Behaviour of Escherichia coli O157:H7 and Listeria monocytogenes in Normal and DFD Beef of an Autochthonous Portuguese Breed

This study was carried out to identify the behaviour of Escherichia coli O157:H7 and of Listeria monocytogenes inoculated in Maronesa breed beef with different ultimate pH (pHu) (Normal and DFD), and stored at two different temperatures (4 and 9 °C), during 28 days post mortem (pm). The main objective was to illustrate the problematic feature of dealing with beef showing high pHu and stored at mild abusive temperatures (9 °C). Beef steaks (ms. longissimus dorsi) were inoculated with low levels (2–3 log CFU/g) of those both pathogens and packed in air, vacuum and three gaseous mixtures with decreasing O2 and increasing CO2 concentrations (MAP70/20, MAP50/40 and MAP30/60). At 4 °C, the growth of E. coli O157:H7 presented the same pattern on Normal and DFD meat. On the contrary, the growth of L. monocytogenes was higher in DFD meat, revealing the effect of the pHu and its psychotropic character. At abusive temperatures, both pathogens grew, achieving high levels in DFD meat. In these cases, the MAP with the highest CO2 concentration (60%) was revealed to be more effective against the development of E. coli O157:H7, therefore, not exceeding levels of 5 log CFU/g at the end of storage, while in L. monocytogenes, it reaches 8 log CFU/g under the same conditions.


Introduction
Beef from the Maronesa cattle has high commercial value due to its autochthonous breed, environmentally sustainable production system, and it has Protected Designation of Origin (PDO) [1,2]. However, in order to exploit the full potential of meat products, it is important to ensure that products reach the consumer in the best condition [3]. The quality of the meat can be affected by intrinsic and extrinsic parameters, such as age, sex, breed [4], feeding [5], enzyme activity [6], meat-modified atmosphere packaging (MAP) [7], or meat contamination [8]. The hygienic quality, which is a result of beef processing and handling, is particularly important because it has direct implications for consumer confidence [9]. Contamination of beef occurs throughout slaughtering, deboning, cutting, packaging, and storage, during which time temperature and hygiene are important parameters affecting the shelf-life of fresh meat for industrial use or retail [10,11].
Microbial pathogens are usually associated with the most serious meat safety issues in product recalls and foodborne diseases [12][13][14]. Nowadays, besides the considerable knowledge about the microbiota responsible for foodborne diseases, a high number of outbreaks and incidents keep occurring, many of them associated with meat and meat divided into two pH groups: Normal (pH ≤ 5.8, n = 4) and DFD (pH ≥ 6.2, n = 4). After that, muscles were cut into pieces of approximately 200 g, packed in a vacuum and stored at −80 • C until the beginning of the experiment. On day 1 of the experiment, cuts of muscles were kept at 2 • C for 2 h. After this time, approximately 1 cm of the external surface of the meat was aseptically removed, and cuts were sliced. At the end of this process, pieces of meat (0.5 cm thick, surface 2 × 2.5 cm, ≈5 g) were obtained. Immediately after this preparation, meat samples were analysed (24 h pm) for meat characterisation and discarded L. monocytogenes and E. coli O157:H7 contamination prior to inoculation. Experiments were performed using four animals in each experimental unit per each pH group, and a control sample was prepared in all conditions of the experimental design.

