Survival of Arcobacter butzleri During Production and Storage of Artisan Water Buffalo Mozzarella Cheese

Abstract

Water buffalo mozzarella cheese (WBMC) is a fresh stretched cheese produced from whole chilled buffalo milk. Although pasteurization of milk and the use of defined starter cultures are recommended, traditional technology involving unpasteurized milk and natural whey cultures is still employed for WBMC production in Italy. The purpose of this study was to assess the behavior of Arcobacter butzleri during WBMC production and storage under different temperature conditions (5, 10, and 20°C). Raw milk was experimentally inoculated with one reference strain and two isolates of A. butzleri, and the count was monitored during WBMC production and storage. The bacterial count of A. butzleri decreased during curd ripening (from 7.83 log colony-forming units (CFU)/g to 4.14 log CFU/g in about 4 h) and a further decrease (>4 log CFU/g) was observed at the end of curd stretching. During storage testing, A. butzleri was never detected by direct plating, whereas it was recovered from 12 of the total 162 WBMC until the end of storage testing by enrichment. The results revealed that A. butzleri is able to survive during WBMC production and storage at different temperature conditions. Consequently, traditional WBMC produced from raw milk could represent a potential source of Arcobacter infection for humans.

Introduction

T he genus Arcobacter was proposed and included in the family Campylobacteraceae in 1991 (Vandamme et al., 1991) and currently includes 17 species (Figueras et al., 2012) of which Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii have been associated with human and animal illness (Ho et al., 2006). A. butzleri is the most important and prevalent species of the genus: It has been classified as a serious hazard to human health by the International Commission on Microbiological Specifications for Foods (ICMFS, 2002) and as a significant zoonotic pathogen (Cardoen et al., 2009; Collado and Figueras, 2011).

The broad distribution of A. butzleri has been associated with phenotypic differences relating to growth capabilities in terms of metabolic differences and with a high degree of genetic heterogeneity among isolates, with multiple genotypes being found in a single location, or even in a single animal (Houf et al., 2003; Son et al., 2006; Ferreira et al., 2013; Merga et al., 2013). In addition to vast distribution and variability, resistance to common antimicrobial agents was also observed for Arcobacter, constituting a concern among the Campylobacteraceae family (Ferreira et al., 2013).

The consumption of raw contaminated food of animal origin was hypothesized as a route of transmission of Arcobacter spp. to humans (Van Driessche et al., 2005), and the initial source seems to be fecal contamination during the various stages of production processes (Ongör et al., 2004; Scullion et al., 2006; Van Driessche and Houf, 2008). Worldwide there is increasing evidence that livestock animals are significant reservoirs of Arcobacter spp.: these bacteria have frequently been isolated from the intestinal tracts and fecal samples of different farm animals including water buffalo (Piva et al., 2013) and may contaminate milk during milking. Water buffalo mozzarella cheese (WBMC) is a fresh stretched cheese produced from whole chilled water buffalo milk. Although pasteurization of milk and the use of defined starter cultures are recommended, traditional technology involving unpasteurized milk and natural whey cultures is still employed for WBMC production in Italy. In the artisan mozzarella cheese factory, mozzarellas are stored at room temperature in a conditioning liquid commonly composed of water resulting from stretching, acidified with whey from the previous manufacture (Villani et al., 1996) or, more recently, by tap water salted and acidified with lactic or citric acid; for the storage some producers claim a shelf-life of 5 days keeping the product at room temperature, others from 5 to 10 days storing the product at room temperature for 1–3 days and afterwards in the refrigerator, and yet others for up to 3 weeks at refrigerator temperature (Finazzi et al., 2011).

Based on evidence that several foodborne pathogens—specifically Salmonella Typhimurium, Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli O157:H7—are able to survive thermal treatment during stretching or remain viable during the shelf-life of artisan WBMC (Oliviero et al., 2010; Serraino et al., 2012; Serraino et al., 2013a), this study aimed to assess the survival of A. butzleri during WBMC production and storage, under different temperature conditions, assuming product contamination due to the presence of A. butzleri in raw milk.

