Salmonella choleraesuis subspecies arizonae (IIIa) and subspecies diarizonae (IIIb)(formerly Arizona hinshawii) were originally isolated from reptiles (Caldwell and Ryerson, 1939), but have since been isolated from a wide variety of animals ( Weiss and Hall). Subspecies II, IIIa, IIIb and IV are most commonly found in cold-blooded animals ( Le Minor, 1984)

Infections in man caused by S. choleraesuis subspecies diarizonae are predominantly food-borne (Edwards et al., 1959), often caused by serovars found in domestic animals such as cattle (61:1,v:1,5,(7)) and sheep (61:1,v:1,5,(7); 61:k:1,5,(7)) ( Weiss and Hall).

Several reports indicate that Salmonella choleraesuis subspecies diarizonae serovar 61:k:1,5,(7) [S. IIIb 61:k:1,5,(7)] has become adapted to sheep (Ryff; Edwards; Harvey and Greenfield). Strains of S. IIIb 61:k:1,5,(7) are assumed to become invasive when the host animals (sheep) are stressed or debilitated (Greenfield and Long). The monophasic serovar 61:-:1,5 is commonly diagnosed in sheep ( Weiss et al., 1986).

1.1. Occurrence in Norway

The Salmonella serovars IIIb 61:-:1,5 and IIIb 61:k:1,5 have been sporadically isolated from sheep in Norway. In 1993, Salmonella IIIb 61:-:1,5 was isolated from an aborted foetus and a stillborn lamb (Mork et al., 1994). In this paper, we assume these isolates to be identical to the tabulated serovar S. IIIb 61:k:1,5,(7) (Popoff and Le Minor, 1997). Five infections in man caused by different serovars of the subspecies diarizonae have been verified in Norway between 1989 and 1998, of which one was S. IIIb 61:k:1,5,(7) (National Institute of Public Health, Oslo).

1.2. Growth in foods

The ability of some salmonellae to grow at low temperatures may pose a public health hazard if the chilling temperature is not under adequate control. Several strains have been observed growing below 10°C(D’Aoust, 1991). Animals with depleted muscle glycogen when killed, produce meat that is characterised as dark, firm and dry (DFD). DFD muscle has pH above 6.0 whereas the pH in normal meat is about 5.5 ( Devine and Chrystall, 1988). However, little is known about the influence of temperature and pH on growth of S. IIIb 61:k:1,5,(7) in meat and meat products.

The aim of this study was to describe the growth characteristics in the broth of three serovars of Salmonella choleraesuis (IIIb 61:k:1,5,(7), Enteritidis and Dublin) under different pH and temperature conditions. Furthermore, the aim was to describe the growth at low temperature of S. IIIb 61:k:1,5,(7) in competition with the indigenous microflora in both minced meat and chops from both normal and DFD-meat.

2. MATERIALS AND METHODS

2.1. Strains

The experiments were undertaken using three strains of S. IIIb 61:k:1,5,(7) (492/91, 2431/93 and 2748/93). They were all isolated from Norwegian sheep. The strains were supplied by the National Institute of Public Health, Oslo. For comparison, strain SLV-242 of Salmonella choleraesuis subspecies choleraesuis serovar Dublin (S. Dublin) and strain CCUG 34136 of Salmonella choleraesuis subspecies choleraesuis serovar Enteritidis (S. Enteritidis) were used.

For the meat experiments the three strains of serovar 61:k:1,5,(7) were made resistant to rifampicin (Foegeding et al., 1992). The adapted strains (S. IIIb 61:k:1,5,(7)_Rif) grew, although markedly reduced, on Tryptone Soy Agar (TSA; Difco, Detroit, USA) when the agar concentration of rifampicin was 400 g/ml. The same procedure was repeated again using modified Brilliant Green Agar (BGA; Oxoid, Basingstoke, UK). BGA containing 25 g/ml rifampicin was chosen for further experiments.

2.2. Growth of Salmonella in broth

2.2.1. Inocula

All the strains were cultivated in Tryptone Soy Broth (TSB; Oxoid CM129) for 24 h at 30°C followed by 16 h at 25°C. The three broths S. IIIb 61:k:1,5,(7) strains were mixed in the ratio 1:1:1, while S. Dublin and S. Enteritidis were used in pure culture.

2.2.2. Media

The three bacterial suspensions were diluted with peptone broth (PB; 8.5 g of NaCl, 1 g of Oxoid Bacteriological Peptone in 1000 ml of distilled water, pH adjusted to 7.2 with 1.0 M NaOH). The suspensions were inoculated into TSB, with pH adjusted to 5.5 and 6.2.

2.2.3. Absorbance validation

S. IIIb 61:k:1,5,(7) was grown in TSB at pH 6.2 and incubated at 37°C for 48 h to validate the absorbance measurements against the number of colony forming units (CFU) on Tryptone Glucose Extract Agar (TGEA; Oxoid CM127).

