**3.6. Sulphite reducing clostridia**

332 Olive Germplasm – The Olive Cultivation, Table Olive and Olive Oil Industry in Italy

completely colourless colonies.

**3.5.** *Staphylococcus aureus*

Mac Conkey's MUG agar and Eosin Methylene Blue agar (EMB) are differential and selective media, suitable to obtain at the same time the isolation of *Salmonella, Shigella* and coliform bacteria, in particular *E. coli*. In the first medium, lactose-negative colonies are colourless, lactose-positive colonies are red and often surrounded by a turbid zone due to the precipitation of bile acids. *E. coli* can be identified by fluorescence in UV due to its β-Dglucoronidase production. In EMB agar, lactose-fermenters form colonies with dark-blue centres (*E. coli* may also have a green metallic sheen) while the non-lactose fermenters form

The foods most associated with staphylococcal poisoning are meat products, dairy products and cream filled bakery products. In processed foods in which *S. aureus* is destroyed by processing, its presence usually indicates post-processing contamination from human skin, mouth, nose or food handlers (Lancette & Bennett, 2003). Due to the high salt tolerance of *S. aureus*, it can grow in table olives even though the low pH and the olive phenols may represent natural inhibitors (Tassou & Nychas, 1994). However, it may be isolated and enumerated in table olives for the same above mentioned reason (as contamination index).

A variety of coagulase-positive and coagulase-negative staphylococci are able to produce enterotoxins, but *S. aureus* still plays a predominant role in staphylococcal food poisoning.

Baird-Parker agar and Rabbit Plasma Fibrinogen agar are the media recommended by the International Organisation for Standardisation (ISO). Moreover, Baird-Parker agar is also used in the official AOAC method in the United States (Zangerl &Asperger, 2003). Tellurite reduction, egg yolk reaction and a high level of sodium chloride are the most applied

*S. aureus* colonies in Baird-Parker agar are black, with an opaque zone around the colony because of the egg yolk reaction. Another medium frequently used is the Mannitol Salt agar,

selective chemicals added in media for *S. aureus* isolation and enumeration.

**Figure 3.** *S. aureus* colonies on Mannitol Salt Agar.

Clostridia are a widespread heterogeneous group of bacteria showing metabolic and nutritional differences. They easily contaminate foods because they produce resistant spores which can survive under mild processing conditions. The group of sulphite reducing clostridia may be used as marker of raw material quality and hygiene of manufacturing practices. Of particular concern for public health are *C. botulinum*, which forms a deadly toxin in foods, and *C. perfringens* which causes enteritis when present in high numbers. Other species or strains are also known to be toxinogenic or neurotoxinogenic (Bredius & Ree, 2003). *C. perfringens* is the agent of a food poisoning usually associated with consumption of cooked meats or poultry products. Sporulating cells of *C. perfringens* produce a heat-labile enterotoxin which appears to be released *in vivo* in the intestine (Labbe, 2001).

Most isolation media include sulphite and an appropriate iron salt. Sulphite is reduced to sulphide by the clostridial enzyme sulphite reductase; the sulphide will then precipitate as a black deposit in the presence of iron salt. This causes a blackening of the liquid media and clostridia will appear as black colonies. To obtain a higher selectivity for *C. perfringens*  isolation, the Oleandomycin Polymixin Sulphadiazine Perfringens (OPSP) agar medium was performed. It contains selective antibiotics and utilises sodium metabisulphite and liver

extract as sources of H2S with ferric ammonium citrate as the indicator. In any case, subsequent confirmation tests such as motility, reduction of nitrate, lactose fermentation, gelatin liquefaction (Labbe, 2001) should be necessary. The plates are incubated anaerobically at 37°C for 18-48 hours.

Microbiological Aspects of Table Olives 335

(McClain & Lee, 1988) 25 mL of brine sample are added to 225 mL of Listeria Enrichment broth base for the resuscitation of stressed cells. After blending, the bag is incubated at 30°C and then, at intervals from 4 hours to 7 days, an aliquot is plated for enumeration in Listeria Selective agar base and incubated at 37°C for 24 h. Black colonies on selective agar base

The presumptive *Listeria* isolates are to be tested for sugar fermentation, tumbling motility, hemolytic reaction and growth at different salt concentrations (Caggia et al., 2004). Alternatively to this medium, ALOA agar contains the chromogenic compound for the detection of β-glucosidase, common to all *Listeria*, which appear as blue coloured colonies. While *L. monocytogenes,* which possesses a specific phospholipase, hydrolyses the specific substrate added to the medium producing an opaque halo around the colonies (Beumer &

