In vitro antimicrobial activity of essential oils against enterotoxigenic Escherichia coli found in a nation-wide commercial farm survey

The antibacterial activity of some natural essential oils (EO) have the potential to reduce reliance on antibiotics to prevent post-weaning diarrhoea (PWD) caused by enterotoxigenic Escherichia coli (ETEC). However, a systematic screening of EO against the most common ETEC strains in Australia has never been established and is the main objective of the current research. We hypothesised that the sensitivity to the EO (alone or combined) by the selected ETEC strains prevalent in Australian piggeries was serotype-dependent or pathotype-dependent.

Ninety-one different E. coli isolates were collected from Victoria, New South Wales, Queensland, Western Australia and Southern Australia pig nurseries and characterised by the principal ETEC virulence genes (LT, Sta, Stb, stx₁, stx₂, eaeA, ehxA, F4, F5, F6 and F18) using multiplex PCR. An initial screening of 18 EO using a broth microdilution method against the two isolates with the most common ETEC serotypes (O:141ab and O:149) was done to find the minimum inhibitory concentrations (MIC). The EO with a MIC ≤0.1% for non-natives and ≤0.65% for native EO were selected and subjected to a chequerboard method to study potential synergies between EO combinations. A fractional inhibitory concentration index (FICI; defined as (MIC_EO1 in the combination/MIC_EO1) + (MIC_EO2 in the combination/MIC_EO2) of ≤0.5 was set as an indicator of synergistic activity in the EO combinations tested. Finally, the 91 isolates were tested against the most successful combinations of oils (Do et al. 2015).

Our results showed that the most common virulence gene was Stb present in 42 isolates followed by LT (33) and Sta (31). Twelve of the E. coli strains isolates had no ETEC virulence genes. Oregano, clove, thyme and cinnamon for non-natives and lemon myrtle, lemon ironbark, peppermint gum and nerolina for natives presented average MIC of 0.02%, 0.08%, 0.08%, 0.07% and 0.1%, 0.4%, 0.55%, 0.65%, respectively. The synergies between different EO combinations are shown in Table 1. Based on the MIC we observed resistance, low, medium, high, and very high sensitivities for 38.3%, 28.3%, 13.3%, 6.6% and 13.3% respectively. No relationship between sensitivity with serotypes and pathotypes was found.

Table 1.  Synergistic antimicrobial activity of essential oils in combinations against E. coli serotypes O:141 and O:149

In conclusion, our results confirmed that the virulence gene profile of ETEC found in Australian pig nurseries is similar to previous publications from elsewhere. Different ETEC serotypes showed different sensitivities to EO (Gutierrez et al. 2008). The non-native EO presented a lower MIC compared with native EO; however, the native EO had a better synergistic potential hypothetically due to a differential mode of action. Additional studies are warranted.