Microorganisms and Growth Conditions
Pieces of meat were inoculated with 20 µL of a suspension of E. coli O157:H7 (NCTC 12900) and L. monocytogenes (ATCC 7973) for an inoculation level of 2-3 log (CFU/g) per strain and package.
In air packaging, meat pieces were tray-packaged in air overwrapped with polyethylene film and in a vacuum: they were individually vacuum packaged in COMBITHERM bags (WIPAK Walsrode, HAFRI) which have an oxygen transmission rate (OTR) of 63 cm 3 m −2 d −1 atm −1 at 23 • C, 0% RH and water vapour transmission (WVT) of 1 g m −2 d −1 at 23 • C, 85% RH. For MAP, pieces of meat were individually placed in COM-BITHERM XX bags (WIPAK Walsrode, HAFRI) 0.115 mm thick and OTR of 1 cm 3 m −2 d −1 atm −1 at 23 • C, 0% RH and WVT of 1 g m −2 d −1 at 23 • C, 85% RH. The atmosphere in the MAP was first removed and then flushed with the appropriate gas mixture (Praxair, Portugal) using a SAMMIC V-420 SGA. The final ratio between gas and meat was approximately 3:1.
Samples were stored at 4 ± 0.5 • C and 9 ± 0.5 • C and examined for microbiological counts on days 1 (2 h after packaging), 3, 7, 10, and 14 pm. For air packaging, microbiological counts were not carried out at 14 days pm. On the contrary, on vacuum packaging, microbiological counts were also carried out at 21 and 28 days pm.

Microbial Analysis
Meat pieces were aseptically collected at each interval. Samples were homogenized with sterile buffered peptone water for 90 s in a Stomacher (IUL, Barcelona, Spain). Serial decimal dilutions were prepared in the same solution and were plated on CT-SMAC (Biokar BK147 + BS037) for E. coli O157:H7 counts (37 • C for 24 h) and Compass L. mono agar (Biokar BM06508) in case of L. monocytogenes (37 • C for 24-48 h). After incubation, typical colonies were counted, and results were expressed in log CFU/g.

Data Analysis
One-way analysis of variance (ANOVA) was conducted to test the effect of pHu (Normal and DFD) and of the temperature of storage (4 ± 0.5 • C and 9 ± 0.5 • C), for each

Behaviour of Escherichia coli O157:H7
The results of counts of E. coli O157:H7 in inoculated beef samples, according to pHu (Normal and DFD) and the type of packaging for each storage temperature, are presented in Table 1. Two hours after inoculation, the Normal and DFD samples had a similar count for E. coli O157:H7. These counts were slightly greater than the programmed level of inoculation, though this was a very slight excess and never attained a logarithmic unit. The final pH of the meat was not decisive on the growth of E. coli O157:H7 in beef stored at 4 ± 0.5 • C. The highest E. coli O157:H7 counts for DFD meat and temperature of 9 • C were observed in vacuum packaging for all storage times (4.39 ± 0.65 log CFU/g at 3 days pm; 6.57 ± 0.26 log CFU/g at 7 days pm; 7.77 ± 0.32 log CFU/g at 10 days pm and 8.35 ± 0.21 log CFU/g at 21 days pm). However, no significant differences were observed between Normal and DFD pHu at 3 days pm. At 10 days pm, the largest difference between the DFD and Normal meat was observed in the vacuum atmosphere (4.05 log CFU/g; p < 0.001) followed by the air atmosphere (3.61 log CFU/g; p < 0.01). In the MAP70/20, differences between the DFD and Normal meat reached 3.04 log CFU/g (p < 0.001). The MAP packaging with more concentration of CO 2 , namely MAP30/60, showed the lowest counts with no significant differences between pH groups at 10 and 14 days pm.
The counts of E. coli O157:H7 on Normal and DFD beef were similar at a temperature of 4 • C during the storage period, maintaining the initial levels. However, at 9 • C, growth levels were achieved for DFD meat of~8 log CFU/g in vacuum from day 10 pm, reaching the highest value of 8.76 ± 0.64 log CFU/g at day 28 pm. In the MAP with the high CO 2 concentration (MAP30/60), counts were not higher than 5 log CFU/g, which appears to reveal the inhibitory effect of CO 2 in the development of E. coli O157:H7 ( Figure 1). The pathogen E. coli O157:H7 packed in MAP70/40 and MAP30/60 and stored at mild abusive temperature (9 • C) followed the same pattern and showed similar counts over time.