2.2.4. Absorbance measurements

The bacterial suspension was diluted 1:100 in TSB, 400 l of this dilution being dispersed into micro-titre plate wells (Honeycomb 2, Labsystems Corp., Helsinki, Finland). The experiments were undertaken at pH 5.5 and 6.2 and at incubation temperatures of 4, 6, 8 and 12°C. Three replicates were analysed for each factor combination. The bacterial absorbance was continuously measured and recorded using Bioscreen C (Labsystems Corp., Helsinki, Finland) and the data were processed with Biolink software (Labsystems Corp.). The absorbance measurements were performed at wavelengths of 450–580 nm (wide band).

2.2.5. Growth measurements

Maximum growth rates (_max) were estimated by adapting the equation of Baranyi and Roberts (1994). Lag periods were estimated manually from plots of the growth curves as the time from the start of incubation to the time when the tangent from the maximum derivative of the growth curve crossed the extrapolated lag level.

2.3. Growth of S. IIIb 61:k:1,5,(7) in mutton

2.3.1. Mutton

Two sheep were used in the experiment. One sheep received subcutaneous adrenaline (1 mg/ml adrenalin; Nycomed Pharma AS, Oslo, Norway) injections before killing to induce a DFD condition in the muscle tissue (Hedrick; Braggins; Watanabe and Watanabe). Three subcutaneous injections of adrenaline were administrated: 0.3 mg/kg body weight 53 and 42 h before death and 0.2 mg/kg body weight 4 h before death. The sheep was housed with free access to water, but deprived of food after the first injection.

The animals were slaughtered at Fellesslakteriet, Oslo, and the carcasses were chilled over night, following the procedure of the abattoir. The initial temperature in the cold storage chamber was set at about 2°C and airflow was >2 m/s (Frøystein and Torsen, 1997). The final meat temperature the morning after was 3.5°C. Meat pH was directly measured approximately a centimetre beneath the muscular surface of the carcasses 24 h after killing by means of a pH-meter (Knick Portamess 751 calimatic). Both carcasses were deboned, and the meat frozen to −20°C, transported for 10 h to Swedish Meats R&D, Sweden, where it was stored at −20°C. Chops, 1 cm thick, were cut from the double-loin (fore- and hind-saddle) the day after, while still frozen. Discs with a diameter of approximately 2.3 cm were incised from the chops using a circular knife. The trimmings were minced in a kitchen mincer (Bosch, Stuttgart, Germany). The meat was again stored for 24 h at −20°C and thereafter kept at −1.5°C for 3 days. Qualitative tests for Salmonella were performed on the meat prior to inoculation according to the method described by the Nordic Committee on Food Analysis (NMKL) no. 71. Quantitative analyses for fat (NMKL no. 131, 1989), water (NMKL no. 23, 1991) and glycogen determination with amyloglucosidase (D. Keppler Boeringer Mannheim GmBH Biochemika, Germany) were carried out on samples of frozen meat. The pH was measured prior to the start of the experiments (PHM82 Standard pH-meter, Radiometer Copenhagen, Denmark).

2.3.2. Inocula

The three isolates of S. IIIb 61:k:1,5,(7)_Rif were transferred separately from bovine blood agar plates to three tubes with 10 ml of Brain Heart Infusion (BHI; Difco, Detroit, MI, USA) and incubated with ambient air at 30°C overnight. After incubation, aliquots of 0.1 ml were transferred separately to three other tubes with 10 ml of BHI and incubated with ambient air at 25°C overnight. A mixture of the three isolates of S. IIIb 61:k:1,5,(7)_Rif was made by mixing 100 l of each broth in 750 l PB. The mixture was diluted tenfold with PB to produce detectable low-level inocula resembling faecal contamination.

2.3.3. Inoculation

Two groups of meat-discs were put in two different bowls containing PB with about 1000 CFU/ml of S. IIIb 61:k:1,5,(7)_Rif for about 5 min. The discs were randomly retrieved and paired in Petri dishes. The two groups of minced meat were inoculated with approximately 100 CFU/g S. IIIb 61:k:1,5,(7)_Rif and mixed for 2–3 min with the kitchen mincer. The inoculated minced meat was dispersed in Petri dishes with approximately 30 g in each. Inoculation was performed at about 20°C. The Petri dishes were incubated at 8°C with ambient air in ventilated plastic bags.

2.3.4. Growth measurements

Two replicate samples of meat-discs and minced meat were analysed each day during the experiment. Each sample was weighed in a filter stomacher bag (Seward Stomacher 400), diluted 10⁻¹ with PB and mixed in a stomacher (Colworth Stomacher 400) for 30 s. Tenfold dilutions were made and analysed for S. IIIb 61:k:1,5,(7)_Rif and aerobic microorganisms.