Materials and Methods

The experimental design consisted of four trials: three replicates of inoculated batches and one noninoculated control batch.

Bacterial strains

One reference strain (A. butzleri strain DSM 8739, Leibniz Institute DSMZ, Braunschweig, Germany) and two different A. butzleri field isolates, showing different pulsed-field gel electrophoresis patterns (IA/FED 1/2011 and IA/FED 4/2011 isolated from feces of two lactating water buffaloes within the same herd), were used in the study; they were grown separately on nutrient agar supplemented with 5% laked horse blood (Oxoid, Basingstoke, UK) incubated at 30°C for 48 h microaerobically, by evacuating 80% of the normal atmosphere and introducing a gas mixture of 8% CO₂, 8% H₂, and 84% N₂ into the jar. All bacterial colonies present in the plates were collected by a sterile swab and suspended separately in saline (NaCl 0.85%, VWR International, Milan, Italy); the suspensions obtained were cultured separately in 1 L of Arcobacter broth supplemented with 5% laked horse blood (Oxoid, Basingstoke, UK) incubated in continuous agitation at 30°C for 48 h aerobically, then centrifuged at 3000×g for 1 h; the pellets were resuspended separately in 1 L of saline (NaCl 0.85%, VWR International, Milan, Italy). For the bacterial inocula, the three isolate suspensions of A. butzleri were quantified spectrophotometrically and serially diluted in Butterfield's phosphate buffer in order to obtain approximately the same concentration. An equal volume of the three suspensions (1 L each) was pooled and used to inoculate raw milk in order to obtain a final concentration in milk of about 10⁷ colony-forming units (CFU)/mL. The number of viable cells of the three suspensions was verified by 10-fold dilution and direct plating on nutrient agar supplemented with 5% laked horse blood (Oxoid, Basingstoke, UK) incubated at 30°C for 48 h microaerobically.

WBMC production

Raw milk was purchased from the Veterinary Medicine Faculty (University of Bologna) cheese factory and reported, by the routine control, fat, protein and lactose content of 8.58, 4.78, and 4.97 g/100 mL, respectively, somatic cell count of 179.000/mL, and total bacterial count of 175,000 CFU/mL. Mozzarella was produced according to the traditional technology, as described by Addeo and Coppola (1983) and Villani et al. (1996), using 50 L of unpasteurized milk and natural whey culture as starter. Briefly, the raw milk was heated to 37–38°C, and the whey and rennet were added and were left to ripen at 37–38°C for about 4 h. Afterwards, the curd was extracted from the whey and stretched with hot water (90°C) for about 3 min and then molded into the traditional round shape. Each mozzarella weighed about 250 g. The conditioning liquid was prepared with sterilized tap water, salt up to 2°Bé, and lactic acid 80% to a final pH of 2.79 and 5.5°SH (Soxhlet-Henckel)/50 mL. Single 250-g WBMC were vacuum packed in conditioning liquid at room temperature (about 22°C). The temperature during production and stretching was measured by a Hobo H08-002-02 data logger.

Storage testing

For each batch, 45 packed WBMC were divided into three groups (15 WBMC for each group) for the storage tests at three different temperatures (5, 10, and 20°C) for 10 days. The storage conditions were chosen to simulate optimal storage conditions (5°C), domestic storage (10°C) (Beaufort et al., 2008), and thermal abuse (20°C).

