Three

.',

Salmonella

1998. Eels contaminated at farm level in Italy were the vehicle of infection after having undergone smoking at four different smokehouses (125). Besides animal products, a wide variety of other agricultural commodities may from time to time contain salmonellae and this contamination can lead to widespread or localised human infection. For example, contaminated chocolate has caused four outbreaks in North America and Europe (85). Foods and food additives as diverse as coconut, grain, rye flour, chilli powder, peanut butter, sunflower seeds, egg noodles, pasta, yeast, cottonseed, sesame seed products, soy based products, cider, and gelatine have contained salmonellae. Table 8.11 lists the occurrence of salmonellae in a few surveys of fruit, vegetables and spices. Salmonellae can be found on a small percentage of melons. Contaminated watermelon and cantaloupe have caused outbreaks in the US and England (85). As a result of cross contamination from external contamination to freshly cut internal surfaces, the incidence of Salmonella contamination can be higher at retail than at wholesale (339). The serovars found on raw vegetables are usually similar to those found in local surface waters (339, 370). Though numbers of salmonellae on vegetables are usually less than 1/g, numbers as high as 240/g have been found on Dutch endive (370). Salmonellae have been found in paprika and pepper (Table 8.11) and in other spices such as coriander, fenugreek, parsley, and peppermint (85). Pepper has caused at least two outbreaks - one in Canada with Salmonella Weltevreden and another in Norway with S. Oranienburg. Contaminated paprika and paprika powdered potato chips were responsible for an estimated 1000 cases in a nationwide outbreak of salmonellosis in Germany in 1993 (252). The outbreak was notable in that a variety of serovars including monophasic and non-motile rare serovars were involved and that the number

of salmonellae in the food was low (2.5 Salmonella cells/gram paprika; 0.04-0.45 Salmonella cells/ gram of chips). Salmonellae were detected in Australian mung beans which were implicated in an outbreak of salmonellosis from bean sprouts in England (293). Human infection in Norway and Sweden has also been associated with eating raw bean sprouts. Salmonellae have been also detected in alfalfa sprouts in Finland and Sweden. Alfalfa sprouts have been responsible for several Salmonella outbreaks in the United States in recent years. Recent outbreaks associated with fresh produce in Australia include 36 cases from iceberg lettuce contaminated with S. Bovismorbificans PT 32, 47 cases of S. Stanley acquired from imported peanuts, and 500 cases of S. Typhimurium from unpasteurised orange juice.

Survival

and growth

Salmonellae have simple nutritional need~ and most are able to grow in a glucose-ammonium ionmineral salts mIXture. A wide range of other substrates can be used as sources of energy and carbon and of nitrogen. Being facultative anaerobes, salmonellae are able to grow in the absence of oxygen. On beef muscle stored at 20°C, the growth rate under nitrogen is only s~tly less than that obtained in air (160). High concentrations of carbon dioxide (50-60%) strong~it growth on crab meat,beeIStm and ground beef at 10-11°C (200, 250) but there is little inhibition at -- 20°C (344). Temperature On foods, most salmonellae do not grow at temperatures below 7°C. Growth has been reported in chicken a la king at 6.7°C (10), however other studies have failed to detect growth at 7°C on beef slices, ground beef, ground

Table 8.11. Incidence of salmonellae in some fruit, vegetables and spices. Country

Number sampled

Percent positive

Reference

Melons

USA

3660

0.96

252

Cantaloupe

USA

211

1.9

85 339

Food

Leafy vegetables Stem and root vegetables

Spain

346

7.5

Spain

204

4.9

339

Spain

299

3.3

339

Vegetables

Holland

180

6.7

370

Paprika

Greece

35

2.9

85

Various

548

8.8

85

Other vegetables.

