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Journal of Food Protection, Vol. 69, No. 2, 2006, Pages 308±314 Copyright ᮊ, International Association for Food Protection

Growth of Aeromonas hydrophila in the Myzithra, , and during Storage at 4 and 12؇C

DEMETRIOS K. PAPAGEORGIOU,* DIMITRIOS S. MELAS, AMIN ABRAHIM, AND APOSTOLOS S. ANGELIDIS

Laboratory of Milk Hygiene and Technology, Department of Food Hygiene and Technology, School of Veterinary Medicine, Aristotle University of Thessaloniki, 54124 Thessaloniki,

MS 05-340: Received 15 July 2005/Accepted 19 September 2005

ABSTRACT Downloaded from http://meridian.allenpress.com/jfp/article-pdf/69/2/308/1677981/0362-028x-69_2_308.pdf by guest on 02 October 2021

The fresh whey cheeses Myzithra, Anthotyros, and Manouri were inoculated with Aeromonas hydrophila strain NTCC 8049 (type strain) or with an A. hydrophila strain isolated from food (food isolate) at levels of 3.0 to 5.0 ϫ 102 CFU/g of and stored at 4 or 12ЊC. Duplicate samples of cheeses were tested for levels of A. hydrophila and pH after up to 29 days of storage. At 4ЊC, A. hydrophila grew in Myzithra and Anthotyros with a generation time of ca. 19 h, but no growth was observed in Manouri. In Myzithra, average maximum populations of 8.87 log CFU/g (type strain) and 8.79 log CFU/g (food isolate) were recorded after 20 and 22 days of storage at 4ЊC, respectively. The average maximum populations observed in Anthotyros stored at 4ЊC were 6.72 log CFU/g (food isolate) and 6.13 log CFU/g (type strain) and were observed after 15 and 16 days of storage, respectively. A. hydrophila grew rapidly and reached high numbers in cheeses stored at 12ЊC. The average generation times were 3.7 and 3.9 h (Myzithra), 4.1 and 6.1 h (Anthotyros), and 8.0 and 9.2 h (Manouri) for the type strain and the food isolate, respectively. Among the different trials, the highest A. hydrophila population recorded (10.13 log CFU/g) was in Myzithra that had been inoculated with the food isolate after 8 days of storage at 12ЊC. To prevent A. hydrophila growth in whey cheeses, efforts must be focused on preventing postprocessing contamination and temperature abuse during transportation and storage.

