.

', Salmonella

1998. Eels contaminated at farm level in Italy of salmonellae in the food was low (2.5 Salmonella
were the vehicle of infection after having under- cells/gram paprika; 0.04-0.45 Salmonella cells/
gone smoking at four different smokehouses (125). gram of chips). Salmonellae were detected in
Besides animal products, a wide variety of Australian mung beans which were implicated in
other agricultural commodities may from time to an outbreak of salmonellosis from bean sprouts in
time contain salmonellae and this contamination England (293). Human infection in Norway and
can lead to widespread or localised human Sweden has also been associated with eating raw
infection. For example, contaminated chocolate bean sprouts. Salmonellae have been also
has caused four outbreaks in North America and detected in alfalfa sprouts in Finland and
Europe (85). Foods and food additives as diverse Sweden. Alfalfa sprouts have been responsible for
as coconut, grain, rye flour, chilli powder, peanut several Salmonella outbreaks in the United
butter, sunflower seeds, egg noodles, pasta, yeast, States in recent years. Recent outbreaks
cottonseed, sesame seed products, soy based associated with fresh produce in Australia include
products, cider, and gelatine have contained 36 cases from iceberg lettuce contaminated with
salmonellae. S. Bovismorbificans PT 32, 47 cases of S. Stanley
Table 8.11 lists the occurrence of salmonellae acquired from imported peanuts, and 500 cases of
in a few surveys of fruit, vegetables and spices. S. Typhimurium from unpasteurised orange juice.
Salmonellae can be found on a small percentage of
melons. Contaminated watermelon and cantaloupe
have caused outbreaks in the US and England Survival and growth
(85). As a result of cross contamination from
external contamination to freshly cut internal
surfaces, the incidence of Salmonella contami- Salmonellae have simple nutritional need~ and
nation can be higher at retail than at wholesale most are able to grow in a glucose-ammonium ion-
(339). The serovars found on raw vegetables are mineral salts mIXture. A wide range of other
usually similar to those found in local surface substrates can be used as sources of energy and
waters (339, 370). Though numbers of carbon and of nitrogen. Being facultative
salmonellae on vegetables are usually less than anaerobes, salmonellae are able to grow in the
1/g, numbers as high as 240/g have been found on absence of oxygen. On beef muscle stored at 20°C,
Dutch endive (370). Salmonellae have been found the growth rate under nitrogen is only s~tly
in paprika and pepper (Table 8.11) and in other less than that obtained in air (160). High
spices such as coriander, fenugreek, parsley, and concentrations of carbon dioxide (50-60%)
peppermint (85). Pepper has caused at least two strong~it growth on crab meat,beeIStm
outbreaks - one in Canada with Salmonella and ground beef at 10-11°C (200, 250) but there is
Weltevreden and another in Norway with little inhibition at
-- 20°C (344).
S. Oranienburg. Contaminated paprika and
paprika powdered potato chips were responsible Temperature
for an estimated 1000 cases in a nationwide On foods, most salmonellae do not grow at
outbreak of salmonellosis in Germany in 1993 temperatures below 7°C. Growth has been
(252). The outbreak was notable in that a variety reported in chicken a la king at 6.7°C (10),
of serovars including monophasic and non-motile however other studies have failed to detect
rare serovars were involved and that the number growth at 7°C on beef slices, ground beef, ground

