ammonia salt is broken down to ammonia it creates alkalinity
* Indicator: **bromthymol blue** **(**turns from green to blue for +)
* differential media, selective for citrate utilizers

HE agar

* selective and differential (isolation and differentiation of gram -)
* Differentia agents: lactose, sucrose, salicin
* Indicator: Bromothymol blue and acid fuchsin
* Colonies of Salmonella and Shigella spp. are green to bluish-green in color.
* H2S producers are black at the center of the colonies
* ph indicators turn yellow under acidic conditions
* selective agent: bile salt
* differential agent: sodium thiosulfate
* Indicator: ferric ammonium citrate

Ferric ammonium citrate is added to HE agar why?

react with H2S and form a black precipitate

XLD agar

* Selective Agent: bilesalts Deoxycholate
* indicates lactose fermentation and H2S production
* Differential agent: lactose, sucrose, salicin
* Indicator: bromthymol blue,acid fuchsin –pH indicators that will turn yellow under acidic conditions
* Differential Agent: sodiumthiosulfate
* Indicator: ferric ammoniumcitrate
* Selective and differential

In the XLD agar what is used to detect sulfer reduction

sodium thiosulfate and ferric ammonium citrate

Enterobacteriaceae

* all ferment glucose
* all are oxidase negative
* all reduce nitrates to nitrites
* Many are catalase positive

Opprotunistic Enterobacteriaceae

* Often part of the humans normal intestinal flora
* Outside of normal habitat can cause serious infections
* ex. E.coli- can cause septicemia if its in the blood

E. coli normal flora

* Normal GI tract flora
* female genital tract

E. coli transmission

* for non-gastrointestinal- endogenous or direct contact for gastrointestinal (it varies with strain)
* fecal-oral spread
* contaminated food or water (uncooked beef or unpasteurized milk)

Edwardsiella tarda normal flora

* Gi tract of cold-blooded animals (reptiles)

Edwardsiella tarda transmission

* contaminated water
* Contact with animal carrier

Citrobacter, Enterobacter, Klebsiella, Morganella, Proteus, Providencia, and Serratia Normal flora

Normal GI tract flora

Citrobacter, Enterobacter, Klebsiella, Morganella, Proteus, Providencia, and Serratia transmission

* Endogenous- direct contact
* Enterobacter-medical devices
* Serratia- healthcare associated

Ecoli extraintestinal infection

* Urinary tract infections
* Bacteremia
* Neonatal meningitis
* Most common G- causing nosocomial infections

Citrobacter, Enterobacter, Klebsiella, Morganella, Proteus, Providencia, and Serratia pathogenesis

* Nosocomial infections of the respiratory tract
* urinary tract (==Proteus, Citrobacter==) infections
* blood infections
* ==Enterobacter==- top 10 in healthcare-related infections
* med. devices
* ==K. pneumoniae== CC23
* pyogenic liver abscess

E.coli vireulence factors

* Adhesions Endotoxin
* Capsule production
* Pili- attachment

Citrobacter, Enterobacter, Klebsiella, Morganella, Proteus, Providencia, and Serratia virulence factors

* Endotoxins
* Capsules
* Adhesion proteins
* Resistance to multiple antimicrobial agents (panresistant strains of K. pneumoniae)

Enterotoxigenic (ETEC) infection

Traveler’s and childhood diarrhea (food and water)

Enterotoxigenic (ETEC) virulence factor

* Pili
* GI colonization
* Heat-liable
* Heat-stable Enterotoxins
* (secretion of water and • electrolytes into bowel)

Enteroinvasive (EIEC) infection

* Dysentery (necrosis, ulceration,inflammation of the bowel)

Enteroinvasive (EIEC) virulence factor

* Invade enterocytes lining large intestine (Shigella-like)

Enteropathogenic (EPEC) infection

* Diarrhea in infants
* Can cause chronic diarrhea

Enteropathogenic (EPEC) virulence factors

* Bundle-forming pilus
* intimin
* other factors that mediate attachment to mucosal cells of the small bowel (loss of microvilli)

Enterohemorrhagic (EHEC) (STEC) infection

* Inflammation
* bleeding of the mucosa of the large intestine.
* Hemolytic-uremic syndrome possible (toxin)

Enterohemorrhagic (EHEC) (STEC) virulence factors

* Similar to Shiga toxin of Shigella.
* E. coli O157:H7

Enteroaggregative (EAEC) infection

* Watery diarrhea (can be prolonged)

Enteroaggregative (EAEC) virulence factors

* Binding by pili.
* Shiga-like toxin
* hemolysin- like toxins.