Aerobic microorganisms were determined on TGEA incubated at 28°C for 3 days. BGA containing 25 g/ml rifampicin was used as quantitative and indicative media for the S. IIIb 61:k:1,5,(7)_Rif. The BGA plates were incubated at 37°C for 24 h. On the 6th day, quantitative analyses for Pseudomonas spp. and Enterobacteriaceae were undertaken. CFC-agar plates (Pseudomonas Agar Base; Oxoid CM559), supplemented with Cetrimide-Fucidin-Cephaloridine (Oxoid SR103), were incubated at 25°C for 48 h. After incubation, the plates were overlayed with 1% N′,N′,N′,N′-tetramethyl-p-phenylene diamine dihydrochloride (Fluka Chemie AG, Buchs, Switzerland) and oxidase positive colonies were counted. Enterobacteriaceae were analysed according to NMKL no. 144, 1992.

3. RESULTS

3.1. Growth of Salmonella in broth

3.1.1. Absorbance validation

There was a linear association between absorbance values and log CFU/ml. When log CFU/ml and absorbance were plotted against time, the growth curves followed each other closely (Fig. 1).

 

 

Fig. 1. Growth in broth of 61:k:1,5,(7) at 37°C and pH 6.2. Measurements of absorbance plotted against log CFU/ml from plate counts of S. IIIb 61:k:1,5,(7).

 

3.1.2. Absorbance measurements

Data concerning lag periods, _max and maximum absorbance are presented in Table 1. The bacterial concentration in each well was initially between 4×10⁶ and 10⁷ CFU/ml, the corresponding absorbance values being 0.18 and 0.23. Small increases of absorbance were registered at 4 and at 6°C for S. IIIb 61:k:1,5,(7) at pH 6.2, and similar findings were recorded at 6°C for serovar Enteritidis. Although growth was recorded for all three serovars at 8°C, the growth of serovar Dublin was markedly slower than the growth of the serovars IIIb 61:k:1,5,(7) and Enteritidis.

 

 

Table 1. Lag period, _max and maximum obtained absorbance for the salmonella serovars IIIb 61:k:1,5,(7), Dublin and Enteritidis grown in broth at 4, 6, 8 and 12°C at pH 5.5 and 6.2^a
 

 

At 8°C, the lag periods varied depending on serovar and pH. The lag period of the serovar 61:k:1,5,(7) was reduced from 55 h at pH 5.5 to 15 h at pH 6.2, while the lag period of serovar Enteritidis was reduced from 35 to 20 h. This effect of pH on the lag period was not seen for serovar Dublin and neither at 12°C for any of the serovars.

The impact of pH on stationary levels and growth rates at both 8 and 12°C is illustrated in Fig. 2.

 

 

Fig. 2. Growth curves of S. IIIb 61:k:1,5,(7) in broth at pH 5.5 and 6.2 at (a) 12°C and (b) 8°C. Values are means of three replicate absorbance readings.

 

3.2. Growth of S. IIIb 61:k:1,5,(7) in mutton

3.2.1. Performance of the plates

The BGA containing 25 g/ml rifampicin reduced the natural microbial flora by 5–6 log units without an impact on the growth of the rifampicin-adapted strains compared with growth on TSA and BGA.

3.2.2. Mutton characteristics

The meat pH measured at the abattoir 24 h post-mortem ranged from 5.47 to 5.86 for the normal-meat and from 6.77 to 7.07 for the DFD-meat. The pH in M. longissimus dorsi was 5.47 and 6.84 in the normal meat and the DFD-meat, respectively. When the pH was checked before the experiments started, the normal meat pH was 6.0 and DFD-meat pH 7.0.

Results from the analyses of fat, water and glycogen are given in Table 2. The qualitative tests for Salmonella from the meat prior to inoculation yielded negative results.

 

 

Table 2. Fat, water and glycogen content in the mutton

 

3.2.3. Growth in mutton

Growth curves for S. IIIb 61:k:1,5,(7)_rif and aerobic microorganisms in minced meat and chops at 8°C are shown in Fig. 3. In minced meat, Salmonella levels increased by about two log units in 7 days, while in chops the level remained at inoculum level throughout the period. Also the aerobic microorganisms increased faster in minced meat than in chops. The lag periods were shorter, while the growth rates and stationary levels were similar in minced meat and chops. After 6 days, the microbial flora of the meat was dominated by Pseudomonas spp. (>5×10⁸ CFU/g), while the level of Enterobacteriaceae ranged from 300 to 14.5×10⁵ CFU/g.

 

 

Fig. 3. Growth of S. IIIb 61:k:1,5,(7) and aerobic microorganisms in minced meat and chops at pH 6.0 and pH 7.0 at 8°C. The curves represent the mean of two replicates for each value of pH.

