Enterobacteriaceae (Coliforms) Gram Negative Rods Enterobacteriaceae (Coliforms)

API 20E Test Strip All Negative All Positive The fast way to run many tests on an organism is to inoculate either an APT 20E test strip ($12 each) or an Enterotube ($45 each). The organism is placed into each well and the results are read two days later.

API 20E Test Record Sheets

Enterotube: more expensive than API-20E media

Symbols that indicate test results: A: acid has formed. This usually indicates a sugar nutrient was broken down. ALK: means the media is alkaline after the test. This usually also indicates a negative test, so we just abbreviate it to “K”. Therefore: K: means the test was negative G: means gas formed

You could also perform each of those tests in individual test tubes or plates, as shown in the following slides.

Thio Broth: Determines oxygen requirements This test is done to see if the organism is aerobic (growth only at the top), anaerobic (growth only at the butt), or facultative (growth throughout). A facultative organism can live with or without air, so that is considered a virulence factor.

Thio Broth Not shown: anaerobic; growth only at the butt Aerobic Facultative

Litmus milk This is done to see if the organism has the enzyme lactase. If it does, it will ferment lactose, the milk sugar. The waste product will be an acid, so it will change the pH indicator from bluish to pink. If gas is produced during this process, gas bubble will also appear. Some organisms ferment lactose and produce gas, and some ferment lactose without making gas. Since milk also contains a protein called casein, this test will also determine if the organism has the enzyme caseinase to break that protein down. If it breaks the protein down only partially, curds (solid milk) will form. If it can break the milk protein all the way down to amino acids, a brown ring appears at the top.

Litmus Milk Test P A A/C A/CG A A K White (R) means litmus was reduced and became clear. Pink (A) means it fermented lactose and is acidic. Bluish (K) means the pH is basic, so lactose was not fermented Curds formed (C) Gas in the curd (G) Brown ring (P) means the casein protein in the milk was broken down (proteolysis)

Litmus Milk A/CG

Motility Test Positive

Triple Sugar Iron (TSI) Slants A = acid (sugar was fermented) K = negative result G = gas formation H2S = sulfur was used, iron was reduced to black color K/AG K/K K/A H2S A/AG A/AG

MR Test: pH indicator Negative Positive (acid present)

VP Test Negative Positive

IMViC (+ + - -) E. coli Indole MR Citrate VP Positive Positive Negative Citrate Negative

IMViC (- - + +) Enterobacter Indole MR Citrate VP Negative Negative Positive Citrate Positive

IMViC (+ - - -) Indole Citrate MR VP Positive Negative Negative

Urea Test Negative Positive Negative

Citrate Positive Negative

Antibiotic Sensitivity

Antibiotic Sensitivity Organism is resistant to the antibiotic Organism is susceptible to the antibiotic

Antibiotic Sensitivity: Check zone diameters on given table Organism has intermediate resistance to the antibiotic Organism is resistant to the antibiotic Organism is susceptible to the antibiotic

Positive Coagulase Test: Differentiates S. Aureus from S epidermidis

Catalase

Oxidase Test

Citrobacter freundii

Citrobacter freundii Uses citrate as their sole source of carbon. These bacteria can be found almost everywhere in soil, water, wastewater, etc. It can also be found in the human intestine. They are rarely the source of illnesses, except for infections of the urinary tract and infant meningitis.

Citrobacter freundii Motility: negative MR: positive - - - - + + +

Escherichia coli Motile species Non-motile species

Escherichia coli

Escherichia coli One of the main species of bacteria living in the lower intestines of mammals, known as gut flora. There are about 1 trillion excreted in the feces daily. However, the bacteria are not confined to this environment, and specimens have also been located, for example, on the edge of hot springs. The E. coli strain O157:H7 is one of hundreds of strains of the bacterium that causes illness in humans. E. coli are unable to sporulate. Thus, treatments which kill all active bacteria, such as pasteurization or simple boiling, are effective for their eradication, without requiring the more rigorous sterilization which also deactivates spores.

Escherichia coli E. coli can generally cause several intestinal and extra-intestinal infections such as urinary tract infections, meningitis, peritonitis, septicemia and pneumonia. Although it is more common in females due to the shorter urinary tract, urinary tract infection is seen in both males and females. Poor toilet habits can predispose to infection (doctors often advise women to "wipe front to back, not back to front") but other factors are also important (pregnancy in women, prostate enlargement in men). If E. coli bacteria escape the intestinal tract through a perforation (a hole or tear, for example from an ulcer, a ruptured appendix, or a surgical error) and enter the abdomen, they usually cause peritonitis that can be fatal without prompt treatment. However, E. coli are extremely sensitive to such antibiotics as streptomycin or gentamycin.

Escherichia coli Certain strains of E. coli, such as Escherichia coli O157:H7, produce a toxin and can cause food poisoning usually associated with eating cheese and contaminated meat (contaminated during or shortly after slaughter or during storage or display). The usual countermeasure is cooking suspect meat "well done"; the alternative of careful inspection of slaughtering and butchering methods (to make sure that the animal's colon is removed and not punctured).

