Article Analysis: Antibiotic Resistant Bacteria in Hedgehogs

Article Reviewed: Emergence of methicillin resistance predates the clinical use of antibiotics, published online via Nature, January 2022.

In a previous blog, we discussed phage therapy as a potential treatment option for antibiotic resistant bacterial infections. As mentioned in that post, most well-known and documented antibiotic resistant bacterial species is MRSA (methicillin-resistant Staphylococcus aureus), which has been linked to hundreds of thousands of infections in the United States and Europe annually.

First, what is methicillin? Methicillin is a member of a group of antibiotics known as β-lactam antibiotics[2], named after the beta-lactam ring common to their structures (refer to square-shaped ring with the double oxygen bond in following image). Their mode of action is to prevent the development of the cell wall outside a bacteria's plasma membrane by disrupting the development of the peptidoglycan layer.[2] This layer is made up of amino acids (the building blocks of proteins and sugars.

Methicillin was developed after previous drugs such as penicillin lost effectiveness due to evolving bacterial resistance. In these earlier iterations of antibiotics, the drug was subjected to hydrolyses by enzymes created by the bacteria called β-lactamases. These enzymes essentially use water to break the beta-lactam ring and render the administered antibiotic harmless to the target bacterium.[2] As the number of bacteria that survive the antibiotic dosage increases, the conferred resistance to the drug also increases.

Methicillin, despite having a beta-lactam ring, was found to be resistant to degradation through β-lactamase hydrolyses and thus was used readily as a weapon against penicillin-resistant infections. Without getting too far into the weeds of the mechanism of methicillin, it affects the development of the cell wall by inhibiting peptidoglycan from crosslinking and forming the wall's structure.[3] The process of developing the cell wall is the product of a crosslinking reaction performed by a group called penicillin-binding proteins (PBPs).[3] In normal S. aureus cells there are four PBPs, but in methcillin-resistant S. aureus, a fifth, PBP2a, is present.[3] This extra penicillin-binding protein provides resistance to the mechanism of action by methicillin via a new gene, mecA, acquired from another bacteria of unknown origin.[4] Since the discovery of PBP2a, a second novel protein, PBP2c, has also been discovered.[4]

The generally agreed upon theory for the rise in MRSA stems from the overuse of antibiotics in people and livestock, with some evidence pointing to dairy cows as the initial group infected. According to Larsen, et al[4], with regards to mecC antibiotic-resistant S. aureus, the development of resistance was driven by natural selection in the wild hedgehog population.

Hedgehogs are a group of seventeen species of small mammal that are covered in spines for protection and can roll into a tight ball when threatened. They are native to Asia, Africa and Europe and have been labeled intrusive pests in other parts of the globe.[5] The African pygmy hedgehog is a crossbreed and common household pet that has been made illegal in areas where it can survive and threaten the native flora and fauna.

For this study, Larsen, et al. identify a dermatophyte called T. erinacai as a potential antagonist for S. aureus and the reason for the development of the PBP2c proteins that convey methicillin resistance.[4] Dermatophytes are a class of fungi that are common on the skin of animals because they require the protein keratin to survive. Keratin is the material that makes up hair and nails and hedgehog quills. Because of the large source of keratin present on hedgehogs, they are a common target for the fungus T. erinacai, which researchers have found produces a penicillinase-labile penicillin-like substance called penicillin G.[4]

These preliminary findings indicate that may hedgehogs have S. aureus (which can become MRSA) and a fungus that releases a penicillin analogue coexisting on their skin.[4] The proposed mechanism is that hedgehog skin is an environment where T. erinacai is essentially releasing an antibiotic that has subjected S. aureus to a selection process where bacteria equipped to survive contact with the antibiotic thrive and those that are susceptible perish.[4]

To investigate their belief that some form of MRSA may have a natural origin from the skin of hedgehogs, the researchers took swabs from the skin, feet, and nasal cavities of 276 hedgehogs throughout Europe and New Zealand.[4] These were then analyzed for the presence of MRSA.