Aeromonas spp. are gram-negative facultative anaero- in raw milk in Greece as well as in the Greek whey cheeses bic rod-shaped bacteria widely distributed in nature (20, 31, Anthotyros and Manouri (30). A high frequency of A. hy- 38). Aeromonas spp. are widespread in foods and the en- drophila isolation has also been reported from the Italian vironment and were recognized as pathogens of aquatic and whey cheese (45). Upon postprocessing contami- amphibian animals long before they were considered path- nation, these cheeses are susceptible to bacterial prolifera- ogenic for humans (3, 6, 19). Aeromonas spp. are com- tion because of their high moisture content (up to 70%) and monly isolated from water, fresh vegetables, and foods of their high pH (6.1 to 6.5). Therefore, the objective of this animal origin such as seafood, meat, raw milk, and cheese study was to determine the potential of growth for A. hy- (8, 11±13, 23, 25, 28, 30, 33, 39, 44, 45). Refrigeration of drophila in the whey cheeses Myzithra, Anthotyros, and these foods is not adequate for control of Aeromonas, as Manouri stored at 4 and 12ЊC. some of the strains isolated from foods can grow at refrig- eration temperatures and produce enterotoxins, cytotoxins, MATERIALS AND METHODS or hemolysins (5, 7, 18, 21, 22, 26, 41). Bacterial strains and preparation of inocula. Two A. hy- The taxonomy of Aeromonas spp. is an area of ongoing drophila strains were used in this study. One was the strain A. research (1, 15, 43) and, to date, there are 14 recognized hydrophila NTCC 8049, provided by the Irish National Food Cen- species within the genus Aeromonas (16). Only ®ve species ter (Qunsined, Dublin, Ireland), and the other was an A. hydro- are thought to be of clinical importance to humans, and of phila strain isolated from raw ewe's milk (food isolate) in our these, two species and one biotype of another species laboratory (30). Freeze-dried A. hydrophila cultures were regen- (Aeromonas hydrophila, Aeromonas caviae, and Aeromon- erated in brain heart infusion broth, which was incubated aero- bically at 28ЊC for 18 h. Intermediate cultures were then prepared as veronii biovar sobria) account for more than 85% of all by transferring the regenerated cultures to brain heart infusion clinical isolates (14, 15, 42). Gastroenteritis due to Aero- broth (28ЊC for 18 h), and working cultures were prepared by monas spp. is primarily a problem in young children, the transferring 0.3 ml of the intermediate culture into 200 ml of ster- elderly, and the immunocompromised (2, 10, 14, 19). ile skim milk in screw-top Erlenmeyer ¯asks, which were incu- A. hydrophila has been incriminated for 16.3% of 881 bated aerobically at 28ЊC for 18 h. For each trial, the appropriate cases of diarrhea in children in Greece between 1991 and volume of a working culture was added to the cheese to generate 1993 (24). Previous work in our laboratory has documented an initial inoculum of ca. 3.0 to 5.0 ϫ 102 CFU/g. the presence of A. hydrophila and other motile aeromonads Inoculation and storage of Myzithra, Anthotyros, and Manouri. All three types of whey cheese used in the experiments * Author for correspondence. Tel: ϩ30 2310 999806; Fax: ϩ30 2310 (Myzithra, Anthotyros, and Manouri) were manufactured in a 999803; E-mail: [email protected]. commercial dairy facility in central , Greece, by a pro- J. Food Prot., Vol. 69, No. 2 GROWTH OF A. HYDROPHILA IN WHEY CHEESES 309 cedure described elsewhere (36). For each of the three whey RESULTS AND DISCUSSION cheeses, eight experimental trials were performed (8 ϫ 3 ϭ 24 trials total). Four trials (two for each strain) were prepared and Composition of whey cheeses. The Greek regulatory stored at 4ЊC, and an additional four trials were prepared and standards for whey cheeses specify that the moisture con- stored at 12ЊC for up to 29 days. For each trial, ca. 3 kg of whey tent of Myzithra (standard version) and Anthotyros must cheese was inoculated. The inoculation was performed in a grad- not exceed 70% and that the fat in dry matter (FDM) of ual and sequential manner to ensure optimum distribution of A. these products must not be less than 50 and 65%, respec- hydrophila cells in the cheese: 0.5 kg of whey cheese was inoc- tively. Manouri must not contain more than 60% moisture ulated with a calculated volume of A. hydrophila culture and or less than 70% FDM. The addition of salt is allowed in mixed thoroughly for 3 min in a sterile 14-liter open-mouth round- these cheeses, and commercially produced whey cheeses in glass container. The metal stirring rods of the mixer had been also Greece usually contain 1 to 2% salt, but no standards are sterilized. A second 0.5-kg portion of whey cheese was next added in place to regulate its concentration. Myzithra can be found to the glass container and mixed thoroughly for 4 min; then, two in the market as a dry, salted cheese. However, it is also more 1-kg portions were added. After each 1-kg addition of