Salmonella enterica Occurance

Only in humans/mammals (not normal flora)

Salmonella bongori occurance

Widely spread in nature (animals)

Salmonella bongori transmission

* Contaminated food processed from animals
* Fecal-oral route in healthcare setting when handwashing guidelines not followed

Salmonella enterica transmission

• Fecal-oral route (Contaminated food and water)

Shigella sp. occurance

Only in humans/primates (not normal flora)

Shigella sp transmission

* Person-to-person
* Contaminated food or water

Yersinia sp occurance

Rodents-(Y. pestis) (not normal flora)

Yersinia sp. transmission

* Bite of flea vectors
* Pneumonic by airborne droplets
* Other Yersinia sp. by undercooked meat or contact with infected animals

Salmonella sp. infection

* Gastroenteritis and diarrhea where infection is limited to mucosa and submucosa of GI tract
* Bacteremia and extra-intestinal infections
* Enteric fever (typhoid) prolonged fever and multisystem involvement blood, lymph nodes, liver, & spleen

Salmonella sp. virulence factors

* allow survival in and destruction of phagocytes, and facilitate spread.

Shigella sp. infection

Dysentery- defined by acute inflammatory colitis and bloody diarrhea characterized by:

* cramps
* bloody and mucoid stools

Shigella sp. virulence

* escape from phagocytic vesicles.
* Intercellular spread and inflammation.
* Shiga toxin.

Yersinia pestis infection

* Bubonic plague
* High fever and buboes proceeding to rapid and severe bacteremia
* Pneumonic plague
* Involves the lungs and characterized by malaise and pneumonia symptoms.
* Both are rapidly fatal.

Yersinia pestis virulence factor

* Many - Adapt for intracellular survival.
* Produce antiphagocytic capsule
* exotoxins
* endotoxins
* coagulase
* fibrinolysin

Y. enterocolitica, Y. pseudotuberculosis infection

* Enterocolitis is characterized by fever, diarrhea, and abdominal pain.
* Can cause acute mesenteric lymphadenitis (presents as appendicitis).

Y. enterocolitica, Y. pseudotuberculosis virulence factors

Encoded on a virulence plasmid which allows attachment and invasion of intestinal mucosa and spread to lymphatic tissue.

Microbiome:

\
the community of microorganisms (such as fungi, bacteria and viruses) that exists in a particular environment” (NHGRI – genome.gov)

Gut Microbiome roles

* Gut microbes known to convert bile salt and bile acids to unconjugated bile acids and secondary bile acids.
* Gut microbes ferment starch and other polysaccharides the host cannot process.
* Gut microbes produce short chain fatty acids and vitamins for the host.
* Help development of the naïve immune system.
* Can work with host functions and alter regulation of host responses

IBD

Crohn’s disease and ulcerative colitis no identified pathogenic cause

Type I diabetes

\- flora affecting handling of nutrients in the intestine.

Antibiotics- (microbiome)

need to use more focused antibiotics (fidaxomicin- C. difficile). • Bacteriophage therapy

Probiotics-

* try to add back the “missing” important microbe.
* Often Lactobacillus and Bifiddobacterium.
* Yogurt and kefir- some effect on C. difficile infections
* Best results for acute gastroenteritis in children

Prebiotics and diet therapy-

* supply nutrients to favor growth of beneficial microbes.
* Generally non-digestable carbohydrates (microbe will digest)
* Best results seen with Crohn’s disease in children
* exclusive enteral nutritional (EEN) therapy

Microbial restoration

* transplantation of an intact microbial community.
* FMT- fecal material transplant
* Shown very effective in C. difficile treatment.
* Not shown effective in treatment for IBD or obesity yet.

What organisms are + for MR test and VP?

Lysine decarboxylase and ornithine decarboxylase test

* indicator is bromocresol purple and cresol red
* Works best when oxygen is excluded( hence mineral oil on top)
* enzyme removes a carboxyl group which leaves an alkaline end product

Listeria monocytogenes found where

* Found in animals, birds, sewage, soil, milk, milk products, and vegetable matter.
* Found in the human GI tract and in the vagina of healthy humans

Listeria disease is spread by:

In humans, the disease is spread by:

* Transmission- direct contact


* Ingestion of contaminated food (meat, vegetables, and diary)

Listeria monocytogenes pathogenesis

* Bacteremia
* CNS infections- meningitis, encephalitis, spinal cord infections.
* Focal infections (endocarditis, arthritis, osteomyelitis, etc) is less common
* ==Neonatal==- Early- granulomatosis infantisepiticum (in utero infection disseminated systemically that causes stillbirth) -
* ==Late onset==- bacterial meningitis.

