Staph test: Profiling bacterial infection in wounds

Infection spreading

Image: ARS/USDA

The bacterium Staphylococcus aureus lives on human skin as well as other regions like the nasal passages and is responsible for many minor ailments like pimples, boils and carbuncles. However, most of us will recognise this organism as being the cause of many serious infections, especially since the emergence of antibiotic-resistant strains which seem to take hold in hospitals.

Staph, as it is commonly known, is also one of the most common sources of infections in wounds with the potential for serious, if not fatal, consequences. But despite its prevalence, scientists know very little of the nature of the infection process at the wound site. How does the bacterium take hold and what changes occur as a result?

Now, a team of scientists based in Taiwan have investigated the growth of Staph in wounds in mice over a period of time. Hui-Fen Wu, Jayaram Lakshmaiah Narayana and Judy Gopal from the National Sun Yat-Sen University employed the novel step of taking swabs from the wound site for analysis by mass spectrometry in order to map the molecular changes that evolved.

Wound testing

Two mice were given a small incision on their backs which was infected with a suspension of Staphylococcus aureus. A third mouse was cut but not infected for use as a control. Sterile swabs were used to take samples from the wound site immediately after the incision, then at intervals over the next two weeks.

All of the samples were analysed by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI MS) to identify the compounds at the site that were affected by the infection. They were supported by bacterial counting using the standard agar plate method.

A sample collected two weeks after infection was also analysed in two ways to distinguish between compounds originating from the mouse response to infection and from the bacteria. One part was analysed directly by MALDI MS while the other was incubated for 24 hours on an agar plate before MALDI MS analysis.

Markers of Staph infection

The wound in one mouse showed no signs of infection and was completely healed after about one week. The second mouse did become infected and this was confirmed by the bacterial count.

The mass spectra from the infected mouse displayed the expected characteristic peaks for proteins from the bacterium, as well as other peaks originating from the mice. One peak in particular changed in abundance in a way that mirrored the degree of infection. It was identified as alpha-defensin which is a known antimicrobial peptide with broad activity against Gram-positive and Gram-negative bacteria. So, it might be possible to monitor its appearance as an indication of wound infection by Staphylococcus aureus.

A mass spectral peak corresponding to beta-defensin was also detected but this is less significant because this protein is expressed at many different locations around the body.

The researchers also examined low-molecular-weight compounds, typically with m/z values below 1000. A number of peaks with m/z values above 400 appeared after infection which were not characteristic bacterial peaks. Although they could not be explained at this time, they might also be useful as indicators of infection.

One peak that was identified was attributed to adenosine and its intensity mirrored the degree of infection. Adenosine is produced by adenosine synthase A and is a critical factor in regulating the host immune system. A second peak was attributed to lysophosphatidylserine, which is expressed to aid the defence against pathogens. A third, unidentified peak also correlated with the bacterial count.

So, a number of high- and low-molecular-weight compounds appear to be generated in response to the infection of a wound by Staphylcoccus aureus, although they will need to be confirmed in a much wider study. This is the first time that MALDI MS has been used in this type of study on wound infections. It is a simple technique with relatively little sample preparation and “could reduce the analysis time for analyzing both the immunological and bacterial protein and nonprotein parameters,” said Hu.