Escherichia coli Escherichia coli O157:H7 is the strain is believed to be associated with the 2006 United States E. coli outbreak linked to fresh spinach. Severity of the illness varies considerably; it can be fatal, particularly to young children, the elderly or the immunocompromised E. coli are susceptable to amoxicillin, cephalosporins, aminoglycosides, streptomycin, or gentamycin. Antibiotic resistance is a growing problem. Some of this is due to overuse of antibiotics in humans, but some of it is probably due to the use of antibiotics as growth promoters in food animals.

Escherichia coli - -

Enterobacter aerogenes

Enterobacter aerogenes Several strains of the these bacteria are pathogenic and cause opportunistic infections in immunocompromised (usually hospitalized) hosts. The urinary and respiratory tract are the most common sites of infection. Enterobacter can be distinguished from other Gram neg rods by virtue of being a 'fast fermenter' of lactose (as are E. coli and Klebsiella).

Enterobacter Methyl Red: negative + +

Klebsiella pneumoniae

Klebsiella pneumoniae This is a nonmotile, encapsulated, lactose-fermenting, and facultatively anaerobic organism. It is found in the normal flora of the mouth, skin, and intestines. It naturally occurs in the soil and about 30% of strains can fix nitrogen in anaerobic conditions.

Klebsiella pneumoniae K. pneumoniae can cause bacterial pneumonia, typically due to aspiration by alcoholics, though it is more commonly implicated in hospital-acquired urinary tract and wound infections, particularly in immunocompromised individuals. Klebsiella ranks second to E. coli for urinary tract infections in older persons. Feces are the most significant source of patient infection, followed by contact with contaminated instruments. Klebsiella is resistant to ampicillin but susceptible to aminoglycosides and cephalosporins.

Klebsiella pneumoniae

Proteus vulgaris

Proteus vulgaris Responsible for many human urinary tract infections. Proteus species do not ferment lactose, are oxidase negative, and urease positive; some species are motile.

Proteus vulgaris

Providencia stuartii

Providencia stuartii This organism is motile. They are opportunistic pathogens in humans and can cause urinary tract infections, particularly in patients with long-term indwelling urinary catheters or extensive severe burns. Some strains are sensitive to ampicillin.

Providencia stuartii IMViC: + + - + ODC: - ONPG: + H2S: - Mannitol: -

Salmonella enterica

Salmonella enterica

Salmonella enterica This organism causes typhoid fever and foodborne illness. Salmonella species are motile and produce hydrogen sulfide. They usually do not ferment lactose.

Salmonella enterica Food poisoning from Salmonella is usually from poultry and raw eggs and from food that has been cooked or frozen, and not eaten straight away. The overuse of antibiotics in both the poultry and beef industries have created a strain of salmonella which is potentially resistant to antibiotics. It can also be caught by handling reptiles, such as iguanas.

Salmonella enterica The prevention of Salmonella as a food illness involves effective sanitizing of food contact surfaces. Alcohol has proven to be an effective topical sanitizer against Salmonella.

Salmonella enterica

Shigella somnei

Shigella somnei This organism is non-motile, non-spore forming. It is H2S positive. It is the causative agent of human shigellosis. Shigella infection is typically via ingestion (fecal–oral contamination). 10 bacterial cells can be enough to cause an infection.

Shigella somnei The stool may contain blood, mucus, or pus (eg dysentery). Shigella cause dysentery that results in the destruction of the epithelial cells of the intestine. Some strains produce an enterotoxin similar to E. coli O157:H7 Symptoms usually begin two to four days after ingestion. Symptoms usually last for several days, but can last for weeks. Severe dysentery can be treated with ampicillin.

Shigella somnei IMViC: - + - - or + + - - ODC: + or - LDC: - Urease: - H2S: - Sucrose: - TSI: K/A

Serratia marcescens

Serratia marcescens This organism is involved in nosocomial infections, particularly urinary tract infections and wound infections. It is motile. Due to its preference for damp conditions, it is commonly found growing in bathrooms (especially on tile grout), where it manifests as a pink discoloration. Once established, complete eradication of the organism is often difficult, but can be accomplished by application of a bleach-based disinfectant.

Serratia marcescens S. marcescens can cause conjunctivitis and tear duct infections. It is common in the respiratory and urinary tracts of adults and the gastrointestinal system of children.

Serratia marcescens IMViC: - + + + or - - + + LDC: + ARA: - ORN: +

Lactose Negative Perform an Indole test: Positive Negative Proteus vulgaris (H2S negative) Providencia stuartii (H2S positive) E. coli (Citrate negative) Citrobacter freundii (H2S positive, Citrate positive) Negative Perform a urease test (all should be negative) Perform the MR-VP test

Urease negative Perform a motility test Positive Negative Salmonella enterica (H2S positive) Serratia marcescens (H2S negative) Negative Shigella somnei

MR-VP Tests +/- -/+ Citrobacter freundii Klebsiella pneumoniae (non-motile) Enterobacter aerogenes (motile)