 

 

4. DISCUSSION

The close relationship between CFU g⁻¹ and absorbance confirms that absorbance measurement is a reliable way to measure bacterial growth in broth (Fig. 1).

The broth experiments clearly demonstrate differences between the serovars’ ability to grow at 4, 6 and 8°C, and how this growth depends on pH-values normally found in meat and meat products. Although small increases of absorbance were registered at 4 and 6°C for S. IIIb 61:k:1,5,(7), the data do not allow any definite inference to be made about growth. However, the overall tendency was that serovar IIIb 61:k:1,5,(7) grew better than serovar Enteritidis, which again grew markedly better than serovar Dublin at the low temperatures employed.

According to Ingraham and Marr (1996), Escherichia coli and Salmonella choleraesuis serovar subspecies choleraesuis serovar Typhimurium grow at a maximum rate between pH 6.0 and 8.0 and more slowly at half a pH unit or so beyond these limits. S. IIIb 61:k:1,5,(7) responded concordantly in monocultural broth and reached an exponential phase within 55 h at 8°C and pH 5.5, with the time reduced to 15 h at pH 6.2. The obtained maximum absorbance and the stationary level at pH 5.5 were also lower than at pH 6.2.

The chops represented a surface environment where the number of injured muscle cells exposed to the bacteria is markedly reduced compared to minced meat. When meat is minced, the bacteria are mixed into the mass of damaged tissue. Both experimental conditions should provide more than enough nutrients and humidity for bacterial growth (Dainty and Mackey, 1992).

Mackey and Kerridge (1988) found that Salmonella could compete with the background flora in minced beef and reach similar stationary levels when incubated aerobically between 10 and 35°C. They reported small effects of inoculum to background flora ratios between 15 and 35°C, and even a shorter generation interval of the salmonellae when the inoculated number of salmonellae was low relative to the background flora at 10°C. Alford and Palumbo (1969) found that Salmonella strains were able to compete with the background flora at 10°C, but also showed that Salmonella serovars were decimated at 4°C.

Under aerobic conditions, pseudomonades were expected to dominate the background flora (Blickstad and Dainty) and Pseudomonas spp. were not regarded to be sensitive to pH differences between 7.0 and 6.0 (Blickstad et al., 1981). Pseudomonas spp. dominated the background flora in minced meat and chops with pH 7.0, but no growth was registered on the 7th day on the CFC-plates from chops with pH 6 because we plated out on CFC-agar from the 10⁻⁷ and 10⁻⁸ dilutions and counts lower than 10⁷ CFU/g could not be registered. The aerobic microorganisms appeared to be inhibited in chops at pH 6.0 compared to chops at pH 7.0, but the exponential phases were equally steep. The reason for this apparently prolonged lag period of the aerobic microorganisms might be the lower initial number of the psychrotrophic bacterial flora. The long generation intervals at 8°C amplified that effect.

Some of the delayed growth in chops compared to the minced meat could be due to a different homogenising effect in the stomacher. The minced meat was getting mixed thoroughly, while the chops were only washed with the solution. Even though the chops were of small size and therefore with a large surface relative to the mass, the dilutional effect per g of meat had to be smaller for the minced meat than for the chops.

We assume that Norwegian sheep carcasses will occasionally be contaminated with S. IIIb 61:k:1,5,(7) and that some consumers are exposed to the bacteria. One single human case in Norway in the last 8 years indicates a low virulence of the strains in humans. S. IIIb 61:k:1,5,(7) is often reported to be more prevalent in adult sheep than in lambs (Mork and Valheim). Lambs represent the vast majority of the animals slaughtered, and this could also contribute to the low number of human cases. Mutton from adult sheep is mainly conserved and processed in traditional ways such as salting, drying, smoking and fermentation, either alone or in combination. These processes give products which provide a hostile environment to Salmonella, e.g., salted and fermented sausages with low a_w (0.87–0.91) and pH of about 5.1. Fresh products such as chops, steaks and, indeed, minced meat represent the critical products and demand careful hygienic attention. Contaminated minced meat will harbour the bacteria within the mass and these could avoid decimation when the mince is heated.

5. CONCLUSION

The experiments showed that S. IIIb 61:k:1,5,(7) grows slowly at 8°C. The mutton experiments indicate that S. IIIb 61:k:1,5,(7) will probably not be able to multiply in meat to an extent representing a serious public health hazard, given its apparent low virulence and normal hygienic handling.

ACKNOWLEDGEMENTS

Financial support from the Norwegian Research Council and Norwegian Meat Research Centre is gratefully acknowledged. The authors wish to thank Mrs Britt Arvidsson, Dr Elisabeth Borch and Dr Terje Frøystein for skilful assistance.
 

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