A. butzleri detection and count

For each batch, detection and count of A. butzleri were performed in triplicate from raw milk (before and after A. butzleri inoculation), natural whey starter, conditioning liquid, curd at the end of ripening, and curd after stretching (mozzarella cheese at time 0) and during storage after 1, 2, 3, 6, and 10 days. A total of 54 WBMC were analyzed for each batch. Detection was performed using the enrichment procedure described by Houf et al. (2001); briefly, 25 mL for liquid samples or 25 g for solid samples were inoculated into 225 mL of Arcobacter broth (Oxoid Ltd.) supplemented with 5% lysed horse blood and a mix of cefoperazone (16 mg/L), amphotericin B (10 mg/L), 5-fluorouracil (100 mg/L), novobiocin (32 mg/L), and trimethoprim (64 mg/L) as a selective supplement. All antimicrobial substances were obtained as laboratory standard powders from Sigma (St. Louis, MO). After 48 h of incubation, an aliquot of 10 μL of the enrichment broth was streaked onto selective agar plates prepared by suspending 24 g of Arcobacter broth (Oxoid Ltd.) and 12 g of Agar Technical No. 3 (Oxoid Ltd.) and supplemented with selective supplement as described above. The plates were incubated at 28±1°C under microaerobic conditions, as described above, and after 48 h of incubation were checked daily up to 5 days. The A. butzleri count was performed by 10-fold dilution and direct plating (0.1 mL in duplicate) on agar plates prepared as described above and incubated microaerobically at 28°C for 48 h. Colonies were counted and a selection of 10 colonies for each plate were subcultured and examined for presumptive identification such as growth under aerobic condition, cellular morphology, oxidase, and catalase activity. The DNA of colonies was extracted using the REDExtract-N-Amp Tissue polymerase chain reaction (PCR) Kit (Sigma, St. Louis, MO) and subjected to species confirmation by multiplex PCR (Douidah et al., 2010).

Lactic acid bacteria (LAB) count, pH, and a_w determination

The following analyses were additionally made on each sample: mesophilic LAB count by 10-fold dilution and inclusion in M17 and MRS agar plates (Oxoid, Basingstoke, UK) incubated respectively aerobically and under microaerobic conditions at 35°C for 48 h; pH was measured by an instrument with automatic temperature compensation (Hanna Instruments HI 223); a_w was determined by AquaLab model series 3.

Statistical analysis

The results relating the A. butzleri and LAB count and pH values were performed by t-test, whereas the results relating the number of samples positive to A. butzleri at different storage temperatures were analyzed using chi-square test. Statistical significance was set at p<0.05. All analyses were carried out with PRISM 5.0 software.

Results

A. butzleri was not detected in noninoculated raw milk, natural whey starter, conditioning liquid, or in any samples of the noninoculated batch. The change in A. butzleri count, pH, and LAB population count during WBMC production are summarized in Table 1. In inoculated batches, A. butzleri count significantly (p=0.01) decreased during curd ripening from 7.83±0.06 log CFU/g to 4.14±0.27 log CFU/g in about 4 h and a further decrease was observed at the end of curd stretching when A. butzleri was not detectable either by direct plating or by enrichment. The maximum temperature reached during curd stretching was 71.2°C±1.3 (mean±SD of four batches); the temperature remained over 60°C for at least 2 min and over 55°C for at least 3 min in all four batches (data not shown). From inoculation of raw milk to the end of ripening, pH decreased from 6.79 to 5.07 and LAB count increased from 4.36 log CFU/g to 8.10 log CFU/g, and from 6.62 log CFU/g to 9.07 log CFU/g, respectively, on MRS and M17 agar (Table 1).

Table 1.

Change of pH and of Arcobacter butzleri and Lactic Acid Bacteria (LAB) Count (Log Colony-Forming Units/g) in the Traditional Water Buffalo Mozzarella Cheese Production Process (Mean of 9 Samples: 3 Sample Units for 3 Batches±Standard Deviation)

Sample	Arcobacter butzleri	pH	LAB (MRS agar)	LAB (M17 agar)
Contaminated raw milk after natural whey starter and rennet addition	7.83±0.06^a	6.79±0.01^a	4.36±0.08^a	6.62±0.12^a
Curd at the end of ripening	4.14±0.27^b	5.07±0.01^b	8.10±0.23^b	9.07±0.15^b
Curd after stretching	n.d.	5.09±0.02^b	7.03±0.1^c	7.17±0.15^c

a–c

Mean values in columns bearing different letters are significantly different by chi-square test (p<0.05).

n.d., not detected.