Behaviour of Listeria Monocytogenes
The results of counts of L. monocytogenes in inoculated beef samples, according to pHu (Normal and DFD) and the type of packaging for each storage temperature, are presented in Table 2. At 1 day pm, in the five packagings, DFD and Normal samples had a similar count for L. monocytogenes with a score closer to that desired in all packaging. Two more days after storage, the consequences of temperature started to show that L. monocytogenes increased considerably in DFD compared to Normal meat, and these differences were more pronounced, particularly in MAP70/20 compared to the other two MAP. L. monocytogenes achieved at 3 days pm and 7 days pm average counts of 3.41 ± 0.92 log CFU/g and 6.49 ± 0.83 log CFU/g in DFD meat and 2.61 ± 0.65 log CFU/g and 1.74 ± 0.76 log CFU/g in Normal meat, which generally maintained the same pattern during the remaining storage time.
In Normal pH, the effect of abusive storage temperature was not noticed in the development of L. monocytogenes, contrarily to DFD meat which presented very significant differences in most situations in which the meat was in abusive temperatures.
The MAP30/60, with the higher content of CO 2, seems to inhibit this microorganism a little more, showing inferior counts for all storage times when compared with MAP70/20 and MAP50/40.
At a temperature of storage of 4 • C, the growth of L. monocytogenes was not observed for Normal meat. On DFD meat, the counts achieved levels of 6 log CFU/g, 7 log CFU/g and 8 log CFU/g in vacuum at day 14 pm, 21 pm and 28 pm, respectively. The lowest counts were obtained on meat packaged in the MAP30/60 for a long time.
At abusive temperature (9 • C) and Normal pHu, L. monocytogenes presented similar counts during the storage period. On the contrary, for DFD meat, L. monocytogenes devel-Foods 2023, 12, 1420 5 of 12 oped very quickly after day 3 pm, achieving values higher than 8 log CFU/g in air and vacuum at day 10 pm. These levels were attained in MAPs later at day 14 pm. ds 2023, 12, x FOR PEER REVIEW reveal the inhibitory effect of CO2 in the development of E. coli O157 pathogen E. coli O157:H7 packed in MAP70/40 and MAP30/60 and sto temperature (9 °C) followed the same pattern and showed similar cou

Behaviour of Listeria Monocytogenes
The results of counts of L. monocytogenes in inoculated beef sam pHu (Normal and DFD) and the type of packaging for each storag presented in Table 2. At 1 day pm, in the five packagings, DFD and N

