Temperature abuse and Salmonella Typhimurium colonization disrupt the indigenous bacterial communities of pasteurized bovine milk over time

Abstract

Even though the population structure of the bovine milk residential bacterial population is known, the alterations in the population structure associated with food safety issues, such as temperature abuse/pathogen colonization, are unknown. Here, alterations of the bacterial population, either incubated at 37 °C (temperature abuse) or inoculated with Salmonella Typhimurium (pathogen colonization), were characterized using full-length 16S rRNA sequencing. At zero hour, the bacterial population of milk primarily constituted of Firmicutes and Thermi. Of the 218 genera identified, Thermus (37%) and Streptococcus (34%) were the most dominant. The 12-h incubation at 37 °C replaced almost 96% of the population by Firmicutes, exemplified by a remarkable increase in the abundance of the genus Bacillus. Concurrently, only 36 genera survived, with an abundance of Bacillus, which showed a 98-fold increase during the 12-h incubation. Similarly, only 150 genera remained after 12 h in Salmonella-inoculated milk. Both temperature abuse and Salmonella inoculation significantly reduce bacterial diversity and richness. Nonmetric multidimensional scaling analysis between the control and Salmonella inoculated samples was significantly distinct at all times, confirming alterations in the bacterial population during Salmonella colonization. Even though the load of Firmicutes increased temporally, bacteria belonging to the genera Bacillus, Macrococcus, and Serratia genera were depleted in Salmonella inoculated milk samples. Taken together, both experimental conditions, viz. temperature abuse and Salmonella contamination, demonstrated a significant drop in residential milk bacterial diversity. This general drop in bacterial diversity could allow Salmonella to occupy and colonize the milk matrix.

Keywords: Salmonella Typhimurium; 16S rRNA amplicon sequencing; Bacterial community; Bovine milk; Milk microbiology.

Fig. 1

Relative abundances of the bacterial taxa (at Phylum level) of Control and Inoculated milk samples at different time points.

Fig. 2

Relative abundances of the top 30 families of bacterial taxa present in Control and Inoculated milk samples at different time points.

Fig. 3

Heatmap depicting the top 20 genera of bacteria found in Control and Inoculated milk samples at different time points. The intensity of the colors in the heatmap indicates a relatively higher or lower abundance (scale 0–0.8).

Fig. 4

Effect of temperature abuse-37 °C (red colored) and Salmonella inoculation (blue colored) over time on the bacterial alpha diversity of pasteurized bovine milk depicted by (A) bacterial richness; (B) Shannon diversity index; (C) Pielou’s evenness index. Statistical inference is highlighted within each panel of the plot and assessed using ANOVA analyses followed by Tukey’s HSD post hoc test (P < 0.001). The box spans the interquartile range (IQR; first quartile to the third) with the median indicated by a dark horizontal line, the whiskers show the 1.5xIQR. Significant differences are indicated by the presence of different lowercase letters above each box plot.

Fig. 5

The bacterial community structure in the NMDS ordination for Control and Inoculated milk samples at different time points. The significant effect on the ordination is given by P < 0.001. Circles indicate Control milk samples and triangles indicate Inoculated milk samples. Different time points are represented by respective colors.

Fig. 6

Linear discriminant analysis effective size (LEfSe) bar plot (LDA > 3) showing enriched taxa in Control and Inoculated milk samples.