Pepper

, Because the sample sizes differ, a comparison of the incidence between products cannot be made. b

Bean,broadbean,cucumber,eggplant,marrow,pepper,tomato. 227

Stephen Jay, Dianne Davos, Mark Dundas, Elizabeth Frankish and Diane Lightfoot

specific sigma factor (sigma38). There are also two RpoS- independent systems that are both low pH inducible. One is an acid tolerance induced in log phase cells by exposure to mild acid (pH 6-5.5) (139). The other is induced in stationary phase cells by exposure to a pH of less than 5.0 for some hours. However, in spite of these systems, Salmonella appear to be significantly less tolerant of low pH (pH 2.5; hydrochloric acid) than Shigella spp. or Escherichia coli (159). These latter two organisms possess additional acid survival systems that are not present in salmonellae (244). The lethal effects of low pH and organic acids (mainly acetic) are important in destroying any salmonellae that may be present in mayonnaise and salad dressing (155, 249, 354). In the production of Cheddar cheese, lactose is fermented principally to lactic acid. Any salmonellae present can grow in the early stages of production since the pH fall is, by itself, insufficient to stop growth. Growth ceases during salting from the combination of lowered pH and aw (158, 304). Slow acid production results in a higher pH, Salmonella growth during the early stages of ripening, and extended survival of salmonellae. During the ageing and maturation of the cheese, salmonellae die slowly at rates that decrease with decrease in ageing temperature and increase with decrease in pH (158, 415). Ageing cannot be relied on to prevent human infection from contaminated cheese and, in naturally contaminated cheddar cheese, salmonellae can still be present after 8 months storage at 5°C (95). In the production of fermented sausages, salt is added along with sugar and starter culture. The presence of salt in the sausage mix restricts the growth of salmonellae for a short time during the initial stages of fermentation (156), but is insufficient, by itself, to prevent growth (initial aw 0.97-0.96). An active fermentation is important so that the pH is lowered to prevent Salmonella growth (347, 350, 351). During the drying and ageing phase, salmonellae gradually die at a rate which increases with increasing storage temperature, increases with decreasing pH of the sausage, and depends on the strain and the final aw reached. An example is shown in Figure 8.2 where the numbers of S. Typhimurium are plotted against time during the production of pepperoni. In this experiment (350), fermentation was at 35°C for one day for the lowest pH set and for two days for the other two trials. In pepperoni with the highest pH (pH 5.8), salmonellae grew in the first few days. In these sausages, the lack of a pH decrease also resulted in slower drying and this slower rate of drying was also a factor in the growth and later slower decline in viable numbers. 230

Water activity ~Aerobip.::! lly. most salmonellae are able to grow in laboratory mp-di::!from an aw of 0.99
(6.,5). '(he

m;nimHffi

aw Cllluwiug

Ctel"Ubic

growth in broth is 0.945. In~ome dried foods (e.g. dried meat, dehydrated soup) growth has been observed at an aw of 0.93. As the temperature and pH are decreased below the optimum, the percentage of salt required to prevent the growth of salmonellae decreases (151, 259). Nitrite, particularly at pH 5.6, also reduces the amount of salt needed to stop growth. At aw levels below those allowing growth, salmonellae die slowly. The rate of death decreases as the aw is lowered and also decreases as the temperature is reduced (387). The effect of water activity is illustrated in the study by Crumrine and Foltz (77) where wheat, inoculated with salmonellae, was stored at 25°C and at constant relative humidities ranging from 7% to 98%. As the relative humidity of storage increased, the death rate of the salmonellae increased. Salmonellae are known to survive for long periods in a large range of dried products, e.g. non-fat milk, egg products, rendered animal byproducts, environmental dusts in factories, soil, litter and faeces. Rayman and coworkers (325) estimated that the time for a 90% reduction in the numbers of two strains of Salmonella in pasta and egg pasta was 90-360 days when the dried pasta was stored at ambient temperature. Other workers have found salmonellae still present in naturally contaminated pasta after 18 months storage at room temperature. When Salmonella Eastbourne was incorporated into chocolate bars from contaminated spray dried milk, about 1% of the cells were still viable after the chocolates had been stored for 19 months at room temperature (371). Salmonella inoculated into halva confection were recovered after eight months of storage (225). In honey stored at lOoC, inoculated salmonellae have been found to survive for over two years, and, in commercial dry seasonings containing 50% salt, inoculated salmonellae survived for more than 29 weeks at 22°C. Five Salmonella serotypes were found to survive 24 weeks storage in3 peanut butter at 5°C but were recovered from only one peanut butter product after the same storage time at 21°C (54). Differences in product composition were considered responsible for the survival variations observed. Highly salted foods, where there is some fluid (e.g. salted sausage casings), or brines are usually lethal to salmonellae, particularly when they are at ambient temperatures. However, S. Typhimurium can survive in cheese brines for several weeks (201). Survival is better at lower temperatures and at higher temperatures the degree of survival is pH dependant.