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

Food Country Number sampled Percent positive Reference
Melons USA 3660 0.96 252

Cantaloupe USA 211 1.9 85

Leafy vegetables Spain 346 7.5 339

Stem and root vegetables Spain 204 4.9 339

Other vegetables. Spain 299 3.3 339

Vegetables Holland 180 6.7 370

Paprika Greece 35 2.9 85

Pepper Various 548 8.8 85

, 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 Water activity
RpoS- independent systems that are both low pH ~Aerobip.::!
lly. most salmonellae are able to grow in
inducible. One is an acid tolerance induced in log laboratory mp-di::!from an aw of 0.99<L..t()hp-Iow
phase cells by exposure to mild acid (pH 6-5.5) 0.960 (6.,5). '(he m;nimHffi aw Cllluwiug Ctel"Ubic
(139). The other is induced in stationary phase growth in broth is 0.945. In~ome dried foods (e.g.
cells by exposure to a pH of less than 5.0 for some dried meat, dehydrated soup) growth has been
hours. However, in spite of these systems, observed at an aw of 0.93. As the temperature and
Salmonella appear to be significantly less tolerant pH are decreased below the optimum, the
of low pH (pH 2.5; hydrochloric acid) than Shigella percentage of salt required to prevent the growth
spp. or Escherichia coli (159). These latter two of salmonellae decreases (151, 259). Nitrite,
organisms possess additional acid survival particularly at pH 5.6, also reduces the amount of
systems that are not present in salmonellae (244). salt needed to stop growth.
The lethal effects of low pH and organic acids At aw levels below those allowing growth,
(mainly acetic) are important in destroying any salmonellae die slowly. The rate of death
salmonellae that may be present in mayonnaise decreases as the aw is lowered and also decreases
and salad dressing (155, 249, 354). as the temperature is reduced (387). The effect of
In the production of Cheddar cheese, lactose water activity is illustrated in the study by
is fermented principally to lactic acid. Any Crumrine and Foltz (77) where wheat, inoculated
with salmonellae, was stored at 25°C and at
salmonellae present can grow in the early stages
constant relative humidities ranging from 7% to
of production since the pH fall is, by itself,
98%. As the relative humidity of storage
insufficient to stop growth. Growth ceases during
increased, the death rate of the salmonellae
salting from the combination of lowered pH and increased. Salmonellae are known to survive for
aw (158, 304). Slow acid production results in a long periods in a large range of dried products, e.g.
higher pH, Salmonella growth during the early non-fat milk, egg products, rendered animal by-
stages of ripening, and extended survival of products, environmental dusts in factories, soil,
salmonellae. During the ageing and maturation of litter and faeces. Rayman and coworkers (325)
the cheese, salmonellae die slowly at rates that estimated that the time for a 90% reduction in the
decrease with decrease in ageing temperature and numbers of two strains of Salmonella in pasta and
increase with decrease in pH (158, 415). Ageing egg pasta was 90-360 days when the dried pasta
cannot be relied on to prevent human infection was stored at ambient temperature. Other
from contaminated cheese and, in naturally workers have found salmonellae still present in
contaminated cheddar cheese, salmonellae can naturally contaminated pasta after 18 months
still be present after 8 months storage at 5°C (95). storage at room temperature. When Salmonella
In the production of fermented sausages, salt Eastbourne was incorporated into chocolate bars
is added along with sugar and starter culture. The from contaminated spray dried milk, about 1% of
presence of salt in the sausage mix restricts the the cells were still viable after the chocolates had
growth of salmonellae for a short time during the been stored for 19 months at room temperature
initial stages of fermentation (156), but is (371). Salmonella inoculated into halva confection
insufficient, by itself, to prevent growth (initial aw were recovered after eight months of storage
0.97-0.96). An active fermentation is important so (225). In honey stored at lOoC, inoculated
salmonellae have been found to survive for over
that the pH is lowered to prevent Salmonella
growth (347, 350, 351). During the drying and two years, and, in commercial dry seasonings
containing 50% salt, inoculated salmonellae
ageing phase, salmonellae gradually die at a rate survived for more than 29 weeks at 22°C. Five
which increases with increasing storage
Salmonella serotypes were found to survive 24
temperature, increases with decreasing pH of the
weeks storage in3 peanut butter at 5°C but were
sausage, and depends on the strain and the final recovered from only one peanut butter product
aw reached. An example is shown in Figure 8.2 after the same storage time at 21°C (54).
where the numbers of S. Typhimurium are Differences in product composition were
plotted against time during the production of considered responsible for the survival variations
pepperoni. In this experiment (350), fermentation observed. Highly salted foods, where there is some
was at 35°C for one day for the lowest pH set and fluid (e.g. salted sausage casings), or brines are
for two days for the other two trials. In pepperoni usually lethal to salmonellae, particularly when
with the highest pH (pH 5.8), salmonellae grew in they are at ambient temperatures. However, S.
the first few days. In these sausages, the lack of a Typhimurium can survive in cheese brines for
pH decrease also resulted in slower drying and several weeks (201). Survival is better at lower
this slower rate of drying was also a factor in the temperatures and at higher temperatures the
growth and later slower decline in viable numbers. degree of survival is pH dependant.