Listeria virulence factors

* Listeriolysin O-hemolytic and cytotoxic toxin that allows survival within phagocytes
* Internalin- Cell surface protein that induces phagocytosis
* Act A- Induces actin polymerization on the surface of host cells
* Produces cellular extensions- facilitates cell-to-cell spread
* Siderophores- scavenges iron from human transferrin.

Vibrio species normal habitat

* Habitat- brackish or marine water
* Not part of the normal human flora

Vibrio cholerae pathogenesis

* profuse, watery diarrhea leading to dehydration, hypotension, and often death. (occurs in epidemics and pandemics)
* May cause nonepidemic diarrhea and occasionally extra-intestinal infections of wounds, septicemia, and eye and ear infections.

Vibrio cholerae virulence factors

* Cholera toxin- causes mucosal cells to hyper-secrete water and electrolytes into the GI tract lumen Zot toxin
* accessory cholera toxin
* hemolysins/cytotoxins Motility and chemotaxis- mediate the distribution of the organisms
* Mucinase- allows penetration of the mucosal layer
* Toxin coregulated pili (TCP)- means by which the organism attaches to mucosal cells for cholera toxin release

Acinetobacter sp occurance

* Widely distributed in nature (soil, water, food) including hospitals
* Can be found on skin
* UR tract in extended stay hospitalized patients

Acinetobacter sp. transmission

* Medical instruments
* IV
* catheters

Pseudomonas aeruginosa occurance

* Survives well in domestic and hospital environments
* Rarely found as normal flora

Pseudomonas aeruginosa occurance

* Survives well in domestic and hospital environments
* Rarely found as normal flora

Pseudomonas aeruginosa transmission

* Contaminated food and water
* Contaminated medical devices
* Introduced by penetrating wounds

Pseudomonassp. occurance

* Environmental
* not normal flora

Pseudomonas sp. transmission

Medical devices

Alcaligenessp occurance

* Soil and water


* Hospital environments

Alcaligenessp. transmission

* Contaminated medical devices
* solutions

Acinetobacter sp disease

* Usually nosocomial
* During warm seasons
* Most commonly- genitourinary tract, respiratory tract, wounds, bacteremia

Pseudomonas aeruginosa disease

* Folliculitisotitis externa
* eye infections
* Following trauma
* osteomyelitis
* endocarditis
* Nosocomially RTI, UTI, wounds, bacteremia, and CNS infections

Pseudomonas aeruginosa virulence factors

* Exotoxin A
* endotoxins
* proteolytic enzymes
* alginate
* pilli
* Antimicrobial resistance

Pseudomonas sp. disease

Uncommon in UTI, RTI, wounds, and bacteremia

Alcaligenes sp. disease

* Usually immunocompromised patients
* Often a contaminant
* Found in blood, RT, and urine

Oxidation-Fermentation Test

* Fermenters use pyruvate and NADH (from glycolysis) to produce acids that acidify medium (lowers pH)
* organisms that cannot use sugar may degrade amino acids alkalinizing medium (raises pH)
* Glucose oxidizers- in the absence of oxygen glucose cannot be utilized
* Fermentation generates more acid and will turn media more yellow than oxidation
* Contains bromthymol blue as pH indicator

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The Enterotube II

* 15 biochemical tests
* (glucose, gas production, lysine decarboxylase, ornithine decarboxylase, H2S, indole, adonitol, lactose, arabinose, sorbitol, Voges-Proskauer, dulcitol, phenylalanine deaminase, urea and citrate)
* After 18 to 24 hours of incubation, interpret all reactions, with the exception of indole and VP.

The API (Analytical Profile) 20E

* Biochemical panel for identification and differentiation of members of the family Enterobacteriaceae
* It holds 20 mini-test chambers containing dehydrated media having chemically-defined compositions for each test.
* They usually detect enzymatic activity, mostly related to fermentation of carbohydrate or catabolism of proteins or amino acids by the inoculated organisms.

Spores and toxins are killed using

bleach