Salmonella

sulphapyridine, sulphathiazole and novobiocin, or on various combinations of inhibitors. A description of the inhibitory mechanisms of these media and a report on their performance with different foods under various conditions have been well documented in comprehensive reviews by Litchfield (248) and Fagerberg and co-workers (123, 124), Harvey and Price (172) and Vassiliadis (398). The evidence in the literature relating to the relative efficiency of selective enrichment media in the recovery of salmonellae from foods is often contradictory and confusing. Historically, the two groups of media most widely used were those based on tetrathionate or selenite. More recently, the medium proposed by Rappaport and Vassiliadis (402) has gained wider acceptance and is often used as a replacement for a tetrathionate based medium. Usually two dissimilar selective media are used to maximise recovery of salmonellae. Martin and Katz in 1991 (255) proposed to use a single medium for preenrichment and selective enrichment. The medium was tryptone soya broth (TSB) supplemented with ingredients to assist in the resuscitation of salmonellae. Mter 4 h incubation at 35°C, the medium was made selective by the addition of selective agents and incubation continued for another 16 h. The selective agents were selenite and L-cystine or tetrathionate broth. This procedure was more productive in the recovery of salmonellae than the then current BAM method (6th edition, 1984). Although the exact mechanism of tetrathionate inhibition is not fully understood, it is known that when it is combined with thiosulphate it is toxic to actively growing non-

Salmonella enteric bacteria (301). This toxicity is related to the concentrations of tetrathionate and thiosulphate, both of which inactivate enzymes by reacting with sulphydryl groups. The selective properties of tetrathionate broths can be increased by the inclusion of bile salts and brilliant green to inhibit Gram positive bacteria (220). Moreover, the presence of brilliant green suppresses the growth of Gram negative lactose fermenters. The addition of calcium carbonate provides a highly buffered medium so that pH is of no concern. The mechanism of selenite toxicity is also not known. It has been postulated that the inhibitory effects of selenite broth are due to the incorporation of selenium ions into analogues of sulphur containing amino acids (seleno-amino acids), and to the reaction of selenite with sulphydryl groups of cellular components such as enzymes (412). In broth culture, the growth of bacteria causes the reduction of selenite to selenium. This reaction can lead to an increase in pH which may reduce the toxicity of the selenite and result in overgrowth of competitors. Leifson (236) included lactose in selenite media so that the acid produced by lactose fermenting organisms in the sample would stabilise the pH at around neutrality. In several other selenite based media, lactose has been substituted by other carbohydrates such as mannitol and dulcitol which are fermented by salmonellae and hence favour their growth. Dulcitol selenite broth, for example, was developed by Raj (318) to improve the recovery of salmonellae from seafood. Another notable example of such media is the more commonly used mannitol selenite cystine broth. The addition of cystine to selenite media has been

Table 8.13. Selective enrichment media for the isolation of salmonellae Medium

Selective

Gram negative (GN) broth

Reference

agents Hajna (164)

Sodium citrate, desoxycholate Magnesium

chloride, malachite green

Rappaport et al. (321)

Rappaport-Vassiliadis

medium (RV)

Magnesium

chloride, malachite green

Vassiliadis

Rappaport-Vassiliadis with soya peptone

medium (RV)

Magnesium

chloride, malachite green

van Schothorst and Renaud (392)

Rappaport's medium

et al. (401, 404)

Selenite-F broth

Selenite

Leifson (236)

Selenite cystine broth

Selenite

North and Bartram (287)

Selenite brilliant green broth

Selenite, sodium taurocholate,

brilliant green

Osborne and Stokes (299)

Selenite brilliant green sulphonamide broth

Selenite, sodium taurocholate, sulphapyridine

brilliant green,

Stokes and Osborne (362)

Tetrathionate

broth

Tetrathionate,

bile salts

Muller (279)

Tetrathionate brilliant green broth

Tetrathionate,

bile salts, brilliant green

Kauffmann (220)

Strontium chloride medium

Strontium

Strontium selenite medium

Strontium ion, selenite

ion

Iveson and MacKay-Scollay

(208)

Iveson and MacKay-Scollay

(208)

239 -

\.