The change of pH and LAB population count during storage testing are summarized in Table 2. A. butzleri was never detected by direct plating (<50 CFU/g) but was detected in 12 out of 162 WBMC samples by enrichment procedure until the end of storage testing in mozzarellas stored at all temperatures (Table 2). Positive samples were detected more frequently in mozzarellas stored at 20°C than in mozzarellas stored at 10 and 5°C, but no significant differences were shown by chi-square test in the prevalence of positive samples at different storage temperatures (p=0.1884).

Table 2.

Presence Arcobacter butzleri (Number of Positive Samples/Total Samples) Detected by Enrichment and Values of Lactic Acid Bacteria (LAB) Count and pH (Mean of 9 Samples: 3 Sample Units for 3 Batches±Standard Deviation) in Water Buffalo Mozzarella Cheese Stored at Different Temperatures

	Storage temperature 5°C
Days after packing	A. butzleri	pH	LAB (MRS agar)	LAB (M17 agar)
0	0/9	5.09±0.02^a	7.03±0.11^a	7.17±0.15^a
1	1/9	5.22±0.06^b	6.31±1.58^a	7.31±1.18^a
2	0/9	5.18±0.06^a,b	5.67±0.40^b	6.21±0.22^b
3	0/9	5.17±0.05^a,b	6.36±1.17^a	7.66±0.95^a
6	2/9	5.13±0.04^a,b	6.51±1.17^a	7.39±1.09^a
10	2/9	5.22±0.05^b	7.17±0.09^a	7.59±0.86^a

	Storage temperature 10°C
Days after packing	A. butzleri	pH	LAB (MRS agar)	LAB (M17 agar)
0	0/9	5.09±0.02^a	7.03±0.11^a	7.17±0.15^a
1	1/9	5.10±0.02^a	6.53±1.48^a	6.02±1.17^a
2	0/9	5.18±0.05^a	6.65±1.12^a	6.91±1.06^a
3	0/9	5.12±0.05^a	7.12±0.13^a	7.60±0.60^a
6	0/9	5.02±0.01^b	8.21±0.16^b	8.49±0.14^b
10	0/9	5.16±0.03^a,c	8.30±0.28^b	8.54±0.06^c

	Storage temperature 20°C
Days after packing	A. butzleri	pH	LAB (MRS agar)	LAB (M17 agar)
0	0/9	5.09±0.02^a	7.03±0.11^a	7.17±0.15^a
1	1/9	5.03±0.12^a	7.13±1.01^a	6.91±0.49^a
2	1/9	4.99±0.11^a	8.01±0.12^b	8.27±0.66^b
3	0/9	4.86±0.05^b	8.21±0.09^b	8.38±0.12^b,c
6	3/9	4.73±0.18^c	8.46±0.12^c	8.63±0.10^b,c
10	1/9	4.71±0.06^c	8.33±0.18^b	8.33±0.18^b,c

a–c

Mean values in columns bearing different letters are significantly different by t-test (p<0.05).

During storage at 5°C, LAB count and pH values remained substantially unchanged, whereas pH showed a decrease in WBMC stored at 10 and 20°C, which was more pronounced at higher storage temperature (Table 2). LAB count, on both MRS agar and M17 agar, showed an increase during storage testing at 10 and 20°C; a_w values remained substantially unchanged during storage testing at all temperatures ranging from 0.988 to 0.984.

No significant differences were observed in the LAB count, pH, and a_w values of the inoculated and noninoculated batch (data not shown).

Discussion

Although Arcobacter spp. have been isolated from healthy cow and water buffalo feces and may reasonably contaminate milk during milking, this is the first study to investigate the behavior of A. butzleri in a raw-milk cheese, and no data are available to compare our results.

