Isolation of Biofilm-Forming Staphylococci from the Bulk-Tank Milk of Small Ruminant Farms in Greece

Abstract

The objectives of this study were (i) to describe staphylococcal isolates recovered from bulk-tank raw milk collected from sheep and goat farms during a countrywide study performed in Greece, (ii) to study management factors potentially associated with their presence in bulk-tank milk and (iii) to provide evidence regarding their association with the quality of the milk. In total, 312 staphylococcal isolates, recovered from samples of bulk-tank raw milk from 444 small ruminant farms in Greece, were evaluated in this work. The in vitro formation of biofilm by the isolates was tested by combining the findings of (a) culture appearance on Congo Red agar plates and (b) results of a microplate adhesion test. The most frequently identified species was Staphylococcus aureus (75 isolates); other frequently recovered species were S. simulans (44 isolates), S. equorum (34 isolates) and S. haemolyticus (26 isolates); in total, 23 species were identified. In total, 224 (71.8%) isolates were biofilm-forming and were recovered from the bulk-tank milk samples of 148 sheep flocks (45.5%) and 55 goat herds (46.2%). There was evidence of seasonality in the isolation of staphylococci: during spring, mostly biofilm-forming isolates were recovered, whilst during summer, mostly non-biofilm-forming isolates were recovered. Among farms applying machine-milking, the proportion of farms from which biofilm-forming isolates were recovered was higher where water with temperature < 50 °C or ≥90 °C was used to clean the milking parlour. In the multivariable analyses, for farms applying machine-milking, the temperature of the water emerged as the only significant variable (p = 0.024), whilst in farms applying hand-milking, the only tendency that emerged was for the frequency of collection of milk from the farm tank (p = 0.08). In sheep flocks, recovery of biofilm-forming staphylococci from the bulk-tank milk was associated with higher somatic cell counts and higher total bacterial counts in the milk. The study identified abiotic factors related to the presence and isolation of these bacteria, specifically the temperature of water used for the cleaning of the milking parlour (in farms where machine-milking is applied) and the frequency of milk collection from the farm tank. These factors apply after the production of milk, and they could thus be regulated appropriately in order to reduce bacterial load and improve the quality of milk delivered to dairy plants. In sheep farms, an association was also seen between recovery of biofilm-forming staphylococci and high somatic cell counts in milk.

Keywords: Staphylococcus; biofilm; goat; mastitis; milk; sheep; slime; somatic cell counts.

Figure 1

Locations (red dots) of 444 small ruminant farms around Greece, in the 13 administrative regions of the country, which were visited during a countrywide investigation for bulk-tank milk sampling for recovery of staphylococcal isolates.

Figure 2

Location (black dots) of small ruminant farms (n = 203) around Greece, from the bulk-tank milk of which biofilm-forming staphylococcal isolates were recovered during a countrywide investigation.

Figure 3

Proportion of farms from which biofilm-forming staphylococcal isolates were (solid bars) or were not (pattern bars) recovered, in accord with the season of the year when the visit was made: during spring, isolation of biofilm-forming isolates from 54.3% of farms visited during that season, during summer, isolation of non-biofilm forming isolates from 62.4% of farms visited during that season.

Figure 4

Proportion of farms applying machine-milking, from which biofilm-forming staphylococcal isolates were recovered from bulk-tank milk (dashed line) and average number of isolates from these farms (solid line), in accord with the temperature of the water used to clean the milking parlour (proportion of farms from which biofilm-forming staphylococci were isolated according to temperature ranges <49 °C: 88.9%, 50–69 °C: 47.9%, 70–89 °C: 39.0%, ≥90 °C: 56.0%; median number of biofilm-forming staphylococci isolated per farm according to temperature ranges <49 °C: 1, 50–69 °C: 0, 70–89 °C: 0, ≥90 °C: 1).

Figure 5

Differences (%) in overall results of quality-related parameters (somatic cell counts, total bacterial counts, fat content, protein content, lactose content) of the bulk-tank in samples from which biofilm-forming staphylococci were isolated in comparison to samples from which biofilm-forming staphylococci were not isolated (p-values: for somatic cell counts 0.0002, for total bacterial counts 0.028, for fat content 0.29, for protein content 0.56, for lactose content 0.83).