Quiz 3

Methyl red, citrate test, new bacteria, etc.

Methyl red test

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

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