ELSEVIER 

lnlemational Journa

of Food Microbiology 

International Journal of Food Microbiology 40 (1998) 117–121 

Research Communication 

Behavior of Salmonella typhimurium DT104 during the 

manufacture and storage of pepperoni 

a a a a Anne M. Ihnot , Ann M. Roering , Rachel K. Wierzba , Nancy G. Faith , 

a ,b , John B. Luchansky *

a 

Department of Food Microbiology and Toxicology, Food Research Institute, University of Wisconsin, Madison, Wisconsin 53706, USA b

Department of Food Science, University of Wisconsin, Madison, Wisconsin 53706, USA 

Received 10 September 1997; received in revised form 17 November 1997; accepted 8 January 1998 

*Corresponding author. Addresss for correspondence: Food 

Research Institute, 1925 Willow Drive, Madison, WI 53706, USA. 

Tel.: 1 1 608 2637777; fax: 1 1 608 2631114; e-mail: 

jbluchan@facstaff.wisc.edu 

0168-1605/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. 

PII S0168-1605(98)00018-X 

Abstract 

Pepperoni batter (ca. 70% pork:30% beef) was prepared and subsequently inoculated with a six-strain cocktail (ca. 

4.4 3 107 per gram batter) of Salmonella typhimurium DT104. After fermentation at 368C and 92% relative humidity (RH) 

to # pH 4.8, counts of the pathogen decreased by about 1.3 log units. An additional 1.6 log unit decrease was observed 10 10 

following drying at 138C and 65% RH to a moisture protein ratio (M/Pr) of 1.6:1. After storage of pepperoni sticks for 56 

days under vacuum at 4 or 218C, counts of the pathogen were about 4.6 and 6.6 log10 units lower, respectively, compared 

with starting levels in the batter. These data establish that fermentation and drying result in about a 3.0 log10 reduction in 

numbers of S typhimurium DT104 in pepperoni sticks and that storage of pepperoni sticks under vacuum at ambient 

temperature is more severe on the pathogen than refrigerated storage. © 1998 Elsevier Science B.V. 

Keywords: Salmonella typhimurium DT104; Pepperoni; Multidrug; Resistant; Pathogen; Fermentation 

1. Introduction 

Isolates of Salmonella typhimurium defnitive 

phage type 104 (DT104) were frst observed in 1984 

in the United Kingdom (UK). Subsequently, the 

widespread and frequent association of DTlO4 isolates with farm animals or foods derived therefrom 

has been observed (Hollingsworth, 1997). Phage 

type 104 strains of Salmonella are resistant to 

multiple antibiotics, usually displaying resistance to 

ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (R-type ACSSuT) (Threlfall 

et al., 1996). More recently, some strains have been 

isolated which also display resistance to trimethoprim and fuoroquinolones (Threlfall et al., 1996). In 

the UK, DT104 strains of S. typhimurium were 

second only to phage type 4 strains of S. enteritidis 

as the most prevalent Salmonella strains isolated

(Threlfall et al., 1996). In the United States (US), S.

typhimurium was also the second most commonly 

reported serotype of Salmonella (Hosek et al., 1997). 
Among S. typhimurium strains in the US, the DT104

phenotype was identifed in 32% (90 of 282 isolates)

of isolates tested during 1996, as compared to a

prevalence of 28% (273 of 976 isolates) in 1995 and

7.0% in 1990 (Hosek et al., 1997). 

Contact with ill farm animals and consumption of

beef, chicken, pork sausage, and meat paste were

identifed as risk factors for DT104 infections in the

UK (Davies et al., 1996; Wall et al., 1994). Although

humans may have been the original source that

subsequently spread this bacterium to animals

(Anonymous, 1997), far less is known about the

reservoir or risk factors for S. typhimurium in the

US. A 1996 outbreak of salmonellosis among Nebraska elementary school children was attributed to S

typhimurium DT104 (Hosek et al., 1997). This US

outbreak was linked to consumption of expired

chocolate milk, but the organism was never cultured

from the remaining cartons nor was the organism

isolated from a kitten or turtle handled by children

who became ill (Anonymous, 1997; Hosek et al.,

1997). 

In 1995, the United States Department of Agriculture (USDA) Food Safety and Inspection Service

(FSIS) began requiring manufacturers of dry and

semi-dry fermented sausage to validate processes to

insure a 5-log10-unit reduction in numbers of Escherichia coli O157:H7 (Reed, 1995). In mid-1997,

the USDA/FSIS indicated that when a plant could

not demonstrate compliance with that requirement,

more frequent sampling of products from the plant

for E. coli O157:H7, as well as for Salmonella

species and Listeria monocytogenes, would be required (Billy, 1997). Although information on the

behavior of S. typhimurium in fermented meats was

published some years ago (Goepfert and Chung,

1970; Masters et al., 1981; Smith et al., 1975),no

information exists on the fate of S. typhimurium

DT104 in fermented meats. However, there has been

at least one report confrming the recovery of

S.typhimurium DT104 from a stick of salami that

displayed a plasmid profle identical to the plasmid

profle from an isolate from a patient who consumed

the same salami and became ill (Wall et al., 1994).