Results (Positive for MRSA):
- 66% England & Wales
- 50% Czech Republic
- 50% Denmark
- 29% Portugal
- 6% New Zealand
- 0% Greece, Romania, Italy, France, and Spain

NOTE: New Zealand was included because hedgehogs have a large population there following introduction events in the late 1800s

From this data, the researchers concluded that the MRSA-carrying hedgehogs had 6 different clonal complexes (essentially different strains) that could be used to track evolutionary history.[4] They then tested the samples negative for MRSA for methicillin-susceptible strains of S. aureus.

Having identified that hedgehogs are carriers for MRSA and hypothesizing that T. erincai is causing natural selection for resistant bacteria, the researchers investigated the antibiotic properties of T. erincai. To do this, they grew cultures of T. erincai and checked for the presence of antibiotics, where they found penicillin G.[4] The researchers then added both MRSA and 'normal' strains of S. aureus to the cultures of T. erincai and observed the results. The results are seen in the following image.

How to read this image:
1. Each of the twelve circles above represents a plate (think petri dish) that has bacteria grown on it. When bacteria covers the entire plate, we call this a lawn. In the above plates, each is split into four quadrants and each quadrant has a different culture added to it via antibiotic disk (the smaller white circles). The darker circles around these antibiotic disks are zones of inhibition, or areas where bacteria cannot grow. The larger the dark circle, the farther from the antibiotic disc that bacterial growth is impeded.
2. Each line is a different culture of S. aureus. The top row is normal (or non-resistant) and the second and third rows are two cultures of MRSA.
3. The columns represent different versions of the bacterial culture. For the MRSA cultures, column one is the wild type (the culture as taken from the hedgehog) while the next three columns are the same cultures with different genes knocked out. Note that when these genes are knocked out, antibiotic resistance goes down.
4. The top row shows the inverse, with the normal S. aureus strain having genes added and conveying additional antibiotic resistance.
5. Finally, the triangle you see in the names of the cultures is a delta sign which is used to indicate the gene is knocked out. So MRSA with delta mecC means the gene that encodes for the PBP2c has been disrupted and the protein is no longer manufactured.

The data shown in the culture tested indicates that the dermatophyte T. erincai does produce penicillin G and it does function as an antibiotic. This also supports the notion that it could be the selection mechanism for antibiotic resistance S. aureus on hedgehogs.

To determine if hedgehogs do serve as local reservoirs for MRSA strains, the researchers then looked into the evolutionary lineages of the observed strains. This involved detailed investigations into current and historical samples searching for specific strains of MRSA. The science is pretty confusing on this, but essentially they show that there are numerous strains present prior to the introduction of modern antibiotics and these are the same strains found in hedgehog samples from the past and present.

In this image, the researchers are showing the three lineages of mecC MRSA they investigated along with their branching paths. The trees on the left show where each distinct strain branches and the filled in circle on the plot is the estimated last common ancestor. This can be thought of as the time where the split occurred that led to the new strains. The black and white boxes also show whether the the strain was detected in hedgehogs. As you can see, there are numerous strains of methicillin resistant S. aureus present in the wild, within the hedgehog population prior to the introduction of antibiotics.[4]

Finally, the researchers wanted to show that the variability found in mecC MRSA strains by comparing the observed strains found among humans and hedgehogs in Denmark. By looking at the above image, we see that the researchers divided Denmark into two regions, Jutland and the Major Islands. The results show that there are distinct patterns of dispersal based upon location and this was shown to be significant (p=0.0149).[4]  This implies the distribution is unlikely random chance and likely implies a correlation between the two.

With all the data provided here, the researchers have shown a compelling case for antibiotic resistant bacteria having origins beyond selection caused by human antibiotic usage. This would indicate that an approach to combat antibiotic resistance requires research beyond the human realm.

I think it is also important to note that this does not mean hedgehogs are dangerous to human health or a carrier of disease that can be readily spread to humans. Rather, hedgehogs have demonstrated that some other organisms may be providing the perfect environment for bacteria to evolve antibiotic resistance.

As the owner of a pygmy hedgehog, I'm not going to change my behavior when handling her, but rather find this to be a very interesting development.

References:
1. PubChem - Methicillin
2. Wikipedia - Methicillin
3.  Penicillin-Binding Protein 2a of Methicillin-Resistant Staphylococcus aureus
4. Emergence of methicillin resistance predates the clinical use of antibiotics
5. Wikipedia - Hedgehog