N2 Gas Flushing Limits the Rise of Antibiotic-Resistant Bacteria in Bovine Raw Milk during Cold Storage

Abstract

Antibiotic resistance has been noted to be a major and increasing human health issue. Cold storage of raw milk promotes the thriving of psychrotrophic/psychrotolerant bacteria, which are well known for their ability to produce enzymes that are frequently heat stable. However, these bacteria also carry antibiotic resistance (AR) features. In places, where no cold chain facilities are available and despite existing recommendations numerous adulterants, including antibiotics, are added to raw milk. Previously, N₂ gas flushing showed real potential for hindering bacterial growth in raw milk at a storage temperature ranging from 6 to 25°C. Here, the ability of N₂ gas (N) to tackle antibiotic- resistant bacteria was tested and compared to that of the activated lactoperoxidase system (HT) for three raw milk samples that were stored at 6°C for 7 days. To that end, the mesophiles and psychrotrophs that were resistant to gentamycin (G), ceftazidime (Ce), levofloxacin (L), and trimethoprim-sulfamethoxazole (TS) were enumerated. For the log₁₀ ratio (which is defined as the bacterial counts from a certain condition divided by the counts on the corresponding control), classical Analyses of Variance (ANOVA) was performed, followed by a mean comparison with the Ryan-Einot-Gabriel-Welsch multiple range test (REGWQ). If the storage "time" factor was the major determinant of the recorded effects, cold storage alone or in combination with HT or with N promoted a sample-dependent response in consideration of the AR levels. The efficiency of N in limiting the increase in AR was highest for fresh raw milk and was judged to be equivalent to that of HT for one sample and superior to that of HT for the two other samples; moreover, compared to HT, N seemed to favor a more diverse community at 6°C that was less heavily loaded with antibiotic multi-resistance features. Our results imply that N₂ gas flushing could strengthen cold storage of raw milk by tackling the bacterial spoilage potential while simultaneously hindering the increase of bacteria carrying antibiotic resistance/multi-resistance features.

Keywords: N2 gas; Ryan-Einot-Gabriel-Welsch test; antibiotic resistance (AR); cold storage; lactoperoxidase system; raw milk.

Figure 1

Mesophilic (M) and psychrotrophic (P) bacterial counts (expressed in log cfu/ml) from raw milk samples S1, S2, and S3 that were stored for 7 days at 6°C (C), cold-stored while the lactoperoxidase system was activated (HT) or cold-stored while flushed with N₂(N). The colonies were enumerated on Mueller-Hinton agar plates that contained no antibiotics (“no AB”) or one of the following ABs: G (gentamycin), Ce (ceftazidime), L (levofloxacin) and TS (trimethoprim-sulfamethoxazole) (the error bars correspond to standard deviations).

Figure 2

REGWQ results depicting the impact of either 3 or 7 days of storage at 6°C on mesophilic (M) and psychrotrophic (P) populations that were recovered from the raw milk samples S1, S2, and S3. Means with different letters indicate significant differences (alpha risk = 0.05).

Figure 3

REGWQ results describing the overall inhibitory efficiency of HT (the activated lactoperoxidase system) and N (N₂ gas flushing) treatments, compared to the control (C) for the raw milk samples S1, S2, and S3 when stored at 6°C for 7 days. Means with the same letter are not significantly different (alpha risk = 0.05).

Figure 4

REGWQ results showing the impact of the HT (the activated lactoperoxidase system) and N (N₂ flushed milk) treatments, compared to the control (C), on the three raw milk samples S1, S2, and S3 when considering the mesophiles (S1M, S2M, and S3M) and the psychrotrophs (S1P, S2P, and S3P) for the “no AB” condition, and in the presence of the antibiotics G (gentamycin), Ce (ceftazidime), L (levofloxacin), and TS (trimethoprim-sulfamethoxazole).