manufactured as a low-fat, fresh cheese, without the addi- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/69/2/308/1677981/0362-028x-69_2_308.pdf by guest on 02 October 2021 cheese, the mixture was thoroughly mixed for 5 min. The inoc- ulated cheese was distributed (in ca. 50-g portions) into each of tion of salt (17). Low-fat, salt-free, fresh Myzithra (as in 60 sterilized 90-g plastic urine-collection containers (Latex Inc., the experiments) is preferred by consumers because of con- Arnea, Greece). The containers' well-®tted plastic caps prevented temporary nutritional practices. The moisture content, moisture loss. Each ®lled container was sealed and kept refriger- FDM, SWP, and pH of the whey cheeses used in the ex- ated until the last container had been prepared (for each trial, the periments are presented in Table 1. The chemical compo- entire packaging process was completed within 30 min), and then sition of Manouri and Anthotyros cheeses used in this study the sealed containers were stored at 4 or 12ЊC. were in accordance with the Greek standards with respect to moisture and FDM. We purposely chose the low-fat, un- Sampling schedule and enumeration of A. hydrophila. Pri- salted version of Myzithra for our experiments to evaluate or to the inoculation of cheeses, duplicate 10-g cheese samples the response of A. hydrophila in a whey cheese that is sub- from all 24 trials were checked for the presence of A. hydrophila stantially different from the other two Greek whey cheeses by means of direct plating and cold enrichment (7ЊC for 72 h) as previously described (30, 34). The lower detection limit of direct in terms of its chemical composition. plating was 10 CFU/g (0.2 ml from the 10Ϫl dilution spread onto The average initial pH values of Myzithra, Anthotyros, each of ®ve plates), and that of cold enrichment was less than 1 and Manouri at the beginning of the experiments (time 0) CFU/10 g. From each container, duplicate samples were used for were 6.31, 6.56, and 6.10, respectively (Table 1). In the the enumeration of A. hydrophila and the determination of pH Myzithra trials, the pH of cheese remained relatively con- immediately postinoculation and thereafter at 24-h intervals (4ЊC stant throughout the experiments, with an overall minor de- trials) or at 12- to 24-h intervals (12ЊC trials). crease of up to 0.45 (Figs. 1A and 2A). A substantial pH For the enumeration of A. hydrophila, 10 g of cheese was decrease was noted in the Anthotyros trials, which was homogenized with 90 ml of sterile quarter-strength Ringer's so- more pronounced at 12ЊC (Figs. 1B and 2B). In the Man- lution in sterile 500-ml stomacher bags with a stomacher 400 ouri trials, the pH dropped considerably only when stored (Seward Medical, London, UK) for 3 min (initial 1:10 dilution). at 12ЊC and particularly when inoculated with strain NTCC Appropriate serial decimal dilutions were made in Ringer's solu- 8049 (Figs. 1C and 2C). tion, and 0.2 ml was spread onto starch ampicillin agar (35) and incubated aerobically at 28ЊC for 24 h. After incubation, starch Growth of A. hydrophila in whey cheeses at 4؇C. The ampicillin agar plates were ¯ooded with ca. 5 ml of Lugol iodine results of the preliminary microbiological analyses (with solution, and yellow-to-honey±colored, 2- to 4-mm-diameter, am- direct plating and cold enrichment) showed that all 24 sets ylase-positive colonies were counted (23, 34). Selected colonies of whey cheeses were free of Aeromonas (below the lower from plates inoculated from the highest countable dilutions (prior detection limit) prior to their inoculation. The growth of to the addition of Lugol solution) were con®rmed as A. hydrophila both strains of A. hydrophila in Myzithra and Anthotyros, as previously described (32, 37, 38). along with the changes in the pH of the cheeses during Chemical analyses of cheeses. For each trial, moisture, fat, storage at 4ЊC, is shown in Figure 1A and 1B. The lag salt, and pH determinations were performed in duplicate. Moisture phase of A. hydrophila was shorter in Myzithra than in and fat determinations were made according to the standard meth- Anthotyros, and in both cheeses, the lag phase of the food ods for the examination of dairy products (27). The pH of cheeses isolate was shorter than that of the type strain. In the My- was monitored with a pH meter (pH 320, WTN, Weiheim, Ger- zithra and Anthotyros trials, A. hydrophila grew with sim- many), and salt determinations were made by the Quantab chlo- ilar generation times of ca. 19 h. Maximum populations of ride titrator test strip method (Miles Laboratory, Inc., Elkhart, Myzithra (average Ϯ SD) of 8.87 Ϯ 0.06 log CFU/g (type ϭ Ind.). Salt in water phase (SWP) was calculated as % SWP (% strain) and 8.79 Ϯ 0.06 log CFU/g (food isolate) were re- ϫ ϩ salt 100)/(% salt % moisture). corded after 20 and 22 days of storage at 4ЊC, respectively. Calculation of growth characteristics. Growth curves were The maximum populations observed in Anthotyros were constructed to describe the growth of each strain in each cheese lower than those noted in Myzithra by more than 2 logÐ at each storage temperature. The lag phase and generation time of 6.72 Ϯ 0.04 log CFU/g (food isolate) and 6.13 Ϯ 0.30 log A. hydrophila under the different experimental conditions were CFU/g (type strain)Ðand were observed after 15 and 16 calculated by ®tting the growth data to the Gompertz equation as days of storage, respectively. In both cheeses, these elevat- described elsewhere (9). ed A. hydrophila populations remained relatively un- 310 PAPAGEORGIOU ET AL. J. Food Prot., Vol. 69, No. 2

