Antibacterial activity of polyunsaturated fatty acids related to chronic wound infections

Eshlak, MS

Abstract

Wound infections present major health problems worldwide. It has been revealed that
bacterial biofilm communities frequently colonise chronic wounds.Biofilms with Multidrugresistant organisms (MDRO) such as Methicillin Resistant Staphylococcus aureus (MRSA)
and Pseudomonas aeruginosa are increasingly implicated in chronic wound infections. The
limited therapeutic options are further compromised by the fact that wound-infecting bacteria
often co-exist within a polymicrobial biofilm community, which enhances bacterial tolerance
or resistance to antimicrobials. The polyunsaturated fatty acid (PUFA) compounds including
Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) offer both
immunomodulatory and antimicrobial properties that could enhance wound healing.

This study evaluated the activity and efficiency of these compounds against wound
pathogens and compared them to common antibiotics Linezolid (LZD) and vancomycin
(VAN). Standard antibiotic sensitivity assays were used to determine the ability of PUFAs to
inhibit growth and biofilm formation. Most activity was observed against S. aureus strains.
The bacteriostatic activity (minimum inhibitory concentration - MIC) of DHA and EPA for S.
Aureus SA3 and P. aeruginosa PA01 was 65 and 117 μgml-1
respectively. Meanwhile, the bacteriocidal activity required higher concentrations (MBC) of 260 and 468 μgml-1
respectively. The antibiotic LZD was most active against Gram positive bacteria (MIC range
from 1.9 µg ml-1 to 3.9 µg ml-1). However, vancomycin was the most potent biofilm disrupter, with MBECs ranging from 5-12.5 µg ml-1. These concentrations were 35-fold lower than the MBECs recorded for linezolid (125 -500 µg ml-1). Considerably higher concentrations of
PUFA compounds were required to disrupt biofilms. The MBECs of DHA ranged between
750 and 3125 µg ml-1. EPA was found to be more disruptive than DHA (MBECs ranging
between 500 and 2250 µg ml-1), while the MBECs of combination treatment with both
PUFAs (DHA+EPA) ranged from 750 - 1500 µg ml-1.

The antimicrobial activity of different concentrations of PUFAs was further
Assessed by monitoring inhibition of growth of a clinical wound isolate of S. aureus (SA3)for
24 h. PUFAs were shown to differentially affect expression of global gene regulators using
RT-qPCR. DHA and EPA were found to upregulate agrA and icaA but downregulate sarA at
the same concentrations and exposure times.

Two different infection models were used to assess the activity of PUFAs in vivo (Galleria mellonella) and on ex-vivo skin compared to common antibiotics (LZD and
VAN). PUFAs reduced virulence of SA3 in G.mellonella, protecting the larvae from death.
Furthermore, prolonged exposure to sub-inhibitory concentrations affected growth
Efficiency of SA3 in vivo. Whole genome sequencing of parent vs PUFA and antibiotics adapted strains of S. aureus SA3 revealed some common mutations in sarA and clfB genes, that
may contribute to altered virulence and susceptibility to treatment. Preliminary studies with
the ex-vivo skin model suggested that this may be a useful system for testing the efficacy of
antimicrobial compounds for wound-treatment. Differences in SA3 survival and growth were
measurable in the presence of PUFAs or absence of PUFAs.

In conclusion, PUFAs (DHA and EPA) showed weak antimicrobial activity
compared to common antibiotics against common wound-associated bacteria. However, this
study shows evidence that PUFAs have additional desirable properties, including disruption
of global gene regulation, reduction of virulence and low potential to promote development
of resistance.

Citation

Eshlak, M. (in press). Antibacterial activity of polyunsaturated fatty acids related to chronic wound infections. (Thesis). University of Salford

• Thesis Type: Thesis
• Acceptance Date: Apr 30, 2019
• Deposit Date: May 7, 2019
• Publicly Available Date: May 7, 2019