Lab 8:

1. Lab 8: Isolation of Enterococcus in foods

2. Identifying characteristics • Gram positive. • Cocci shape. • Non-motile. • Occur in pairs or short chains. • Cells are one micrometer in diameter. • Predominately inhabit human intestines. • 7. facultative anaerobes (prefer anaerobic). • 8. Complex and variable nutritional requirements. • 9. Resistant to many Gram-positive antibiotics. • 10. members of the lactic acid bacteria (LAB) group. • Perform simple fermentation.

3. Mechanism of pathogenicity unknown. • Used as indicators of fecal pollution in the purification of water and dried and frozen foods. • Members of genus streptococcus. • Belong to Lancefield's serologic group D Streptococcus. • Catalase negative. • Can grow in 6.5% NaCl. • Can grow at a pH range of 9.6 to 4.6. • Can grow at temperatures ranging from 10 to 45°C. • Optimum growth at 37°C. • Sensitive to chlorination.

4. Taxonomic description • The enterococcus group is a subgroup of the fecal streptococci that includes at least five species: S. faecalis, S. faecium, S. durans, S. gallinarum, and S. avium. • The enterococci are differentiated from other streptococci by their ability to grow at high pH (9.6 at 10), high temperature (45°C) and in high salt concentrations (6.5% sodium chloride). • The enterococcus are generally resistant to many Gram positive antibiotics such as the tetracyclines, aminoglycosides, sulfonamides, some penicillins, and lincosamides. E. faecalis and E. faecium are the most frequent species found in humans. E. faecalis is the only enterococcus species that has been • genetically characterized. • Its genome is 3 mb in length. The two genetic mechanisms first discovered in the enterococci were conjugative transposons and sex pheromone plasmids. Some strains require vitamin B and amino acids for growth.

5. Isolation and ecology • Most procedures employ presumptive media followed by confirmatory tests. Primary selective agents can be azide, tellurite, bile, neomycin, taurocholate, selenite, NaCl, alcohol, phenylethyl, and thallium. For isolation, the Association of Food and Drug Officials of the United States recommends KF agar medium. This is selective differential agar that contains sodium azide, that inhibits catalase positive organisms, and tetrazolium chloride which produces a red color in the colonies. • Ethyl violet azide (EVA) broth can be used as a confirmation. Fecal enterococci from water can be isolated, cultivated, and enumerated in this broth. Growth of fecal enterococci in EVA results in turbidity and a purple sediment in the bottom of liquid cultures.

6. Enterococci are able to grow in the presence of bile and hydrolyze the esculin; the liberated diphydroxycourmarin complexes (Esculetin) with ferric citrate present in the media to form a dark brown/black soluble compound. • Enterococci occur naturally in soil and can be readily isolated from most plant roots as well. They are also found routinely in frozen seafood, cheese, dried whole egg powder, raw and pasteurized milk, frozen fruits, fruit juices, and vegetables. Occasionally they are used as starter cultures for making hard cheese. Some strains produce high levels of the amines tyramine and histamine.

7. Tyramine may be involved in causing migraines. They are capable of producing extracellular proteinases and peptidases to hydrolyze large peptides and transport them into the cell to convert them to amino acids. • Blood agar is often used as a diagnostic test for the enterocococci, especially when isolations are made from food or clinical samples. • Two of the five enterococcal species (faecalis and durans) will usually produce hemolysis on blood agar.

8. Public health significance • The enterococci are used as a bacterial indicator for determining the extent of fecal contamination in foods and in recreational surface waters. Water quality guidelines based on enterococcal density have been proposed for recreational waters. • The guideline is 33 enterococci/100 mL for recreational fresh waters. For marine waters, the guideline is 35 enterococci/100 mL. The guidelines are based on the geometric mean of at least five samples per 30-d period during the swimming season. • There are two types of selection methods. The membrane filter technique is used for samples of fresh and saline waters; however, • it is unsuitable for highly turbid waters. • The multiple-tube technique is also applicable to fresh and marine waters, but is primarily used for raw and chlorinated wastewater.

9. For the presumptive test procedure of the multiple-tube technique, a series of azide dextrose broth tubes are inoculated and incubated. If not turbid, tubes are reincubated. Tubes showing turbidity are streaked onto Pfizer selective enterococcus (PSE) agar. • Brownish-black colonies with brown halos confirm the presence of fecal streptococci. These colonies are transferred to a tube of brain-heart infusion broth containing 6.5% NaCl. Growth indicates colonies of the enterococcus group. • In the membrane filter technique, the sample is filtered, the filter containing the colonies are transferred to an agar medium which is incubated. The filter is transferred to EIA medium containing esculin and ferric acid as selective agents.

10. Pink to red enterococci colonies develop a black or reddish-brown precipitate. A well isolated colony from brain-heart infusion agar is then transferred onto a brain-heart infusion broth tube and incubated. • After growth, a sample of the culture is transferred to bile esculin agar, brain-heart infusion broth, and brain-heart infusion broth with 6.5% NaCl. Growth at 45°C in 6.5% NaCl indicates presence of enterococcus group. • For clinical or food samples, additional tests that may be conducted include bile solubility.

11. Enterococcus organisms have been detected by plate counts using either Packers crystal violet azide blood agar or maltose azide agar ( KF streptococcus agar). • Alternatively, for smaller concentration of organisms, an MPN technique can be used employing maltose azid broth ( KF streptococcus broth ) or glucose azide broth. • The media used also tend to permit the growth of a number of enterococci and other group D streptococci that are not necessarily of fecal origin. • The presence of azide in the media helps to inhibit staphylococci, as a result of its inhibitory action against the cytochromes.

12. Glucose azide broth • This procedure employs the multiple tube technique, so that small numbers of enterococci can be detected. • Pipette aseptically 1 ml of each of the prepared dilutions of the sample into each of 3 or 5 tubes of glucose azide broth. • If very low concentrations of organisms are expected, 10 ml or even 100 ml amounts of the lowest dilution may be added to equal volumes of double strength medium, three or five bottles being prepared at each dilution. • Incubate the inoculated glucose azide broths at 35C for 72h and examine for the production of acid. 5. Record those tubes that are positive and subculture a loopful from each positive tube into fresh single strength glucose azide broth ( 5ml per tube ) and incubate at 45C in a water bath for 48h, examining the tubes after 18 and 48h. 6. The production of acid at 45C within 18h indicate s enterococci, and can be confirmed rapidly by microscopies examination for the presence of short –chained streptococci. 7. The most MBN of the enterococci can be determined using probability tables.

13. Kanamycin aesculinazide agar • Surface inoculate plates of well dried medium by spreading 0.1ml amounts of dilutions over the medium. • Incubate at 37C or 42C for 24h. • This medium is made selective by the use of azide and kanamycin, incubating at 42C increase its selectivity. • Colonies of enterococci are small, white or grey, and surrounded by large black haloes • Some strains of mesophilic lactobacilli may grow on this medium, and some of these are capable of splitting aesculin and therefore producing black haloes.

14. SF broth contains sodium azide, which inhibits most bacteria other than enterococci. • The enterococci will grow in SF broth and ferment the dextrose, turning the pH indicator from violet to a yellow brown color. • Bile Esculin agar slant: • The enterococci will grow in the presence of the bile salts in the medium. • They hydrolyze the esculin, producing esculetin which reacts with the iron salts in the medium turning the agar black.

16. END OF LECTURE