TABLE 1. Moisture, fat in dry matter (FDM), salt in water phase (SWP), and pHa of Myzithra, Anthotyros, and Manouri cheeses at the start of the experiments Cheese Storage temp (ЊC) Strain % moisture (mean Ϯ SD) % FDM (mean Ϯ SD) % SWP (mean Ϯ SD)

Myzithra 4 NTCC 8049 67.7 17.02 Ðc 68.1 15.67 Ð FIb 68.4 15.82 Ð 68.3 17.35 Ð 12 NTCC 8049 67.8 17.08 Ð 68.7 15.97 Ð FI 69.1 16.18 Ð 67.9 15.57 Ð (68.25 Ϯ 0.48) (16.33 Ϯ 0.71) Anthotyros 4 NTCC 8049 66.9 66.46 2.01 66.5 65.67 2.02 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/69/2/308/1677981/0362-028x-69_2_308.pdf by guest on 02 October 2021 FI 67.2 65.54 1.74 67.4 65.95 1.99 12 NTCC 8049 66.2 65.08 1.77 65.8 64.32 2.29 FI 65.7 65.59 2.04 67.6 64.81 1.73 (66.66 Ϯ 0.72) (65.43 Ϯ 0.67) (1.95 Ϯ 0.19) Manouri 4 NTCC 8049 51.2 70.69 4.17 51.7 71.42 4.44 FI 52.1 70.98 4.10 50.8 71.13 4.21 12 NTCC 8049 51.6 70.24 3.84 52.2 71.12 4.10 FI 52.7 70.82 4.36 53.1 70.36 4.03 (51.93 Ϯ 0.76) (70.85 Ϯ 0.40) (4.16 Ϯ 0.19) a The initial pH values of whey cheeses (mean Ϯ SD) were 6.31 Ϯ 0.02 (Myzithara), 6.56 Ϯ 0.05 (Anthotyros), and 6.10 Ϯ 0.00 (Manouri). b FI, food isolate. c Myzithra was manufactured without the addition of salt. changed throughout the experiment (Fig. 1A and 1B). The at 12ЊC, the populations of strain NTCC 8049 began to two Aeromonas strains failed to grow in Manouri stored at decline and dropped to undetectable levels at day 28 of 4ЊC. After the inoculation of Manouri, the A. hydrophila storage (Fig. 2C). This difference in the fate of the two A. populations remained viable for ca. 5 days and subsequent- hydrophila strains in Manouri after 2 weeks of storage at ly began to decline, reaching undetectable levels after 10 12ЊC may be due to the different changes in the pH of the to 12 days of storage (Fig. 1C). cheeses in the corresponding trials. In the food-isolate trials, Growth of A. hydrophila in whey cheeses at 12؇C. the pH of the cheeses did not change substantially during The behavior of A. hydrophila was different in the three the course of the experiment, showing an overall decrease whey cheeses stored at 12ЊC when compared to storage at of ca. 0.6. In contrast, in the type-strain trials, although the 4ЊC. Both strains of A. hydrophila grew in Manouri at 12ЊC, pH was relatively unchanged (decrease of 0.3) during the with an average lag phase and generation time of 37.9 Ϯ ®rst 15 days of storage, it dropped by 1.4 during the sub- 5.9 h and 9.2 Ϯ 1.4 h (food isolate) and 28.2 Ϯ 5.8 h and sequent 13 days of storage, reaching a value of 4.38 at day 8.0 Ϯ 0.9 h (type strain), respectively (Table 2). After the 29 of the experiment. This drop in pH accompanied the ®rst week of storage, the growth pattern of the two strains observed 5.39-log CFU/g decline (from 5.39 log CFU/g to during the subsequent 7-day storage period changed: A. hy- undetectable levels) in the populations of A. hydrophila drophila NTCC 8049 entered a stationary phase, whereas NTCC 8049 (Fig. 2C). A. hydrophila is known to be fairly the food isolate continued to grow with a distinctly slower, sensitive to low pH (34). Previous work in our laboratory, but steady rate, resulting in a population increase of ca. 1 with rice pudding that was inoculated with the same strains log (from ca. 4.5 log CFU/g to ca. 5.5 log CFU/g) (Fig. of A. hydrophila and stored at 12ЊC, has shown that both 2C). The fate of the two strains was distinctly different after strains are fairly pH sensitive and that they become inac- that. The food isolate followed 2 days of intense growth tivated at pH values of 5.5 or below (37). Also, A. hydro- that resulted in an additional 1.2-log increase, reaching 6.7 phila is inactivated during the manufacture and ripening of log CFU/g, and then its population remained relatively un- cheese, as soon as the pH of Feta drops to 4.8 or below changed. In contrast, following the ®rst 15 days of storage (29). J. Food Prot., Vol. 69, No. 2 GROWTH OF A. HYDROPHILA IN WHEY CHEESES 311 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/69/2/308/1677981/0362-028x-69_2_308.pdf by guest on 02 October 2021