Our study found a decrease in A. butzleri count (about 3.5 log CFU/g) in the curd after 4 h of ripening; this reduction was associated with an increased LAB count and a decrease of pH from 6.79 to 5.07. The minimum pH growth limit reported for A. butzleri is 5.5, even if some strains can tolerate pH 5.0 (D'sa and Harrison, 2005). The pH growth limit was shown to be temperature dependent: D'sa and Harrison (2005) observed that A. butzleri was able to tolerate a lower pH in Ellinghausen McCullough Johnson Harris medium at 25°C than at 37°C (temperature conditions comparable to those of the WBMC curd ripening). Similar results were obtained by Hilton et al. (2001), who reported a minimum pH growth limit of 5.0 at 30°C and of 5.5 at 37°C in brain heart infusion (BHI). During curd ripening, the significant Arcobacter count reduction could be explained both by the fact that A. butzleri is probably under unfavorable pH/temperature conditions and by an additional effect due to LAB competition that cannot be excluded.

The A. butzleri count reduction observed during stretching (>4 log CFU/g) seems to be in line with previously reported data: D'Sa and Harrison (2005) reported a D-value of 0.30–0.42 min in phosphate-buffered saline at pH 5.5 at 55°C and a D-value of 0.03–0.11 min at 60°C; Hilton et al. (2001) reported a similar D-value (0.4 min at 55°C in BHI) for A. butzleri in the stationary phase, but a higher D-value (1.1 min) in the exponential growth phase. The comparison of these data must be interpreted with caution, considering both the protective effect of the organic material on A. butzleri as shown by Van Driessche and Houf (2008), who demonstrated that Arcobacter spp. survive thermal treatments longer by incorporating organic material in water, and the likelihood of uneven heat distribution during stretching.

Throughout storage testing, A. butzleri was never detected by direct plating, probably because it was always under the detection limit of the plate count method used in the test; by contrast, viable A. butzleri were recovered by enrichment at all storage temperatures until the end of storage testing (Table 2). The reported ability of A. butzleri to grow at temperatures as low as 10°C and to survive at lower temperatures seems to be dependent on the test medium used (Hilton et al., 2001; Van Driessche and Houf, 2008; Kjeldgaard et al., 2009), but no data on the behavior of A. butzleri in fresh cheeses during storage are available in the literature to compare our results; considering the pH values observed during the storage test and the temperature conditions performed, A. butzleri could be more or less stressed at the different temperatures of storage, but nevertheless it was able to survive until the end of the storage test. Indeed, Isohanni et al. (2013) recently demonstrated an increased resistance to acid stress in heat-stress-adapted A. butzleri cells, and it is possible that the heat stress during stretching could play a role in the ability of A. butzleri to survive to nonoptimal pH conditions during mozzarella cheese storage.

The results of this study, performed by contaminating water buffalo milk with a reference strain and field isolates from dairy water buffaloes, demonstrated that A. butzleri is able to survive during WBMC production and storage at different temperature conditions and consequently may represent a potential source of infection for humans as reported for raw cow's milk (Scullion et al., 2006; Pianta et al., 2007; Ertas et al., 2010; Shah et al., 2012; Serraino et al., 2013b). Furthermore, we must emphasize that the presence of A. butzleri in water buffalo milk does not influence the traditional WBMC production process.

Although a high number of A. butzleri are unlikely to be present in milk used to produce WBMC, no information on the level of naturally contaminated water buffalo milk is available, and only qualitative data are reported in the literature for cow's milk. For this reason, the true role of contaminated raw milk and raw-milk products as vehicles for A. butzleri infection needs to be assessed.

Disclosure Statement

No competing financial interests exist.

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Survival of Arcobacter butzleri During Production and Storage of Artisan Water Buffalo Mozzarella Cheese

Abstract

Introduction

Materials and Methods

Bacterial strains

WBMC production

Storage testing

A. butzleri detection and count

Lactic acid bacteria (LAB) count, pH, and aw determination

Statistical analysis

Results

Discussion

Disclosure Statement

References

Lactic acid bacteria (LAB) count, pH, and a_w determination