Discussion
The pHu and storage temperature significantly influenced the growth of both E. coli O157:H7 and L. monocytogenes.
At 4 • C of storage, the counts of L. monocytogenes for all packages in DFD beef were below 5 log CFU/g (10 days pm), with the higher counts observed in the air (4.47 log CFU/g; p < 0.05) and vacuum (4.55 log CFU/g; p < 0.05) packages (Table 2). On the contrary, at 4 • C the counts of E. coli H157:H7 were about 3 log CFU/g during all times of storage (3, 7, 10, 14, 21 and 28 days pm) in all types of packaging, showing no significant differences (Table 1). Thus, the low storage temperature (4 • C) associated with the DFD condition was not enough to produce an extensive inhibition of L. monocytogenes as observed in E. coli O157:H7. These results can be justified by the fact that L. monocytogenes is a psychrotrophic bacterium, capable of surviving and multiplying at low temperatures, both under aerobic and anaerobic conditions and adhering to various surfaces [36]. Moreover, L. monocytogenes has the ability to grow at a pH of 6 or higher, as observed in DFD meat [37] and has a high tolerance to low pH and high salt concentration [38]. According to Nissen et al. [39], at 4 • C, L. monocytogenes and Y. enterocolitica are considered the most serious pathogens in meat. Low temperatures induce enzymes such as RNA helicase, which improves the activity of L. monocytogenes, as well as replication at low temperatures. Moreover, the capacity to produce biofilms enhances L. monocytogenes' ability to survive harsh environments and also use flagella at lower temperatures which enables the ability to propel itself [40]. Elevated CO 2 and reduced O 2 levels are commonly used to extend the shelf-life of food products through the inhibition of microbial growth and oxidative changes [41]. The use of O 2 -free atmospheres in packages has been suggested for different meat products [42]. In the present study, MAPs inhibited, compared to the air and vacuum atmospheres, the development of L. monocytogenes mainly in the MAP with the highest concentration of CO 2 . The lower OTR (1.0 cm 3 m −2 day −1 ) of the packaging film used in this experiment for MAP, in comparison to films of greater O 2 permeability (OTR = 4.5 cm 3 m 2 day −1 ) used in the study conducted by Tsigarida et al. [43] can justify these results. Saraiva et al. [41], using the same packaging film as the present study, found that L. monocytogenes in beef may be reduced by~1.0 log in vacuum packaging and by~1.5 log on average in the MAPs. Generally, under an anaerobicmodified atmosphere, the LAB compete with the support microflora and have shown to be effective in inhibiting the growth of pathogenic bacteria such as L. monocytogenes in meat products [44]. The combination of selected LAB strains with antimicrobial compounds, for instance, acid and sodium lactate or the use of active packaging, could be the next step strategy for eliminating the risk of L. monocytogenes in meat and dairy-ripened products [45]. Many food-spoiling LABs are facultative aerobic and quite resistant to CO 2 . This contributes to the fact that LABs can be found as the main spoilers on high-oxygen packaged meat [41]. Therefore, L. monocytogenes can represent a risk when stored at temperatures higher than 0 • C, and the efficacy of thermal treatments is limited by the ability to survive and actively replicate at temperatures between −0.4 and 45 • C [46]. Air and vacuum atmospheres had similar counts for this pathogen (Figure 2), which could be related to the O 2 permeability rate of the packaging film used for the vacuum atmosphere (OTR = 63 cm 3 m −2 day −1 ), not so efficient in inhibiting the L. monocytogenes [41].
A higher proportion of CO 2 in MAP50/40 and even more in MAP30/60 have led to lower growth of L. monocytogenes compared to other types of packaging. This result is consistent with other research whose results confirmed that the growth of L. monocytogenes was inhibited with increasing concentrations of CO 2 [39,47]. According to Saraiva et al., [41] L. monocytogenes requires CO 2 levels of 40% v/v or higher for effective inhibition. In view of maintaining the cytoplasmic pH within a range that is consistent with growth and survival, the decarboxylation reaction needs to occur to help maintain the cytoplasmic pH, though a high concentration of CO 2 can inhibit the decarboxylation reaction by which CO 2 is released through feedback mechanisms [41,48].