FIGURE 2. Growth of A. hydrophila and changes in pH in the whey cheeses Myzithra (A), Anthotyros (B), and Manouri (C) at 12ЊC. Data points represent the mean of two separate trials (open FIGURE 1. Growth of A. hydrophila and changes in pH in the symbols, pH; closed symbols, A. hydrophila; triangles represent whey cheeses Myzithra (A), Anthotyros (B), and Manouri (C) at trials with A. hydrophila NTCC 8049; squares represent trials 4ЊC. Data points represent the mean of two separate trials (open with the A. hydrophila food isolate). Values below 1 log CFU/g symbols, pH; closed symbols, A. hydrophila; triangles represent represent positive results obtained after cold enrichment, whereas trials with A. hydrophila NTCC 8049; squares represent trials values at 0 log CFU/g represent negative results obtained after with the A. hydrophila food isolate). Values below 1 log CFU/g cold enrichment. represent positive results obtained after cold enrichment, whereas values at 0 log CFU/g represent negative results obtained after strains (Table 2). In the Anthotyros trials, the maximum cold enrichment. populations reached by the two strains after 5.5 and 7.5 days of storage at 12ЊC were 7.16 Ϯ 0.13 log CFU/g (type Increasing the storage temperature of Anthotyros and strain) and 6.68 Ϯ 0.01 log CFU/g (food isolate), respec- Myzithra cheeses from 4 to 12ЊC caused a decrease in the tively. The A. hydrophila populations began to decline, ei- duration of lag phase (Table 2). Also, reductions (up to ther immediately after the maximum population was ®vefold) were observed in the generation times of both reached (type strain) or after entering a brief stationary 312 PAPAGEORGIOU ET AL. J. Food Prot., Vol. 69, No. 2