'''''1

Salmonella

selective enrichment and/or selective plating media which include this agent as an ingredient. The use of multiple selective agents was an important consideration in the development of the Australian Standard Method (AS 1766.2.5). The usual incubation conditions for selective enrichment are 18-24 h at 35-37°C or 41-43°C. Harvey and Thomson (175) and Harvey and Price (169) showed that the isolation efficiency of selenite F broth could be increased by incubating at an elevated temperature of 43°C. The rationale underlying this technique is that while most salmonellae can multiply profusely at around 41-43°C, many competing contaminants are suppressed. It was also suggested that 42°C may be safer as 43°C possibly represented the upper end of the useful temperature range. Other investigators have since endorsed the advantage of elevated incubation temperatures for the recovery of salmonellae from a variety of sample types, particularly raw meat (57, 113, 120, 148, 209, 276, 343, 355). Elevated incubation temperature is specifically recommended for RV medium (398, 404). In view of the possible inhibition of some salmonellae at 43°C, Litchfield (248) recommended parallel testing of samples at 37°C and 43°C. Similarly, the SAA specifies parallel incubation of selective enrichment media at 37°C and 42°C (359). On the other hand, the ICMSF recommends incubation of two selective media both at 43°C (203). A single incubation temperature was considered necessary in order to restrict the number of selective variables. The use of RV medium at 42°C has been recommended by the AOAC (216), however the American Public Health Association (APHA) (134) currently do not recommend elevated temperatures of incubation of enrichment media in their procedures for the examination of foods for salmonellae. The choice of 35-37°C as the incubation temperature for all enrichment media was probably influenced by evidence produced by several investigators that certain media may be lethal to salmonellae when incubated at 43°C, e.g. tetrathionate (264, 395, 403). In addition to the possible detrimental effect of elevated incubation temperatures on the recovery of salmonellae from some selective enrichment broths, the brand and method of media preparation have been shown to be important factors in obtaining reliable and reproducible results (170, 171). The optimum time of incubation of a selective enrichment broth is a function of the number of salmonellae present in the sample (270). It has been reported that the subculture of selective enrichment broths after at least two different incubation periods, such as 24 and 48 h, will

result in the isolation of salmonellae from more samples (145, 264). McCoy (264) concluded that for qualitative examination of samples, subculture for 24 and 48 h, which will detect up to 85% of positive samples, should suffice. For quantitative examinations (MPN procedures), daily subculture for 4 d was considered essential. In the examination of samples for multiple serovars, Harvey and Price (171) showed that more serovars could be isolated by repeat subculture from enrichment broths at 18, 24, 48 and 72 h. When using selenite or tetrathionate media, it is generally accepted that the inoculation- ratio of pre-enrichment culture to selective medium should be 1:10 (v/v) (134, 398). For RV medium a ratio of 1:100 is preferred (398, 400). Kafel and Pogorzelska (218) found that dilutions of 1:100 to 1:10000 were optimal for isolation of salmonellae from raw ground beef using tetrathionate and selenite cystine broths. Other important factors that should also be considered in enrichment methodology are agitation during incubation, addition of emulsifying agents, and secondary enrichment procedures. Evidence reported in the literature on the relative advantages and disadvantages of aeration of enrichment broths is unclear (123). Although the work of Alford and Knight (1) suggested that aeration of selenite cystine broth may enhance the isolation of salmonellae, it was concluded that aeration should be explored for each different sample material. Improved oxygenation of selective broths can often be achieved by simply reducing the depth of the medium using wide diameter containers, and by loosely securing the lids of the containers. The addition of emulsifying agents to selective media has been variously claimed to increase, decrease or have no effect on the recovery rate of salmonellae from high fat samples (123). The nature of the sample, the type of enrichment media, and the temperature of incubation appear to be important contributing factors. Secondary enrichment of negative selective enrichment broths in either the primary medium or a different medium has been shown to frequently produce a higher yield of salmonellae (210). Hence in the non-routine examination of samples where precise information on the presence of salmonellae is required, secondary enrichment procedures may be valuable. Selective motility Several methods have been devised for isolating salmonellae from other microorganisms on the basis of their motility through semi-solid agar. Craigie (74) described a technique using semisolid nutrient agar in a specially designed narrow 241 \