Where counts < 100 CFU/g are expected, it is necessary to use the Most Probable Number

In order to rapidly perform additional confirmation tests, systems of strips are commercially

Propionibacteria are often associated with food spoilage. The "zapateria" spoilage, which can occur in brined olives, is characterized by a malodorous fermentation due to propionic acid produced by certain species of *Propionibacterium* (Jay, 2000), alone or together with *Clostridium* spp.. The propionibacteria may be considered as marker of the end of shelf life of table olives, because the growth of these bacteria cause an increase in pH values creating conditions for the growth of spoilage or pathogen bacteria and the arising of off-odors

Unfortunately, propionibacteria are difficult to isolate because they grow very slowly on solid media. The complex medium usually used is not able to suppress competing organisms. This medium is Sodium Lactate agar (1% typticase, 1% yeast extract, 1% sodium lactate, 0.025% dipotassium phosphate, 1.5% agar) in which the propionibacteria growth appears in 5-7 days at 32°C under anaerobic or microaerophilic conditions (Richter &

A successful olive treatment depends on different factors. The olive cultivars show different fermentation behaviours when directly brined. In fact, besides the correct application of manufacturing practices, the knowledge of the chemical and physical characteristics of olive cultivar used and its attitude to treatments are decisive. A more complete knowledge of the olive cultivars is necessary to bring fermentation to a successful condition and attain a good final product. A correlation between each method and chemical-physical composition of

Curtis, 2003). Another chromogenic medium used is the Rapid' L. mono agar.

medium were considered presumptive *Listeria* colonies.

available for *Listeria* (API Listeria) as for other pathogens.

(MPN) technique (USDA, 2001).

**3.8. Propionibacteria** 

(Plastourgos & Vaughn, 1957).

Vedamuthu, 2001).

**4. Conclusion** 

### **3.7.** *Listeria monocytogenes*

Listeria species are ubiquitous organisms widely distributed in the environment, especially in plant matter and soil. The microorganism is established as an important foodborne pathogen, which can grow at high salt concentration (up to 10% of NaCl) and at refrigerated temperatures. A study carried out by the U.S. Food and Drug Administration, U.S. Department of Agriculture (USDA, 2001), indicated that vegetables are able to support *L. monocytogenes* growth and, after a few years, Caggia and co-workers (2004) demonstrated that *L. monocytogenes* can survive and grow in green table olives. As indicated by the most recent scientific report of the European Food Safety Authority (2012), the number of listeriosis cases in humans in the EU slightly decreased, and 1,601 confirmed human cases were reported in 2010.

The minimum infective dose of *L. monocytogenes* has not yet been established and many authorities require that the organism must be absent in 25 g of product. This has led to the development of methods following the sequence of pre-enrichment, selective enrichment and diagnostic plating. The whole procedure may take about five days. Then, presumptive positive results need to be confirmed adding further time to complete the examination (Beumer & Curtis, 2003).

**Figure 5.** *L. monocytogenes* colonies on ALOA medium.

Most of the isolation media differentiate *Listeria* spp. by means of aesculin hydrolysis which, in the presence of iron, forms a black phenolic compound. According to the USDA method (McClain & Lee, 1988) 25 mL of brine sample are added to 225 mL of Listeria Enrichment broth base for the resuscitation of stressed cells. After blending, the bag is incubated at 30°C and then, at intervals from 4 hours to 7 days, an aliquot is plated for enumeration in Listeria Selective agar base and incubated at 37°C for 24 h. Black colonies on selective agar base medium were considered presumptive *Listeria* colonies.

The presumptive *Listeria* isolates are to be tested for sugar fermentation, tumbling motility, hemolytic reaction and growth at different salt concentrations (Caggia et al., 2004). Alternatively to this medium, ALOA agar contains the chromogenic compound for the detection of β-glucosidase, common to all *Listeria*, which appear as blue coloured colonies. While *L. monocytogenes,* which possesses a specific phospholipase, hydrolyses the specific substrate added to the medium producing an opaque halo around the colonies (Beumer & Curtis, 2003). Another chromogenic medium used is the Rapid' L. mono agar.

Where counts < 100 CFU/g are expected, it is necessary to use the Most Probable Number (MPN) technique (USDA, 2001).

In order to rapidly perform additional confirmation tests, systems of strips are commercially available for *Listeria* (API Listeria) as for other pathogens.