TABLE 2. Lag phase, generation time, and maximum populations (average Ϯ SD) of two Aeromonas hydrophila strains in the whey cheeses Myzithra, Anthotyros, and Manouri stored at 4 and 12ЊC Strain NTCC 8049 Food isolate

Storage Generation Maximum popula- Generation Maximum popula- Whey cheeses temp (ЊC) Lag phase (h) time (h) tions (log CFU/g) Lag phase (h) time (h) tions (log CFU/g)

Myzithra 4 73.2 Ϯ 0.8 18.3 Ϯ 0.7 8.87 Ϯ 0.06 36.0 Ϯ 12.9 19.8 Ϯ 0.8 8.79 Ϯ 0.06 12 13.2 Ϯ 4.1 3.7 Ϯ 0.1 9.95 Ϯ 0.00 0 3.9 Ϯ 0.1 10.13 Ϯ 0.02 Anthotyros 4 123.0 Ϯ 20.0 19.3 Ϯ 0.5 6.13 Ϯ 0.30 81.6 Ϯ 17.8 19.8 Ϯ 3.4 6.72 Ϯ 0.04 12 34.3 Ϯ 0.6 4.1 Ϯ 0.0 7.16 Ϯ 0.13 63.6 Ϯ 10.9 6.1 Ϯ 0.5 6.68 Ϯ 0.01 Manouri 4 NGa NG NG NG NG NG 12 28.2 Ϯ 5.8 8.0 Ϯ 0.9 5.39 Ϯ 0.20 37.9 Ϯ 5.9 9.2 Ϯ 1.4 6.65 Ϯ 0.23 a