230
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Salmonella

sulphapyridine, sulphathiazole and novobiocin, or Salmonella enteric bacteria (301). This toxicity is
on various combinations of inhibitors. A related to the concentrations of tetrathionate and
description of the inhibitory mechanisms of these thiosulphate, both of which inactivate enzymes by
media and a report on their performance with reacting with sulphydryl groups. The selective
different foods under various conditions have properties of tetrathionate broths can be
been well documented in comprehensive reviews increased by the inclusion of bile salts and
by Litchfield (248) and Fagerberg and co-workers brilliant green to inhibit Gram positive bacteria
(123, 124), Harvey and Price (172) and Vassiliadis (220). Moreover, the presence of brilliant green
(398). suppresses the growth of Gram negative lactose
The evidence in the literature relating to the fermenters. The addition of calcium carbonate
relative efficiency of selective enrichment media provides a highly buffered medium so that pH is
in the recovery of salmonellae from foods is often of no concern.
contradictory and confusing. Historically, the two The mechanism of selenite toxicity is also not
groups of media most widely used were those known. It has been postulated that the inhibitory
based on tetrathionate or selenite. effects of selenite broth are due to the
More recently, the medium proposed by incorporation of selenium ions into analogues of
Rappaport and Vassiliadis (402) has gained wider sulphur containing amino acids (seleno-amino
acceptance and is often used as a replacement for acids), and to the reaction of selenite with
a tetrathionate based medium. Usually two sulphydryl groups of cellular components such as
dissimilar selective media are used to maximise enzymes (412). In broth culture, the growth of
recovery of salmonellae. Martin and Katz in 1991 bacteria causes the reduction of selenite to
(255) proposed to use a single medium for pre- selenium. This reaction can lead to an increase in
enrichment and selective enrichment. The pH which may reduce the toxicity of the selenite
medium was tryptone soya broth (TSB) and result in overgrowth of competitors. Leifson
supplemented with ingredients to assist in the (236) included lactose in selenite media so that
resuscitation of salmonellae. Mter 4 h incubation the acid produced by lactose fermenting
at 35°C, the medium was made selective by the organisms in the sample would stabilise the pH at
addition of selective agents and incubation around neutrality. In several other selenite based
continued for another 16 h. The selective agents media, lactose has been substituted by other
were selenite and L-cystine or tetrathionate carbohydrates such as mannitol and dulcitol
broth. This procedure was more productive in the which are fermented by salmonellae and hence
recovery of salmonellae than the then current favour their growth. Dulcitol selenite broth, for
BAM method (6th edition, 1984). example, was developed by Raj (318) to improve
Although the exact mechanism of the recovery of salmonellae from seafood. Another
tetrathionate inhibition is not fully understood, it notable example of such media is the more
is known that when it is combined with commonly used mannitol selenite cystine broth.
thiosulphate it is toxic to actively growing non- The addition of cystine to selenite media has been

Table 8.13. Selective enrichment media for the isolation of salmonellae

Medium Selective agents Reference
Gram negative (GN) broth Sodium citrate, desoxycholate Hajna (164)
Rappaport's medium Magnesium chloride, malachite green Rappaport et al. (321)
Rappaport-Vassiliadis medium (RV) Magnesium chloride, malachite green Vassiliadis et al. (401, 404)
Rappaport-Vassiliadis medium (RV) Magnesium chloride, malachite green van Schothorst and Renaud (392)
with soya peptone
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 Selenite, sodium taurocholate, brilliant green, Stokes and Osborne (362)
broth sulphapyridine
Tetrathionate broth Tetrathionate, bile salts Muller (279)

Tetrathionate brilliant green broth Tetrathionate, bile salts, brilliant green Kauffmann (220)
Strontium chloride medium Strontium ion Iveson and MacKay-Scollay (208)
Strontium selenite medium Strontium ion, selenite Iveson and MacKay-Scollay (208)