There has also been at least two recalls of fermented

meats due to Salmonella contamination, a recall of

Lebanon bologna in 1995 (United States Department

of Agriculture, 1995) and a recall of cervelat in 1997

(United States Department of Agriculture, 1997).

. 

The association of S. typhimurium DT104 with 

exposure to live animals and foods derived therefrom 

and the higher hospitalization and mortality rates for 

DT104 isolates compared with non-typhoid salmonellae, as well as the likelihood of more aggressive testing of fermented meats for Salmonella, 

prompted us to evaluate the viability of this bacterium after fermentation, drying, and storage of 

pepperoni. 

2. Materials and methods 

Six clinical isolates of Salmonella typhimurium 

were used. Those were: (i) strains H3380 and H3402 

(phage type DT104); (ii) strains H3278 and H2662 

(phage type DTlO4b); and (iii) strains G8430 and 

G7601 (phage type U302). All six isolates displayed 

an indistinguishable genomic fngerprint as determined by pulsed-feld gel electrophoresis (data not 

shown). The salmonellae were maintained at 2 208C 

in brain heart infusion (BHI; Difco Laboratories Inc., 

Detroit, MI) broth plus 10% glycerol and transferred 

twice in BHI broth prior to use. The Pediococcus 

acidilactici starter culture (Saga 200, Quest International, Rochester, MN) was prepared by adding 12 

ml of the thawed commercial culture, brought up to 

100 ml with sterile double distilled water, to about 

23 kg of raw meat to deliver about 107 cfu/g of 

batter. 

The S. typhimurium strains were separately grown 

overnight at 378C in 10 ml of BHI broth. A 750 ml 

portion of each of the overnight cultures was transferred to a separate fask containing 750 ml BHI 

broth and incubated for 18 h at 378C. The cells were 

harvested by centrifugation, the six cell suspensions 

were combined, and the fnal volume was adjusted to 

75 ml with 0.1% peptone (Difco). 

Pepperoni was manufactured essentially as described (Faith et al., 1997; Hinkens et al., 1996) and 

outlined in Fig. 1. Meat was tested for viable S. 

typhimurium by direct plating prior to stuffng, after 

fermentation, during drying, and during storage using 

triplicate samples of meat and duplicate platings of 

each dilution. At each sampling interval, three 25-g 

portions of the batter or a 25-g cross section from the 

center of the three different sticks were aseptically 

transferred to separate Stomacher bags (Seward 

Medical, London, UK) containing 0.85% saline and 
Pepperoni Manufacture 

Inoculate Batter 

! 

Ferment 

{36°C, 92% RH, pH ::;4.8) 

! 

Dry 

{13°C, 65% RH, M/Pr 1.6:1) 

! 

21°c 

! 

Vacuum 

Package/Store 

! 

! 

4°c 

! 

Vacuum 

Fig. 1. Pepperoni manufacture. Flow diagram depicting the 

pepperoni processes which were evaluated. 

macerated for 2 min in a stomacher (model 400; 

Tekmar, Cincinnati, OH). Next, 0.1 ml portions, with 

and without prior dilution were spread onto XLD 

(Remel, Ltd., Lenexa, KS) agar plates. 

When numbers of the pathogen decreased below 

detection by direct plating ( , 101 cfu g), the presence/absence of the pathogen was determined by 

enrichment. A 25 g portion from each of three sticks 

was aseptically transferred to separate Stomacher 

bags containing 225 ml lactose broth (Difco). After 

maceration, the samples were incubated overnight at 

378C and an inoculation loop was used to streak a 

portion of the contents of each bag onto XLD agar 

plates that were then incubated at 378C for 24 h. The 

non-inoculated batter was also macerated using a 

stomacher to test for S. typhimurium and total 

aerobic bacterial numbers by spreading 0.1 ml 

portions, with and without prior dilution, onto XLD 

and trypticase soy (Difco) agar plates, respectively. 

Immediately after grinding, the batter was also 

macerated using a Stomacher to test for viable 

pediococci by spreading 0.1 ml portions, with and 

without prior dilution, onto Lactobacilli MRS 

(Difco) agar plates. All plates were incubated at 

378C for 24 to 48 h before colonies were counted. 

Chemical analyses were performed by a commercial testing laboratory on a 250-g composite sample 

from three sticks following Association of Offcial 

Analytical Chemists (AOAC) procedures to determine the moisture (AOAC procedure 950.46) and 

protein (AOAC procedure 928.06) contents as 

specifed for meat products (McNeal, 1990). 