NG, no growth. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/69/2/308/1677981/0362-028x-69_2_308.pdf by guest on 02 October 2021 phase (food isolate), and dropped to undetectable levels af- products can occur during their storage in domestic refrig- ter 17 days of storage at 12ЊC. The decline in the popula- erators. In a study by Sergelidis et al. (40), temperatures in tions of both strains of A. hydrophila began when the pH excess of 10ЊC were recorded in 25 and 13.6% of the do- of the cheeses dropped below 6.0 and continued to decrease mestic and retail refrigerators examined, respectively. Also, as the pH of the cheeses continued to drop (Fig. 2B). Unlike in a more recent survey in , 27 and 10% of the in the Anthotyros trials, no substantial reduction in the domestic refrigerators examined had temperatures ranging maximum numbers of A. hydrophila was observed in the from 8.1 to 10.0ЊC and 10.1 to 12.0ЊC, respectively (4). Myzithra trials until the end of the experiment. Maximum The storage temperature of 12ЊC in our experiments was populations of 9.95 Ϯ 0.00 log CFU/g (type strain) and therefore chosen to simulate improper storage temperature 10.13 Ϯ 0.02 log CFU/g (food isolate) were observed after conditions. Our study documents that A. hydrophila can 7 and 8 days of storage at 12ЊC, and these high levels of grow and reach high numbers in the whey cheeses Myzith- A. hydrophila persisted in the cheeses, with only a minor ra, Anthotyros, and Manouri stored at 12ЊC. reduction, until the end of the experiment. This may be At 4ЊC, growth of A. hydrophila was observed in the partly because, in the Myzithra trials, there was no sub- Anthotyros and Myzithra cheese trials, with maximum pop- stantial decrease in pH (Fig. 2A) and because no salt is ulations of up to 6.72 and 8.87 log CFU/g after 2 and 3 added during the manufacture of this product (Table 1). weeks of storage, respectively, but growth was not ob- Whey cheeses are traditionally produced in Greece served in Manouri. Among the three whey cheeses studied, with milk whey recovered during Feta cheese production. Manouri had the lowest percentage of moisture content Whey cheeses receive adequate heat treatment during the (51.93 Ϯ 0.76), the lowest initial pH (6.1), and the highest whey-protein denaturation step (82 to 92ЊC for 15 to 30 percentage of SWP (4.16 Ϯ 0.19). It is therefore likely that min); therefore, these cheeses are in excellent microbiolog- these factors acted synergistically with the low temperature ical status immediately following manufacture (17), as no of storage to inhibit the growth of A. hydrophila. known foodborne pathogen or spoilage microorganisms can In conclusion, the results of this work indicate that, if survive this heat treatment process, with the exception of present in unsalted low-fat Myzithra or in Anthotyros, A. heat-resistant bacterial spores. On the other hand, they are hydrophila can grow and reach very high numbers, even made without the addition of starter cultures, have a high under refrigerated storage. Growth was not observed in moisture content (up to 70%), and exhibit pH values be- Њ tween 6.0 and 6.5. Therefore, the high pH and high mois- Manouri stored at 4 C, but the microorganism showed am- Њ ture content render these products suitable substrates for ple growth when the cheese was stored at 12 C. The typical rapid growth of contaminating microorganisms, which, in shelf lives given by the manufacturers for whey cheeses are whey cheeses, can be lactic acid bacteria originating from ca. 10 days for fresh, unsalted Myzithra, ca. 40 days for the dairy facility environment as well as other pathogenic Anthotyros, and ca. 4 to 6 months for Manouri. With the Њ or nonpathogenic organisms (17). Indeed, such products exception of Manouri stored at 4 C, A. hydrophila grew to have been shown to support ample growth of the psychro- high numbers in all other whey cheese trials in our study trophic foodborne pathogen Listeria monocytogenes, even in a time frame that was within the products' intended re- under refrigerated storage (36). frigerated shelf life without resulting in any noticeable off- Following their manufacture in the dairy facility, whey odors or changes in the physical appearance of the cheeses. cheeses are usually packaged at 12 to 14ЊC, temperatures Even though the A. hydrophila mechanism (or mechanisms) that retard the multiplication of many bacteria but that, at of pathogenesis has not been fully elucidated and the in- the same time, are tolerable for the workers. Also, in fectious dose for humans remains unknown, every effort Greece, cheeses are occasionally subjected to temperature should be made to prevent the contamination of whey abuse during their transportation from the dairy factory to cheeses. Postpackaging pasteurization of these products the refrigerator shelves of the retail stores, particularly dur- may be a sensible solution to postprocessing contamination. ing the summer months. Further temperature abuse of these Also, the application of hazard analysis critical control J. Food Prot., Vol. 69, No. 2 GROWTH OF A. HYDROPHILA IN WHEY CHEESES 313 points in all stages of manufacture and distribution of whey microbiology: fundamentals and frontiers. ASM Press, Washington, cheeses will help ensure their safety for the public. D.C. 21. Kirov, S. M., E. K. Ardestani, and L. J. Hayward. 1993. The growth ACKNOWLEDGMENTS and expression of virulence factors at refrigeration temperature by Aeromonas strains isolated from foods. Int. J. Food Microbiol. 20: This research was supported by the General Secretariat of Research 159±168. and Technology of the Greek Ministry of Industry and Commerce and the 22. Kirov, S. M., and F. Brodribb. 1993. Exotoxin production by Aero- Aristotle University of Thessaloniki. The authors thank Dr. Konstantinos monas spp. in foods. Lett. Appl. Microbiol. 17:208±211. Koutsoumanis, Lecturer in the Department of Food Science and Technol- 23. Kirov, S. M., D. S. Hui, and L. J. Hayward. 1993. Milk as a potential ogy, Faculty of Agriculture in the Aristotle University of Thessaloniki, for source of Aeromonas gastrointestinal infection. J. Food Prot. 56: his help in calculating the kinetic parameters of growth, and ®nally, the 306±312. Macedonian Milk Industry, MEVGAL S.A., for providing the whey chees- 24. Kouppari, G., A. Za®ropoulou, N. Xenos, E. Papadomanolaki, and es for the experiments. B. Deligianni. 1994. Enteropathogenic microorganisms in children's diarrhea and their resistance to antibiotics, p. 137. Abstr. 16th Natl. REFERENCES Congr. Microbiol., Greek Society of Microbiology, Patra, Greece.

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