Biofilm/Persister/Stationary Phase Bacteria Cause More Severe Disease Than Log Phase Bacteria II Infection with Persister Forms of Staphylococcus aureus Causes a Chronic Persistent Skin Infection with More Severe Lesion that Takes Longer to Heal and is not Eradicated by the Current Recommended Treatment in Mice

Staphylococcus aureus is commonly associated with various wound infections [1]. Despite treatments, many patients experience recurrence of infection (see [2] for a review) due to the presence of persister cells (see [3] for a review on persister cells and infectious diseases). Persister cells downregulate their metabolic activities in stressed conditions (like saturated cultures or biofilms), entering a dormant-like stage, and hence are usually missed by antibiotics. Since antibiotics work by targeting specific physiological activity of the bacterial cells, they can only target cells that are active and dividing.

Numerous skin infections have been associated with a biofilm lifestyle of the infecting bacteria [4,5] and S. aureus biofilms have been a major area of study in skin wound infections. Persister cells are found within biofilms too, since the nutrient and oxygen limiting biofilm environment represents a stressed environment. Persister cells thus form a clinically relevant aspect of wound infections and it is important to be able to incorporate them into the models used to study wound infections. This study uses a mouse model to study persister cells of S. aureus in skin infections.

 

Experimental setup:. Infections were done by subcutaneous injection of the bacteria onto a shaved patch of skin with the same number of bacteria (10⁸ CFU). The skin lesion sizes were measured post infection upto 4 weeks. Histological comparison of the different infections was done by skin tissue dissection. To study the effect of treatment with doxycycline and rifampin, mice were treated one week post infection for seven days. Another system used to study the effect of persister cell infections was the C. elegans model. A nematode killing assay was performed to study to effect of the different infections on the lifespan of the worm.

 

Important Results:

 

Persister cells cause more severe infections compared to log phase cultures
In order to establish a model for persister cell infections, the authors infected mice with persister forms (saturated cultures and biofilm bacteria) and compared them to log phase culture infected mice. Mice infected with saturated culture and biofilm bacteria showed bigger lesions (almost 3 times in size) and more bacteria were recovered from these skin lesions than those infected with log phase cultures. In general, biofilm bacteria showed the most persistent infection, requiring at least one week longer to heal. The histopathology of these persister cell infections was also more severe with more crust formation, immune cell infiltration and focal lesions forming. The log phase bacterial infection only showed low levels of immune cell infiltration.

Thus, the persistent forms of bacteria cause a more severe and persistent infection as compared to log phase growing bacteria.

Treatment with the clinically recommended drug combination: A combination of doxycycline + rifampin is recommended for treatment of such recurrent skin infections. In order to test the efficacy of this combination on persister cell infections, mice infected with biofilm bacteria were exposed to this treatment for 7 days post infection. The treatment had no affect on the bacterial load, with the number of bacteria isolated from the infected tissue being similar for treated and non-treated mice. However, the skin lesion size did stall from growing in the treated mice, which showed only a 15% increase in lesion size whereas non-treated mice showed a 200% increase. Thus, although the treatment though doesn’t heal the lesions, it does prevent further development of the lesion.

Persister cell infections in C. elegans cause lower survival rate: Using a nematode killing assay, the persistent forms of S. aureus were found to be more virulent causing a lower survival rate in the case of the saturated phase culture infections as compared to log phase infections. At 2 days post infection, stationary phase bacteria-infected worms had a survival rate of 60%, whereas log phase bacteria-infected worms had a survival rate of 82%. This difference was seen in both cases of higher as well as lower inoculum (10⁶ vs 10⁴ CFU).

 

Interesting aspects of the study: Persister cells have been found to play a role in pathogenesis and have been described for numerous pathogens [6]. Thus, developing an understanding of these forms of bacteria and incorporating them in infection studies is relevant. This study aims at developing such a persistent infection model using saturated cultures and biofilm bacteria, which have higher persister cells, for mice skin infections. The study shows that these forms of bacteria can be used to mimic more severe and persistent forms of infections as compared to using log phase bacteria. This can have important implications in understanding S. aureus persistent infections and testing treatments that can tackle such infections. This study thus provides a potential model that can mimic such infections and be used as a platform to study them further.

 

References/Further Reading:

1. Tong, Steven YC, et al. “Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management.” Clinical microbiology reviews3 (2015): 603-661.
2. Creech, C. Buddy, Duha N. Al-Zubeidi, and Stephanie A. Fritz. “Prevention of recurrent staphylococcal skin infections.” Infectious Disease Clinics3 (2015): 429-464.
3. Lewis, Kim. “Persister cells, dormancy and infectious disease.” Nature Reviews Microbiology1 (2007): 48.
4. Zhao, Ge, et al. “Biofilms and inflammation in chronic wounds.” Advances in wound care7 (2013): 389-399.
5. Brandwein, Michael, Doron Steinberg, and Shiri Meshner. “Microbial biofilms and the human skin microbiome.” NPJ biofilms and microbiomes1 (2016): 3
6. Fisher, Robert A., Bridget Gollan, and Sophie Helaine. “Persistent bacterial infections and persister cells.” Nature Reviews Microbiology8 (2017): 453.

Tags: persistent skin infections, skin infections, staphylococcus aureus

doi: https://doi.org/10.1242/prelights.6617

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