The absence ( , 10 cfu/g) of the pathogen on 

XLD plates by direct plating was scored as 9 for 

regression analyses. When cells of the pathogen were 

not detected by enrichment, a value of 1.0 was used 

for regression analyses. 

3. Results and discussion 

Previous studies have evaluated the fate of Salmonella in fermented meats. As one example, Smith 

et al. (1975) reported about a 1.3 log10 reduction in 

numbers of S. typhimurium after fermentation (358C, 

85% RH, pH 4.5) and about a 3.0 log10 reduction in 

pathogen numbers after drying (128C, 65% RH, 22 

days) of pepperoni. As other examples, Goepfert and 

Chung (1970) reported a 0.75 to 2.5 log10 reduction 

of S. typhimurium after fermentation (308C, 90% 

RH, # pH 4.8) of Thuringer sausage and Masters et 

al. (1981) reported about a 2.0 log10 reduction of S. 

typhimurium after fermentation (358C, 90% RH, 5.0 

pH 5.0) of summer sausage. All three studies utilized 

a commercial lactic starter culture. These data are 

similar to results obtained in the present study for S. 

typhimuriun DT104. More specifcally, fermentation 

and drying of pepperoni resulted in a 2.9 log10 unit 

decrease in numbers of S. typhimurium. In related 

experiments, monitoring the fate of E. coli O157:H7 

in pepperoni, fermentation and drying resulted in 

about a 1.8 log10 unit decrease in pathogen numbers 

(Faith et al., 1997; Hinkens et al., 1996). As such, S. 

typhimurium DT104 appears less viable during fermentation and drying of pepperoni than E. coli 

O157:H7. Storage of pepperoni under vacuum for 56 

days at 48C resulted in an additional 1.7 log10 unit 

reduction of S. typhimurium DT104 over what was 

achieved by fermentation and drying. After storage 

for 56 days at 218C under vacuum, pathogen numbers decreased an additional 3.7 log10 cfu/g over 
8 

- 21°C 

7 D 4°c 

E 6 

ca 

... 

a, 5 ... 

Cl) 

CL 4 

:I 

-u 

0 3 ... a, 

.3 2 

lime 0 day 0.4 day 1 day 4 day 10 day18 day 1 day 7 day 14 day 28 day 68 

j- Fermentation -f i-1---- Drying ----➔ -------Storage-------

Fig. 2. S. typhimurium counts (log10 cfu per gram; n 5 2) from pepperoni after fermentation at 368C to pH 4.8, drying at l38C and 65% RH 

to a M/Pr of # 1.6:1, and storage under vacuum at room or refrigeration temperatures. Black bars are results for storage at 218C and white 

bars are results for storage at 48C. 

what was achieved by fermentation and drying. After 

28 days at 218C, it was not possible to recover the 

pathogen by direct plating. However, it was possible 

to recover the pathogen by enrichment in 5 of 6 

sticks from both trials after 28 days and in 1 of 6 

sticks from both trials after 56 days. The greater 

decrease in numbers of DT104 at 218C compared to 

48C during storage were similar to results obtained 

with E. coli O157:H7 in our previous study (Faith et 

al., 1997). Lastly, microbiological analyses of the 

raw meat revealed no indigenous Salmonella species 

and a total aerobic plate count and lactic acid 

bacteria count of 3.5 3 105 and 8.1 3 103 cfu/g, 

respectively. The LAB count after the addition of the 

starter culture was 5.9 3 107 cfu/g. (Fig. 2) 

The incidence of salmonellae in over 1110 samples from 40 sausage-manufacturing plants in the US 

was estimated at 12 to 29% (Johnston et al., 1982). 

A recent survey in the UK of fresh sausage batter 

prior to fermentation identifed a prevalence of 2 to 

11% for salmonellae (reported by Wall et al., 1994), 

whereas a previous survey between 1969 and 1974 

reported the prevalence for salmonellae in sausages 

at about 60% (Roberts et al., 1975). The results of 

the present study indicate that phage type 104 

isolates of S. typhimurium are generally less viable 

in pepperoni than serotype O157:H7 isolates of E. 

coli. As such, manufacturing processes of fermented 

meats already validated for E. coli O157:H7 should 

be suffcient to also deliver a 5 log10 unit reduction 

in counts of S. typhimurium DT104. 

Acknowledgements 

We extend our appreciation to Tim Lorang for 

invaluable suggestions and assistance, to Doskocil 

Food Service Company for providing meat, spices, 

and casings, and to Peggy Hayes and Bala 

Swaminathan of the CDC for providing isolates of S. 

typhimurium. This work was supported, in part, by 

contributions to the University of WisconsinMadison Food Research Institute from the Food 

Industry and, in part, by the College of Agricultural 

and Life Sciences of the University of WisconsinMadison. 
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