is limited by the ability to survive and actively replicate at temperatures between −0.4 an 45 °C [46]. Air and vacuum atmospheres had similar counts for this pathogen (Figure 2 which could be related to the O2 permeability rate of the packaging film used for th vacuum atmosphere (OTR = 63 cm 3 m −2 day −1 ), not so efficient in inhibiting the monocytogenes [41]. A higher proportion of CO2 in MAP50/40 and even more in MAP30/60 have led t lower growth of L. monocytogenes compared to other types of packaging. This result consistent with other research whose results confirmed that the growth of L. monocytogen was inhibited with increasing concentrations of CO2 [39,47]. According to Saraiva et a [41] L. monocytogenes requires CO2 levels of 40% v/v or higher for effective inhibition. I view of maintaining the cytoplasmic pH within a range that is consistent with growth an At a temperature of storage of 4 • C, the counts of E. coli O157:H7 on Normal and DFD, as well as for abusive temperature on Normal beef, were similar during the storage period, maintaining the initial levels ( Figure 1). Regarding DFD meat and temperature of storage of 9 • C, the E. coli O157:H7 achieved the highest growth levels in a vacuum atmosphere (~8 log CFU/g), showing statist differences from Normal meat (p < 0.001) and from a temperature of storage of 4 • C (p < 0.001) at 7, 10, 14, 21 and 28 days pm.
The pathogen E. coli O157:H7 in air packaging multiplied rapidly; however, MAP30/60 showed lower growth levels during the storage period. In this study, this was the atmosphere with high CO 2 content (60%), and E. coli O157:H7 counts did not attain 5 log CFU/g at 14 days pm. These results can be due to the inhibitory effect of CO 2 . In previous experiments, E. coli O157:H7 in atmospheres with high concentrations of CO 2 (ratio of 30%CO 2 :70%O 2 or 0.4%CO:60%CO 2 :39.6%N 2 ) have been reported as being inhibitory to the multiplication of E. coli O157:H7 at an abusive storage temperature of 10 • C [38]. As mentioned above, for L. monocytogenes, E. coli uses the same mechanism of decarboxylation systems to protect the cell from a precipitous drop in pH. These systems depend on the activity of cytoplasmic pyridoxal-5 -phosphate (PLP)-containing amino acid decarboxylases, which consume one proton and release one CO 2 for every molecule of substrate amino acid, thus helping maintain the cytoplasmic pH [49]. At refrigeration temperatures from 0 to 2 • C, high concentrations of CO 2 (ratio of 20%CO 2 :80%O 2 or 0.4%CO:30%CO 2 :69.6%N 2 ) can lead to a reduction of one logarithmic unit in the levels from E. coli O157:H7 [50]. In accordance with the present study, Nissen et al. [39] reported that E. coli O157:H7 could be inhibited at 10 • C at high CO 2 concentration and pHu inferior to 6. Moreover, even in MAP with a high CO 2 and low CO mixture, meat acquires a stable colour, and the shelf-life can be extended.
The behaviour of L. monocytogenes in abusive temperatures differed between DFD and Normal pHu meats for all types of packaging after 7 days pm. For instance, at 10 day pm, Normal and DFD meat showed, respectively, counts of 2.07 log CFU/g and 8.25 log CFU/g in the air (p < 0.001); 2.54 log CFU/g and 8.24 log CFU/g (p < 0.001) on Vacuum; 1.69 log CFU/g and 7.91 log CFU/g in MAP70/20 (p < 0.001); 1.37 log CFU/g and 7.79 log CFU/g in MAP50/40 (p < 0.001); and 1.42 log CFU/g and 7.01 log CFU/g in MAP30/60 (p < 0.001). However, regarding the stored temperatures, for instance, at 14 day pm, count obtained at 4 • C and 9 • C of storage for DFD meat were respectively 5.71 log CFU/g and 8.33 log CFU/g (p < 0.001) in Vacuum; 4.82 log CFU/g and 8.55 log CFU/g in MAP70/20 (p < 0.001); and 4.07 log CFU/g and 8.53 log CFU/g in MAP50/40 (p < 0.001). Thus, even with meat stored at temperatures within recommended limits, the development of L. monocytogenes occurred markedly in meat with a high pHu, though when comparing both temperatures, the effect was not as intense as was the effect of pHu. However, unlike E. coli O157:H7, the effect of pH was not felt when storage was carried out at the appropriate temperature. Also, in this pathogen, at 9 ± 0.5 • C of storage, significantly greater growth was noted in the DFD meat samples.

Conclusions
The present study highlights the importance of maintaining the cold chain under strict surveillance conditions is confirmed to avoid abuses, which can have even more serious consequences in the case of DFD meat. As referred to above, the occurrence of DFD condition was more frequently observed in the L. dorsi muscle of the Maronesa breed, mainly in males. The results revealed that this condition associated with abusive storage temperatures allowed the growth of E. coli O157:H7 at higher levels, which is especially more evident in air and vacuum packages. For L. monocytogenes, the low storage temperature (4 • C) associated with the DFD condition was not enough to inhibit the growth of L. monocytogenes, as observed in E. coli O157:H7. This is due to the characteristic psychotropic of L. monocytogenes.
The use of special packing conditions, such as vacuum or MAP, in order to increase the shelf-life of meat or for technological and sensorial reasons can have consequences in terms of the development of some of the pathogens present in meat. Overall, the MAP were the most effective in controlling the development of E. coli O157:H7 and L. monocytogenes. In MAP, the effect of the CO 2 level was observed, being even more noticeable in pH that better supported microbial growth.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.