239

- \.
 '''''1

Salmonella

selective enrichment and/or selective plating result in the isolation of salmonellae from more
media which include this agent as an ingredient. samples (145, 264). McCoy (264) concluded that
The use of multiple selective agents was an for qualitative examination of samples, subculture
important consideration in the development of the for 24 and 48 h, which will detect up to 85% of
Australian Standard Method (AS 1766.2.5). positive samples, should suffice. For quantitative
The usual incubation conditions for selective examinations (MPN procedures), daily subculture
enrichment are 18-24 h at 35-37°C or 41-43°C. for 4 d was considered essential. In the examination
Harvey and Thomson (175) and Harvey and Price of samples for multiple serovars, Harvey and
(169) showed that the isolation efficiency of Price (171) showed that more serovars could be
selenite F broth could be increased by incubating isolated by repeat subculture from enrichment
at an elevated temperature of 43°C. The rationale broths at 18, 24, 48 and 72 h.
underlying this technique is that while most When using selenite or tetrathionate media,
salmonellae can multiply profusely at around it is generally accepted that the inoculation- ratio
41-43°C, many competing contaminants are of pre-enrichment culture to selective medium
suppressed. It was also suggested that 42°C may should be 1:10 (v/v) (134, 398). For RV medium a
be safer as 43°C possibly represented the upper ratio of 1:100 is preferred (398, 400). Kafel and
end of the useful temperature range. Other Pogorzelska (218) found that dilutions of 1:100 to
investigators have since endorsed the advantage 1:10000 were optimal for isolation of salmonellae
of elevated incubation temperatures for the from raw ground beef using tetrathionate and
recovery of salmonellae from a variety of sample selenite cystine broths.
types, particularly raw meat (57, 113, 120, 148, Other important factors that should also be
209, 276, 343, 355). Elevated incubation considered in enrichment methodology are
temperature is specifically recommended for RV agitation during incubation, addition of
medium (398, 404). emulsifying agents, and secondary enrichment
In view of the possible inhibition of some procedures. Evidence reported in the literature on
salmonellae at 43°C, Litchfield (248) recommended the relative advantages and disadvantages of
parallel testing of samples at 37°C and 43°C. aeration of enrichment broths is unclear (123).
Similarly, the SAA specifies parallel incubation of Although the work of Alford and Knight (1)
selective enrichment media at 37°C and 42°C suggested that aeration of selenite cystine broth
(359). On the other hand, the ICMSF recommends may enhance the isolation of salmonellae, it was
incubation of two selective media both at 43°C concluded that aeration should be explored for
(203). A single incubation temperature was each different sample material. Improved
considered necessary in order to restrict the oxygenation of selective broths can often be
number of selective variables. The use of RV achieved by simply reducing the depth of the
medium at 42°C has been recommended by the medium using wide diameter containers, and by
AOAC (216), however the American Public Health loosely securing the lids of the containers. The
Association (APHA) (134) currently do not addition of emulsifying agents to selective media
recommend elevated temperatures of incubation has been variously claimed to increase, decrease
of enrichment media in their procedures for the or have no effect on the recovery rate of
examination of foods for salmonellae. The choice salmonellae from high fat samples (123). The
of 35-37°C as the incubation temperature for all nature of the sample, the type of enrichment
enrichment media was probably influenced by media, and the temperature of incubation appear
evidence produced by several investigators that to be important contributing factors. Secondary
certain media may be lethal to salmonellae when enrichment of negative selective enrichment
incubated at 43°C, e.g. tetrathionate (264, 395, broths in either the primary medium or a
403). In addition to the possible detrimental effect different medium has been shown to frequently
of elevated incubation temperatures on the produce a higher yield of salmonellae (210). Hence
recovery of salmonellae from some selective in the non-routine examination of samples where
enrichment broths, the brand and method of precise information on the presence of
media preparation have been shown to be salmonellae is required, secondary enrichment
important factors in obtaining reliable and procedures may be valuable.
reproducible results (170, 171).
The optimum time of incubation of a selective Selective motility
enrichment broth is a function of the number of Several methods have been devised for isolating
salmonellae present in the sample (270). It has salmonellae from other microorganisms on the
been reported that the subculture of selective basis of their motility through semi-solid agar.
enrichment broths after at least two different Craigie (74) described a technique using semi-
incubation periods, such as 24 and 48 h, will solid nutrient agar in a specially designed narrow

241